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Biochemical studies on the expression of overdominance at the phosphoglucomutase-2 locus in the Pacific… Pogson, Grant H. 1988

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BIOCHEMICAL STUDIES ON THE EXPRESSION OF OVERDOMINANCE AT THE PHOSPHOGLUCOMUTASE-2 LOCUS IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS (THUNBERG) by GRANT H. POGSON B.Sc. C a r l e t o n U n i v e r s i t y , Ottawa 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA December 1988 © Grant H. Pogson, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology The University of British Columbia Vancouver, Canada Date December 20, 1988 DE-6 (2/88) i i ABSTRACT Numerous s t u d i e s have documented s i g n i f i c a n t a s s o c i a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and f i t n e s s - r e l a t e d t r a i t s i n n a t u r a l p o p u l a t i o n s , but the e x p l a n a t i o n s f o r these p a t t e r n s remain unknown. The o b j e c t i v e of the p r e s e n t s tudy was to examine the m e r i t s of the overdominance h y p o t h e s i s as the mechanism r e s p o n s i b l e f o r a p o s i t i v e c o r r e l a t i o n between a d u l t body weight and h e t e r o z y g o s i t y i n v o l v i n g the phosphoglucomutase-2 (Pqm-2) l o c u s i n the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s . The k i n e t i c and s t r u c t u r a l p r o p e r t i e s of seven Pqm-2 genotypes were examined over p h y s i o l o g i c a l ranges of temperature and p H . S i g n i f i c a n t d i f f e r e n c e s were d e t e c t e d between Pqm-2 genotypes i n a v a r i e t y of enzymic p a r a m e t e r s , but these were l a r g e l y c o n f i n e d to genotypes p o s s e s s i n g the Pqm-2-92 a l l e l e , and h e t e r o z y g o t e s d i s p l a y e d s t r i c t i n t e r m e d i a c y for a l l f u n c t i o n a l and s t r u c t u r a l p r o p e r t i e s examined. The e x p r e s s i o n of m a r g i n a l overdominance a t the Pqm-2 l o c u s was c o n s i d e r e d u n l i k e l y because of the l i m i t e d scope of the observed v a r i a t i o n between a l l o z y m e s , and i t s i n c o m p a t i b i l i t y w i t h a l l e l i c f r e q u e n c i e s i n n a t u r a l p o p u l a t i o n s . The t h r e e most common h e t e r o z y g o t e s at the Pqm-2 l o c u s d i s p l a y e d the ex tremely unusual p r o p e r t y of overdominant enzyme a c t i v i t i e s . The magnitude of t h i s overdominance was s i m i l a r in the mant le and adduc tor muscle t i s s u e s , and was c o n s i s t e n t l y o b s e r v e d i n p o p u l a t i o n samples from two i n t e r t i d a l p o s i t i o n s in t h r e e d i f f e r e n t seasons . A p h y s i o l o g i c a l impact of the Pqm-2 polymorphism was demonstrated on the metabol i sm of g l y c o g e n , the b i o c h e m i c a l pathway in which PGM f u n c t i o n s . Pgm-2 genotypes e x h i b i t e d d i f f e r e n t c o n c e n t r a t i o n s of g lycogen i n t h e i r m a n t l e , but not t h e i r adduc tor muscle t i s s u e s , -which were d i r e c t l y a s s o c i a t e d w i t h v a r i a t i o n i n t h e i r PGM a c t i v i t y l e v e l s . I t was sugges ted t h a t Pgm-2 genotype-dependent enzyme a c t i v i t y v a r i a t i o n may a f f e c t r a t e s of g lycogen s y n t h e s i s by a p a r t i t i o n i n g e f f e c t at the g l u c o s e - 6 - p h o s p h a t e b r a n c h p o i n t . Non-random a s s o c i a t i o n s were d e t e c t e d between the PGM a c t i v i t i e s of Pgm-2 g e n o t y p i c groups and the a c t i v i t i e s of a d j a c e n t g lycogen s y n t h e s i s pathway enzymes, but none that c o u l d c l e a r l y account for the d i f f e r i n g g lycogen c o n c e n t r a t i o n s observed between genotypes . The e x p r e s s i o n of overdominance f o r PGM a c t i v i t y , and i t s impact on mantle g lycogen l e v e l s , p r o v i d e d d i r e c t ev idence f a v o r i n g the overdominance e x p l a n a t i o n as the cause of the l a r g e r body weights of h e t e r o z y g o t e s at the Pgm-2 l o c u s i n C r a s s o s t r e a g i g a s . i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES . ix ACKNOWLEDGEMENTS .' x i CHAPTER 1: GENERAL INTRODUCTION 1 The Study Animal 8 The Study Enzyme 11 Format of the T h e s i s 14 CHAPTER 2: BIOCHEMICAL CHARACTERIZATION OF PGM-2 GENOTYPES .15 INTRODUCTION .15 MATERIALS AND METHODS 21 RESULTS 33 E l e c t r o p h o r e s i s and Pqm-2 A l l e l e F r e q u e n c i e s 33 B i o c h e m i c a l P r o p e r t i e s of Pqm-2 Genotypes 41 DISCUSSION 71 CHAPTER 3: ENVIRONMENTAL AND GENOTYPIC EFFECTS ON PGM ACTIVITY 87 INTRODUCTION 87 MATERIALS AND METHODS 92 RESULTS 95 E f f e c t s of Season and I n t e r t i d a l P o s i t i o n 99 E f f e c t of Pqm-2 Genotype > 103 P r e d i c t e d E f f e c t s of a Pgm-2 N u l l A l l e l e 115 DISCUSSION 121 CHAPTER 4: PHYSIOLOGICAL EFFECTS OF THE PGM-2 LOCUS ON GLYCOGEN METABOLISM 140 INTRODUCTION 140 MATERIALS AND METHODS 145 RESULTS 149 E f f e c t s of Season and I n t e r t i d a l P o s i t i o n 153 E f f e c t of Pqm-2 Genotype 157 DISCUSSION 171 CHAPTER 5: ACTIVITY STRUCTURE OF THE GLYCOGEN SYNTHESIS PATHWAY 195 INTRODUCTION 195 MATERIALS AND METHODS 198 RESULTS - 202 E l e c t r o p h o r e s i s 202 C o r r e l a t i o n s between Pathway Enzyme A c t i v i t i e s 203 Pathway A c t i v i t i e s of Pqm-2 G e n o t y p i c Groups 206 M u l t i p l e R e g r e s s i o n A n a l y s e s 218 DISCUSSION 223 CHAPTER 6: GENERAL DISCUSSION 236 LITERATURE CITED . .248 v i L I S T OF TABLES T a b l e I . Pqm-2 a l l e l e f r e q u e n c i e s , c o n f o r m i t y to H a r d y -Weinberg e x p e c t a t i o n s , and h e t e r o z y g o t e d e f i c i e n c i e s a t the four sampl ing da te s . . . . 3 9 T a b l e I I . P u r i f i c a t i o n summary f o r the Pgm-2-104 a l l o z y m e . . 42 T a b l e I I I . E f f e c t of temperature on the Vmax(f ) /Vmax(r) r a t i o s of four Pgm-2 homozygotes 58 T a b l e I V . F - r a t i o s from a n a l y s e s of v a r i a n c e on PGM s p e c i f i c a c t i v i t y , PGM a c t i v i t y / g t i s s u e , and s o l u b l e p r o t e i n e x t r a c t e d / g t i s s u e i n the mantle and a d d u c t o r muscle t i s s u e s 97 T a b l e V . Seasona l v a r i a t i o n i n the mantle s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) of seven Pgm-2 genotypes .104 T a b l e V I . Seasona l v a r i a t i o n i n the adductor muscle s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) of seven Pqm-2 genotypes 106 T a b l e V I I . Decompos i t ion of Pqm-2 s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) i n t o enzyme a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s expres sed on a gram wet t i s s u e weight b a s i s .110 T a b l e V I I I . Comparison of enzyme a c t i v i t i e s ( u n i t s / m g p r o t e i n and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) of homozygote and h e t e r o z y g o t e c l a s s e s p o s s e s s i n g or l a c k i n g the Pgm-2-100 a l l e l e 113 T a b l e I X . P r e d i c t e d r e d u c t i o n s of enzyme a c t i v i t i e s ( u n i t s / m g and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) i n Pgm-2 homozygote c l a s s e s assuming a n u l l a l l e l e i s pre sen t a t a f requency of 0.044 117 T a b l e X . C o l l a p s e of the m u l t i - a l l e l i c Pgm-2 s t r u c t u r a l l o c u s polymorphism by a h y p o t h e t i c a l t i g h t l y - l i n k e d r e g u l a t o r y l o c u s s e g r e g a t i n g f o r two a l l e l e s 132 T a b l e X I . F - r a t i o s from a n a l y s e s of v a r i a n c e on g lycogen c o n c e n t r a t i o n s and PGM s p e c i f i c a c t i v i t i e s i n the mantle and adduc tor muscle t i s s u e s 151 T a b l e X I I . Combined e f f e c t s of season and i n t e r t i d a l p o s i t i o n on the mantle g lycogen c o n c e n t r a t i o n s (jxmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s 163 T a b l e X I I I . Combined e f f e c t s of season and i n t e r t i d a l p o s i t i o n on the adductor muscle g lycogen c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s . . . 1 6 7 T a b l e X I V . Glycogen c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) and s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) of homozygotes and h e t e r o z y g o t e s p o s s e s s i n g or l a c k i n g the Pqm-2-1 00 a l l e l e 169 T a b l e X V . K i n e t i c parameters i n the mantle t i s s u e s of the four homozygotes and the three h e t e r o z y g o t e s for the Pqm -2-100 a l l e l e 178 T a b l e X V I . Product-moment c o r r e l a t i o n c o e f f i c i e n t s between the a c t i v i t i e s of enzymes i n the g lycogen s y n t h e s i s pathway 204 v i i i T a b l e X V I I . F - r a t i o s from the a n a l y s e s of v a r i a n c e on the mant le a c t i v i t i e s of the g lycogen s y n t h e s i s pathway enzymes and mantle g lycogen c o n c e n t r a t i o n s 207 T a b l e X V I I I . A c t i v i t i e s ( u n i t s / g s o l u b l e p r o t e i n ) of HK, PGM, UDPGP, and GS of four Pqm-2 g e n o t y p i c c l a s s e s i n the low i n t e r t i d a l zone 210 T a b l e X I X . A c t i v i t i e s ( u n i t s / g s o l u b l e p r o t e i n ) of HK, PGM, UDPGP, and GS of four Pqm-2 g e n o t y p i c c l a s s e s i n the h i g h i n t e r t i d a l zone 212 T a b l e XX. R e s u l t s from the m u l t i p l e r e g r e s s i o n a n a l y s i s of the g lycogen s y n t h e s i s pathway enzyme a c t i v i t i e s on g lycogen l e v e l s i n the low i n t e r t i d a l sample 219 T a b l e X X I . R e s u l t s from the m u l t i p l e r e g r e s s i o n a n a l y s i s of the g lycogen s y n t h e s i s pathway enzyme a c t i v i t i e s on g lycogen l e v e l s in the h i g h i n t e r t i d a l sample 221 LIST OF FIGURES F i g u r e 1. E l e c t r o p h o r e t i c s t a i n i n g p a t t e r n s of o y s t e r PGM under (A) s t a n d a r d and (B) c a t a l y t i c r u n n i n g c o n d i t i o n s .35 F i g u r e 2. E f f e c t of temperature on the apparent M i c h a e l i s c o n s t a n t s ( i n MM) f o r g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and g l u c o s e - 1 , 6 - d i p h o s p h a t e (2; lower h a l f ) of seven Pqm-2 genotypes . . . 4 5 F i g u r e 3. E f f e c t of pH on the apparent M i c h a e l i s c o n s t a n t s ( i n MM) f o r g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and g l u c o s e - 1 , 6 - d i p h o s p h a t e (2; lower h a l f ) of seven Pqm-2 genotypes 49 F i g u r e 4. E f f e c t of temperature on the Vmax/Km r a t i o s of seven Pgm-2 genotypes for the forward r e a c t i o n d i r e c t i o n 54 F i g u r e 5. E f f e c t of pH on the Vmax/Km r a t i o s of seven Pgm-2 genotypes f o r the forward r e a c t i o n d i r e c t i o n . . . . . 5 6 F i g u r e 6. Apparent M i c h a e l i s c o n s t a n t s ( i n MM) f or g l u c o s e -6-phosphate of four Pqm-2 homozygotes e s t i m a t e d from the Haldane e q u a t i o n . . . 6 1 F i g u r e 7. PH-dependent a c t i v i t i e s of four Pqm-2 homozygotes a t 2 0 ° C 64 F i g u r e 8. Thermal i n a c t i v a t i o n p l o t s of seven Pqm-2 genotypes at 5 0 ° C 67 F i g u r e 9. E f f e c t of magnesium ion on the a c t i v i t i e s of four Pqm-2 homozygotes 69 X F i g u r e 10. E f f e c t of temperature on the e s t i m a t e d in v i v o apparent M i c h a e l i s c o n s t a n t s ( i n MM) f or g l u c o s e - 1 -phosphate (1; upper h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; lower h a l f ) of seven Pqm-2 genotypes 75 F i g u r e 11. E f f e c t of pH on the e s t i m a t e d rn v i v o apparent M i c h a e l i s c o n s t a n t s ( i n jiM) f o r g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; lower h a l f ) of seven Pqm-2 genotypes 77 F i g u r e 12. E f f e c t of season and i n t e r t i d a l p o s i t i o n on s p e c i f i c a c t i v i t y ( u n i t s / m g p r o t e i n ) , PGM a c t i v i t y ( u n i t s / g t i s s u e ) , and s o l u b l e p r o t e i n (mg/g t i s s u e ) i n the mantle and adduc tor muscle t i s s u e s 100 F i g u r e 13. Seasona l v a r i a t i o n i n the mantle and adduc tor muscle g lycogen c o n c e n t r a t i o n s (/nmoles g l u c o s y l u n i t s / g t i s s u e ) at the two i n t e r t i d a l p o s i t i o n s 154 F i g u r e 14. Seasona l v a r i a t i o n i n the mantle and adductor muscle g lycogen c o n c e n t r a t i o n s (jxmoles g l u c o s y l u n i t s / g t i s s u e ) of Pqm-2 homozygote and h e t e r o z y g o t e c l a s s e s . . . 1 5 9 F i g u r e 15. P r e d i c t e d f l u x advantage of Pgm-2 h e t e r o z y g o t e s and percent f l u x to g lycogen as a f u n c t i o n of the PGI/PGM a c t i v i t y r a t i o . . . . 1 8 4 x i ACKNOWLEDGEMENTS I would l i k e to thank P. M a c C l e l l a n d f o r p r o v i d i n g access to h i s o y s t e r l e a s e and to S. Tamm and K. Mesa f o r t h e i r a s s i s t a n c e i n the c o l l e c t i o n of samples . T . Mommsen, B . D e v l i n , E . Matsumoto, and J . G . H a l l p r o v i d e d v a l u a b l e a d v i c e on the p u r i f i c a t i o n of o y s t e r PGM, and on the e x p e r i m e n t a l d e s i g n and s t a t i s t i c a l a n a l y s i s of the enzyme k i n e t i c d a t a . S p e c i a l thanks are i n o r d e r to P.W. Hochachka f o r k i n d l y p e r m i t t i n g the use of h i s l a b o r a t o r y f a c i l i t i e s , and for welcoming me i n t o h i s r e s e a r c h g r o u p . Numerous s t u d e n t s and f a c u l t y members o f f e r e d v a l u a b l e s u g g e s t i o n s d u r i n g v a r i o u s s tages of my r e s e a r c h . I wish to express my g r a t i t u d e i n p a r t i c u l a r to B . D e v l i n , R. S u a r e z , and D. S c h l u t e r . I would l i k e to e s p e c i a l l y thank my s u p e r v i s o r , C . F . Wehrhahn, f o r h i s a d v i c e and c o n t i n u e d encouragement throughout my s t u d y . F i n a l l y , I would l i k e to acknowledge the f i n a n c i a l a s s i s t a n c e p r o v i d e d by a N . S . E . R . C . P o s t g r a d u a t e S c h o l a r s h i p , U n i v e r s i t y of B r i t i s h Columbia Graduate F e l l o w s h i p s , and N . S . E . R . C . O p e r a t i n g g r a n t s to C . F . Wehrhahn. 1 CHAPTER 1 GENERAL INTRODUCTION The e v o l u t i o n a r y p r o c e s s may be d e f i n e d as the c o n v e r s i o n of v a r i a t i o n among i n d i v i d u a l s w i t h i n a p o p u l a t i o n i n t o v a r i a t i o n between p o p u l a t i o n s and s p e c i e s , both s p a t i a l l y and t e m p o r a l l y (Lewontin 1974, p . 12) . I r r e s p e c t i v e of mechanism, a l l e v o l u t i o n a r y change has , and w i l l c o n t i n u e , to depend on the e x i s t e n c e of g e n e t i c v a r i a t i o n a f f e c t i n g the p h y s i o l o g i c a l , m o r p h o l o g i c a l and b e h a v i o r a l a t t r i b u t e s tha t c o n s t i t u t e the d i s t i n c t i v e n e s s of b i o l o g i c a l s p e c i e s . As argued e l o q u e n t l y by Mayr (1982) , the c o n c e p t u a l r e v o l u t i o n p i o n e e r e d by Darwin (1859) i n v o l v e d the replacement of a " t y p o l o g i c a l " w i t h a " p o p u l a t i o n a l " w o r l d - v i e w tha t r e c o g n i z e d the importance of t h i s n a t u r a l l y - o c c u r r i n g v a r i a t i o n between i n d i v i d u a l s as one of the fundamental c h a r a c t e r i s t i c s of b i o l o g i c a l l i f e . The c r u c i a l r o l e p l a y e d by g e n e t i c v a r i a t i o n in the a d o p t i o n of t h i s new p e r s p e c t i v e i s a prime reason tha t i t s s tudy has been, and w i l l a lways be , a p r i n c i p a l focus of e v o l u t i o n a r y s t u d i e s . A l l e v o l u t i o n a r y t h e o r i e s r e l y d i r e c t l y on the presence of i n t r a - or i n t e r - p o p u l a t i o n a l v a r i a t i o n , but d i f f e r i n how these changes o c c u r . A major source of pas t and presen t c o n t r o v e r s i e s concerns the r e l a t i v e importance of D a r w i n i a n n a t u r a l s e l e c t i o n as an agent of the e v o l u t i o n a r y p r o c e s s . E a r l y i n the neo-s y n t h e t i c p e r i o d a s i g n i f i c a n t r o l e was a t t r i b u t e d to random 2 g e n e t i c d r i f t i n p r o d u c i n g what were then i n t e r p r e t e d as non-a d a p t i v e d i f f e r e n c e s between subspec i e s and geograph ic races ( e . g . Robson and R i c h a r d s 1936). These sent iments were r e f l e c t e d i n the w r i t i n g s of the founders of the n e o - D a r w i n i s t movement a t t h i s t ime ( e . g . Dobzhansky 1937; Simpson 1944). G o u l d (1983) has p o i n t e d out how l a t e r p u b l i c a t i o n s of these same a u t h o r s s h i f t e d from a p l u r a l i s t i c to a s t r i c t a d a p t a t i o n i s t i n t e r p r e t a t i o n of the same p a t t e r n s tha t he termed the "hardening of the modern s y n t h e s i s " . A s i m i l a r t r e n d o c c u r r e d i n the work of S e w a l l Wright over t h i s same p e r i o d ( P r o v i n e 1986). Only r e c e n t l y has the a d a p t a t i o n i s t paradigm ( c f . Gould and Lewont in 1979) g i v e n way to a l l o w the c o n s i d e r a t i o n of a l t e r n a t i v e modes of e v o l u t i o n a r y change . In a d d i t i o n to d i sagreements over i t s r e l a t i v e importance i n d i r e c t i n g the e v o l u t i o n a r y p r o c e s s , another l o n g - s t a n d i n g d i s p u t e has c e n t e r e d on how s e l e c t i o n o p e r a t e s i n n a t u r a l p o p u l a t i o n s . Dobzhansky (1955) summarized these oppos ing views i n what he termed the " c l a s s i c a l " and the "balanced" hypotheses of p o p u l a t i o n s t r u c t u r e . Proponents of the c l a s s i c a l s c h o o l c o n t e n d tha t n a t u r a l s e l e c t i o n m a i n l y s erves a " p u r i f y i n g " f u n c t i o n , by removing d e l e t e r i o u s mutants from p o p u l a t i o n s . In c o n t r a s t , the b a l a n c e d view h o l d s tha t the major r o l e of n a t u r a l s e l e c t i o n i s to a c t i v e l y m a i n t a i n g e n e t i c v a r i a t i o n w i t h i n p o p u l a t i o n s through v a r i o u s types of b a l a n c i n g s e l e c t i o n ( e . g . overdominance , f requency-dependent s e l e c t i o n , v a r i a b l e s e l e c t i o n over time and s p a c e ) . Genie h e t e r o z y g o s i t y i s thus viewed by the 3 "balanced" s c h o o l as a d a p t i v e and s t a b l e (g iven s p e c i f i c e n v i r o n m e n t a l c i r c u m s t a n c e s ) , whereas the " c l a s s i c a l " s c h o o l p r e d i c t s tha t h e t e r o z y g o s i t y i s t r a n s i e n t and c o n t r i b u t e s l i t t l e to p o p u l a t i o n adaptedness . Lewont in (1974) has argued t h a t the c u r r e n t c o n t r o v e r s y over the a d a p t i v e s i g n i f i c a n c e of the h i g h l e v e l s of enzyme polymorphism observed i n n a t u r a l p o p u l a t i o n s , uncovered by e l e c t r o p h o r e t i c p r o c e d u r e s , i s s i m p l y a c o n t i n u a t i o n of t h i s d i s p u t e , but now waged at the m o l e c u l a r l e v e l . Arguments based on the importance of p u r i f y i n g s e l e c t i o n i n d e t e r m i n i n g v a r i o u s a s p e c t s of p r o t e i n s t r u c t u r e and f u n c t i o n form a c o r n e r s t o n e of the n e u t r a l theory of m o l e c u l a r e v o l u t i o n (Kimura 1983). N e u t r a l theory has s t i m u l a t e d heated d i s c u s s i o n in the f i e l d s of p o p u l a t i o n and e v o l u t i o n a r y g e n e t i c s . Comparisons of the q u a n t i t i e s and d i s t r i b u t i o n s of a l l o z y m i c ( i . e . a l l e l i c isozymes) v a r i a t i o n w i t h i n and between s p e c i e s to p r e d i c t i o n s of v a r i o u s n e u t r a l and s e l e c t i v e models has dominated these areas of s tudy f o r many years (see d i s c u s s i o n s in Lewont in 1974; A y a l a et a l . 1974; Ne i 1975; Ewens 1977; G i l l e s p i e 1978; Nevo 1978; W i l l s 1981; Kimura 1983; Nei and Koehn 1983; Ohta and Aoki 1985). A review of the ev idence tha t has been g a r n e r e d for and a g a i n s t v a r i o u s n e u t r a l p r e d i c t i o n s w i l l not be p r e s e n t e d h e r e . S u f f i c e i t to say t h a t i n d i r e c t ( c f . McDonald 1983) or s t a t i s t i c a l approaches have not been s u c c e s s f u l i n r e s o l v i n g t h i s c o n t r o v e r s y , s i n c e both n e u t r a l and s e l e c t i o n t h e o r y are s u f f i c i e n t l y robust to account f o r v i r t u a l l y any observed 4 d i s t r i b u t i o n of a l l e l i c po lymorphism. An a l t e r n a t i v e r e s e a r c h s t r a t e g y t h a t e v o l v e d over the same p e r i o d i n v o l v e d the d i r e c t measurement of n a t u r a l s e l e c t i o n on a l l o z y m i c v a r i a t i o n through the comparison of the b i o c h e m i c a l and p h y s i o l o g i c a l a t t r i b u t e s of enzyme genotypes which , through t h e i r i n t e r a c t i o n s w i t h e n v i r o n m e n t a l v a r i a b l e s , may g i v e r i s e to f i t n e s s d i f f e r e n c e s r e l e v a n t to the a d a p t i v e s i g n i f i c a n c e of the polymorphism s t u d i e d ( c f . C l a r k e 1975; Koehn 1978). A l t h o u g h concerned i n i t i a l l y w i t h t e s t i n g the v a l i d i t y of n e u t r a l t h e o r y , t h i s approach has deve loped i n t o a s e p a r a t e d i s c i p l i n e of e v o l u t i o n a r y g e n e t i c s (see Watt 1985a) and has been s u c c e s s f u l l y a p p l i e d to polymorphisms a t the aminopept idase -1 l o c u s i n the b lue m u s s e l , M y t i l u s e d u l i s (Koehn, Newel l and Immerman 1980; Koehn and Immerman 1981; Koehn and S i e b e n a l l e r 1981; H i l b i s h , Deaton and Koehn 1982; H i l b i s h and Koehn 1985; reviewed by Koehn and H i l b i s h 1987), the l a c t a t e dehydrogenase-B l o c u s in the k i l l i f i s h , Fundulus h e t e r o c l i t u s ( P l a c e and Powers 1979, 1984a, 1984b; D i M i c h e l e and Powers 1982a, 1982b; rev iewed by Powers, D i M i c h e l e and P l a c e 1983), and the phosphoglucose isomerase l o c u s i n C o l i a s b u t t e r f l i e s (Watt 1977, 1983; Watt , C a s s i n and Swan 1983; Wat t , C a r t e r and Blower 1985; rev iewed i n Watt 1985a, 1985b). The g r e a t s t r e n g t h of these s t u d i e s l i e in t h e i r m e c h a n i s t i c l i n k i n g of a l l e l i c v a r i a t i o n onto p h e n o t y p i c " c h a r a c t e r s t a t e s " ( c f . Lewont in 1972) that may i n t u r n be exposed to the s e l e c t i v e p r o c e s s . 5 E x a m i n a t i o n of the impact of enzyme polymorphisms on p h e n o t y p i c c h a r a c t e r s has r e c e n t l y expanded to c o n s i d e r m u l t i p l e - l o c u s e f f e c t s . In these s t u d i e s , i n d i v i d u a l s a r e s c o r e d f o r t h e i r genotypes at a s m a l l number of e l e c t r o p h o r e t i c l o c i ( u s u a l l y 5 to 7) and p o o l e d i n t o a s e r i e s of d i s c r e t e h e t e r o z y g o s i t y c l a s s e s . R e l a t i o n s h i p s between the degree of enzyme h e t e r o z y g o s i t y and v a r i o u s p h e n o t y p i c and p h y s i o l o g i c a l parameters are then examined by s t a n d a r d l i n e a r r e g r e s s i o n p r o c e d u r e s . F o l l o w i n g t h i s b a s i c p r o t o c o l , a l a r g e number of s t u d i e s have demonstrated s i g n i f i c a n t c o r r e l a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and d i f f e r e n t m o r p h o l o g i c a l , p h y s i o l o g i c a l and f i t n e s s - r e l a t e d t r a i t s in a wide d i v e r s i t y of organisms (reviewed by M i t t o n and G r a n t 1984; Zouros and F o l t z 1987). These a s s o c i a t i o n s have been most e x t e n s i v e l y documented in marine b i v a l v e s . In these o r g a n i s m s , m u l t i p l e - l o c u s h e t e r o z y g o s i t y has been p o s i t i v e l y c o r r e l a t e d wi th growth r a t e (S ingh and Zouros 1978; Z o u r o s , S i n g h and M i l e s 1980; F u j i o 1982; Green et a l . 1983; Koehn and Gaf fney 1984; Koehn, D i e h l and S c o t t 1988), v i a b i l i t y (Zouros et a l . 1983; D i e h l and Koehn 1985), and f e c u n d i t y (Rodhouse et a l . 1986), and n e g a t i v e l y c o r r e l a t e d w i t h r a t e s of oxygen consumption (Koehn and Shumway 1982; G a r t o n , Koehn and S c o t t 1984; D i e h l et a l . 1985), p r o t e i n t u r n o v e r (Hawkins, Bayne and Day 1986), and weight l o s s under n u t r i t i v e s t r e s s (Rodhouse and Gaf fney 1984). The u n d e r l y i n g c a u s e ( s ) of these r e l a t i o n s h i p s i s unknown. One p o s s i b i l i t y i s t h a t h e t e r o z y g o t e s a t these enzyme l o c i are 6 f u n c t i o n a l l y s u p e r i o r to homozygotes (overdominance ) . The overdominant e f f e c t s at each l o c u s are a d d i t i v e , and hence when poo led t o g e t h e r r e s u l t i n a more e f f i c i e n t and b u f f e r e d phenotype . An a l t e r n a t i v e e x p l a n a t i o n i s tha t these c o r r e l a t i o n s are not caused by the e l e c t r o p h o r e t i c l o c i themse lves , but i n s t e a d by t i g h t l y - l i n k e d l o c i tha t are s e g r e g a t i n g f o r d e l e t e r i o u s r e c e s s i v e a l l e l e s ( a s s o c i a t i v e overdominance ) . I f l i n k a g e d i s e q u i l i b r i u m e x i s t s between these l o c i , a p r o p o r t i o n of homozygotes for these d e l e t e r i o u s a l l e l e s are expected to be presen t w i t h i n the homozygous, but not the h e t e r o z y g o u s , geno typ ic groups at the e l e c t r o p h o r e t i c l o c i examined. T h e r e f o r e , d e t r i m e n t a l p h e n o t y p i c e f f e c t s would be m a n i f e s t e d i n the homozygote c l a s s e s tha t are r e f l e c t e d in the m u l t i p l e - l o c u s r e l a t i o n s h i p , but no d i r e c t advantage i s expected by h e t e r o z y g o s i t y per se . A r e l a t e d h y p o t h e s i s i s t h a t the p a t t e r n s are caused by i n b r e e d i n g , which i n s e v e r a l s p e c i e s of marine b i v a l v e s has been shown to r e s u l t i n decreased l a r v a l v i a b i l i t y and growth ( e . g . Longwel l and S t i l e s 1973; B e a t t i e et a l . 1987). A c c o r d i n g to t h i s e x p l a n a t i o n , the reduced performance of enzyme homozygotes i s s i m p l y a m a n i f e s t a t i o n of i n b r e e d i n g d e p r e s s i o n . O b v i o u s l y , t h i s h y p o t h e s i s r e q u i r e s the e x p r e s s i o n of s i g n i f i c a n t i n b r e e d i n g c o e f f i c i e n t s i n the s tudy p o p u l a t i o n . T h i s c o r o l l a r y i s c e r t a i n l y met by marine b i v a l v e s , i n which marked h e t e r o z y g o t e d e f i c i e n c i e s at e l e c t r o p h o r e t i c l o c i are a common f e a t u r e of t h e i r p o p u l a t i o n s t u c t u r e s , p a r t i c u l a r l y at the e a r l y 7 p o s t - s e t t l e m e n t s tage (see Zouros and F o l t z 1984; S ingh and Green 1984). A f i n a l e x p l a n a t i o n f o r these p a t t e r n s i s that they are caused by the undetec ted presence of n u l l a l l e l e s ( p r o d u c i n g n o n - f u n c t i o n a l enzyme p r o d u c t s ) at the e l e c t r o p h o r e t i c l o c i s t u d i e d . The m i s c l a s s i f i c a t i o n of n u l l h e t e r o z y g o t e s as homozygotes c o u l d thus account f o r 1) the h e t e r o z y g o t e d e f i c i e n c i e s , and 2) the s u p e r i o r p r o p e r t i e s of h e t e r o z y g o t e s for two f u n c t i o n a l a l l e l e s , because homozygote c l a s s e s would c o n t a i n a percentage of these low a c t i v i t y n u l l h e t e r o z y g o t e s . E s t a b l i s h i n g the cause of r e l a t i o n s h i p s between m u l t i p l e -l o c u s h e t e r o z y g o s i t y and f i t n e s s - r e l a t e d t r a i t s i s a matter of t h e o r e t i c a l and p r a c t i c a l i m p o r t a n c e . On the t h e o r e t i c a l s i d e , d e m o n s t r a t i n g tha t the e l e c t r o p h o r e t i c l o c i are r e s p o n s i b l e c o u l d s i m u l t a n e o u s l y prove the f u n c t i o n a l s i g n i f i c a n c e of a l a r g e number of enzyme polymorphisms tha t would s t r a i n the c r e d i b i l i t y of the n e u t r a l t h e o r y . U n d e r s t a n d i n g the p o t e n t i a l s e l e c t i v e b a s i s f o r the maintenance of t h i s g e n e t i c v a r i a t i o n i s p a r t i c u l a r l y r e l e v a n t , c o n s i d e r i n g the l a r g e d i v e r s i t y of s p e c i e s i n v o l v e d in these s t u d i e s . D i s t i n g u i s h i n g between these a l t e r n a t i v e hypotheses i s a l s o of v i t a l importance for s e l e c t i v e b r e e d i n g programs. B r e e d i n g methodolog ies for the improvement of c o m m e r c i a l l y v a l u a b l e t r a i t s would d i f f e r g r e a t l y i f overdominance , r a t h e r than a s s o c i a t i v e overdominance , was expres sed at the l o c i examined in these s t u d i e s . The o b j e c t i v e of my study was to determine i f ev idence 8 f a v o r i n g the overdominance h y p o t h e s i s c o u l d be o b t a i n e d for an enzyme l o c u s i n v o l v e d i n a m u l t i p l e - l o c u s h e t e r o z y g o s i t y r e l a t i o n s h i p through an examinat ion of the b i o c h e m i c a l and p h y s i o l o g i c a l p r o p e r t i e s of homozygous and he terozygous geno types . I chose to s tudy the phosphoglucomutase-2 l o c u s i n the P a c i f i c o y s t e r , C r a s s o s t r e a q i g a s . THE STUDY ANIMAL The Japanese , or P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s (Thunberg) i s a t emperate , i n t e r t i d a l s p e c i e s n a t i v e to the western P a c i f i c where i t s geograph ic range extends from China to the southern U . S . S . R . , and i n c l u d e s the Japanese a r c h i p e l a g o ( S t e n z e l 1971). T h i s s p e c i e s was i n t r o d u c e d to the west coas t of N o r t h Amer ica a t the t u r n of the c e n t u r y for commerc ia l purposes and has become w e l l e s t a b l i s h e d in i s o l a t e d pocket s of B r i t i s h Columbia and Washington s t a t e (Quayle 1969). The p r i n c i p a l f a c t o r l i m i t i n g the c o l o n i z a t i o n of t h i s new h a b i t a t by C . q i g a s has been i t s i n a b i l i t y to spawn s u c c e s s f u l l y . R e p r o d u c t i o n i n the P a c i f i c o y s t e r r e q u i r e s warmer summer water t emperatures ( 2 1 ° C or h i g h e r ) than u s u a l l y e x p e r i e n c e d i n t h i s geographic r e g i o n (Quayle 1969). The study p o p u l a t i o n was l o c a t e d on a p r i v a t e o y s t e r l e a s e i n Nanoose Bay, s i t u a t e d on the e a s t e r n s i d e of Vancouver I s l a n d 20 km n o r t h of Nanaimo, B r i t i s h C o l u m b i a . O r i g i n a l l y e s t a b l i s h e d from P e n d r e l l Sound b r e e d i n g s t o c k , c o n t i n u o u s p r o d u c t i o n from t h i s l e a s e i s dependent on supplement ing the n a t u r a l r e p r o d u c t i o n of the bay w i t h seed from 9 l o c a l s u p p l i e r s (P. M a c C l e l l a n d , p e r s o n a l c o m m u n i c a t i o n ) . Sex d e t e r m i n a t i o n i n C r a s s o s t r e a g i g a s , as i n o ther o y s t e r s , i s complex, r e s p o n d i n g to both g e n e t i c and e n v i r o n m e n t a l i n f l u e n c e s ( G a l t s o f f 1964; Haley 1977; Buroker 1983). The sexes are u s u a l l y s e p a r a t e , but h e r m a p h r o d i t i c i n d i v i d u a l s are g e n e r a l l y d e t e c t e d a t low f r e q u e n c i e s . The p r e v a i l i n g p a t t e r n of sex change i n the genus C r a s s o s t r e a i s p r o t a n d r o u s ; young an imal s b e g i n n i n g l i f e as males but s w i t c h i n g to females as they grow o l d e r . F e r t i l i z a t i o n o c c u r s e x t e r n a l l y , and a f t e r a p l a n k t o n i c l a r v a l s tage r a n g i n g from 15 to 30 d a y s , the l a r v a e s e l e c t a s u i t a b l e s u b s t r a t e to which they adhere , metamorphose, and beg in t h e i r s e s s i l e a d u l t l i f e . E x c e l l e n t d e s c r i p t i o n s of the b i o l o g y of t h i s s p e c i e s , and the c l o s e l y r e l a t e d C . v i r q i n i c a , may be found i n Yonge (1960) , G a l t s o f f (1964) , and Quayle (1969) . The P a c i f i c o y s t e r i s a f a c u l t a t i v e anaerobe , capab le of s u r v i v i n g f o r p r o l o n g e d p e r i o d s i n the complete absence of oxygen. The a b i l i t y of marine b i v a l v e s to t o l e r a t e anox ic c o n d i t i o n s i s a c c o m p l i s h e d by 2 0 - f o l d r e d u c t i o n s i n t h e i r b a s a l m e t a b o l i c r a t e s and the u t i l i z a t i o n of nove l m e t a b o l i c pathways g e n e r a t i n g ATP by s u b s t r a t e - l e v e l p h o s p h o r y l a t i o n s (reviewed by de Zwaan 1983). V i r t u a l l y a l l a s p e c t s of metabol i sm i n C . g igas undergo pronounced seasona l f l u c t u a t i o n s which are i n t e g r a t e d w i t h p r e v a i l i n g a b i o t i c c o n d i t i o n s , the a v a i l a b i l i t y of f o o d , and the annua l c y c l e of r e p r o d u c t i o n (reviewed by Gabbott 1983). 10 A dominant s e a s o n a l c y c l e in marine b i v a l v e s , c l o s e l y t i e d to r e p r o d u c t i o n , i n v o l v e s the s y n t h e s i s and d e g r a d a t i o n of g l y c o g e n . The t i m i n g of t h i s c y c l e v a r i e s between d i f f e r e n t s p e c i e s depending on t h e i r spawning season . In C . g i g a s , g lycogen accumulates i n the mantle and d i g e s t i v e g l a n d i n the f a l l and e a r l y s p r i n g and i s degraded i n the e a r l y summer for gametogenesis p r i o r to spawning in l a t e J u l y and August (Quayle 1969). S i n c e phosphoglucomutase f u n c t i o n s i n g lycogen m e t a b o l i s m , a p h y s i o l o g i c a l e f f e c t of the Pgm-2 polymorphism must be expres sed through the d i f f e r e n t i a l a b i l i t i e s of genotypes to s y n t h e s i z e or degrade g l y c o g e n . F i t n e s s - r e l a t e d d i f f e r e n c e s between Pqm-2 genotypes may in t u r n be e x p r e s s e d through these e f f e c t s on t i s s u e g lycogen l e v e l s because of the c e n t r a l r o l e p l a y e d by t h i s c a r b o h y d r a t e i n the energy metabol i sm of o y s t e r s (Gabbott 1975). As g e n e r a l l y found for o ther marine i n v e r t e b r a t e s , C r a s s o s t r e a g i g a s possesses s u b s t a n t i a l l e v e l s of a l l o z y m i c v a r i a t i o n . In f i v e e l e c t r o p h o r e t i c s t u d i e s on t h i s s p e c i e s summarized by O z a k i and F u j i o (1985) , an average of 53% of the enzyme l o c i examined were po lymorphic and the mean h e t e r o z y g o s i t y per i n d i v i d u a l was 19.4%. C i r c u m s t a n t i a l ev idence f a v o r i n g a net h e t e r o z y g o t e advantage for growth a n d / o r v i a b i l i t y has been suggested for a number of po lymorph ic l o c i i n C . g i g a s . These i n c l u d e l e u c i n e aminopept idase (Nagaya, S a s a k i and F u j i n o 1978), c a t a l a s e ( F u j i o , Nakamura and S u g i t a 1979), an u n i d e n t i f i e d muscle p r o t e i n (Buroker 1979), and a s p a r t a t e 11 a m i n o t r a n s f e r a s e ( S u g i t a and F u j i o 1982). M u l t i p l e - l o c u s r e l a t i o n s h i p s have not been e x t e n s i v e l y s t u d i e d i n C . g i g a s . However, a f t e r p o o l i n g da ta from 20 w i l d Japanese p o p u l a t i o n s , F u j i o (1982) observed a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between h e t e r o z y g o s i t y at f i v e enzyme l o c i and a d u l t body we ight . The Pgm-2 l o c u s was i n v o l v e d i n t h i s r e l a t i o n s h i p ; h e t e r o z y g o t e s e x h i b i t e d g r e a t e r body weights than homozygotes i n 18 of the 20 p o p u l a t i o n s . Based on the r e s u l t s of F u j i o ' s (1982) s t u d y , I set out to determine i f overdominance was indeed r e s p o n s i b l e for the h i g h e r growth r a t e s of Pqm-2 h e t e r o z y g o t e s . THE STUDY ENZYME Phosphoglucomutase (PGM, E . C . 2 . 7 . 5 . 1 , a l p h a - D - g l u c o s e - 1 -phosphate : a l p h a - D - g l u c o s e - 1 , 6 - d i p h o s p h a t e p h o s p h o t r a n s f e r a s e ) c a t a l y z e s the i n t e r c o n v e r s i o n of g l u c o s e - 1 - p h o s p h a t e (G1P) and g l u c o s e - 6 - p h o s p h a t e (G6P) i n presence of g l u c o s e - 1 , 6 - d i p h o s p h a t e and magnesium i o n . A comprehensive review of i t s s t u c t u r a l and f u n c t i o n a l p r o p e r t i e s may be found i n Ray and Peck (1972) . PGM i s a monomeric enzyme, e x h i b i t i n g m o l e c u l a r weights from d i f f e r e n t organisms r a n g i n g from 62,000-67,000 d a l t o n s . The r a b b i t muscle enzyme's complete sequence of 561 amino a c i d s has r e c e n t l y been de termined by Ray et a l . (1983) . N a j j a r and Pullman" (1954) f i r s t demonstrated tha t PGM may e x i s t i n p h o s p h o r y l a t e d and d e p h o s p h o r y l a t e d s t a t e s , r e p r e s e n t i n g a c t i v e and i n a c t i v e forms of the enzyme, r e s p e c t i v e l y . The d i s c o v e r y t h a t g l u c o s e - 1 , 6 - d i p h o s p h a t e ' s s o l e f u n c t i o n was to c o n v e r t the 12 dephospho i n t o the phosphoenzyme l e d to u n c e r t a i n t y s u r r o u n d i n g PGM's r e a c t i o n mechanism. Ray and R o s c e l l i (1964a) showed tha t the dephosphoenzyme of r a b b i t muscle PGM was formed once every 20 c a t a l y t i c c y c l e s , hence , the r e a c t i o n i n t h i s s p e c i e s approx imates a " u n i - u n i " mechanism. However, Hanabusa et a l . (1966) found tha t PGM e x t r a c t e d from b a c t e r i a d i s p l a y e d a " p i n g -pong" mechanism: the d i phosphat e d i s s o c i a t e d f r e q u e n t l y from the enzyme's c e n t r a l r e a c t i o n complex, thus a c t i n g as the " f i r s t produc t" and the "second s u b s t r a t e " i n the scheme proposed by C l e l a n d (1963) . S i m i l a r p a t t e r n s have been d e s c r i b e d d u r i n g the l e s s e f f i c i e n t i n t e r c o n v e r s i o n of o t h e r 5- and 6 -carbon sugar phosphates by PGM (Passonneau et a l . 1969). T h e r e f o r e , phosphoglucomutase f u n c t i o n s a l o n g a cont inuum between these extremes depending on i t s p h y l o g e n e t i c o r i g i n , the s u b s t r a t e s i n v o l v e d , and the p a r t i c u l a r assay c o n d i t i o n s (Ray and Peck 1972). The r e a c t i o n c a t a l y z e d by PGM i s f r e e l y r e v e r s i b l e , but i t s e q u i l i b r i u m c o n s t a n t s t r o n g l y f a v o r s the c o n v e r s i o n of g l u c o s e -1-phosphate to g l u c o s e - 6 - p h o s p h a t e (Keq = [G6P]/ [G1P] = 1 7 . 2 ) . Because of i t s r e v e r s i b i l i t y , PGM p a r t i c i p a t e s i n both the s y n t h e s i s and d e g r a d a t i o n of g l y c o g e n . L a c k i n g any r e g u l a t o r y p r o p e r t i e s , PGM's m e t a b o l i c r o l e i s to respond e f f i c i e n t l y to f l u x r a t e s de termined by t h r e e c l o s e l y - p o s i t i o n e d enzymes whose a c t i v i t i e s are under s t r i n g e n t r e g u l a t o r y c o n t r o l : g lycogen s y n t h e t a s e , g lycogen p h o s p h o r y l a s e , and p h o s p h o f r u c t o k i n a s e . The enzymic parameter most r e l e v a n t for PGM's c a t a l y t i c f u n c t i o n i s 13 i t s Vmax/Km r a t i o , s i n c e the enzyme i s u n l i k e l y to be s a t u r a t e d i n v i v o (see A t k i n s o n 1977), and v i a i t s compos i te na ture Vmax s i m u l t a n e o u s l y i n c o r p o r a t e s s t e a d y - s t a t e a c t i v i t y l e v e l s (Hoffman 1981; Watt 1983). T h e r e f o r e , f u n c t i o n a l d i f f e r e n c e s between Pqm-2 genotypes of p h y s i o l o g i c a l s i g n i f i c a n c e are most l i k e l y to be expres sed through these Vmax/Km r a t i o s . Phosphoglucomutase i s a w i d e l y po lymorph ic enzyme, g e n e t i c v a r i a n t s h a v i n g been r e p o r t e d i n a d i v e r s i t y of organisms (see G a u l d i e 1984). A l l e l i c isozymes of PGM have been c h a r a c t e r i z e d from D r o s o p h i l a melanoqaster ( F u c c i et a l . 1979) and the anemone, M e t r i d i u m s e n i l e (Hoffman 1985). The b i o c h e m i c a l d i f f e r e n c e s d e t e c t e d between Pgm genotypes i n both s p e c i e s were l i m i t e d and thus p r o v i d e d l i t t l e support f o r the a d a p t i v e s i g n i f i c a n c e of e i t h e r po lymorphism. PGM i s a h i g h l y po lymorph ic enzyme i n marine b i v a l v e s , and has been i n c l u d e d i n v i r t u a l l y a l l s t u d i e s i n v o l v i n g m u l t i p l e - l o c u s h e t e r o z y g o s i t y in these o r g a n i s m s . In two of these s t u d i e s , l o c u s - s p e c i f i c comparisons of homozygotes and h e t e r o z y g o t e s d e t e c t e d s i g n i f i c a n t d i f f e r e n c e s o n l y between Pgm genotypes ( e . g . Rodhouse and Gaf fney 1984; D i e h l et a l . 1985). The s i g n i f i c a n t e f f e c t s of h e t e r o z y g o s i t y for t h i s enzyme i n o ther b i v a l v e s p e c i e s suggests t h a t b i o c h e m i c a l d i f f e r e n c e s c o u l d e x i s t between a l l e l i c v a r i a n t s a t the Pqm-2 l o c u s in C . g igas that may account for the l a r g e r body weights of h e t e r o z y g o t e s r e p o r t e d by F u j i o (1982) . 1 4 FORMAT OF THE THESIS The t h e s i s has been o r g a n i z e d i n t o a s e r i e s of d i s c r e t e c h a p t e r s . Chapter 2 p r e s e n t s the p u r i f i c a t i o n and b i o c h e m i c a l p r o p e r t i e s of seven Pqm-2 genotypes . Chapter 3 compares the mantle and adductor muscle s p e c i f i c a c t i v i t i e s of Pgm-2 genotypes sampled from two i n t e r t i d a l p o s i t i o n s i n t h r e e seasons from the Nanoose Bay study p o p u l a t i o n . An assessment of the p h y s i o l o g i c a l e f f e c t s of t h i s polymorphism on g lycogen metabol i sm i s the s u b j e c t of Chapter 4. Chapter 5 a n a l y z e s the a c t i v i t y s t r u c t u r e of the g lycogen s y n t h e s i s pathway to a l l o w a more d e t a i l e d examinat ion of the e f f e c t s of the Pqm-2 l o c u s on the s y n t h e s i s of g l y c o g e n . A g e n e r a l d i s c u s s i o n of the major f i n d i n g s of each c h a p t e r and t h e i r i m p l i c a t i o n s for m u l t i p l e -l o c u s h e t e r o z y g o s i t y r e l a t i o n s h i p s i s p r e s e n t e d i n Chapter 6. 15 CHAPTER 2 BIOCHEMICAL CHARACTERIZATION OF PGM-2 GENOTYPES INTRODUCTION Overdominance , or h e t e r o z y g o t e s u p e r i o r i t y , has remained a c o n t r o v e r s i a l e x p l a n a t i o n f o r the maintenance of g e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s s i n c e i t s t h e o r e t i c a l f o r m u l a t i o n by F i s h e r (1922) . The p o p u l a r i t y of overdominance peaked i n the 1940s and 1950s l a r g e l y through the e m p i r i c a l work of Dobzhansky and h i s c o l l e a g u e s ( e . g . Dobzhansky and Levene 1948, 1951), the p r o v o c a t i v e ideas of L e r n e r (1954) , and the B r i t i s h e c o l o g i c a l g e n e t i c s s c h o o l l e d by E . B . F o r d ( F o r d 1965). A f t e r the d i s c o v e r y and examinat ion of the p a t t e r n s of a l l o z y m i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s , a v a r i e t y of e x p e r i m e n t a l and-t h e o r e t i c a l s t u d i e s have d i s c r e d i t e d the r o l e p l a y e d by overdominance i n m a i n t a i n i n g b a l a n c e d po lymorphisms . E v i d e n c e a g a i n s t overdominance has i n c l u d e d : d i s c r e p a n c i e s from p r e d i c t e d a d d i t i v e and dominance components of v i a b i l i t y e s t i m a t e s ( e . g . Mukai et a l . 1974); the unobserved l a r g e d e p r e s s i o n s i n f i t n e s s expected upon i n b r e e d i n g ( e . g . Lewont in 1974, p . 207); the a s y m m e t r i c a l f requency d i s t r i b u t i o n s of e l e c t r o p h o r e t i c a l l e l e s a t p o l y m o r p h i c l o c i ( e . g . Yamazaki and Maruyama 1972; Coyne 1976); the h i g h l e v e l s of g e n e t i c v a r i a t i o n d i s p l a y e d by h a p l o i d s such as E . c o l i ( e . g . S e l a n d e r and L e v i n 1980); the i n h e r e n t d i f f i c u l t i e s in e s t a b l i s h i n g s t a b l e m u l t i - a l l e l i c 16 e q u i l i b r i a by overdominance ( e . g . L e w o n t i n , G i n z b u r g and T u l j a p u r k a r 1978); the p o t e n t i a l r o l e s of e n v i r o n m e n t a l h e t e r o g e n e i t y and frequency-dependent s e l e c t i o n i n m a i n t a i n i n g polymorphisms ( H e d r i c k , Ginevan and Ewing 1976; C l a r k e 1979; H e d r i c k 1986); and , perhaps most i m p o r t a n t l y , a p a u c i t y of examples d e m o n s t r a t i n g i t s o c c u r r e n c e . The case a g a i n s t overdominance appears so overwhelming t h a t Kimura (1983, p . 282) s t a t e d tha t "only b l i n d f a i t h can m a i n t a i n i t " . Renewed i n t e r e s t i n overdominance has been sparked by recen t s t u d i e s , on a wide v a r i e t y of organ i sms , documenting s i g n i f i c a n t c o r r e l a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and v a r i o u s m o r p h o l o g i c a l , p h y s i o l o g i c a l and f i t n e s s - r e l a t e d t r a i t s (rev iewed by M i t t o n and Grant 1984; Zouros and F o l t z 1987). In marine b i v a l v e s , a s s o c i a t i o n s i n v o l v i n g m u l t i p l e - l o c u s h e t e r o z y g o s i t y , and the p o t e n t i a l l y r e l a t e d d e f i c i e n c i e s of h e t e r o z y g o t e s , have been most e x t e n s i v e l y s t u d i e d and d i s c u s s e d i n the American o y s t e r C r a s s o s t r e a v i r g i n i c a (S ingh and Zouros 1978; Z o u r o s , S ingh and M i l e s 1980; S ingh 1982; Koehn and Shumway 1982; Zouros et a l . 1983; F o l t z , Newkirk and Zouros 1983; Zouros and F o l t z 1984; S ingh and Green 1984; F o l t z 1986a, 1986b). D e s p i t e e x t e n s i v e work, the u n d e r l y i n g cause of these r e l a t i o n s h i p s remains unknown. A l t h o u g h c o n s i s t e n t w i t h s i n g l e -l o c u s overdominance , these r e s u l t s c o u l d a l s o be produced by i n b r e e d i n g , the u n d e t e c t e d presence of n u l l a l l e l e s , or a s s o c i a t i v e overdominance . 17 I n b r e e d i n g was o r i g i n a l l y d i s c o u n t e d as an e x p l a n a t i o n by Z o u r o s , S ingh and M i l e s (1980) because the h e t e r o g e n e i t y of the i n b r e e d i n g c o e f f i c i e n t s , c a l c u l a t e d f o r each of the seven l o c i s t u d i e d , was s i g n i f i c a n t l y g r e a t e r than expec ted from a p r o c e s s t h a t s h o u l d a f f e c t a l l l o c i e q u a l l y . F u r t h e r ev idence a g a i n s t the i n b r e e d i n g h y p o t h e s i s was p r o v i d e d by M i t t o n and P i e r c e (1980) and C h a k r a b o r t y (1981) who showed t h a t i n d i v i d u a l h e t e r o z y g o s i t y a t a s m a l l subset of l o c i does not a c c u r a t e l y r e f l e c t o v e r a l l genomic h e t e r o z y g o s i t y , a c o n c l u s i o n a l s o emphasized by Smouse (1986) (see however d i s c u s s i o n i n Zouros and F o l t z 1987). R e c e n t l y , F o l t z (1986a, 1986b) d e t e c t e d the s e g r e g a t i o n of n u l l a l l e l e s at two l o c i i n C . v i r q i n i c a , thus p r o v i d i n g support f o r a h y p o t h e s i s o r i g i n a l l y c o n s i d e r e d u n l i k e l y by Z o u r o s , S i n g h and M i l e s (1980) . However, the e x p l a n a t o r y power of n u l l a l l e l e s s t i l l appears l i m i t e d because they were observed at one l o c u s (Lap-2) but not at four o t h e r s i n v o l v e d in these e a r l i e r s t u d i e s ( P q i , G o t , Pgm, X d h ) . The n u l l a l l e l e e x p l a n a t i o n a l s o s u f f e r s from an important t h e o r e t i c a l l i m i t a t i o n : the absence of n u l l homozygotes i n the l a r g e sample of Zou ros , S ingh and M i l e s (1980) i m p l i e s an u n r e a l i s t i c a l l y h i g h s e l e c t i v e advantage of n u l l h e t e r o z y g o t e s over genotypes p o s s e s s i n g two f u n c t i o n a l a l l e l e s tha t i s d i f f i c u l t to r e c o n c i l e . At p r e s e n t , n e i t h e r the i n b r e e d i n g or n u l l a l l e l e hypotheses appear capab le of p r o v i d i n g a g e n e r a l e x p l a n a t i o n for the r e l a t i o n s h i p s between h e t e r o z y g o s i t y and f i t n e s s - r e l a t e d t r a i t s , at l e a s t f o r o y s t e r s . 18 D i s t i n g u i s h i n g between the overdominance and a s s o c i a t i v e overdominance hypotheses i s much more d i f f i c u l t . Are c o r r e l a t i o n s i n v o l v i n g m u l t i p l e - l o c u s h e t e r o z y g o s i t y produced by overdominance at the enzyme l o c i s c o r e d , or a r e they a consequence of a s s o c i a t i v e overdominance caused by the presence of d e l e t e r i o u s r e c e s s i v e a l l e l e s s e g r e g a t i n g at a number of t i g h t l y - l i n k e d l o c i ( c f . Ohta 1971)? The d i s t i n c t i o n between these a l t e r n a t i v e s i s d i r e c t l y ana logous to the d i s p u t e between the "dominance" and "overdominance" e x p l a n a t i o n s f o r h e t e r o s i s , or h y b r i d v i g o r (Zouros and F o l t z 1987), which remains u n r e s o l v e d d e s p i t e decades of s tudy (see Gowen 1952; Wright 1977, p . 9-46; Sedco le 1981). I n d i r e c t approaches to d e t e r m i n i n g the cause of h e t e r o z y g o s i t y - g r o w t h r a t e r e l a t i o n s h i p s i n s e v e r a l P inus s p e c i e s have not h e l p e d to s o l v e t h i s q u e s t i o n . Comparison of i n b r e d and c r o s s b r e d progeny of P . a t t e n u a t a by S t r a u s s (1986, 1987) or the a p p l i c a t i o n of the "adapt ive d i s t a n c e " model of Smouse (1986) to h e t e r o z y g o s i t y r e l a t i o n s h i p s i n a number of P . r i g i d a p o p u l a t i o n s by Bush, Smouse and L e d i g (1987) o n l y t e s t e d the p r e d i c t i o n s of the i n b r e e d i n g v e r s u s the overdominance hypotheses . Any e v i d e n c e f o r overdominance p r o v i d e d by these approaches cannot d i s c r i m i n a t e between genuine or a s s o c i a t i v e overdominance . Perhaps the o n l y way to p r o v i d e c o n c r e t e ev idence f o r the overdominance e x p l a n a t i o n and s i m u l t a n e o u s l y d i s t i n g u i s h between these compet ing hypothese s , i s to study these enzyme polymorphisms d i r e c t l y and produce the b i o c h e m i c a l and 19 p h y s i o l o g i c a l support f o r these' r e s u l t s i n m e c h a n i s t i c terms ( c f . C l a r k e 1975; Koehn 1978; Watt 1985b). To succeed , t h i s r e s e a r c h s t r a t e g y must meet t h r e e r e q u i r e m e n t s . F i r s t , i t must demonstrate tha t f u n c t i o n a l d i f f e r e n c e s a r e expres sed at the b i o c h e m i c a l l e v e l between a l l e l i c v a r i a n t s at the l o c u s chosen f o r s t u d y . These d i f f e r e n c e s must be s u f f i c i e n t i n magnitude to c o n f e r a net advantage to h e t e r o z y g o t e s through e i t h e r 1) m a r g i n a l overdominance a r i s i n g from h e t e r o z y g o t e i n t e r m e d i a c y ( c f . W a l l a c e 1959), or 2) genuine overdominance produced by s u p e r i o r k i n e t i c p r o p e r t i e s of h e t e r o z y g o t e s , thus e l i m i n a t i n g the p o t e n t i a l c o n t r i b u t i o n of unknown t i g h t l y - l i n k e d l o c i . Second, the f u n c t i o n a l s u p e r i o r i t y of h e t e r o z y g o t e s must be m a n i f e s t e d a t the p h e n o t y p i c l e v e l through some p h y s i o l o g i c a l e f f e c t a t t r i b u t a b l e to the l o c u s i n q u e s t i o n . The p h y s i o l o g i c a l impact of t h i s a l l e l i c v a r i a t i o n must be c a p a b l e of e x p l a i n i n g the involvement of the l o c u s i n the h e t e r o z y g o s i t y r e l a t i o n s h i p . T h i r d , the f i t n e s s a r r a y of genotypes at the l o c u s under s tudy must be c o m p a t i b l e w i t h c o n d i t i o n s p r o d u c i n g a s t a b l e e q u i l i b r i u m s t a t e . Given the d i f f i c u l t i e s of m a i n t a i n i n g s t a b l e m u l t i - a l l e l i c e q u i l i b r i a by overdominance ( c f . L e w o n t i n , G i n z b u r g and T u l j a p u r k a r 1978), t h i s requirement i s e s p e c i a l l y p e r t i n e n t because most enzyme l o c i i n these s t u d i e s i n v o l v i n g o y s t e r s possess 3 to 5 a l l e l e s a t moderate f r e q u e n c i e s . I have taken t h i s d i r e c t approach wi th the phosphoglucomutase-2 l o c u s (PGM; E . C . 2 . 7 . 5 . 1 ) in the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s . PGM c a t a l y z e s the i n t e r c o n v e r s i o n of 20 g l u c o s e - 1 - p h o s p h a t e and g l u c o s e - 6 - p h o s p h a t e i n the presence of the c o f a c t o r s g l u c o s e - 1 , 6 - d i p h o s p h a t e and magnesium i o n , and f u n c t i o n s m e t a b o l i c a l l y i n the s y n t h e s i s and d e g r a d a t i o n of g l y c o g e n . In the P a c i f i c o y s t e r , F u j i o (1982) observed a s i g n i f i c a n t p o s i t i v e r e l a t i o n s h i p between h e t e r o z y g o s i t y and a d u l t body weight in a study on 20 w i l d p o p u l a t i o n s around J a p a n . The Pgm-2 l o c u s was was one of the f i v e enzymes i n v o l v e d i n t h i s c o r r e l a t i o n . Pgm-2 h e t e r o z y g o t e s e x h i b i t e d g r e a t e r mean weights than homozygotes i n 18 of the 20 p o p u l a t i o n s sampled. The o b j e c t i v e of t h i s c h a p t e r was to determine i f b i o c h e m i c a l d i f f e r e n c e s e x i s t between the four most common a l l o z y m e s at the Pqm-2 l o c u s in C . g i g a s t h a t p r o v i d e ev idence i n support of the overdominance h y p o t h e s i s . To a s se s s the presence of these f u n c t i o n a l d i f f e r e n c e s , the k i n e t i c p r o p e r t i e s of seven Pgm-2 genotypes were measured over p h y s i o l o g i c a l l y important ranges of temperature and pH. O y s t e r s s i t u a t e d i n the i n t e r t i d a l zone e x p e r i e n c e l a r g e f l u c t u a t i o n s i n both t e m p e r a t u r e , through s e a s o n a l v a r i a t i o n and d a i l y changes a s s o c i a t e d w i t h the t i d a l c y c l e , and i n t r a c e l l u l a r pH, as a consequence of the t r a n s i t i o n s between a e r o b i c and a n a e r o b i c metabol i sm ( e . g . Wijsman 1975; Walsh , McDonald and Booth 1984). K i n e t i c d i f f e r e n c e s may e x i s t between Pgm-2 a l l o z y m e s over these broad ranges of temperature or pH t h a t c o u l d be r e s p o n s i b l e f o r i m p a r t i n g a net advantage to h e t e r o z y g o t e s tha t c o u l d p r o v i d e a f o u n d a t i o n for e x p l a i n i n g t h e i r s u p e r i o r r a t e s of growth r e p o r t e d by F u j i o (1982) . 21 MATERIALS AND METHODS C h e m i c a l s . A l l b u f f e r s , s u b s t r a t e s , c o f a c t o r s , p r o t e i n s , and c o u p l i n g enzymes used for enzymatic as says were o b t a i n e d from Sigma. Sephadex G-100 and G-25 were s u p p l i e d by P h a r m a c i a , DEAE-C e l l u l o s e (DE32, m i c r o g r a n u l a r ) from Whatman, and u l t r a f i l t r a t i o n membrane cones (CF 25) from Amicon. Coomassie b lue G-250 and s tandards used f o r p r o t e i n d e t e r m i n a t i o n s were p r o v i d e d by B i o - R a d , and the e l e c t r o s t a r c h f o r e l e c t r o p h o r e s i s from Connaught L a b o r a t o r i e s . A n i m a l s . O y s t e r s were c o l l e c t e d over a t h r e e year p e r i o d from the i n t e r t i d a l zone of a p r i v a t e o y s t e r farm l o c a t e d i n Nanoose Bay on Vancouver I s l a n d , B r i t i s h C o l u m b i a . Sampl ing s t a t i o n s were e s t a b l i s h e d at two t i d a l h e i g h t s on the l e a s e ; a low water s i t e s i t u a t e d at 0 . 5 - 0 . 8 m above mean low water (MLW), and a h i g h water s i t e at 1 .7-2 .0 m above MLW. An imal s were r e t u r n e d to the l a b o r a t o r y on i c e , and a f t e r a s m a l l p i e c e of mantle was d i s s e c t e d f o r e l e c t r o p h o r e s i s , immediate ly f r o z e n at - 4 0 ° C . E l e c t r o p h o r e s i s . H o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s was performed on 12% g e l s (w/v) a c c o r d i n g to the methods of K r i s t j a n s s o n (1963) . A p p r o x i m a t e l y 0.5 g of f r o z e n mantle t i s s u e was homogenized in an e q u a l volume of 10 mM T r i s , 10 mM m a l e i c a c i d , 1 mM EDTA, 1 mM MgC12, pH 7.4 ( b u f f e r A of F u c c i et a l . 1979) and c e n t r i f u g e d f o r 5 min at top speed i n a c l i n i c a l c e n t r i f u g e . Supernatant was a p p l i e d to the g e l s w i th Whatman No. 22 2 f i l t e r paper w i c k s . G e l s were run at 200 V (35 mA) f o r a p p r o x i m a t e l y 5 h and PGM was s t a i n e d v i s u a l l y as o u t l i n e d by Shaw and Prasad (1970) . For improved r e s o l u t i o n of the a l l o z y m i c banding p a t t e r n s , two d i f f e r e n t r u n n i n g c o n d i t i o n s were r o u t i n e l y employed. Under "s tandard" c o n d i t i o n s the e l e c t r o d e b u f f e r was 100 mM T r i s , 100 mM b o r a t e , 5 mM EDTA, pH 8 . 3 . The g e l b u f f e r was a 1 0 - f o l d d i l u t i o n of the e l e c t r o d e b u f f e r . Under " c a t a l y t i c " c o n d i t i o n s , the f o l l o w i n g s a t u r a t i n g c o n c e n t r a t i o n s of s u b s t r a t e and c o f a c t o r s were added to the s t a n d a r d g e l and e l e c t r o d e b u f f e r s to make the PGM a l l o z y m e s c a t a l y t i c a l l y a c t i v e w h i l e m i g r a t i n g in the e l e c t r i c f i e l d : 1 mM g l u c o s e - 1 - p h o s p h a t e , 5 M M g l u c o s e - 1 , 6 - d i p h o s p h a t e , and 1.5 mM MgC12. Two t e c h n i q u e s were used to examine the presence of h idden v a r i a t i o n w i t h i n the PGM e l e c t r o m o r p h i c c l a s s e s as d e s c r i b e d f o r both D r o s o p h i l a melanoqaster ( T r i p p a , L o v e r r e and Catamo 1976; T r i p p a et a l . 1978) and M y t i l u s e d u l i s (Beaumont and Bever idge 1983). F o l l o w i n g T r i p p a et a l . (1978) , two s l i c e s of the same g e l , run under s t a n d a r d c o n d i t i o n s , were t r e a t e d in the f o l l o w i n g manner. One s l i c e , s e r v i n g as a c o n t r o l , was i n c u b a t e d f o r 15 min i n a water bath at room temperature a n d , the o t h e r , f o r the same l e n g t h of t ime i n a water bath a t 5 0 ° C . The s l i c e s were immediate ly t r a n s f e r r e d to s t a i n i n g s o l u t i o n s and s u b s e q u e n t l y s c o r e d f o r d i f f e r e n t i a l s t a i n i n g i n t e n s i t i e s . 300 homozygotes f o r the most common a l l e l e were t r e a t e d i n t h i s f a s h i o n . The pH-dependent r e s o l u t i o n of c r y p t i c a l l e l e s was examined w i t h the T r i s - m a l e i c a c i d b u f f e r system d e s c r i b e d i n 23 Beaumont and Bever idge (1983) . A sample of 120 o y s t e r s , composed of the 8 most f requent Pqm-2 genotypes , was examined e l e c t r o p h o r e t i c a l l y employ ing a 10 mM T r i s , 10 mM m a l e i c a c i d , pH 7.4 g e l b u f f e r , and a 100 mM T r i s , 100 mM m a l e i c a c i d e l e c t r o d e b u f f e r a d j u s t e d to e i t h e r pH 7.4 or 6 . 0 . G e l s run at each pH were compared under the above and " c a t a l y t i c " c o n d i t i o n s d e s c r i b e d p r e v i o u s l y . P u r i f i c a t i o n . A l l p u r i f i c a t i o n s teps were c a r r i e d out on i c e or at 4 ° C . The f o l l o w i n g procedure was i d e n t i c a l f or the 4 Pgm-2 a l l o z y m e s p u r i f i e d , and s t a r t i n g m a t e r i a l f or each r e p r e s e n t e d p o o l e d t i s s u e from 5-8 i n d i v i d u a l s ,homozygous f o r the a p p r o p r i a t e a l l e l e . A p p r o x i m a t e l y 70-80 g of mantle and adduc tor muscle t i s s u e was minced and homogenized w i t h an U l t r a - T u r r a x homogenizer i n 3 volumes of b u f f e r A and s t i r r e d for 30 m i n . The homogenate was c e n t r i f u g e d a t 42,000 x g f o r 1 h and the supernatant passed through a f i l t e r of g l a s s woo l . The crude homogenate was s l o w l y brought to 50% s a t u r a t i o n w i t h an i c e - c o l d s a t u r a t e d s o l u t i o n of ammonium s u l f a t e . A f t e r s t i r r i n g f o r 1 h the suspens ion was c e n t r i f u g e d a t 12,000 x g f o r 20 m i n . The supernatant was brought to 75% s a t u r a t i o n w i t h ammonium s u l f a t e as be fore and e q u i l i b r a t e d w i t h s t i r r i n g f o r 1 h . A f t e r a f i n a l c e n t r i f u g a t i o n at 12,000 x g f o r 20 min , the p e l l e t s were resuspended i n b u f f e r A and d i a l y z e d o v e r n i g h t a g a i n s t t h r e e 100 v o l changes of the same b u f f e r . The d i a l y z e d sample was a d j u s t e d to pH 7.6 and loaded onto 24 a D E A E - C e l l u l o s e column (1 .5 x 30 cm) e q u i l i b r a t e d w i t h 5 mM T r i s , 5 mM m a l e i c a c i d , 0.5 mM MgC12, 0.1 mM EDTA, pH 7.6 ( b u f f e r B) a t a flow r a t e of 5.6 m l / h . A f t e r washing w i t h a minimum of 3 column volumes , the enzyme was e l u t e d w i t h a l i n e a r g r a d i e n t (0-200 mM) of NaCl (200 ml i n t o t a l ) . F r a c t i o n s c o n t a i n i n g the g r e a t e s t s p e c i f i c a c t i v i t y were combined ( t o t a l v o l 15-25 ml) and c o n c e n t r a t e d to a p p r o x i m a t e l y 1.5 ml on an Amicon CF 25 u l t r a f i l t r a t i o n cone by c e n t r i f u g a t i o n at 2,000 x g . The c o n c e n t r a t e d sample was a p p l i e d to a Sephadex G-100 column (1 .5 x 60 cm) e q u l i b r a t e d w i t h b u f f e r A . E l u t i o n was c a r r i e d out at a f low r a t e of 8.4 m l / h and the peak f r a c t i o n s were p o o l e d ( t o t a l v o l 7-9 m l ) . The sample was a d j u s t e d to pH 7.6 and g l u c o s e - 1 - p h o s p h a t e (G1P) was added to a c h i e v e a c o n c e n t r a t i o n of 2 mM. The sample was l oaded onto a second DEAE-C e l l u l o s e column (0 .9 x 15 cm), washed, and e l u t e d w i t h a l i n e a r g r a d i e n t (0- 200 mM) of NaCl ( t o t a l v o l 150 ml) a t a flow r a t e of 7.2 m l / h as b e f o r e , except the e q u i l i b r a t i o n and e l u t i o n b u f f e r (B) a l s o c o n t a i n e d 2 mM G1P. Peak f r a c t i o n s were a g a i n combined ( t o t a l v o l 7-14 ml) and the sample was c o n c e n t r a t e d to a p p r o x i m a t e l y 1 ml by c e n t r i f u g a t i o n a t 2,000 x g on an Amicon CF 25 u l t r a f i l t r a t i o n cone . The p r e p a r a t i o n was passed through a Sephadex G-25 column (0 .9 x 15 cm) e q u i l i b r a t e d w i t h b u f f e r A at a flow r a t e of 8.4 m l / h and peak f r a c t i o n s were p o o l e d . The p u r i f i e d enzyme was brought to 20% g l y c e r o l ( v / v ) and f r o z e n at - 7 0 ° C . The PGM a l l o z y m e s s t o r e d in t h i s manner were extremely s t a b l e , e x h i b i t i n g on average a 10% l o s s in a c t i v i t y over a 2 month p e r i o d . E l e c t r o p h o r e t i c examinat ion of these p a r t i a l l y 25 p u r i f i e d p r e p a r a t i o n s v e r i f i e d the c o r r e c t a l l o z y m i c c o m p o s i t i o n and showed t h a t they were f r e e of enzyme encoded by the Pgm-1 l o c u s . G e n e r a l p r o t e i n c o n c e n t r a t i o n was de termined i n t r i p l i c a t e a t room temperature a f t e r each p u r i f i c a t i o n s t ep on a Pye Unicam SP8-400 U V / v i s i b l e spec trophotometer by the method of B r a d f o r d (1976) u s i n g gamma g l o b u l i n as a s t a n d a r d . Enzyme Assays and K i n e t i c s . A l l enzyme assays were conducted a t 340 nm on a Pye Unicam SP 1800 U V / v i s i b l e s p e c t r o p h o t o m e t e r . Assay temperature was c o n t r o l l e d by a Lauda K - 2 / R D c i r c u l a t i n g water bath a t t a c h e d to a c o n s t a n t temperature c u v e t t e h o l d e r and was m o n i t o r e d by a YSI model 46 thermometer probe i n s e r t e d i n t o a b lank c u v e t t e . PGM a c t i v i t y was measured u s i n g a c o u p l e d assay m o d i f i e d from J o s h i et a l . (1967) . The s t a n d a r d assay medium c o n t a i n e d 50 mM i m i d a z o l e - H C l , 3 mM MgC12, 2 mM g l u c o s e - 1 -phosphate (G1P) , 16 M M g l u c o s e - 1 , 6 - d i p h o s p h a t e ( G l 6 d i P ) , 0.4 mM NADP, 1 u n i t g l u c o s e - 6 - p h o s p h a t e dehydrogenase (G6PDH), pH 7.0 ( 2 0 ° C ) i n a f i n a l volume of 1 m l . The assay m i x t u r e , c o n t a i n i n g an a l i q u o t of PGM, was p r e i n c u b a t e d f o r a minimum of 10 min and the r e a c t i o n s t a r t e d by the a d d i t i o n of G1P. One u n i t of PGM a c t i v i t y i s d e f i n e d as the q u a n t i t y of enzyme r e q u i r e d to c o n v e r t 1 Mmol of s u b s t r a t e to p r o d u c t per minute a t 1 5 ° C . P r e l i m i n a r y k i n e t i c a n a l y s e s were c a r r i e d out on each of the 4 Pgm-2 a l l ozymes f o l l o w i n g e a r l i e r p u r i f i c a t i o n t r i a l s . K i n e t i c parameters for the forward r e a c t i o n (G1P to G6P) were determined by measuring i n i t i a l r a t e s i n t r i p l i c a t e at 11 26 c o n c e n t r a t i o n s of G1P r a n g i n g from 10 M M to 2 mM at a s a t u r a t i n g G16diP c o n c e n t r a t i o n of 16 juM. These exper iments were performed at 5 ° i n t e r v a l s over the temperature range of 5 to 3 0 ° C a l l o w i n g the pH of the i m i d a z o l e b u f f e r to v a r y wi th temperature ( A p K a / ° C = - 0 . 0 1 7 ) . The e f f e c t of pH on the k i n e t i c parameters was s t u d i e d by measur ing i n i t i a l r a t e s as be fore a t pH 6 . 5 , 7.0 and 7.5 a t a c o n s t a n t temperature of 2 0 ° C . Apparent Km and Vmax v a l u e s were e s t i m a t e d by the d i r e c t l i n e a r p l o t of E i s e n t h a l and Cornish-Bowden (1974) . K i n e t i c parameters f o r the c o f a c t o r G16diP were e v a l u a t e d by measur ing i n i t i a l r e a c t i o n r a t e s i n t r i p l i c a t e a t 8 c o n c e n t r a t i o n s of the d iphosphate i n a geometr ic s e r i e s from 0.125 to 16 uM a t s a t u r a t i n g l e v e l s of G1P (1 mM). I n i t i a l r a t e s were measured over the same ranges of temperature and pH as b e f o r e and the k i n e t i c parameters a g a i n e s t i m a t e d by the d i r e c t l i n e a r p l o t . These i n i t i a l k i n e t i c exper iments enab led c o n s t r u c t i o n of an o p t i m a l k i n e t i c d e s i g n tha t m i n i m i z e d the v a r i a n c e s of the parameter e s t i m a t e s ( c f . Duggleby 1979; E n d r e n y i and Chan 1981; C u r r i e 1982). For the forward r e a c t i o n d i r e c t i o n , i n i t i a l r a t e measurements were r e p l i c a t e d 8 t imes at each of two G1P c o n c e n t r a t i o n s (2 mM and 20 uM) a t a d iphosphate c o n c e n t r a t i o n of 16 M M . K i n e t i c parameters f o r the c o f a c t o r G16diP were de termined s i m i l a r l y by r e p e a t i n g i n i t i a l r a t e measurements 8 t imes at each of two d iphosphate c o n c e n t r a t i o n s (16 and 0.70 M M ) at a G1P c o n c e n t r a t i o n of 1 mM. S e v e r a l a d d i t i o n a l p r e c a u t i o n s were taken to min imize e x p e r i m e n t a l e r r o r . For the k i n e t i c s tudy 27 the 4 Pqm-2 a l l o z y m e s were p u r i f i e d s e q u e n t i a l l y , s t o r e d f r o z e n , and subsequent ly s t u d i e d t o g e t h e r under i d e n t i c a l c o n d i t i o n s at each temperature and p H . For a l l i n i t i a l r a t e measurements the a l l o z y m e s were d i l u t e d to equa l a c t i v i t i e s i n b u f f e r A . Three h e t e r o z y g o t e p r e p a r a t i o n s were made by m i x i n g e q u a l a c t i v i t i e s of the most common Pgm-2 a l l ozyme w i t h each of the t h r e e l e s s f requent a l l o z y m e s . A l t h o u g h s y n t h e t i c i n n a t u r e , these h e t e r o z y g o t e p r e p a r a t i o n s ensured t h a t s i m i l a r q u a n t i t i e s of the two monomers were p r e s e n t , a c o n d i t i o n j u s t i f i e d by the s t a i n i n g p a t t e r n s of these genotypes , and not guaranteed by the p u r i f i c a t i o n of enzyme from n a t u r a l l y o c c u r r i n g h e t e r o z y g o t e s . The same s u b s t r a t e s o l u t i o n s , m a i n t a i n e d a t - 4 0 ° C i n s m a l l a l i q u o t s , were used throughout the s t u d y . S u b s t r a t e c o n v e r s i o n was kept to w i t h i n 20% to ensure s t r i c t l y l i n e a r r e a c t i o n time courses (Ray and Peck 1972; Hoffman 1985). S i n c e the k i n e t i c parameters de termined by t h i s e x p e r i m e n t a l d e s i g n r e p r e s e n t s i n g l e e s t i m a t e s , the ex tent of d a y - t o - d a y v a r i a b i l i t y was a s ses sed p r i o r to the s t a r t of the s tudy and was found to be n e g l i g i b l e , p r o v i d i n g the a c t i v i t i e s of the d a i l y p r e p a r a t i o n s were kept c o n s t a n t . R e p l i c a t e Km d e t e r m i n a t i o n s a c r o s s 5 days e x h i b i t e d a c o e f f i c i e n t of v a r i a t i o n of o n l y 2.9%. K i n e t i c parameters f o r G1P and G16diP from t h i s o p t i m a l d e s i g n were e s t i m a t e d by the method of b i w e i g h t r e g r e s s i o n wi th the F o r t r a n program NATO w r i t t e n by Cornish-Bowden (1985) . The k i n e t i c parameters were examined by two-way a n a l y s i s of v a r i a n c e (ANOVA) t r e a t i n g Pgm-2 genotype and e i t h e r temperature or pH as 28 independent v a r i a b l e s . Means expres sed over these ranges of temperature and pH were compared by a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . R a t i o n a l e f o r the s t a t i s t i c a l comparison of the apparent Km v a l u e s of these seven Pqm-2 genotypes at each i n d i v i d u a l pH or temperature i n t e r v a l by the a n a l y s i s of c o v a r i a n c e (ANCOVA) i s as f o l l o w s . F i r s t , f o r a l l genotypes the apparent Km and Vmax v a l u e s were de termined a second t ime by o r d i n a r y l e a s t - s q u a r e s r e g r e s s i o n . These e s t i m a t e s were found to be i d e n t i c a l w i t h those o b t a i n e d by the more robus t b iwe ight p r o c e d u r e . T h i s a n a l y s i s was r e p e a t e d a f t e r chang ing the l i n e a r t r a n s f o r m a t i o n of the M i c h a e l i s - M e n t e n e q u a t i o n in the program from the L i n e w e a v e r - B u r k p l o t (1 /v v s . 1 / [ S ] ) to the E a d i e -Hof s t ee p l o t of v v s . v / [ s ] , which produces a r e g r e s s i o n l i n e w i t h a y - i n t e r c e p t of Vmax and a s l o p e of -Km. These d i f f e r e n t t r a n s f o r m a t i o n s y i e l d e d i d e n t i c a l k i n e t i c parameters f o r both r e g r e s s i o n p r o c e d u r e s , a l t h o u g h the s t a n d a r d e r r o r s o b t a i n e d from the E a d i e - H o f s t e e p l o t were s l i g h t l y l a r g e r . The absence of o u t l i e r s i n the k i n e t i c da ta and the independence of the parameter e s t i m a t e s on the l i n e a r t r a n s f o r m a t i o n a l l o w e d the s t a t i s t i c a l comparison of the apparent Km v a l u e s of d i f f e r e n t genotypes by ANCOVA, t r e a t i n g v / [ s ] as the c o v a r i a t e and v as the dependent v a r i a b l e . D i f f e r e n c e s between the s l o p e e s t i m a t e s (-Km) f o r these Pqm-2 genotypes were t e s t e d by B o n f e r r o n i a p o s t e r i o r i m u l t i p l e range t e s t s . T h i s procedure i s thus analogous to the unplanned comparison of a s e r i e s of r e g r e s s i o n c o e f f i c i e n t s (Soka l and R o h l f 1981, p . 507) , i r r e s p e c t i v e of the v a l i d i t y of t r e a t i n g v / [ s ] as a c o v a r i a t e i n the ANCOVA. 29 K i n e t i c parameters f o r the r e v e r s e r e a c t i o n d i r e c t i o n (G6P to G1P) were e s t i m a t e d by the p roced u re of Ray and R o s c e l l i (1964b) through the c o n s t r a i n t s imposed by the Haldane e q u a t i o n (Haldane 1930): Keq = Vmax(f) app Km (G6P) . . . (1 ) Vmax(r) app Km (G1P) where Keq i s the r e a c t i o n ' s e q u i l i b r i u m c o n s t a n t ( [ G 6 P ] / [ G 1 P ] ) ; Vmax(f) and Vmax(r) are the maximum v e l o c i t i e s of the forward and r e v e r s e r e a c t i o n d i r e c t i o n s , r e s p e c t i v e l y ; and app Km (G6P) and app Km (G1P) are the apparent M i c h a e l i s c o n s t a n t s f o r g l u c o s e - 6 - p h o s p h a t e and g l u c o s e - 1 - p h o s p h a t e , r e s p e c t i v e l y . E q u a t i o n 1 may be r e a r r a n g e d to a l l o w e s t i m a t i o n of app Km (G6P): app Km (G6P) = Keq app Km (G1P) . . . . ( 2 ) Vmax(f ) /Vmax(r) T h e r e f o r e , d e t e r m i n a t i o n of the Vmax(f ) /Vmax(r) r a t i o s " of d i f f e r e n t Pgm-2 genotypes , employing i d e n t i c a l enzyme c o n c e n t r a t i o n s f o r both r e a c t i o n d i r e c t i o n s , enab les i n d i r e c t measurement of t h e i r app Km's f o r G6P, p r o v i d i n g v a l u e s of Keq and app Km (G1P) were a t t a i n e d under s i m i l a r c o n d i t i o n s . Maximum v e l o c i t i e s for both r e a c t i o n d i r e c t i o n s were 30 de termined f o r the four Pqm-2 a l l o z y m e s a t 5 ° i n t e r v a l s over the temperature range of 5 to 3 0 ° C . The assay medium c o n t a i n e d 50 mM i m i d a z o l e - H C l pH 7.0 ( 2 0 ° C ) , 3 mM MgC12, 16 M M G16d iP , and e i t h e r 4 mM G1P ( forward) or G6P ( r e v e r s e ) i n a f i n a l volume of 1 m l . The four a l l o z y m e p r e p a r a t i o n s were d i l u t e d to e q u a l a c t i v i t i e s i n b u f f e r A . E i g h t r e p l i c a t e r e a c t i o n tubes for both r e a c t i o n d i r e c t i o n s were e q u i l i b r a t e d to the a p p r o p r i a t e temperature i n a shak ing water b a t h , and were i n i t i a t e d by the a d d i t i o n of s u b s t r a t e . At 3, 6, and 9 min i n t e r v a l s , 250 jul a l i q u o t s were removed from each r e a c t i o n m i x t u r e . For as says p r o c e e d i n g i n the forward d i r e c t i o n , these were t r a n s f e r r e d i n t o an equa l volume of 1N H C l ; f o r the r e v e r s e d i r e c t i o n i n t o a s i m i l a r volume of 1N NaOH. The q u a n t i t y of G6P produced at each t ime i n t e r v a l from the forward r e a c t i o n was measured i n d u p l i c a t e as d e s c r i b e d by Lang and M i c h a l (1974) . The G1P formed from the r e v e r s e r e a c t i o n was assayed i n a s i m i l a r f a s h i o n by the procedure of Bergmeyer and M i c h a l (1974) . For both r e a c t i o n d i r e c t i o n s , Vmax was e s t i m a t e d as the y -i n t e r c e p t of an unweighted l i n e a r r e g r e s s i o n of the product formed/ t ime v e r s u s t i m e . E q u i l i b r i u m c o n s t a n t s were determined at each temperature by i n c u b a t i n g 10 r e a c t i o n tubes p r e p a r e d as above for 2 h wi th a sample of the Pgm-2-100 a l l o z y m e . The e q u i l i b r i u m was approached from both d i r e c t i o n s w i t h s t a r t i n g c o n c e n t r a t i o n s of 2 mM G1P or G6P. The r e a c t i o n s were s topped by immersion i n a b o i l i n g water bath f o r 2 min , and the c o n c e n t r a t i o n s of G1P and G6P measured i n d u p l i c a t e as d e s c r i b e d 31 above . Apparent M i c h a e l i s c o n s t a n t s f o r G6P f o r the four a l l o z y m e s were e s t i m a t e d from e q u a t i o n 2 by s u b s t i t u t i n g the a p p r o p r i a t e Vmax(f ) /Vmax(r) r a t i o , K e q , and app Km (G1P) e s t i m a t e for the forward r e a c t i o n d i r e c t i o n de termined p r e v i o u s l y . T h e r m o s t a b i l i t y S t u d i e s . The four p u r i f i e d a l l o z y m e s were d i l u t e d to equa l a c t i v i t i e s i n 50 mM sodium phosphate , 0.5 mM MgCl2 , 0.1 mM EDTA, pH 7.0 b u f f e r c o n t a i n i n g 0.1% (w/v) bovine serum a l b u m i n . As i n the k i n e t i c s t u d y , the same three h e t e r o z y g o t e p r e p a r a t i o n s were made and s t u d i e d a l o n g s i d e the four homozygote samples . For each genotype 50 ul a l i q u o t s were p i p e t t e d i n t o a set of 21 g l a s s c u l t u r e tubes (10 x 75 mm). A f t e r b e i n g s e a l e d wi th p a r a f i l m , 18 of the tubes were i n c u b a t e d w i t h s h a k i n g in a water bath at 5 0 ° C . At 5 min i n t e r v a l s over a 30 min p e r i o d t r i p l i c a t e tubes were removed and immediate ly t r a n s f e r r e d to an i c e b a t h . Three 1 c o n t r o l tubes were m a i n t a i n e d i n the i c e bath throughout the e x p e r i m e n t . PGM a c t i v i t i e s were measured i n d u p l i c a t e on a l l samples by the s t a n d a r d assay at 2 5 ° and c o n v e r t e d to the p r o p o r t i o n of a c t i v i t y remain ing at each t ime i n t e r v a l r e l a t i v e to the c o n t r o l . A f t e r P l a c e and Powers (1984a) and H a l l (1985), the thermal d e n a t u r a t i o n of these enzyme genotypes was t r e a t e d as a f i r s t - o r d e r e x p o n e n t i a l decay p r o c e s s . L e a s t - s q u a r e s r e g r e s s i o n of the l o g a r i t h m of f r a c t i o n a l a c t i v i t y remain ing at t ime t a g a i n s t t ime y i e l d e d a s t r a i g h t l i n e w i t h a n e g a t i v e s l o p e e s t i m a t i n g the d e n a t u r a t i o n c o n s t a n t , k d . R e p l i c a t i o n of the experiment produced e s t i m a t e s 32 of kd for a l l genotypes tha t d i d not d i f f e r s i g n i f i c a n t l y from b e f o r e , so the data from both t r i a l s was p o o l e d . D i f f e r e n c e s between genotypes i n these d e n a t u r a t i o n c o n s t a n t s were t e s t e d s t a t i s t i c a l l y by examining the h e t e r o g e n e i t y of these s l o p e s by ANCOVA, and p e r f o r m i n g p a i r - w i s e comparisons w i t h a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . pH o p t i m a . I n i t i a l r e a c t i o n r a t e s were measured under s a t u r a t i n g c o n d i t i o n s a t 0.2 pH u n i t i n t e r v a l s from pH 6.0 to 8.0 at 2 0 ° C by the s t a n d a r d assay on p r e p a r a t i o n s of the four a l l o z y m e s d i l u t e d to e q u a l a c t i v i t i e s i n b u f f e r A . A l l assay components were d i s s o l v e d i n 50 mM i m i d a z o l e - H C l s o l u t i o n s a d j u s t e d to the a p p r o p r i a t e p H . At each pH i n t e r v a l a c t i v i t y measurements were r e p l i c a t e d four t imes a f t e r e n s u r i n g t h a t the c o u p l i n g enzyme G6PDH was not r a t e - l i m i t i n g . The p r o p o r t i o n of a c t i v i t y expressed at each i n t e r v a l r e l a t i v e to tha t observed at the pH optimum was de termined f o r each genotype . The experiment was repeated a second time and because the r e s u l t s d i d not d i f f e r s i g n i f i c a n t l y (by p a i r e d t - t e s t s on the a n g u l a r t r a n s f o r m e d d a t a ) , the da ta from both t r i a l s was p o o l e d . S t a t i s t i c a l comparison of the four genotypes was c a r r i e d out f o r the e n t i r e pH range by two-way ANOVA and at each i n t e r v a l by one-way ANOVA on the a n g u l a r t r a n s f o r m e d p r o p o r t i o n s and t e s t e d by a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . E f f e c t of Magnesium I o n . The four Pgm-2 a l l o z y m e s were d i l u t e d to equa l a c t i v i t i e s in 50 mM i m i d a z o l e - H C l pH 7.0 ( 2 0 ° C ) b u f f e r 33 and passed through a Sephadex G-25 column (0 .9 x 15 cm, e q u i l i b r a t e d w i t h the same b u f f e r ) to remove f r e e magnesium i o n s . I n i t i a l r e a c t i o n r a t e s were measured by the s t a n d a r d assay at 1 5 ° C on four r e p l i c a t e s at magnesium c o n c e n t r a t i o n s of 0 . 5 , 1, 3, 5, and 10 mM. R e a c t i o n v e l o c i t i e s a t each c o f a c t o r c o n c e n t r a t i o n were e x p r e s s e d as p r o p o r t i o n a l a c t i v i t i e s r e l a t i v e to the maximum r a t e measured. The exper iment was r e p l i c a t e d and the data from both t r i a l s was p o o l e d for s t a t i s t i c a l a n a l y s i s . The four a l l o z y m e s were compared by two-way ANOVA on the a n g u l a r t r a n s f o r m e d p r o p o r t i o n s f o r the e n t i r e range of Mg c o n c e n t r a t i o n s , and by one-way ANOVA for each l e v e l s e p a r a t e l y . Means were compared by a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . RESULTS• ELECTROPHORESIS AND PGM-2 A L L E L E FREQUENCIES Two phosphoglucomutase l o c i were d e t e c t e d e l e c t r o p h o r e t i c a l l y i n C r a s s o s t r e a g i g a s . The more c a t h o d a l Pgm-1 l o c u s appeared to be p o l y m o r p h i c , but s i n c e i t s a c t i v i t y was o n l y a f r a c t i o n of t h a t expressed at the Pqm-2 l o c u s i t c o u l d not be r e l i a b l y s c o r e d . Genotypes a t the more a n o d a l Pqm-2 l o c u s e x h i b i t e d double -banded s t a i n i n g p a t t e r n s s i m i l a r to those d e s c r i b e d f o r t h i s enzyme i n o t h e r s p e c i e s ( e . g . Ward and Beardmore 1977; Hoffman 1985). Under s t a n d a r d e l e c t r o p h o r e t i c c o n d i t i o n s homozygotes d i s p l a y e d a two-banded phenotype and 34 h e t e r o z y g o t e s were e i t h e r t h r e e - or four -banded depending on the m o b i l i t i e s of the a l l e l e s c o m p r i s i n g them ( F i g u r e 1A) . T h i s d o u b l e - b a n d i n g was found to r e s u l t from the s imul taneous presence of f a s t e r m i g r a t i n g p h o s p h o r y l a t e d ( a c t i v e ) and s lower m i g r a t i n g d e p h o s p h o r y l a t e d ( i n a c t i v e ) forms of the enzyme, which d i f f e r e d i n m o b i l i t y because of the presence or absence of t h i s n e g a t i v e l y charged phosphate g r o u p . When e l e c t r o p h o r e s i s was c a r r i e d out under c a t a l y t i c r u n n i n g c o n d i t i o n s , known to c o n v e r t PGM e n t i r e l y to the a c t i v e phosphoenzyme (Ray and R o s c e l l i 1964b), t h i s d o u b l e - b a n d i n g was e l i m i n a t e d and the p a t t e r n s became c o n s i s t e n t w i t h those expected f o r a monomeric enzyme ( F i g u r e 1B) . E v i d e n c e c o n s i s t e n t w i t h the c o n v e r s i o n of the presumed s lower m i g r a t i n g dephosphoenzyme to the f a s t e r m i g r a t i n g phosphoenzyme was p r o v i d e d when the c o f a c t o r G16diP a l o n e was added to the g e l b u f f e r a t a c o n c e n t r a t i o n of 5 M M (not shown). T h i s r e s u l t e d in the near e l i m i n a t i o n of the s lower m i g r a t i n g band in a l l genotypes , as p r e d i c t e d from the a b i l i t y of G l 6 d i P to r e a c t w i t h the dephosphoenzyme and c o n v e r t i t to the p h o s p h o r y l a t e d s t a t e ( N a j j a r and Pul lman 1954). These s t a i n i n g p a t t e r n s d i d not r e s u l t as an a r t i f a c t of s torage because f r e s h l y ground t i s s u e and that from o y s t e r s s t o r e d f o r months a t - 4 0 ° C behaved i d e n t i c a l l y . I t i s not known whether the r e s o l u t i o n of these d i f f e r e n t p h o s p h o r y l a t e d forms of PGM was a consequence of the homogenizat ion a n d / o r e l e c t r o p h o r e t i c p r o c e d u r e s , or i f they r e f l e c t a c t u a l iri v i v o p r o p o r t i o n s . 35 F i g u r e 1. E l e c t r o p h o r e t i c s t a i n i n g p a t t e r n s of o y s t e r PGM under (A) s t a n d a r d and (B) c a t a l y t i c r u n n i n g c o n d i t i o n s . Pgm-2 genotypes p r e s e n t e d from l e f t to r i g h t a r e : 92 /92 , 927100, 96 /96 , 96/100, 100/100, 100/104, and 104/104. 37 C a t a l y t i c r u n n i n g c o n d i t i o n s a i d e d i n the s c o r i n g of Pgm-2 genotypes and a l l o w e d u n e q u i v o c a l i d e n t i f i c a t i o n of homozygotes s e l e c t e d f o r enzyme p u r i f i c a t i o n . No h idden v a r i a t i o n was uncovered w i t h i n the most common e l e c t r o m o r p h i c c l a s s at the Pgm-2 l o c u s f o r a t e m p e r a t u r e -s e n s i t i v e (Ts) a l l e l e as demonstrated i n D. melanogaster by T r i p p a , L o v e r r e and Catamo (1976) and T r i p p a et a l . (1978) . I n c u b a t i o n of g e l s c o n t a i n i n g i n d i v i d u a l s homozygous f o r the Pgm -2-100 a l l e l e for 15 min at 5 0 ° C r e s u l t e d i n the c o n s i s t e n t l o s s of a p p r o x i m a t e l y 50% of the PGM a c t i v i t y observed on the c o n t r o l g e l s . No ev idence of Ts h e t e r o z y g o t e s was o b s e r v e d , nor were any Ts homozygotes d e t e c t e d in the sample of 300 o y s t e r s . A l t h o u g h h e t e r o z y g o t e s for a Ts a l l e l e would be hard to i d e n t i f y by t h i s crude method, the complete absence of Ts homozygotes i n d i c a t e s tha t the frequency of such an a l l e l e , i f p r e s e n t , i s so low (p < .08) t h a t i t would not s e r i o u s l y i n f l u e n c e k i n e t i c p r o p e r t i e s of t h i s g e n o t y p i c c l a s s p r e s e n t e d i n the next s e c t i o n . The v a r i a b l e pH system of Beaumont and Bever idge (1983) a l s o f a i l e d to d e t e c t any h e t e r o g e n e i t y w i t h i n the four most common e l e c t r o m o r p h i c c l a s s e s when e l e c t r o p h o r e s i s was c a r r i e d out under e i t h e r s t a n d a r d or c a t a l y t i c c o n d i t i o n s . However, i n agreement w i t h these a u t h o r s the T r i s - m a l e i c a c i d system at pH 7.4 was unable to a c c u r a t e l y r e s o l v e two a l l e l e s (Pgm-2-96 and 104) t h a t c o u l d be more e a s i l y i d e n t i f i e d by the e l e c t r o d e b u f f e r at pH 6 .0 , or by the s t a n d a r d T r i s - b o r a t e system run at a h i g h e r p H . 38 A t o t a l of 8 a l l e l e s were d e t e c t e d at the Pqm-2 l o c u s , i n agreement w i t h that r e p o r t e d by B u r o k e r , Hershberger and Chew (1979a) . A l t h o u g h p a i r c r o s s e s were not performed i n t h i s s t u d y , s e v e r a l of these a l l e l e s have been shown to segregate i n a M e n d e l i a n f a s h i o n i n C . q i g a s by W i l k i n s (1976) , and at the presumably homologous Pgm l o c u s i n C . v i r g i n i c a by F o l t z (1986b). N u m e r i c a l v a l u e s have been a s s i g n e d to these a l l e l e s e x p r e s s i n g t h e i r m o b i l i t i e s r e l a t i v e to the most common 100 a l l e l e . T h e i r observed f r e q u e n c i e s at the low and h i g h water sampl ing s i t e s over a two year p e r i o d are p r e s e n t e d i n T a b l e I . A c o n t i n g e n c y t a b l e a n a l y s i s i n d i c a t e d t h a t these f r e q u e n c i e s were homogeneous over time (x 2 = 34.2 w i t h 35 d f , P > . 50 ) . To examine the p o t e n t i a l e f f e c t of m i c r o e n v i r o n m e n t a l c o n d i t i o n s on these f r e q u e n c i e s , a h e t e r o g e n e i t y C h i - s q u a r e a n a l y s i s was c a r r i e d out comparing a l l e l i c d i s t r i b u t i o n s between the two t i d a l h e i g h t s . No s i g n i f i c a n t d i f f e r e n c e s were found between t i d a l h e i g h t s at any of the four sampl ing d a t e s , on the p o o l e d t o t a l s , or f o r the computed i n t e r a c t i o n t e r m . The C h i - s q u a r e v a l u e s l i s t e d i n T a b l e I t e s t the agreement between the observed g e n o t y p i c p r o p o r t i o n s and Hardy-Weinberg e x p e c t a t i o n s . S t a t i s t i c a l l y s i g n i f i c a n t d e v i a t i o n s were d e t e c t e d for 3 of the 8 samples , and an a d d i t i o n a l two (10/83 High and 11/85 h igh) were n e a r l y s i g n i f i c a n t (P < . 1 0 ) . A h e t e r o g e n e i t y C h i - s q u a r e on these g e n o t y p i c p r o p o r t i o n s y i e l d e d a s i g n i f i c a n t i n t e r a c t i o n term (x 2 = 57.3 w i t h 35 d f , P < . 0 5 ) , caused by the 3/85 sample which e x h i b i t e d the l a r g e s t d e f i c i e n c y of 39 T a b l e I . Pqm-2 a l l e l e f r e q u e n c i e s , c o n f o r m i t y to H a r d y -Weinberg e x p e c t a t i o n s , and h e t e r o z y g o t e d e f i c i e n c i e s at the four sampl ing d a t e s . Frequency at Date and Tidal Height 10/83 6/84 3/85 11/85 Tota ls A l l e l e Low High Low High Low High Low High Low High 84 .004 .002 .003 .003 — - .002 .002 .002 .002 .003 88 .004 .005 .007 .006 — _ .004 .007 .006 .006 .005 92 . 133 . 125 .091 . 146 . 108 . 131 . 118 . 115 . 112 . 129 96 .096 .075 .111 .097 .075 .097 . 100 .094 .094 .093 100 .596 .610 .601 .538 .652 .552 .605 .609 .617 .578 104 . 142 . 133 . 157 . 166 . 141 . 180 . 134 . 155 .141 . 163 108 .025 .050 .027 .038 .019 .032 .034 .013 .027 .027 112 - - - .003 .006 .005 .002 .006 .002 .002 N 121 100 149 160 205 222 220 265 695 747 X 1 9.05 10. 5 4.73 2.40 27.4***20.4** 13.0* 9.72 na na He .597 .586 .593 .651 .537 .635 .591 .583 .577 .613 Ho .579 .550 .557 .631 .454 .550 .550 .562 .528 .572 D - .030 -.061 -.061 -.031 - . 155 - . 134 -.069 - .036 -.085 -.067 na = not a p p l i c a b l e * P < .05 * * P < .01 * * * P < .001 41 h e t e r o z y g o t e s as measured by D = (Ho-He) /He (where Ho and He are the observed and expected h e t e r o z y g o s i t i e s , r e s p e c t i v e l y ) . D v a l u e s c a l c u l a t e d f o r the o ther samples were a l s o n e g a t i v e , but g e n e r a l l y much lower than p r e v i o u s l y r e p o r t e d for t h i s l o c u s [D a v e r a g i n g -0 .122 f o r 6 Washington s t a t e p o p u l a t i o n s (Buroker 1975) and -0 .172 f o r 20 Japanese p o p u l a t i o n s ( F u j i o 1982)] . G i v e n the a l l e l i c f requency d i s t r i b u t i o n s i n T a b l e I , i n d i v i d u a l s a t the Pgm-2 l o c u s are dominated by genotypes homozygous for the Pgm-2-100 a l l e l e and h e t e r o z y g o t e s between t h i s a l l e l e and e i t h e r the Pqm-2-92, 96, and 104 a l l e l e s . T h e r e f o r e , the b i o c h e m i c a l p r o p e r t i e s of these three h e t e r o z y g o t e s and the homozygotes f o r the four common a l l e l e s were s i n g l e d out f o r s tudy to examine the p o t e n t i a l e x p r e s s i o n of overdominance at the Pgm-2 l o c u s . BIOCHEMICAL PROPERTIES OF PGM-2 GENOTYPES A p u r i f i c a t i o n summary f o r the Pgm-2-104 a l l o z y m e i s shown i n T a b l e I I . The four s t ep p roced u re r e s u l t e d i n a 3 0 0 - f o l d p u r i f i c a t i o n and r e c o v e r e d 39% of the s t a r t i n g a c t i v i t y . The presence of s a t u r a t i n g l e v e l s of s u b s t r a t e i n the f i n a l DEAE-s u b s t r a t e s t ep caused an e a r l i e r e l u t i o n of the s a t u r a t e d enzyme i n the s a l t g r a d i e n t , presumably because of a l t e r e d b i n d i n g a n d / o r charge p r o p e r t i e s . H a l l (1985) has r e p o r t e d a s i m i l a r o b s e r v a t i o n in the p u r i f i c a t i o n of two phosphoglucose isomerase a l l o z y m e s from the m u s s e l , M y t i l u s e d u l i s . The f i n a l s p e c i f i c a c t i v i t i e s of the three o ther Pgm-2 a l l o z y m e s were s i m i l a r to T a b l e I I . P u r i f i c a t i o n summary for the Pqm-2-104 a l l o z y m e . 43 F r a c t i o n Volume Spec . A c t i v i t y P u r i f i c a t i o n Recovery (ml) ( u n i t s / m g ) ( f o l d ) (%) Crude 168.0 0.0533 1 100 Homogenate Ammonium 10.0 0.175 3.28 94.3 Su lphate P r e c i p i t a t i o n DEAE- 20.2 2.25 42.2 90.3 C e l l u l o s e Sephadex 6.7 6.94 130 53.2 G-100 D E A E - 13.2 15.9 298 39.3 S u b s t r a t e 44 t h a t shown i n T a b l e II f o r the Pqm-2-104 a l l o z y m e , and a l t h o u g h these p r e p a r a t i o n s were not homogeneous (as de termined by SDS g e l s ) , a l l were found to be f r e e of enzymes c a p a b l e of i n t e r f e r i n g wi th the PGM assay (UDP-glucose p y r o p h o s p h o r y l a s e and phosphoglucose i s o m e r a s e ) . The apparent M i c h a e l i s c o n s t a n t s of seven Pqm-2 genotypes f o r g l u c o s e - 1 - p h o s p h a t e (G1P) and g l u c o s e - 1 , 6 - d i p h o s p a t e (G16diP) over the temperature range of 5 to 3 0 ° C are p r e s e n t e d g r a p h i c a l l y in F i g u r e 2. A two-way a n a l y s i s of v a r i a n c e (ANOVA) on the app Km (G1P) v a l u e s ( F i g u r e 2, upper h a l f ) r e v e a l e d t h a t temperature e x e r t e d a h i g h l y s i g n i f i c a n t e f f e c t (F (5 ,30 ) = 17 .6 , P < . 0 0 1 ) . A p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s i n d i c a t e d tha t the app Km's measured at 5 and 3 0 ° C were s i g n i f i c a n t l y l a r g e r than those at 10, 15, or 2 0 ° C , but the v a l u e s o b t a i n e d at 25 and 3 0 ° C were i n d i s t i n g u i s h a b l e . T h i s a n a l y s i s a l s o uncovered a s i g n i f i c a n t e f f e c t of Pqm-2 genotype (F(6 ,30 ) = 5 .21 , P < . 0 0 1 ) . M u l t i p l e range t e s t s comparing the app Km v a l u e s of d i f f e r e n t genotypes showed tha t t h i s r e s u l t was due e n t i r e l y to the Pqm-2-92/92 homozygote (1A) which e x h i b i t e d s i g n i f i c a n t l y lower app Km's for G1P than the Pgm-2-96/96 and Pgm-2-104/104 homozygotes, and the Pgm-2-100/104 h e t e r o z y g o t e . G e n o t y p i c comparisons at i n d i v i d u a l t emperatures by ANCOVA showed t h a t the s i g n i f i c a n t l y lower app Km's f o r G1P of the Pgm -2 -92 /92 genotype arose from i t s performance over the 15 to 3 0 ° C range . The t h r e e h e t e r o z y g o t e s were s t r i c t l y i n t e r m e d i a t e i n t h e i r app Km (G1P) v a l u e s , as expected f o r a monomer l i k e PGM, 45 F i g u r e 2. E f f e c t of temperature on the apparent M i c h a e l i s c o n s t a n t s ( i n M M ) f or g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and g l u c o s e - 1 , 6 - d i p h o s p h a t e (2; lower h a l f ) of seven Pgm-2 genotypes . In each g r i d the Pgm-2-100/100 homozygote ( c l o s e d c i r c l e ) i s compared to a d i f f e r e n t homozygote (open c i r c l e ) and the c o r r e s p o n d i n g h e t e r o z y g o t e ( p a r t i a l l y c l o s e d c i r c l e ) . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . 28 26 24 22 E 20 18 Q_ D 16 1.6 =1 1.4 E Q. Q. O 1.0 0.8 H 0.6 H O 92/92 O 92/100 • 100/100 O 96/96 O 96/100 • 100/100 O 104/104 O 100/104 • 100/100 1A 1B ; o 1C i i i i t i 2A ' 1 1 1 I 1 2B 1 1 1 1 1 1 2C 0, • ^ - 6 5 10 15 20 25 30 5 10 15 20 25 30 Temperature, °C 5 10 15 20 25 30 -1.6 2 • -1.4 -- "a? -1.2 '~o to -1.0 o -0.8 Q . - CL -0.6 o 47 and were k i n e t i c a l l y i n d i s t i n g u i s h a b l e from each o t h e r except a t 1 5 ° C where the Pgm-2-92/100 h e t e r o z y g o t e expressed a s i g n i f i c a n t l y lower app Km (G1P) than the o ther two. The apparent M i c h a e l i s c o n s t a n t s for the c o f a c t o r g l u c o s e -1 , 6 - d i p h o s p h a t e decreased w i t h i n c r e a s i n g temperature ( F i g u r e 2, lower h a l f ) , and a g a i n a two-way ANOVA found t h i s e f f e c t of temperature to be s i g n i f i c a n t (F (5 ,30 ) = 76 .8 , P < . 0 0 1 ) . M u l t i p l e range t e s t s i n d i c a t e d t h a t these app Km v a l u e s were homogeneous over the upper (20 to 3 0 ° C ) and lower (5 to 1 0 ° C ) temperature ranges , and t h a t both were d i s t i n c t from those measured a t 1 5 ° C . A s i g n i f i c a n t e f f e c t was a g a i n a t t r i b u t a b l e to Pqm-2 genotype (F(6 ,30) = 18 .0 , P < . 0 0 1 ) . As found for the s u b s t r a t e G I F , m u l t i p l e range t e s t s showed t h i s e f f e c t to be caused p r i m a r i l y by the Pgm-2-92/92 homozygote (2A) which d i s p l a y e d s i g n i f i c a n t l y lower app Km ( G l 6 d i P ) v a l u e s than the t h r e e o ther homozygotes . T h i s was found by ANCOVA to h o l d t r u e a t each temperature except 2 5 ° C where the Pqm-2-92/92 homozygote d i d not d i f f e r s i g n i f i c a n t l y from the Pqm-2-100/100 homozygote. O v e r a l l , the Pqm-2-92/100 h e t e r o z y g o t e d i s p l a y e d s i g n i f i c a n t l y lower apparent M i c h a e l i s c o n s t a n t s for G16diP than Pgm-2-100/104, but not Pqm-2-96/100. T h i s o c c u r r e d because the Pgm-2-92/100 and Pqm-2-96/100 h e t e r o z y g o t e s d i f f e r e d at 15, 25, and 3 0 ° C , but not at 5, 10 or 2 0 ° C . S i n c e the pH of the i m i d a z o l e b u f f e r used i n t h i s s tudy v a r i e s i n v e r s e l y w i th temperature (Yancey and Somero 1978), the 48 i n f l u e n c e of temperature on the k i n e t i c f u n c t i o n i n g of these PGM a l l o z y m e s c o n t a i n e d a confound ing e f f e c t of the s imul taneous v a r i a t i o n i n b u f f e r p H . To examine the e f f e c t of pH a l o n e , apparent M i c h a e l i s c o n s t a n t s for G1P and G l 6 d i P were measured a t pH 6 . 5 , 7 . 0 , and 7.5 at a c o n s t a n t temperature of 2 0 ° C ( F i g u r e 3 ) . pH e x e r t e d a h i g h l y s i g n i f i c a n t e f f e c t on the app Km's for both G1P ( F ( 2 , 1 2 ) = 308 .4 , P < .001) and G16diP (F (2 ,12 ) = 517 .0 , P < . 001 ) . For both k i n e t i c p a r a m e t e r s , m u l t i p l e range t e s t s showed that the means expres sed at each pH d i f f e r e d s i g n i f i c a n t l y from each o t h e r . Pqm-2 genotype a l s o c o n t r i b u t e d s i g n i f i c a n t l y to the v a r i a t i o n w i t h pH of both app Km (G1P) (F(6 ,12) = 7 .14 , P < .05) and app Km (G16diP) (F (6 ,12 ) = 6 1 . 4 , P < . 0 0 1 ) . Once more, the Pgm-2-92/92 homozygote was l a r g e l y r e s p o n s i b l e f o r t h i s s i g n i f i c a n t e f f e c t of genotype . M u l t i p l e range t e s t s comparing the app Km (G1P) v a l u e s of the genotypes uncovered a s i g n i f i c a n t d i f f e r e n c e o n l y between the Pgm-2-92/92 and Pgm-2-96/96 homozygotes . For the app Km (G16diP) e s t i m a t e s , the Pgm-2-92/92 genotype d i s p l a y e d s i g n i f i c a n t l y lower v a l u e s than the t h r e e o ther homozygotes . The Pqm-2-100/100 and Pqm-2-96/96 genotypes were i d e n t i c a l , a l t h o u g h each was s i g n i f i c a n t l y d i f f e r e n t from the Pgm-2-104/104 homozygote. For the h e t e r o z y g o t e s , these t e s t s showed Pqm-2-92/100 to be s i g n i f i c a n t l y d i f f e r e n t from the two, which a g a i n were i n d i s t i n g u i s h a b l e over t h i s pH range . A comparison of the p a t t e r n s produced by v a r i a t i o n i n pH on the apparent M i c h a e l i s c o n s t a n t s f o r G1P and G16diP i n F i g u r e 3 49 F i g u r e 3. E f f e c t of pH on the apparent M i c h a e l i s c o n s t a n t s ( i n uM) for g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and g l u c o s e -1 ,6 -d iphosphate (2; lower h a l f ) of seven Pqm-2 genotypes . Genotyp ic comparisons are shown at the top of the f i g u r e . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . Q_ E CL CL D 34 30 26 22 18 14 2 1.4-^. 'ST* 1.2-X I to & 1.0-E 0.8-C L • Q L O 0.6-O 92/92... O 92/100 • 100/100 O 96/96 O 96/100 • 100/100 2A 8 ^ - ° I ' 1 1 ' I 1 1 1 1 I 6.5 7.0 7.5 2B O 104/104.. O 100/104 • 100/100 I ' 1 ' 1 I ' 1 ' 1 I 6.5 7.0 7.5 I 1 1 ' ' I 6.5 7.0 7.5 34 30 ^ 26 t 22 E Q. CL O 18 14 hl.4 1.2 ^ to i.o 5 0.8 ^ C L C L 0.6 ° pH 51 shows tha t one i s v i r t u a l l y a m i r r o r image of the o t h e r . An i n c r e a s e i n pH from 6.5 to 7.0 caused a marked d e c l i n e i n app Km (G1P) but o n l y a s l i g h t i n c r e a s e i n app Km ( G 1 6 d i P ) . A f u r t h e r i n c r e a s e i n pH from 7.0 to 7.5 produced o n l y a s m a l l decrease i n app Km (G1P) but a s u b s t a n t i a l i n c r e a s e in app Km ( G 1 6 d i P ) . T h i s a n t a g o n i s t i c e f f e c t of pH on the a f f i n i t y of the enzyme f o r s u b s t r a t e and c o f a c t o r i s s u g g e s t i v e of a r o l e p l a y e d by the d i s s o c i a t i o n s t a t e of the i m i d a z o l e s i d e c h a i n of a h i s t i d i n e r e s i d u e on the b i n d i n g of these r e a c t a n t s [ the a l p h a - i m i d a z o l e h y p o t h e s i s of Reeves (1972) ] . Under s t a n d a r d p h y s i o l o g i c a l c o n d i t i o n s the i m i d a z o l e moiety i s p r e s e n t i n rough ly e q u a l p r o p o r t i o n s of p r o t o n a t e d and d e p r o t o n a t e d forms (Somero 1981). A c t i v e s i t e sequences of PGM i s o l a t e d from r a b b i t ( M i l s t e i n and Sanger 1961), human ( J o s h i and H a n d l e r 1969), f l o u n d e r (Hashimoto, d e l R i o and Handler 1966), and E . c o l i ( J o s h i and H a n d l e r 1964) a l l possess a h i s t i d i n e a d j a c e n t to the c a t a l y t i c phosphoser ine r e s i d u e . I f o y s t e r PGM a l s o has t h i s h i s t i d i n e p r e s e n t , F i g u r e 3 suggests tha t the d e p r o t o n a t e d form of t h i s r e s i d u e f a v o r s b i n d i n g of G1P to the phosphoenzyme (above pH 7.0) and the p r o t o n a t e d s t a t e f a v o r s b i n d i n g of G16diP to the dephosphoenzyme (below pH 7 . 0 ) , an e f f e c t of pH not p r e v i o u s l y d e s c r i b e d for t h i s enzyme. These p H - i n d u c e d e f f e c t s are c a p a b l e of e x p l a i n i n g some of the v a r i a t i o n i n the app Km (G1P) and (G16diP) e s t i m a t e s i n F i g u r e 2 tha t were a p p a r e n t l y caused by t e m p e r a t u r e . S i n c e the pH of i m i d a z o l e b u f f e r decreases 0.017 pH u n i t s / ° C , and was 52 i n i t i a l l y a d j u s t e d to pH 7.0 at 2 0 ° C , as temperature was i n c r e a s e d from 5 to 3 0 ° C the pH of the assay b u f f e r d e c r e a s e d l i n e a r l y from a p p r o x i m a t e l y 7.3 to 6 . 8 . For the observed v a r i a t i o n i n app Km (G1P) wi th t e m p e r a t u r e , the n e g l i g i b l e changes from 10 to 2 0 ° C (pH 7.2 to 7.0) and the i n c r e a s e s from 20 to 3 0 ° C (as pH f e l l below 7.0) are l a r g e l y p r e d i c t e d from these changes i n pH a l o n e ; on ly the decreases from 5 to 1 0 ° C appear to be an e f f e c t a t t r i b u t a b l e to t e m p e r a t u r e . The v a r i a t i o n i n app Km (G16diP) w i t h temperature i s a l s o c o n s i s t e n t w i th these changes i n p H . The d e c l i n e s in app Km ( G l 6 d i P ) over the temperature range of 5 to 2 0 ° C were expec ted from the drop in b u f f e r pH from 7.3 to 7 . 0 , as were the minor changes observed from 20 to 3 0 ° C as pH decreased below 7 . 0 . These o b s e r v a t i o n s suggest t h a t the i n v i v o f u n c t i o n i n g of these a l l o z y m e s may be more s e n s i t i v e to v a r i a t i o n i n i n t r a c e l l u l a r pH than to changes in ambient t e m p e r a t u r e . Maximum v e l o c i t y (Vmax) e s t i m a t e s o b t a i n e d by b iwe ight r e g r e s s i o n were s t a n d a r d i z e d to a common p r o t e i n content at 5 ° C (to c o r r e c t f o r the s l i g h t d i f f e r e n c e s i n a c t i v i t i e s between a l l o z y m e p r e p a r a t i o n s ) and compared between genotypes over the temperature range of 10 to 3 0 ° C . P l o t s of l o g (Vmax) a g a i n s t temperature were c u r v e d s l i g h t l y downward at h i g h e r temperatures (not shown) as found p r e v i o u s l y for PGM i s o l a t e d from r a b b i t muscle (Ray and Peck 1972) and the sea anemone, M e t r i d i u m s e n i l e (Hoffman 1985). Two-way - a n a l y s i s of v a r i a n c e found no s i g n i f i c a n t d i f f e r e n c e s between genotypes i n the s e n s i t i v i t y of 53 l o g (Vmax) to e i t h e r temperature (F (5 ,24 ) = 1.15, P > .40) or pH (F(6 ,12 ) = 1.56, P > . 2 0 ) . Vmax/Km r a t i o s of the seven Pqm-2 genotypes f o r the forward r e a c t i o n d i r e c t i o n as f u n c t i o n s of temperature and pH are p r e s e n t e d i n F i g u r e s 4 and 5, r e s p e c t i v e l y . S t a t i s t i c a l comparisons of these r a t i o s by two-way ANOVA r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s between genotypes a s s o c i a t e d w i t h both temperature (F (6 ,30 ) = 6 .90 , P < .001) and pH (F (6 ,12 ) = 9 .07 , P < .001) t h a t were produced by the d i f f e r e n c e s i n the apparent M i c h a e l i s c o n s t a n t s for g l u c o s e - 1 -phosphate shown in F i g u r e s 2 and 3, r e s p e c t i v e l y . In F i g u r e 4 t h i s s i g n i f i c a n t r e s u l t i s caused e n t i r e l y by the Pgm-2-92/92 homozygote (A) which d i s p l a y e d a l a r g e r o v e r a l l Vmax/Km r a t i o than a l l o ther genotypes except the Pgm-2-92/100 h e t e r o z y g o t e . Over the pH range of 6.5 to 7.5 the Pgm-2-92/92 homozygote a l s o had a Vmax/Km r a t i o tha t was s i g n i f i c a n t l y g r e a t e r than the Pqm-2-96/96 homozygote but not the Pqm-2-100/100 or Pqm-2-104/104 genotypes . The i n t e r m e d i a t e b e h a v i o r of h e t e r o z y g o t e s a g a i n r e s u l t e d i n the Pgm-2-92/100 genotype b e i n g s i g n i f i c a n t l y d i f f e r e n t from the Pqm-2-96/100 but not the Pqm-2-100/104 h e t e r o z y g o t e . S i n c e Vmax/Km r a t i o s most a c c u r a t e l y r e f l e c t i n  v i v o c a t a l y t i c f u n c t i o n (Hoffman 1981; Watt 1983), these r e s u l t s show tha t o n l y one a l l o z y m e , Pgm-2-92, c o n s i s t e n t l y d i s p l a y s d i v e r g e n t k i n e t i c p r o p e r t i e s . R a t i o s of Vmax(f ) /Vmax(r) for the four Pgm-2 homozygotes from 5 to 3 0 ° C are p r e s e n t e d i n T a b l e I I I . The r a t e of the forward r e a c t i o n exceeded the r e v e r s e by a f a c t o r of t h r e e , 54 F i g u r e 4. E f f e c t of temperature on the Vmax/Km r a t i o s of seven Pqm-2 genotypes f o r the forward r e a c t i o n d i r e c t i o n . Bars represent ±1 standard e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol. E X o O 92/92... O 92/100 • 100/100 O 9.6/96. O 96/100 • 100/100 O 104/104 O 100/104 • 100/100 8 - A B - c : 7 -6 -5 -4 -J / : J/ -3 -2 - -/ -1- ef : i i i i i i 5 10 15 20 25 30 5 10 15 20 25 30 Temperature, °C •••••i —\—V 1 1 1 5 10 15 20 25 30 cn cn 56 F i g u r e 5. E f f e c t of pH on the Vmax/Km r a t i o s of seven Pgm-2 genotypes for the forward r e a c t i o n d i r e c t i o n . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . O 92/92 O 96/96 O 104/104 O 92/100 O 96/100 O 100/104 • 100/100 • 100/100 • 1Q0/100 58 T a b l e I I I . E f f e c t of temperature on the Vmax( f ) /Vmax(r) r a t i o s of four Pgm-2 homozygotes. 59 Pgm-2 Genotype Temperature 92/92 96/96 100/100 104/104 5 3.07+.16 2 . 9 9 ± . 1 0 3 . 0 8 ± . 1 4 2. 97±. 11 10 3 . 0 1 ± . 0 8 2 . 9 2 ± . 0 9 2 . 9 3 ± . 0 9 3. 13±.10 15 3 . 1 2 ± . 0 6 3 . 0 6 ± . 0 6 3 . 0 4 ± . 0 5 3. 07±.07 20 3 . 0 9 ± . 0 5 3.01+.05 3 . 1 0 ± . 0 6 3. 03± .05 25 2 . 8 4 ± . 0 7 2.961.06 3 . 0 2 ± . 0 6 3. 10±.07 30 2 . 8 7 ± . 0 5 2 . 9 3 ± . 0 5 2 . 8 3 ± . 0 5 2. 77±.06 60 s i m i l a r to that r e p o r t e d for r a b b i t muscle PGM by Ray and R o s c e l l i (1964b). Two-way a n a l y s i s of v a r i a n c e on the Vmax r a t i o s d e t e c t e d a s i g n i f i c a n t e f f e c t of temperature (F(5 ,15 ) = 4 .09 , P < . 0 5 ) , t h a t m u l t i p l e range t e s t s found to be caused by the h i g h e r r a t i o s measured a t 1 5 ° C compared to 3 0 ° C . No s i g n i f i c a n t d i f f e r e n c e s were observed between the four Pgm-2 homozygotes (F(3 ,15) = 0 .17 , P > . 9 0 ) . The r e g r e s s i o n s of p r o d u c t formed/ t ime v s . t ime used to c a l c u l a t e these r a t i o s were always h i g h l y s i g n i f i c a n t . The c o e f f i c i e n t s of d e t e r m i n a t i o n f o r the Vmax(f) and Vmax(r) r e g r e s s i o n s averaged 82.0 and 83.4%, r e s p e c t i v e l y . The s l o p e s of the r e g r e s s i o n l i n e s from the forward r e a c t i o n d i r e c t i o n approx imated u n i t y , thus c o n f i r m i n g a l i n e a r c o n v e r s i o n of s u b s t r a t e to p r o d u c t over the assay p e r i o d . However, as found by Ray and R o s c e l l i (1964b) , the s l o p e s from the r e v e r s e d i r e c t i o n were always n e g a t i v e , i n d i c a t i n g d e p a r t u r e s from l i n e a r i t y even though s u b s t r a t e c o n v e r s i o n was 1% or l o w e r . E q u i l i b r i u m c o n s t a n t s for the PGM r e a c t i o n showed a tendency to d e c l i n e w i t h i n c r e a s i n g t e m p e r a t u r e . At 5 ° C , Keq was e s t i m a t e d as 1 8 . 0 ± 0 . 4 5 , wh i l e at 3 0 ° C i t was c a l c u l a t e d to be 1 7 . 3 ± 0 . 3 2 . Because these v a l u e s were s l i g h t l y l a r g e r than p r e v i o u s l y r e p o r t e d f o r t h i s r e a c t i o n , Co lowick and S u t h e r l a n d ' s (1942) e s t imate of 17.2 was a c c e p t e d f o r the e n t i r e temperature r a n g e . E s t i m a t e s of the apparent M i c h a e l i s c o n s t a n t s f o r G6P of the four Pgm-2 homozygotes were c a l c u l a t e d from e q u a t i o n 2 and are p r e s e n t e d in F i g u r e 6. S i n c e the Vmax(f ) /Vmax(r) r a t i o s of F i g u r e 6. Apparent M i c h a e l i s c o n s t a n t s ( i n uM) f or g l u c o s e phosphate of four Pgm-2 homozygotes e s t i m a t e d from the Haldane e q u a t i o n . 160 H 150 H 140 8> 130 A CD. | 120 H C L Q. o 110 H 100 H 9 0 H O 96/96 • iqo/ioo_ • 104/104 T 1 1 1 1 15 20 25 30 Temperature, °C 63 these genotypes were i n d i s t i n g u i s h a b l e , t h e i r app Km (G6P) v a l u e s over t h i s t emperature range were very s i m i l a r to the p a t t e r n s observed for t h e i r app Km's f o r G1P seen i n F i g u r e 2. The a b i l i t y to d e t e c t k i n e t i c d i f f e r e n c e s between Pgm-2 genotypes f o r the r e v e r s e r e a c t i o n d i r e c t i o n by t h i s i n d i r e c t approach i s s e v e r e l y r e s t r i c t e d . T h e r e f o r e , these v a l u e s must be viewed as p r o v i s i o n a l , and a l l t h a t may be c o n c l u d e d i s tha t t h e r e i s no ev idence f o r f u r t h e r d i f f e r e n t i a t i o n through the c o n s t r a i n t s imposed by the Haldane r e l a t i o n s h i p . Over the pH range of 6.0 to 8.0 a l l four Pgm-2 a l l o z y m e s e x h i b i t e d o p t i m a l a c t i v i t y a t pH 7.2 ( F i g u r e 7 ) , which i s s l i g h t l y lower than the pH optima f o r PGM i s o l a t e d from M e t r i d i u m s e n i l e (Hoffman 1985), D . melanoqaster ( F u c c i et a l . 1979), humans ( J o s h i and Handler (1969), f l o u n d e r and shark (Hashimoto and Handler 1966), and two b a c t e r i a l s p e c i e s (Hanabusa et a l . 1966). A l t h o u g h these pH p r o f i l e s were very s i m i l a r i n form, a two-way ANOVA on the a n g u l a r t rans formed p r o p o r t i o n a l a c t i v i t i e s showed tha t s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between these four homozygotes (F(3 ,308) = 15 .3 , P < . 0 0 1 ) . M u l t i p l e range t e s t s r e v e a l e d tha t the o v e r a l l a c t i v i t y of the Pgm-2-92/92 homozygote s i g n i f i c a n t l y exceeded Pgm-2-100/100 and Pqm-2-104/104 but not Pgm-2-96/96. Decompos i t ion of t h i s o v e r a l l a n a l y s i s i n t o 11 i n d i v i d u a l one-way ANOVA's at each pH i n t e r v a l showed that t h i s was caused by the g r e a t e r a c t i v i t y of the Pgm-2-92/92 homozygote over the pH range of 6.0 to 6 . 6 . In the s t a n d a r d p h y s i o l o g i c a l pH range of 7.0 to 7.4 the four 64 F i g u r e 7. PH-dependent a c t i v i t i e s of four Pgm-2 homozygotes at 20°C. Bars represent ±1 standard e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol. Percent Activity 66 a l l o z y m e s were homogeneous. Above pH 7.4 each c u r v e d i s p l a y e d a "shoulder" which for the Pqm-2-92 and Pgm-2-96 a l l o z y m e s o c c u r r e d between pH 7.4 and 7.6 and f o r the Pgm-2-100 and Pgm-2-104 a l l o z y m e s between pH 7.6 and 7 . 8 . Marked d i f f e r e n c e s e x i s t e d between Pgm-2 genotypes in t h e i r temperature s t a b i l i t i e s at 5 0 ° C ( F i g u r e 8 ) . Thermal d e n a t u r a t i o n c o n s t a n t s , de termined f o r each genotype by r e g r e s s i n g l o g ( f r a c t i o n a l a c t i v i t y x 10) a g a i n s t i n c u b a t i o n t i m e , were compared s t a t i s t i c a l l y by ANCOVA and found to be s i g n i f i c a n t l y heterogeneous (F(6 ,448) = 383 .0 , P < . 0 0 1 ) . The Pgm-2-100/100 genotype was the most s t a b l e (kd = - 0 . 0 1 4 3 ) , and m u l t i p l e range t e s t s i n d i c a t e d that i t was s i g n i f i c a n t l y d i f f e r e n t from the Pgm -2-104/104 homozygote (kd = - 0 . 0 1 7 6 ) . These two homozygotes were s i g n i f i c a n t l y more s t a b l e than the Pqm-2-92/92 and Pgm-2-96/96 genotypes ( e x h i b i t i n g k d ' s of -0 .0283 and - 0 . 0 2 9 5 , r e s p e c t i v e l y ) . Because of the i n t e r m e d i a t e p r o p e r t i e s of h e t e r o z y g o t e s , the Pgm-2-100/104 genotype e x h i b i t e d a s i g n i f i c a n t l y lower d e n a t u r a t i o n c o n s t a n t (kd = -0 .0159) than e i t h e r Pgm-2-92/100 (kd = -0 .0206) or Pqm-2-96/100 (kd = -0.0212) w h i c h , as e x p e c t e d , were i n d i s t i n g u i s h a b l e . The e f f e c t of magnesium ion on the i n i t i a l r e a c t i o n v e l o c i t i e s of the four Pgm-2 a l l o z y m e s i s shown i n F i g u r e 9. Each v a r i a n t d i s p l a y e d o p t i m a l a c t i v i t y at 3 mM and a s l i g h t tendency to d e c l i n e at h i g h e r c o f a c t o r c o n c e n t r a t i o n s . S t a t i s t i c a l comparison of the a n g u l a r t rans formed p r o p o r t i o n a l 67 F i g u r e 8. Thermal i n a c t i v a t i o n p l o t s of seven Pgm-2 genotypes a t 5 0 ° C . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . O 92/92 O 96/96 O 104/104 O 92/100 o 96/100 O 100/104 • 100/100 • 100/100 • 100/10Q 0 5 10 15 20 25 30 0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time, minutes 69 F i g u r e 9. E f f e c t of magnesium ion on the a c t i v i t i e s of four Pqm-2 homozygotes . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . 71 a c t i v i t i e s by two-way ANOVA d e t e c t e d no s i g n i f i c a n t d i f f e r e n c e s between Pqm-2 a l l o z y m e s (F(3 ,140) = 1.22, P > . 2 0 ) . DISCUSSION A s e l e c t i o n - b a s e d e x p l a n a t i o n f o r the a s s o c i a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and v a r i o u s p h e n o t y p i c t r a i t s must be founded upon the e x i s t e n c e of b i o c h e m i c a l d i f f e r e n c e s between the a l l e l i c p r o d u c t s of the s p e c i f i c l o c i i n v o l v e d in these r e l a t i o n s h i p s ( c f . C l a r k e 1975; Koehn 1978). Once these f u n c t i o n a l d i f f e r e n c e s have been e s t a b l i s h e d , however, v e r i f i c a t i o n of the overdominance h y p o t h e s i s p l a c e s s t r i n g e n t requ irements on the p r o p e r t i e s of h e t e r o z y g o t e s r e l a t i v e to t h e i r c o n s t i t u e n t homozygotes c o n c e r n i n g both the m a n i f e s t a t i o n of overdominance and the c o n d i t i o n s a l l o w i n g a s t a b l e po lymorph ic e q u i l i b r i u m ( e . g . Mandel 1959; L e w o n t i n , G i n z b u r g and T u l j a p u r k a r 1978). For the Pgm-2 l o c u s in C r a s s o s t r e a g i g a s , k i n e t i c and s t r u c t u r a l d i f f e r e n c e s e x i s t between the four most common a l l o z y m e s . S i g n i f i c a n t d i f f e r e n c e s were observed between Pgm-2 genotypes i n t h e i r apparent M i c h a e l i s c o n s t a n t s f o r g l u c o s e - 1 - p h o s p h a t e and g l u c o s e - 1 , 6 - d i p h o s p h a t e over ranges of temperature ( F i g u r e 2) and pH ( F i g u r e 3 ) , Vmax/Km r a t i o s ( F i g u r e s 4 and 5 ) , pH-dependent a c t i v i t i e s ( F i g u r e 7 ) , and t h e r m o s t a b i l i t i e s ( F i g u r e 8 ) . I t must now be de termined i f these b i o c h e m i c a l d i f f e r e n c e s are s u f f i c i e n t to account f o r the overdominance for a d u l t body weight d e s c r i b e d a t t h i s l o c u s by F u j i o (1982) . S e v e r a l l i n e s of argument suggest tha t they are 72 n o t . F i r s t , c o n s i d e r a t i o n of the c a t a l y t i c r e a c t i o n mechanism of phosphoglucomutase suggests t h a t the observed k i n e t i c d i f f e r e n c e s may not be as pronounced , or even expres sed at a l l , under _in v i v o c o n d i t i o n s . P r e v i o u s s t u d i e s on PGM have e s t a b l i s h e d tha t i t f u n c t i o n s a l o n g a cont inuum between " u n i -u n i " and "ping-pong" k i n e t i c s depending on the s p e c i f i c sugar phosphates i n t e r c o n v e r t e d and the organism from which i t was e x t r a c t e d (reviewed by Ray and Peck 1972). T h i s v a r i a t i o n i s caused by d i f f e r e n c e s i n the d i s s o c i a t i o n r a t e of the d i p h o s p h a t e from the enzyme's c e n t r a l r e a c t i o n complex: i f t h i s o c c u r s f r e q u e n t l y , g l u c o s e - 1 , 6 - d i p h o s p h a t e a c t s as " f i r s t p r o d u c t " as w e l l as "second s u b s t r a t e " i n the c l a s s i c " p i n g -pong" mechanism of C l e l a n d (1963) . D e s p i t e t h i s v a r i a t i o n i n r e a c t i o n mechanism, double r e c i p r o c a l p l o t s of i n i t i a l v e l o c i t y a g a i n s t G1P c o n c e n t r a t i o n at v a r i o u s l e v e l s of G l 6 d i P in the v i c i n i t y of Km ( G l 6 d i P ) produce a s e r i e s of p a r a l l e l l i n e s in accordance wi th the p i n g - p o n g mechanism (Ray and R o s c e l l i 1964a; J o s h i and Handler 1966, 1969; Hashimoto and H a n d l e r 1966). For an enzyme obey ing p i n g - p o n g k i n e t i c s , Ray and R o s c e l l i (1964a) have v e r i f i e d e x p e r i m e n t a l l y t h a t the t r u e or " r e a l i z e d " M i c h a e l i s c o n s t a n t f o r G1P i s r e l a t e d to the apparent Km's f o r both G1P and G16diP by the f o l l o w i n g e q u a t i o n : 73 K'm (G1P) = app Km (G1P)[G16diP] . . . ( 3 ) app Km (G16diP) + [ G l 6 d i P ] where K'm (G1P) i s the r e a l i z e d M i c h a e l i s c o n s t a n t f o r G1P, app Km (G1P) and app Km (G16diP) are the apparent M i c h a e l i s c o n s t a n t s f o r G1P and G l 6 d i P , r e s p e c t i v e l y , and [ G l 6 d i P ] i s the c o n c e n t r a t i o n of G l 6 d i P . In mammalian t i s s u e s , G l 6 d i P c o n c e n t r a t i o n s have been observed to range from 5 /uM ( B e i t n e r , Haberman and Nordenberg 1978) to as h i g h as 80 uM (Passonneau et a l . 1969). These l e v e l s are s e v e r a l o r d e r s of magnitude above PGM's Km f o r G16diP i n these organisms (Ray and R o s c e l l i 1964b; Passonneau et a l . 1969), s u g g e s t i n g that the enzyme e x i s t s _in v i v o e n t i r e l y in the a c t i v e p h o s p h o r y l a t e d s t a t e . However, a number of recent s t u d i e s have demonstrated a c t i v a t i o n s i n the a c t i v i t y of PGM by i n c r e a s e s i n G16diP c o n c e n t r a t i o n s (rev iewed by B e i t n e r 1984) which a r e unexpected i f the c o f a c t o r e x i s t s at s a t u r a t i n g l e v e l s . These r e s u l t s have been i n t e r p r e t e d as a d e i n h i b i t o r y e f f e c t of G 1 6 d i P , but from what i s known about PGM, t h i s can o n l y occur i f some p r o p o r t i o n of the enzyme e x i s t s in the d e p h o s p h o r y l a t e d s t a t e (see Ray and Peck 1972). The e l e c t r o p h o r e t i c d e t e c t i o n of both p h o s p h o r y l a t e d and d e p h o s p h o r y l a t e d forms of o y s t e r PGM i s c o n s i s t e n t w i t h these s u g g e s t i o n s . T h e r e f o r e , assuming an u n s a t u r a t i n g G16diP c o n c e n t r a t i o n of 1 uM the r e a l i z e d M i c h a e l i s c o n s t a n t s for G1P and c o r r e s p o n d i n g Vmax/K'm r a t i o s of the 7 Pgm-2 genotypes as 74 f u n c t i o n s of temperature and pH have been c a l c u l a t e d from e q u a t i o n 3 and are p r e s e n t e d i n F i g u r e s 10 and 11, r e s p e c t i v e l y . Comparison of these m o d i f i e d parameters w i t h those p r e s e n t e d i n F i g u r e s 2, 3, 4 and 5 shows t h a t the K'm (G1P) e s t i m a t e s are reduced by a p p r o x i m a t e l y 50% and hence the Vmax/K'm r a t i o s have i n c r e a s e d by rough ly the same amount. More i m p o r t a n t l y , two-way a n a l y s i s of v a r i a n c e on these new e s t i m a t e s found t h a t the s i g n i f i c a n t e f f e c t s p r e v i o u s l y a t t r i b u t a b l e to Pqm-2 genotype had e i t h e r been e l i m i n a t e d or s u b s t a n t i a l l y r e d u c e d . In c o n t r a s t to the r e s u l t s f or the app Km (G1P) and app Km ( G l 6 d i P ) e s t i m a t e s i n d i v i d u a l l y , these a n a l y s e s were unable to d e t e c t any s i g n i f i c a n t d i f f e r e n c e s between genotypes i n t h e i r r e a l i z e d M i c h a e l i s c o n s t a n t s for G1P (F(6 ,30) = 0 .913 , P > .40) or Vmax/K'm r a t i o s (F(6 ,30) = 1.75, P > .40) over the temperature range o f . 5 to 3 0 ° C ( F i g u r e 10) . Over the pH range s t u d i e d ( F i g u r e 11), s i g n i f i c a n t d i f f e r e n c e s were s t i l l p r e s e n t between genotypes i n these m o d i f i e d K'm v a l u e s (F(6 ,12 ) = 4 .70 , P < .05) and Vmax/K'm r a t i o s (F (6 ,12 ) = 4 .04 , P < . 0 5 ) , but t h e i r magnitude was d i m i n i s h e d and a p o s t e r i o r i m u l t i p l e range t e s t s were unable to d e t e c t any d i f f e r e n c e s between the genotypes . The homogenizat ion of the K'm (G1P) e s t i m a t e s of Pgm-2 genotypes appears to be a consequence of the c o v a r i a t i o n of t h e i r app (G1P) and app Km ( G l 6 d i P ) v a l u e s . A p p a r e n t l y , the amino a c i d s u b s t i t u t i o n ( s ) g i v i n g r i s e to the d i f f e r e n c e s i n F i g u r e 10. E f f e c t of t emperature on the e s t i m a t e d in_ v i v o apparent M i c h a e l i s c o n s t a n t s ( i n MM) f o r g l u c o s e - 1 -phosphate (1; upper h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; lower h a l f ) of seven Pgm-2 genotypes . O 92/92 O 92/100 • 100/100 O ?6/?6 O 96/100 O 104/104 _ O 100/104 9 100/100 1A 1B 1C hie 14 10 £ 8 Q. Q_ D he T r 2B 1 1 1 1 1 1 -5 10 15 20 25 30 14 1 2 £ 10 ^ 8 D h2 i r i r 5 10 15 20 25 30 Temperature, °C "I 1 1 1 1 r -5 10 15 20 25 30 77 F i g u r e 1 1 . E f f e c t of pH on the e s t i m a t e d in_ v i v o apparent M i c h a e l i s c o n s t a n t s ( i n uM) f o r g l u c o s e - 1 - p h o s p h a t e (1; upper h a l f ) and c o r r e s p o n d i n g Vmax/K'm r a t i o s (2; lower h a l f ) of seven Pgm-2 genotypes . • Vmax/K'm app K'm(G1P), fM rTr" "T" I 1 I 1 I cn oo ^ to tn oo =2 O Vmax/K'm app K'm(G1P), yUM 8Z. 79 e l e c t r o p h o r e t i c m o b i l i t y between these a l l o z y m e s a f f e c t the b i n d i n g a n d / o r i n t e r n a l r e a c t i o n pathways for G1P and G l 6 d i P i n a s i m i l a r f a s h i o n . T h i s t r e n d i s most apparent for the Pgm-2-92/92 homozygote which e x h i b i t e d apparent M i c h a e l i s c o n s t a n t s for both G1P and G16diP t h a t were s m a l l e r than most o ther genotypes over the range of t emperatures s t u d i e d (see F i g u r e 2 ) . Upon s u b s t i t u t i o n i n t o e q u a t i o n 3, these lower parameter v a l u e s ac t to c a n c e l each other out such tha t the K'm (G1P) e s t i m a t e s for the Pgm-2-92/92 homozygote become i n d i s t i n g u i s h a b l e from the o ther genotypes . The c o v a r i a t i o n of these k i n e t i c parameters i s a l s o e x h i b i t e d over the pH range of 6.5 to 7.5 ( F i g u r e 3) w i t h the e x c e p t i o n however of the app Km (G1P) v a l u e s of genotypes p o s s e s s i n g the Pqm-2-104 a l l e l e . The anomalous b e h a v i o r of these genotypes may i n p a r t e x p l a i n the s i g n i f i c a n t e f f e c t of Pqm-2 genotype expres sed for these K'm (G1P) e s t i m a t e s and Vmax/K'm r a t i o s over t h i s pH range . However, the important p o i n t be ing s t r e s s e d here i s that by t a k i n g i n t o c o n s i d e r a t i o n the p i n g - p o n g r e a c t i o n mechanism of PGM, and thus the in terdependence of app Km (G1P) and app Km ( G l 6 d i P ) on c a t a l y t i c f u n c t i o n , the k i n e t i c d i f f e r e n c e s expres sed i_n v i v o between these Pgm-2 genotypes may be much l e s s than expected from the d i f f e r e n c e s shown by the parameters c o n s i d e r e d s e p a r a t e l y . I t s h o u l d be emphasized that these k i n e t i c d i f f e r e n c e s may s t i l l h o l d under in v i v o c o n d i t i o n s i f o y s t e r PGM does not conform to the p i n g - p o n g mechanism or the i n t r a c e l l u l a r c o n c e n t r a t i o n of G16diP i s i n f a c t s a t u r a t i n g . However, even i f 80 these f u n c t i o n a l d i f f e r e n c e s are expres sed the b i o c h e m i c a l p r o p e r t i e s of Pgm-2 genotypes do not appear c o m p a t i b l e w i t h e i t h e r the m a n i f e s t a t i o n of overdominance or the maintenance of a s t a b l e po lymorphic e q u i l i b r i u m . M o l e c u l a r c h a r a c t e r i z a t i o n s of phosphoglucomutase from a v a r i e t y of organisms have e s t a b l i s h e d i t as a monomer w i t h a m o l e c u l a r weight r a n g i n g from 62,000 to 67,000 d a l t o n s (rev iewed by Ray and Peck 1972). The e l e c t r o p h o r e t i c s t a i n i n g p a t t e r n s of o y s t e r PGM, i n a d d i t i o n to i t s m o b i l i t y on SDS g e l s and e l u t i o n p a t t e r n s on g e l f i l t r a t i o n co lumns , are a l l i n agreement w i t h these p r e v i o u s s t u d i e s . For a monomeric enzyme, the e x p r e s s i o n of overdominance v i a the s u p e r i o r p r o p e r t i e s of h e t e r o m u l t i m e r s ( e . g . Schwartz and Laughner 1969; S i n g h , Hubby and Lewont in 1974; Watt 1977, 1983), i s not p o s s i b l e . H e t e r o z y g o t e s a t the Pgm-2 l o c u s were expected to d i s p l a y the s t r i c t l y i n t e r m e d i a t e p r o p e r t i e s tha t were i n f a c t observed over the range of c o n d i t i o n s examined. I f b i o c h e m i c a l overdominance i s expres sed at t h i s l o c u s by these d i f f e r e n c e s i n c a t a l y t i c f u n c t i o n , i t must be of the type termed by W a l l a c e (1959) as " m a r g i n a l " , i . e . , the mean performance of h e t e r o z y g o t e s exceeds tha t of homozygotes o n l y when averaged over d i f f e r e n t c o n d i t i o n s . H e t e r o z y g o t e s a t the Pgm-2 l o c u s i n C . g i g a s are dominated by the Pgm-2-92/100, 96/100 and 100/104 genotypes ( r e p r e s e n t i n g 74% of a l l h e t e r o z y g o t e s ) . T h e r e f o r e , i t i s these h e t e r o z y g o t e s t h a t must be r e s p o n s i b l e for overdominant e f f e c t s a t t r i b u t e d to t h i s l o c u s . The e x p r e s s i o n of m a r g i n a l overdominance by the 81 t h r e e most common Pgm-2 h e t e r o z y g o t e s p l a c e s r e s t r i c t i v e c o n d i t i o n s on the b i o c h e m i c a l p r o p e r t i e s of the Pgm-2-92, 96 and 104 a l l o z y m e s r e l a t i v e to the most common Pgm-2-100 a l l o z y m e . Because of the s i m i l a r f r e q u e n c i e s of these l e s s f requent a l l e l e s , the f u n c t i o n a l p r o p e r t i e s of t h e i r r e s p e c t i v e homozygotes must d i v e r g e by r o u g h l y e q u a l amounts from the Pqm-2-100/100 genotype . F u r t h e r m o r e , the f u n c t i o n a l parameters of the l e s s common homozygotes must f l u c t u a t e i n v a l u e above and below tha t of the Pgm-2-100/100 genotype i n such a manner, and w i t h s u f f i c i e n t magni tude , tha t s u p e r i o r p r o p e r t i e s are c o n f e r r e d upon the average b e h a v i o r of the Pgm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s . V a r i a t i o n i n the s e l e c t i v e v a l u e s of these homozygotes c o u l d a r i s e through e i t h e r 1) v a r i a b l e s e l e c t i o n a c r o s s d i f f e r e n t seasons , m i c r o e n v i r o n m e n t a l c o n d i t i o n s , or p h y s i o l o g i c a l s t a t e s ( e . g . Koehn and Immerman 1981), 2) r e v e r s a l s of the k i n e t i c a n d / o r s t r u c t u r a l advantages expres sed over d i f f e r e n t ranges of temperature or pH ( e . g . P l a c e and Powers 1979), or 3) t r a d e - o f f s between d i f f e r e n t c a t a l y t i c or s t r u c t u r a l p r o p e r t i e s ( e . g . Walsh 1981). The o v e r a l l performance of Pgm-2 homozygotes do not seem c a p a b l e of meet ing the above requ irements f o r the p r o d u c t i o n of m a r g i n a l overdominance . The Pgm-2-92 a l l o z y m e possesses a s u i t e of c h a r a c t e r i s t i c s t h a t d i f f e r from the Pgm-2-100 a l l o z y m e , but the same does not h o l d f o r e i t h e r the Pgm-2-96 or Pgm-2-104 a l l o z y m e s . C o n s e q u e n t l y , a case c o u l d be made f o r the e x p r e s s i o n of m a r g i n a l overdominance by the Pgm-2-92/100 h e t e r o z y g o t e by 82 i n v o k i n g a v a r i a b l e s e l e c t i o n reg ime , or from t r a d e - o f f s between c a t a l y t i c f u n c t i o n and t h e r m o s t a b i l i t y . However, the g r e a t e r s i m i l a r i t y of the Pgm-2-96 and Pgm-2-104 a l l o z y m e s r e l a t i v e to the Pgm-2-100 a l l ozyme p r e c l u d e s a s i m i l a r argument f o r the Pgm-2-96/100 and Pgm-2-100/104 h e t e r o z y g o t e s . A l t h o u g h t h e r e i s some ev idence f o r r e v e r s a l s of performance i n v o l v i n g the apparent M i c h a e l i s c o n s t a n t s for G1P and G16diP of Pgm-2-96 and Pgm-2-104 r e l a t i v e to Pgm-2-100 over d i f f e r e n t ranges of temperature and pH (see F i g u r e s 2 and 3 ) , the magnitude of these f l u c t u a t i o n s seem f a r too s m a l l to produce a net h e t e r o z y g o t e advantage . The d i f f e r e n t i a l b e h a v i o r of these h e t e r o z y g o t e s i s a l s o i n c o m p a t i b l e w i t h the maintenance of a s t a b l e m u l t i - a l l e l i c polymorphism by overdominance . In t h e i r n u m e r i c a l a n a l y s e s , L e w o n t i n , G i n z b u r g and T u l j a p u r k a r (1978) found that the c o n d i t i o n s f o r such an e q u i l i b r i u m r e q u i r e tha t the v a r i a n c e i n f i t n e s s of h e t e r o z y g o t e s be much lower than t h a t expres sed between homozygotes and h e t e r o z y g o t e s . A l t h o u g h k i n e t i c d i f f e r e n t i a t i o n may be weakly a s s o c i a t e d w i t h f i t n e s s , the d i s t i n c t i v e n e s s of the Pgm-2-92 a l l o z y m e and hence thve Pgm-2-92/100 h e t e r o z y g o t e compared to both the Pgm-2-96/100 and 100/104 genotypes causes both of these c o n d i t i o n s to be v i o l a t e d . The b i o c h e m i c a l d i f f e r e n c e s uncovered between these a l l o z y m e s are a l s o c o n t r a d i c t o r y to t h e i r observed frequency d i s t r i b u t i o n s . The Pqm-2-92 a l l o z y m e d i s p l a y e d lower app Km (G1P) v a l u e s and t h e r e f o r e l a r g e r c a t a l y t i c a l l y . important 8 3 Vmax/Km r a t i o s from 15 to 3 0 ° C and at a l l pH's compared to the three o t h e r s . T h i s a l l o z y m e a l s o e x h i b i t e d s i g n i f i c a n t l y g r e a t e r a c t i v i t i e s under low pH c o n d i t i o n s , which have been demonstrated to occur i n marine b i v a l v e s as a consequence of p r o l o n g e d a n a e r o b i c metabol i sm (Wijsman 1975; W a l s h , McDonald and Booth 1984). Because of these s u p e r i o r p r o p e r t i e s the Pgm-2-92 a l l e l e might be expected to enjoy a s e l e c t i v e advantage tha t c o u l d e v e n t u a l l y l e a d to i t s f i x a t i o n i n p o p u l a t i o n s of C . g i g a s . The low frequency of t h i s a l l e l e i s i n c o n f l i c t w i t h these advantageous c a t a l y t i c p r o p e r t i e s u n l e s s these are somehow b a l a n c e d by i t s s i g n i f i c a n t l y g r e a t e r t h e r m o l a b i l i t y . T h i s argument however would p r e d i c t the s e l e c t i v e removal of the Pgm-2-96 a l l e l e because t h i s a l l o z y m e was e q u a l l y s e n s i t i v e to thermal d e n a t u r a t i o n , but i n d i s t i n g u i s h a b l e from the Pgm-2-100 a l l ozyme i n a l l o ther p r o p e r t i e s examined. The b i o c h e m i c a l da ta i s s i m i l a r l y unable to e x p l a i n the h i g h f requency of the Pgm-2-100 a l l e l e . R e c e n t l y , Smouse (1986) has p o i n t e d out tha t the most common a l l e l e a t m u l t i - a l l e l i c overdominant l o c i s h o u l d produce the f i t t e s t homozygote. The Pgm -2-100/100 genotype d i s p l a y e d k i n e t i c p r o p e r t i e s tha t were g e n e r a l l y i n t e r m e d i a t e w i t h r e s p e c t to the o t h e r homozygotes. A l t h o u g h the parameter l e v e l s expres sed by the Pgm-2-100/100 homozygote may in f a c t be best s u i t e d f o r in v i v o f u n c t i o n , i t s grea t s i m i l a r i t y to the Pgm-2-96 a l l e l e and the l i m i t e d d i f f e r e n t i a t i o n of the two o ther a l l o z y m e s weakens the argument tha t k i n e t i c i n t e r m e d i a c y at t h i s l o c u s produces the f i t t e s t 84 genotype . The Pgm-2-100/100 genotype d i d e x h i b i t s u p e r i o r i t y over the o ther homozygotes through i t s s i g n i f i c a n t l y lower thermal d e n a t u r a t i o n c o n s t a n t . The r o l e p l a y e d by d i f f e r e n c e s between a l l o z y m e s i n t h e i r temperature s t a b i l i t i e s i n m a i n t a i n i n g enzyme polymorphisms i s u n c l e a r because in s e v e r a l i n s t a n c e s the observed c l i n a l v a r i a t i o n i n a l l e l e f r e q u e n c i e s i s e x a c t l y o p p o s i t e to tha t p r e d i c t e d ( e . g . Oakeshot t et a l . 1981, 1982; P l a c e and Powers 1984a). The i n a b i l i t y of these b i o c h e m i c a l d i f f e r e n c e s to c l e a r l y account f o r the maintenance of these a l l e l e s i n a b a l a n c e d polymorphism i s a dilemma . t h a t has been reached i n p r e v i o u s s t u d i e s of t h i s na ture ( e . g . M e r r i t t 1972; Hoffman 1981; H a l l 1985; Zera 1987). An important l i m i t a t i o n of the c o n c l u s i o n s reached by s t u d i e s of t h i s na ture concern the unde tec ted presence of f u n c t i o n a l d i f f e r e n c e s between Pgm-2 genotypes i n enzymic parameters t h a t were not examined. One p a r t i c u l a r l y important p r o p e r t y tha t c o u l d not be measured was s u b s t r a t e t u r n o v e r number ( k c a t ) . D i f f e r e n c e s i n kcat and kcat /Km r a t i o s between these genotypes c o u l d e a s i l y have gone u n n o t i c e d by the s u b s t i t u t i o n of Vmax f o r k c a t , because Vmax i s a composi te term of the p r o d u c t of kcat and enzyme c o n c e n t r a t i o n . However, s i n c e the Vmax e s t i m a t e s were s t a n d a r d i z e d to a common p r o t e i n c o n c e n t r a t i o n and compared t o g e t h e r under i d e n t i c a l c o n d i t i o n s , the a c t u a l kcat /Km r a t i o s must remain s i m i l a r to those p r e s e n t e d i n F i g u r e s 4 and 5; on ly the r e l a t i v e ' p o s i t i o n s of the c u r v e s f o r d i f f e r e n t genotypes would be changed. A l t h o u g h i t i s 85 p o s s i b l e t h a t the kcat /Km r a t i o s f o r the Pgm-2-92/92, 96/96 and 104/104 homozygotes may a l l be s h i f t e d to a s i m i l a r ex tent r e l a t i v e to the Pqm-2-100/100 genotype , t h i s seems u n l i k e l y and the problem of a c c o u n t i n g f o r the m a r g i n a l overdominance of the t h r e e most common h e t e r o z y g o t e s r e m a i n s . Another important p r o p e r t y of phosphoglucomutase not examined in t h i s s tudy concerns i t s n o n s p e c i f i c i t y : PGM i s c a p a b l e of c a t a l y z i n g the i n t e r m o l e c u l a r t r a n s f e r of a phosphate group between a v a r i e t y of 5 and 6 carbon sugars ( e . g . Lowry and Passonneau 1969). T h e r e f o r e , d i f f e r e n c e s c o u l d e x i s t between Pgm -2 genotypes in t h e i r a f f i n i t i e s f o r d i f f e r e n t s u b s t r a t e s , as demonstrated for the 3 human PGM isozymes by Q u i c k , F i s h e r and H a r r i s (1974) . The r e l a t i v e importance of t h i s f u n c t i o n f o r o y s t e r PGM i s p r o b a b l y l i m i t e d because of the predominant r o l e the p r i m a r y r e a c t i o n p l a y s i n the s e a s o n a l c y c l e of g lycogen s y n t h e s i s and d e g r a d a t i o n ( e . g . Gabbott 1975). In summary, the b i o c h e m i c a l d i f f e r e n c e s observed between the 7 Pgm-2 genotypes examined i n t h i s s tudy do not seem capab le of e x p l a i n i n g the overdominance r e p o r t e d at t h i s l o c u s by F u j i o (1982) . As shown by T u r e l l i and G i n z b u r g (1983) , h e t e r o z y g o t e s u p e r i o r i t y i s expected to be m a n i f e s t e d a t a l l p o l y m o r p h i c l o c i s u b j e c t to some form of b a l a n c i n g s e l e c t i o n . A l t h o u g h the i n t e r p r e t a t i o n of f i t n e s s d i f f e r e n c e s a r i s i n g from iin v i t r o k i n e t i c p r o p e r t i e s i s ex tremely complex, the b i o c h e m i c a l data p r o v i d e l i t t l e i n s i g h t i n t o how t h i s a l l o z y m i c v a r i a t i o n c o u l d 86 be a c t i v e l y m a i n t a i n e d by s e l e c t i o n i n n a t u r a l p o p u l a t i o n s . The next c h a p t e r w i l l examine the p o t e n t i a l c o n t r i b u t i o n of Pgm-2 s p e c i f i c a c t i v i t y v a r i a t i o n in the e x p r e s s i o n of overdominance a t t h i s l o c u s . 87 CHAPTER 3 ENVIRONMENTAL AND GENOTYPIC EFFECTS ON PGM ACTIVITY INTRODUCTION Many s t u d i e s comparing the b i o c h e m i c a l p r o p e r t i e s of genotypes at po lymorphic enzyme l o c i have d e t e c t e d d i f f e r e n c e s i n s p e c i f i c enzyme a c t i v i t y ( e . g . V igue and Johnson 1973; Hay and Armstrong 1976; H i c k e y 1977; D a n f o r t h and Beardmore 1979). In s e v e r a l c a s e s , the cause of t h i s a c t i v i t y v a r i a t i o n when examined by i m m u n o e l e c t r o p h o r e t i c p r o c e d u r e s has been found to r e s u l t from d i f f e r e n c e s i n in v i v o enzyme c o n c e n t r a t i o n s ( e . g . L a i and S c a n d a l i o s 1980; K i n g and McDonald 1983; E s t e l l e and Hodget t s 1984). A l t h o u g h t i s s u e enzyme c o n c e n t r a t i o n s are a f f e c t e d by both e n v i r o n m e n t a l and g e n e t i c f a c t o r s , a growing number of s t u d i e s have demonstrated t h a t these d i f f e r e n c e s are produced by polymorphisms at " r e g u l a t o r y " gene l o c i ( c f . Paigen 1979), i . e . , g e n e t i c e lements tha t c o n t r o l the deve lopmenta l t i m i n g a n d / o r l e v e l s of e x p r e s s i o n of s t r u c t u r a l enzyme l o c i (rev iewed by S c a n d a l i o s and Baum 1982; L a u r i e - A h l b e r g 1985; Pa igen 1986). V a r i a t i o n at these two l e v e l s of gene o r g a n i z a t i o n has p r e s e n t e d a dilemma f o r the i n t e r p r e t a t i o n of the s e l e c t i v e importance of a l l o z y m i c v a r i a t i o n . Do r e g u l a t o r y polymorphisms tha t cause d i f f e r e n c e s i n s t e a d y - s t a t e enzyme a c t i v i t y l e v e l s o v e r r i d e the k i n e t i c a n d / o r s t r u c t u r a l d i f f e r e n c e s t h a t may a l s o e x i s t between a l l o z y m e s ( e . g . W i l s o n , C a r l s o n and White 1977)? 88 Or are both f u n c t i o n a l l y s i g n i f i c a n t and , i f so , what i s the r e l a t i v e importance of each? The answers to these q u e s t i o n s are s t i l l l a r g e l y unknown, even f o r the i n t e n s i v e l y s t u d i e d a l c o h o l dehydrogenase (Adh) and a l p h a - a m y l a s e (Amy) polymorphisms i n D r o s o p h i l a melanogaster (see d i s c u s s i o n in Z e r a , Koehn and H a l l 1985). Marked d i f f e r e n c e s i n s p e c i f i c a c t i v i t y e x i s t between genotypes at both of these l o c i tha t are a t t r i b u t a b l e to both l i n k e d and u n l i n k e d r e g u l a t o r y polymorphisms ( L a u r i e - A h l b e r g 1985). T h e r e f o r e , the k i n e t i c d i f f e r e n c e s observed between these enzyme v a r i a n t s may be of l i t t l e consequence to the maintenance of these polymorphisms i n n a t u r a l p o p u l a t i o n s . However, the h i g h a c t i v i t y A d h - F / F and Amy-4,6 homozygotes a l s o d i s p l a y l a r g e r M i c h a e l i s c o n s t a n t s f o r t h e i r r e s p e c t i v e s u b s t r a t e s ( e . g . McDonald , Anderson and Santos 1980 f o r Adh; Hoorn and S c h a r l o o 1978 f o r Amy), which i n combinat ion w i t h t h e i r i n c r e a s e d a c t i v i t y l e v e l s may p r o v i d e an a d d i t i o n a l advantage f o r f u n c t i o n i n g under h i g h s u b s t r a t e c o n d i t i o n s ( c f . A t k i n s o n 1977). These p a t t e r n s suggest t h a t an important i n t e r a c t i o n may e x i s t between r e g u l a t o r y and s t r u c t u r a l l o c u s po lymorphisms . From these c o n s i d e r a t i o n s , a t tempts to a s se s s the s e l e c t i v e importance of a s p e c i f i c enzyme polymorphism must s i m u l t a n e o u s l y examine the e x i s t e n c e and p o t e n t i a l i n t e r a c t i o n between these d i f f e r e n t types of g e n e t i c v a r i a t i o n . R e g u l a t o r y polymorphisms have been most e x t e n s i v e l y s t u d i e d 89 i n D . melanoqaster (rev iewed by L a u r i e - A h l b e r g 1985). In t h i s s p e c i e s , g e n e t i c v a r i a t i o n a f f e c t i n g enzyme a c t i v i t y l e v e l s appears to be the r u l e r a t h e r than the e x c e p t i o n . F o r example, a l l 23 enzymes s t u d i e d by L a u r i e - A h l b e r g et a l . (1982) i n i s o g e n i c l i n e s e s t a b l i s h e d from 48 second and 48 t h i r d chromosomes e x t r a c t e d from n a t u r a l p o p u l a t i o n s e x h i b i t e d a s i g n i f i c a n t g e n e t i c component to t h e i r observed v a r i a t i o n in a c t i v i t y l e v e l s . In marine i n v e r t e b r a t e s , no r e g u l a t o r y polymorphisms have ye t been c h a r a c t e r i z e d , but t h i s i s not s u r p r i s i n g i n l i g h t of our l i m i t e d knowledge of t h e i r g e n e t i c s t r u c t u r e and o r g a n i z a t i o n . However, tha t r e g u l a t o r y v a r i a t i o n analogous to tha t observed in D r o s o p h i l a e x i s t s i s suggested by v a r i a t i o n i n the s p e c i f i c a c t i v i t i e s of s t r u c t u r a l l o c u s genotypes at the Pg i (Hoffman 1981) and Odh (Walsh 1981) l o c i i n the anemone, M e t r i d i u m s e n i l e , the Gpt l o c u s i n the copepod, T i g r i o p u s c a l i f o r n i c u s (Burton and Feldman 1983), the Pg i l o c u s in the o y s t e r , C r a s s o s t r e a v i r g i n i c a ( M a r t i n 1979), and the aminopept idase -1 l o c u s i n the m u s s e l , M y t i l u s e d u l i s (Koehn and Immerman 1981). The a c t i o n of c i s - or t r a n s - a c t i n g r e g u l a t o r y v a r i a n t s a f f e c t i n g r a t e s of enzyme s y n t h e s i s or d e g r a d a t i o n can be e x c l u d e d as an e x p l a n a t i o n for these a c t i v i t y d i f f e r e n c e s f o r o n l y the aminopept idase -1 l o c u s in M. e d u l i s . H e r e , the observed v a r i a t i o n i n enzyme a c t i v i t i e s were shown by Koehn and S i e b e n a l l e r (1981) to a r i s e from d i f f e r e n c e s between the a l l o z y m e s i n t h e i r s u b s t r a t e t u r n o v e r numbers (kcat v a l u e s ) , s i n c e the measured c o n c e n t r a t i o n of aminopept idase -1 enzyme was s i m i l a r i n a l l genotypes s t u d i e d . 90 In t h i s c h a p t e r , the p a t t e r n s of v a r i a t i o n i n s p e c i f i c a c t i v i t y between genotypes at the po lymorphic Pqm-2 l o c u s in the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s are d e s c r i b e d . In marine b i v a l v e s , the examinat ion of s p e c i f i c a c t i v i t y d i f f e r e n c e s at po lymorph ic l o c i must s i m u l t a n e o u s l y c o n s i d e r t h e i r p o t e n t i a l i n t e r a c t i o n w i t h s e a s o n a l changes i n metabol i sm t h a t r e f l e c t p r e v a i l i n g e n v i r o n m e n t a l c o n d i t i o n s ( i . e . food a v a i l a b i l i t y , t e m p e r a t u r e , s a l i n i t y , e t c . ) and t h e i r annual r e p r o d u c t i v e c y c l e ( L i v i n g s t o n e 1981; Gabbott 1983). In response to one or more of these f a c t o r s the a c t i v i t i e s of a number of enzymes have been observed to f l u c t u a t e on a s easona l b a s i s ( e . g . Chambers et a l . 1975; L i v i n g s t o n e 1976; Gabbott and Head 1980; L i v i n g s t o n e and C l a r k e 1983). One of the dominant annual c y c l e s i n marine b i v a l v e s i n v o l v e s the s y n t h e s i s and d e g r a d a t i o n of g l y c o g e n , t h e i r p r i m a r y energy s torage compound (Gabbott 1975). In B r i t i s h Columbia p o p u l a t i o n s of C . g i g a s , g lycogen i s s y n t h e s i z e d i n the f a l l and s p r i n g , s t o r e d i n the mantle and d i g e s t i v e g l a n d and degraded i n the e a r l y summer p r i o r to spawning (Quayle 1969; Whyte and E n g l a r 1982). S i n c e phosphoglucomutase f u n c t i o n s in g lycogen m e t a b o l i s m , i t s a c t i v i t y l e v e l may v a r y s e a s o n a l l y and e x h i b i t d i f f e r e n t i a l responses between t i s s u e s or m i c r o e n v i r o n m e n t a l c o n d i t i o n s . The p o t e n t i a l i n f l u e n c e of these e n v i r o n m e n t a l and p h y s i o l o g i c a l f a c t o r s on PGM a c t i v i t y c o u l d produce important genotype-by-env ironment i n t e r a c t i o n s r e l e v a n t to the f u n c t i o n a l s i g n i f i c a n c e of t h i s po lymorphi sm, as found f o r the aminopept idase -1 l o c u s in M. e d u l i s (Koehn, Newel l and 91 Immerman 1980; Koehn and Immerman 1981). T h e r e f o r e , the s p e c i f i c a c t i v i t i e s of 7 Pgm-2 genotypes were measured i n two t i s s u e s (mantle and adductor musc le j , a t two i n t e r t i d a l h e i g h t s (low and h i g h water) i n each of three seasons (summer, f a l l and w i n t e r ) . The o b j e c t i v e of t h i s c h a p t e r was to determine i f d i f f e r e n c e s i n s p e c i f i c a c t i v i t i e s , expres sed between genotypes at the Pqm-2 l o c u s i n C . g i g a s , a r e capab le of e x p l a i n i n g the overdominance for a d u l t body weight d e s c r i b e d a t t h i s l o c u s by F u j i o (1982) . In the p r e c e d i n g c h a p t e r , I c o n c l u d e d tha t the k i n e t i c and s t r u c t u r a l d i f f e r e n c e s observed between the four most common Pgm-2 a l l o z y m e s were i n s u f f i c i e n t to c o n f e r a net advantage of h e t e r o z y g o t e s over homozygotes v i a the m a r g i n a l overdominance mechanism ( c f . W a l l a c e 1959) tha t i s r e q u i r e d f o r a monomeric enzyme l i k e PGM. T h i s c o n c l u s i o n d i s c o u n t e d the r o l e p l a y e d by f u n c t i o n a l d i f f e r e n c e s at the s t r u c t u r a l enzyme l o c u s , but l e f t open the p o t e n t i a l e f f e c t s of r e g u l a t o r y gene v a r i a t i o n that produce d i f f e r e n t s t e a d y - s t a t e l e v e l s of PGM a c t i v i t y i n d i f f e r e n t genotypes . A number of t h e o r e t i c a l models have been deve loped t h a t are c a p a b l e of p r o d u c i n g a h e t e r o z y g o t e advantage through s p a t i a l or temporal e n v i r o n m e n t a l h e t e r o g e n e i t y (rev iewed by H e d r i c k , Ginevan and Ewing 1976; F e l s e n s t e i n 1976), some d e a l i n g e x p l i c i t l y w i t h enzyme a c t i v i t y v a r i a t i o n ( e . g . G i l l e s p i e and L a n g l e y 1974; L a t t e r 1975; G i l l e s p i e 1977). The a p p l i c a b i l i t y of these models to the r e s u l t s p r e s e n t e d in t h i s c h a p t e r i s l i m i t e d , however, because they are based on i n t e r m e d i a t e h e t e r o z y g o t e b e h a v i o r ; some Pgm-2 h e t e r o z y g o t e s 92 r e p o r t e d here d i s p l a y the unusua l f e a t u r e of overdominance for s p e c i f i c a c t i v i t y . MATERIALS AND METHODS A n i m a l s . O y s t e r s were c o l l e c t e d i n the summer ( l a t e J u n e ) , f a l l ( e a r l y November), and w i n t e r ( e a r l y March) from the Nanoose Bay, B . C . s tudy s i t e d e s c r i b e d i n Chapter 2. Samples c o n s i s t e d of 150-250 mature o y s t e r s , r a n g i n g i n s i z e from 5-20 cm s h e l l l e n g t h , from each of two sampl ing s t a t i o n s l o c a t e d i n the i n t e r t i d a l zone ( d e s i g n a t e d as "low" and "high" w a t e r ) . An imal s were t r a n s p o r t e d back to the l a b o r a t o r y on i c e i n c o o l e r s where, immediate ly upon a r r i v a l , they were e x c i s e d from t h e i r s h e l l s , b l o t t e d d r y , and weighed. A f t e r a s m a l l s e c t i o n of mantle was d i s s e c t e d f o r e l e c t r o p h o r e s i s the o y s t e r s were f r o z e n at - 4 0 ° C . E l e c t r o p h o r e s i s . S t a r c h g e l e l e c t r o p h o r e s i s was performed as o u t l i n e d i n Chapter 2. F o r each sample, "s tandard" r u n n i n g c o n d i t i o n s were used i n i t i a l l y to i d e n t i f y the Pgm-2 genotype of a l l o y s t e r s . The genotypes of o y s t e r s s c o r e d as Pgm-2-92/92, 96/96 and 104/104 homozygotes were checked a second time under " c a t a l y t i c " r u n n i n g c o n d i t i o n s p r i o r to the d e t e r m i n a t i o n of t h e i r s p e c i f i c a c t i v i t i e s . S p e c i f i c A c t i v i t y Measurements• O y s t e r s from each s e a s o n a l sample were d i v i d e d i n t o four a r b i t r a r i l y a s s i g n e d body weight c l a s s e s (12 .0 -23 .9 g; 2 4 . 0 - 3 5 . 9 g; 3 6 . 0 - 4 7 . 9 g; +48.0 g ) . 93 S p e c i f i c a c t i v i t i e s of the four most common Pqm-2 genotypes (Pgm - 2 - 1 0 0 / 1 0 0 , 92/100, 96/100 and 100/104) were de termined on a subsample of 3-6 i n d i v i d u a l s randomly s e l e c t e d from each of these weight c l a s s e s . Owing to t h e i r r a r i t y , a l l homozygotes f o r the Pgm-2-92, 96 and 104 a l l e l e s (and h e t e r o z y g o t e s for these a l l e l e s examined i n the f a l l sample) were i n c l u d e d f o r s t u d y , i r r e s p e c t i v e of body we ight . PGM s p e c i f i c a c t i v i t i e s were determined i n the mantle and p o s t e r i o r adduc tor muscle of these s e l e c t e d geno types . Un le s s s p e c i f i e d , a l l s teps were c a r r i e d out on i c e or a t 4 ° C . From the mantle a p p r o x i m a t e l y 1 g of t i s s u e was d i s s e c t e d from the most p o s t e r i o r r e g i o n of the l e f t mant le l o b e , b l o t t e d , and weighed to the n e a r e s t m i l l i g r a m . Extreme c a r e was taken to ensure t h a t e x a c t l y the same r e g i o n of the mantle was used i n a l l o y s t e r s . The t i s s u e was homogenized by hand i n a 10 ml Wheaton t i s s u e g r i n d e r in 5 ml of i c e - c o l d e x t r a c t i o n b u f f e r (10 mM T r i s , 1OmM m a l e i c a c i d , 1 mM MgC12, 1 mM EDTA, pH 7 . 4 ) . S i m i l a r l y , a p p r o x i m a t e l y 0.5 g of the "quick" p o r t i o n of the adductor muscle was d i s s e c t e d , weighed, and homogenized i n 3 .5 ml of b u f f e r . The crude homogenates were c e n t r i f u g e d at 12,000 x g f o r 20 min and a 1 ml a l i q u o t of the supernatant was removed for the measurement of PGM a c t i v i t y and s o l u b l e p r o t e i n . Phosphoglucomutase a c t i v i t y was measured a t 1 5 ° C i n the forward r e a c t i o n d i r e c t i o n at 340 nm on a Pye Unicam SP 1800 U V / v i s i b l e spec trophotometer as d e s c r i b e d i n Chapter 2. The 94 r e a c t i o n medium c o n t a i n e d 50 mM i m i d a z o l e - H C l , 3 mM MgC12, 2 mM g l u c o s e - 1 - p h o s p h a t e , 16 uM g l u c o s e - 1 , 6 - d i p h o s p h a t e , 0.4 mM NADP, 1 u n i t g l u c o s e - 6 - p h o s p h a t e dehydrogenase , pH 7.0 ( 2 0 ° C ) i n a f i n a l volume of 1 m l . One u n i t of a c t i v i t y i s d e f i n e d as the q u a n t i t y of enzyme r e q u i r e d to c o n v e r t 1 Mmole of g l u c o s e - 1 -phosphate to g l u c o s e - 6 - p h o s p h a t e per minute under these c o n d i t i o n s . Assays were performed in t r i p l i c a t e u s i n g 20 /xl of the crude homogenate from the mantle and 10 ul from the adductor m u s c l e . A f t e r c o m p l e t i o n of these enzyme assays the samples were f r o z e n a t - 7 0 ° C p r i o r to the d e t e r m i n a t i o n of s o l u b l e p r o t e i n . G e n e r a l p r o t e i n was measured i n t r i p l i c a t e a t room temperature by the method of B r a d f o r d (1976) on a Pye Unicam SP8-400 U V / v i s i b l e spec trophotometer u s i n g gamma g o b u l i n as a s t a n d a r d . S p e c i f i c a c t i v i t y was expres sed i n u n i t s of PGM a c t i v i t y per mg p r o t e i n i n these crude homogenates. PGM a c t i v i t y / g wet t i s s u e weight and s o l u b l e p r o t e i n e x t r a c t e d / g wet t i s s u e weight were c a l c u l a t e d by assuming an i n t r a c e l l u l a r water content of 75% i n the mantle and 50% i n the adduc tor muscle t i s s u e (Walsh, McDonald and Booth 1984). F o r each s e a s o n a l c o l l e c t i o n , the d e t e r m i n a t i o n of s p e c i f i c PGM a c t i v i t y was completed w i t h i n a 4 month p e r i o d . To min imize the d a y - t o - d a y v a r i a b i l i t y i n e x t r a c t i o n a n d / o r assay t e c h n i q u e s and e l i m i n a t e any b i a s i n the r e s u l t s due to the spontaneous l o s s of PGM a c t i v i t y over t ime i n the f r o z e n o y s t e r s , s e v e r a l p r e c a u t i o n s were t a k e n . F i r s t , of the 12 i n d i v i d u a l s s t u d i e d on a t y p i c a l day equa l numbers were s e l e c t e d from each t i d a l 95 h e i g h t . Second, a minimum of four d i f f e r e n t genotypes were chosen for s tudy each day, thus e n s u r i n g a maximum r e p r e s e n t a t i o n of 3 i n d i v i d u a l s of the same genotype . T h i r d , the o r d e r i n which these genotypes were assayed f o r PGM a c t i v i t y and s o l u b l e p r o t e i n was randomized on any g i v e n day . These p r e c a u t i o n s guaranteed t h a t the s p e c i f i c a c t i v i t y measurements f o r a l l genotypes were c a r r i e d out over the same l e n g t h of t ime and s u i t a b l y randomized a c r o s s days so tha t no s y s t e m a t i c e r r o r s would be p r e s e n t i n the r e s u l t s . S t a t i s t i c a l A n a l y s e s . The s p e c i f i c a c t i v i t y d a t a was a n a l y z e d by f a c t o r i a l a n a l y s i s of v a r i a n c e (ANOVA) as d e s c r i b e d i n Soka l and R o h l f (1981) . Homogeneity of v a r i a n c e t e s t s on the raw da ta r e v e a l e d t h a t o n l y the s o l u b l e p r o t e i n e x t r a c t e d / g wet t i s s u e weight data r e q u i r e d t r a n s f o r m a t i o n to the l o g s c a l e to n o r m a l i z e v a r i a n c e s . Means were compared s t a t i s t i c a l l y u s i n g a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . RESULTS The s p e c i f i c a c t i v i t y measurements were o r i g i n a l l y a n a l y z e d f o r each season by a t h r e e - f a c t o r ANOVA w i t h t i d a l h e i g h t , Pqm-2 genotype and body weight c l a s s as independent v a r i a b l e s . Body weight was found to e x p l a i n a s i g n i f i c a n t p o r t i o n of the v a r i a t i o n i n PGM s p e c i f i c a c t i v i t y o n l y in the adduc tor muscle f o r the f a l l sample (F(3 ,144) = 2 . 7 1 , P < . 0 5 ) . H e r e , o y s t e r s i n the t h r e e l a r g e s t weight c l a s s e s (over 24.0 g) e x h i b i t e d g r e a t e r 96 s p e c i f i c a c t i v i t i e s than those i n the l i g h t e s t c l a s s ( 1 2 . 0 - 2 3 . 9 g ) , a l t h o u g h m u l t i p l e range t e s t s were unable to d e t e c t any s i g n i f i c a n t d i f f e r e n c e s between the g r o u p s . Due to the n e g l i g i b l e e f f e c t of body weight on PGM a c t i v i t y , i t was e l i m i n a t e d as a f a c t o r in the o v e r a l l a n a l y s i s which i s summarized i n T a b l e I V . F o r both mantle and adduc tor musc le , season and genotype e x e r t e d h i g h l y s i g n i f i c a n t e f f e c t s . o n PGM s p e c i f i c a c t i v i t y . In each t i s s u e a h i g h l y s i g n i f i c a n t s eason-b y - t i d a l h e i g h t i n t e r a c t i o n term was a l s o o b s e r v e d ; however, no i n t e r a c t i o n s o c c u r r e d between these e n v i r o n m e n t a l f a c t o r s and Pgm-2 genotype . The o v e r a l l a n a l y s e s for mantle and adduc tor muscle t i s s u e were remarkably s i m i l a r . The o n l y d i f f e r e n c e found between these t i s s u e s was the m a r g i n a l l y s i g n i f i c a n t e f f e c t of t i d a l p o s i t i o n a lone f o r the adductor m u s c l e , an e f f e c t t h a t was absent in the mant l e . The s i g n i f i c a n t e f f e c t s of these f a c t o r s on s p e c i f i c a c t i v i t y c o u l d be mediated through d i f f e r e n c e s i n the a c t i v i t y of PGM e x t r a c t e d from these t i s s u e s , s o l u b l e p r o t e i n l e v e l s , or some c o m b i n a t i o n of the two. To determine the r e l a t i v e c o n t r i b u t i o n of e a c h , a n a l y s e s were performed on PGM a c t i v i t y and s o l u b l e p r o t e i n s e p a r a t e l y by e x p r e s s i n g both on a per gram wet t i s s u e weight b a s i s . T a b l e IV shows that the s i g n i f i c a n t e f f e c t s of season , genotype , and the t i d a l h e i g h t - b y - s e a s o n i n t e r a c t i o n term appear l a r g e l y to r e s u l t from t h e i r i n f l u e n c e s on the a c t i v i t y l e v e l of phosphoglucomutase . A l t h o u g h s o l u b l e p r o t e i n l e v e l s in these t i s s u e s d i f f e r e d s i g n i f i c a n t l y between 97 T a b l e I V . F - r a t i o s from a n a l y s e s of v a r i a n c e on PGM s p e c i f i c a c t i v i t y , PGM a c t i v i t y / g t i s s u e , and s o l u b l e p r o t e i n e x t r a c t e d / g t i s s u e i n the mantle and adduc tor muscle t i s s u e s . 98 M a n t l e A d d u c t o r M u s c l e S o u r c e o f df S p e c i f i c PGM S o l u b l e S p e c i f i c PGM S o l u b l e V a r i a t i o n A c t i v i t y A c t i v i t y P r o t e i n A c t i v i t y A c t i v i t y P r o t e i n S e a s o n 2 3 9 . 3 * * * 8 1 . 7 * * * 7 7 . 9 * * * 2 7 1 . 3 * * * 1 6 7 . 1 * * * 2 3 . 0 * " T i d a l 1 2 .52 1 3 . 3 * * * 5 . 4 8 * 5 . 5 6 * 6 . 7 5 * * 3 .46 H e i g h t G e n o t y p e 6 1 1 . 9 * * * 9 . 6 0 * * * 2 . 6 2 * 2 1 . 9 * * * 1 2 . 8 * * * 1.40 G e n o t y p e 12 1.34 0 . 8 3 0 .54 1.09 0 . 5 3 0 . 5 0 x S e a s o n G e n o t y p e 6 0 .54 1.29 0 .24 0 .84 1.06 0 . 4 0 x T i d a l H e i g h t S e a s o n 2 2 3 . 8 * * * 1 7 . 8 * * * 0 .35 1 3 . 7 * * * 5 . 1 2 * * 3 . 7 3 * x T i d a l H e i g h t G e n o t y p e 12 0 .74 0 . 8 0 1.14 1.20 0 .52 0 .37 x T i d a l H e i g h t x S e a s o n E r r o r 456 * P < .05 * * P < .01 * * * P < .001 99 seasons , r e l a t i v e l y minor e f f e c t s were apparent f o r the o ther f a c t o r s and t h e i r r e s p e c t i v e i n t e r a c t i o n t erms . The source of the s i g n i f i c a n t e f f e c t s of these f a c t o r s w i l l now be examined i n d e t a i l . EFFECTS OF SEASON AND INTERTIDAL POSITION The i n f l u e n c e of season and t i d a l p o s i t i o n on the s p e c i f i c a c t i v i t y of phosphoglucomutase and s o l u b l e p r o t e i n i n the mantle and adductor muscle of C . q i g a s are p r e s e n t e d g r a p h i c a l l y i n F i g u r e 12. At a l l t imes of the y e a r , the a d d u c t o r muscle was found to d i s p l a y s i g n i f i c a n t l y h i g h e r l e v e l s of PGM a c t i v i t y and s o l u b l e p r o t e i n than the m a n t l e . However, the s e a s o n a l v a r i a t i o n i n PGM a c t i v i t y , expres sed on a s o l u b l e p r o t e i n or wet t i s s u e weight b a s i s , was s i m i l a r in both t i s s u e s , e x h i b i t i n g a rank o r d e r of f a l l > w in ter > summer. A l t h o u g h s o l u b l e p r o t e i n l e v e l s were a l s o maximal i n the f a l l , s e v e r a l important d i f f e r e n c e s o c c u r r e d between the two t i s s u e s . F i r s t , s e a s o n a l f l u c t u a t i o n s i n s o l u b l e p r o t e i n l e v e l s i n the mantle were more pronounced than i n the adductor muscle and showed a s i g n i f i c a n t l y g r e a t e r a s s o c i a t i o n w i t h the observed PGM a c t i v i t y per gram t i s s u e weight (r = 0.65) than seen in the adductor muscle (r = 0 .43; s t a t i s t i c a l comparison of these Z t rans formed c o e f f i c i e n t s y i e l d s t = 3 .56 , P < .001 , S o k a l and R o h l f 1981, p . 589) . Second, adductor muscle p r o t e i n c o n c e n t r a t i o n i n the summer s i g n i f i c a n t l y exceeded i t s l e v e l i n the w in ter but i n the mantle there were no d i f f e r e n c e s between these seasons . T h i r d , o y s t e r s 100 F i g u r e 12. E f f e c t of season and i n t e r t i d a l p o s i t i o n on s p e c i f i c a c t i v i t y ( u n i t s / m g p r o t e i n ) , PGM a c t i v i t y ( u n i t s / g t i s s u e ) , and s o l u b l e p r o t e i n (mg/g t i s s u e ) i n the mantle and adductor muscle t i s s u e s . Open c i r c l e s = l o w . water; c l o s e d c i r c l e s = h i g h water . Low water sample s i z e s : summer n=64, f a l l n=96, winter n=89. High water sample s i z e s : summer n=57, f a l l n=l00, w i n t e r n=92. Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . Specific Activity, units/mg Specific Activity, units/mg 101 102 from the low water s i t e had g r e a t e r s o l u b l e p r o t e i n c o n c e n t r a t i o n s in t h e i r mantle t i s s u e s than those i n the h i g h i n t e r t i d a l area i n a l l seasons , but o n l y i n the summer and w i n t e r f o r the adductor muscle t i s s u e . Because of the g r e a t e r tendency of PGM a c t i v i t y to c o v a r y w i t h t o t a l p r o t e i n c o n c e n t r a t i o n in the m a n t l e , the s e a s o n a l v a r i a t i o n i n s p e c i f i c a c t i v i t y f o r t h i s t i s s u e was n e a r l y 50% lower than observed i n the a d d u c t o r musc le . F i g u r e 12 shows t h a t the s i g n i f i c a n t e f f e c t of season on PGM s p e c i f i c a c t i v i t y was a consequence of i t s i n f l u e n c e on i n  v i v o enzyme a c t i v i t y , not on s o l u b l e p r o t e i n l e v e l s . In both t i s s u e s the a c t i v i t y of PGM e x t r a c t e d per gram wet t i s s u e weight f l u c t u a t e d s e a s o n a l l y by 64%, whereas the amount of s o l u b l e p r o t e i n v a r i e d by 27% i n the mantle and o n l y 10% i n the adductor musc le . These changes i n t i s s u e p r o t e i n c o n c e n t r a t i o n s between seasons tended to p a r a l l e l the PGM a c t i v i t y / g t i s s u e l e v e l s , and thus a c t e d to e l i m i n a t e r a t h e r than produce s e a s o n a l d i f f e r e n c e s i n s p e c i f i c a c t i v i t y . I t i s o n l y because the r e l a t i o n s h i p between these measures was not e x a c t l y compensatory tha t any s e a s o n a l v a r i a t i o n in PGM s p e c i f i c a c t i v i t y was o b s e r v e d . F i g u r e 12 a l s o shows tha t the i n t e r a c t i o n noted between season and t i d a l p o s i t i o n on s p e c i f i c a c t i v i t y was a t t r i b u t a b l e to t h e i r e f f e c t s on t i s s u e PGM l e v e l s . In the summer and f a l l , o y s t e r s i n the low i n t e r t i d a l zone possessed g r e a t e r a c t i v i t i e s of PGM than an imal s s i t u a t e d h i g h e r i n the i n t e r t i d a l a r e a , but i n the w in ter t h i s p a t t e r n was r e v e r s e d . The i n c r e a s e d PGM a c t i v i t y of 103 o y s t e r s at the h i g h water s i t e i n the w i n t e r , combined w i t h t h e i r lower t i s s u e p r o t e i n l e v e l s , a c c e n t u a t e d the d i f f e r e n c e s i n s p e c i f i c a c t i v i t y observed between t i d a l h e i g h t s i n t h i s s eason . The s i g n i f i c a n t s e a s o n - b y - t i d a l h e i g h t i n t e r a c t i o n seen i n T a b l e IV f o r adductor muscle p r o t e i n c o n c e n t r a t i o n a l s o o c c u r r e d because of a r e v e r s a l of t i d a l r a n k i n g i n the f a l l r e l a t i v e to the o ther two seasons . T h i s can be seen to have no e f f e c t on the s i m i l a r i n t e r a c t i o n between these e n v i r o n m e n t a l f a c t o r s on PGM s p e c i f i c a c t i v i t y . EFFECT OF PGM-2 GENOTYPE The mantle and adductor muscle s p e c i f i c a c t i v i t i e s of seven Pgm-2 genotypes measured i n the summer, f a l l , and win ter are p r e s e n t e d i n T a b l e s V and V I . W i t h i n each season the data from both t i d a l p o s i t i o n s has been p o o l e d owing to the r a r i t y of the Pqm-2-92/92 , 96/96 and 104/104 homozygotes. The c l e a r t r e n d e v i d e n t i n both t a b l e s was t h a t the three Pqm-2 h e t e r o z y g o t e s tended to e x h i b i t g r e a t e r s p e c i f i c a c t i v i t i e s than t h e i r r e s p e c t i v e homozygotes. T h i s apparent overdominance f o r PGM s p e c i f i c a c t i v i t y was extremely c o n s i s t e n t a c r o s s season , t i d a l h e i g h t and t i s s u e . By chance a lone the s p e c i f i c a c t i v i t y of a h e t e r o z y g o t e was expected to exceed both homozygotes by a p r o b a b i l i t y of 1/3. U s i n g data from both t i d a l p o s i t i o n s t h e r e were 36 comparisons and i n 33 of the cases the s p e c i f i c a c t i v i t y of the h e t e r o z y g o t e was g r e a t e r than e i t h e r homozygote ( o c c u r r i n g 16 out of 18 t imes i n the m a n t l e , and 17 out of 18 104 T a b l e V . Seasonal v a r i a t i o n i n the mantle s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) of seven Pgm-2 genotypes . 105 S e a s o n G e n o t y p e N Summer N F a l 1 N W i n t e r N P o o l e d 9 2 / 9 2 5 0 . 085+ .015 8 0 . 128+. 011 9 0 . 085+ .010 22 0 .101+.O06 9 2 / 1 0 0 23 0 . 111 + .007 41 0 . 129+. 005 35 0 . 115 + .005 99 0 .120+.003 9 6 / 9 6 4 0 . 082 + .016 7 0 . 113+. 011 1 1 0 . 066 + .009 22 0. .084+.006 9 6 / 1 0 0 25 0 . 085+, .006 42 0 . 129+. 005 31 0 . 112+ .005 98 0. .112+.003 100/100 29 0 . 086+ .006 42 0 . 1 0 9 ± . 005 40 0 . 091 + .005 111 0. .097+.003 100/104 23 0 . 100+. .007 40 0 . 1 2 8 ± . 005 40 0 . 119+, .005 103 0. , 118+.003 104/104 12 0 . 078+. .009 16 0 . 109+. 008 15 0 . 079+. .008 43 0 . 090+.005 F ( 6 . 7 7 ) = 2. .42* « F ( 6 , 1 4 4 ) = 3 1 .09* F ( 6 , 1 3 2 ) = 8. , 2 3 * * * F ( 6 , 4 5 6 ) = 1 1 . 9 * * * * P < .05 * * * P < .001 106 T a b l e V I . Seasona l v a r i a t i o n i n the adduc tor muscle s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) of seven Pgm-2 genotypes . 107 S e a s o n G e n o t y p e N Summer N F a l l N W i n t e r N P o o l e d 92 /92 5 0 . 127± .013 8 0. .219 + .013 9 0. . 176+ .010 22 0. . 181± .007 9 2 / 1 0 0 23 0 . 155± .006 41 0. . 2 3 3 ± .006 35 0. , 2 3 0 ± .005 99 0. , 2 1 4 ± . .003 9 6 / 9 6 4 0 . 1 3 0 ± . .014 7 0 . 219+. .014 1 1 0. . 175+, .009 22 0. . 181 + .007 9 6 / 1 0 0 25 0 . 1 4 6 ± . .006 42 0 . 236+, .006 31 0 . 226 + .006 98 0. 2 1 0 ± .003 100/100 29 0 . 124+. .005 42 0 . 2 0 6 ± . .006 40 0 . 191 + , .005 11 1 0. 1 7 9 ± , .003 100/104 23 0 . 1 5 3 ± . .006 40 0 . 238+. .006 40 0 . 233+. .005 103 0. 2 1 7 ± . ,003 104/104 12 0 . 1 2 1 ± . .008 16 0 . 204+. .009 15 0 . 181 + . .008 43 0 . 173+. 005 F ( 6 . 7 7 ) = 2 . 4 2 * F ( 6 . 1 4 4 ) = 3 . 0 9 * F ( 6 . 1 3 2 ) = 8 . 2 3 * * * F ( 6 , 4 5 6 ) = 1 1 . 9 * * * * P < .05 * * * P < .001 108 t imes i n the adductor m u s c l e ) . The p r o b a b i l i t y of these r e s u l t s o c c u r r i n g by chance a lone i s a p p r o x i m a t e l y 4 x 1 0 " 1 3 . A l t h o u g h Pgm-2 genotype e x p l a i n e d a s i g n i f i c a n t p o r t i o n of the v a r i a t i o n in s p e c i f i c a c t i v i t y observed in both t i s s u e s i n a l l t h r e e seasons , the e f f e c t was sometimes weak and m u l t i p l e range t e s t s were o f t e n unable to d i f f e r e n t i a t e between the genotypes . However, when the data for each genotype was combined a c r o s s s easons 'and t i d a l p o s i t i o n s the d i f f e r e n c e s became h i g h l y s i g n i f i c a n t . In the m a n t l e , the t h r e e Pgm-2 h e t e r o z y g o t e s behaved as a homogeneous group e x h i b i t i n g s p e c i f i c a c t i v i t i e s tha t s i g n i f i c a n t l y exceeded a l l homozygotes except Pgm-2-92/92. S i m i l a r r e s u l t s were found for the adductor muscle except tha t the t h r e e h e t e r o z y g o t e s were d i s t i n c t from a l l four homozygotes. The e x c e p t i o n a l b e h a v i o r of the Pqm-2-92/92 homozygote's mantle a c t i v i t y was caused by i t s u n u s u a l l y h i g h l e v e l in the f a l l when i t was i n d i s t i n g u i s h a b l e from any of the h e t e r o z y g o t e s . The s i g n i f i c a n c e of t h i s a p p a r e n t l y anomalous r e s u l t appears q u e s t i o n a b l e because the adductor muscle a c t i v i t y of t h i s homozygote at t h i s t ime was not e l e v a t e d and no s i m i l a r t e n d e n c i e s were d i s p l a y e d i n o ther seasons . T h e r e f o r e , I b e l i e v e tha t these he terozygous and homozygous genotypes r e p r e s e n t n e a r l y homogeneous g r o u p s , as found i n the adductor muscle t i s s u e . The magnitude of the d i f f e r e n c e i n s p e c i f i c a c t i v i t y between homozygotes and h e t e r o z y g o t e s was n e a r l y i d e n t i c a l i n both t i s s u e s . The s p e c i f i c a c t i v i t i e s of Pgm-2 h e t e r o z y g o t e s exceeded homozygotes by 24% in the mantle and 20% i n the 109 adductor m u s c l e . The s p e c i f i c a c t i v i t i e s of these genotypes have been decomposed i n T a b l e VII to determine i f the overdominance observed was a t t r i b u t a b l e to d i f f e r e n c e s i n enzyme a c t i v i t y l e v e l s or s o l u b l e p r o t e i n c o n c e n t r a t i o n s . As found for the e f f e c t s of season and t i d a l p o s i t i o n , the i n c r e a s e d s p e c i f i c a c t i v i t i e s of Pgm-2 h e t e r o z y g o t e s was a consequence of t h e i r l a r g e r in v i v o PGM a c t i v i t y l e v e l s . In both t i s s u e s , h e t e r o z y g o t e s posses sed a g r e a t e r PGM a c t i v i t y per gram of t i s s u e r e l a t i v e to homozygotes t h a t was s i m i l a r in magnitude to the d i f f e r e n c e s observed between these groups i n t h e i r s p e c i f i c a c t i v i t i e s (29% and 24% l a r g e r i n the mant le and adduc tor musc l e , r e s p e c t i v e l y ) . S u r p r i s i n g l y , Pgm-2 genotype a l s o e x e r t e d a m a r g i n a l l y s i g n i f i c a n t e f f e c t on the amount of s o l u b l e p r o t e i n e x t r a c t e d from the mantle but not the adduc tor musc le . M u l t i p l e range t e s t s comparing the mantle p r o t e i n c o n c e n t r a t i o n s of these genotypes d e t e c t e d a d i f f e r e n c e o n l y between Pgm-2-100/104 and Pgm-2-104/104. However, i t can be seen t h a t i n both t i s s u e s h e t e r o z y g o t e s c o n s i s t e n t l y d i s p l a y e d s l i g h t l y h i g h e r l e v e l s of s o l u b l e p r o t e i n than homozygotes . S i n c e these d i f f e r e n c e s would ac t in the d i r e c t i o n of l o w e r i n g r a t h e r than i n c r e a s i n g the s p e c i f i c a c t i v i t i e s of h e t e r o z y g o t e s r e l a t i v e to homozygotes, the overdominance observed for these measurements must r e s u l t s o l e l y from the d i f f e r e n c e s between genotypes i n t h e i r PGM enzyme a c t i v i t i e s . 110 T a b l e V I I . Decompos i t ion of Pqm-2 s p e c i f i c a c t i v i t i e s ( u n i t s / m g p r o t e i n ) i n t o enzyme a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s expres sed on a gram wet t i s s u e weight b a s i s . 111 M a n t l e A d d u c t o r M u s c l e G e n o t y p e N S p e c i f i c ' PGM« S o l u b l e 1 S p e c i f i c 1 PGM' S o l u b l e 1 A c t i v i t y A c t i v i t y P r o t e i n A c t i v i t y A c t i v i t y P r o t e i n 9 2 / 9 2 22 0, . 101± .006 2 . 4 6 ± .214 23 . 7 ± 1 . 1 1 0. , 181± .007 7 . 4 0 ± . 370 40 . 5+1.12 9 2 / 1 0 0 99 0, . 1 2 0 ± .003 3 .09+, . 100 25. 5 ± . 5 1 7 0, .214 + .003 8 .62+. . 173 41 . 0 ± . 5 2 6 9 6 / 9 6 22 o . .084+ .006 2. .07+. .214 23 . 2+1.11 0. .181 + .007 7 .21 + . .370 39. 9+1.12 9 6 / 1 0 0 98 0. . 1 1 2 ± .003 2 9 0 ± , . 101 25 . 9+.526 0. , 2 1 0 ± .003 8 .78+. 175 42 . 1+.535 100/100 11 1 0. ,097+. .003 2. .47+, ,095 24. 9 ± . 4 8 9 0. 1 7 9 ± . .003 7, , 3 1 ± . 164 40 . 1 ± . 4 9 7 100/104 103 0 . 1 18+. .003 3. , 0 7 ± . ,099 25 . 9 ± . 5 1 0 0 . 2 1 7 ± . .003 8 . , 7 9 ± . 171 41 . 2+.518 104/104 43 0 . 0 9 0 ± . .005 2. , 0 6 ± . 153 22 . 6 ± . 7 9 0 0 . 173+. .005 7. ,01 + . 264 40 . 6 ± . 8 0 3 F ( 6 , 4 5 6 ) 1 1 . 9 * * * 9 . 6 0 * * * 2 ! .62* 2 1 . 9 * * * 1 2 . 8 * * * 1 .40 1 u n 1 t s / m g p r o t e i n * u n i t s / g wet t i s s u e J mg p r o t e 1 n / g wet t i s s u e * P < .05 * * * P < .001 1 12 Due to the h i g h frequency of the Pgm-2-100 a l l e l e i n the s tudy p o p u l a t i o n , the Pqm-2-92/100, 96/100 and 100/104 genotypes r e p r e s e n t the dominant c l a s s of h e t e r o z y g o t e s at t h i s l o c u s . The next most f requent group of h e t e r o z y g o t e s are formed between the t h r e e l e s s f requent Pgm-2 a l l e l e s , i . e . , Pgm-2-92/96 , 92/104 and 96/104. To determine i f these h e t e r o z y g o t e s a l s o d i s p l a y e d overdominance for s p e c i f i c a c t i v i t y , they were i n c l u d e d f o r s tudy in the f a l l sample . T a b l e V I I I summarizes the r e s u l t s from t h i s season , p o o l e d a c r o s s t i d a l h e i g h t s , f or two c l a s s e s of homozygotes and h e t e r o z y g o t e s tha t have been grouped a c c o r d i n g to the presence or absence of the Pqm-2-100 a l l e l e . In c o n t r a s t to h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e , h e t e r o z y g o t e s l a c k i n g t h i s a l l e l e d i d not e x h i b i t overdominance f o r PGM s p e c i f i c a c t i v i t y i n e i t h e r the mantle or adductor m u s c l e . In each t i s s u e , the Pqm-2-92/96, 92/104 and 96/104 h e t e r o z y g o t e s had s p e c i f i c a c t i v i t i e s tha t were i n d i s t i n g u i s h a b l e from both homozygote c l a s s e s and s i g n i f i c a n t l y lower than the l e v e l expressed i n the o ther h e t e r o z y g o t e g r o u p . The absence of an overdominant e f f e c t in these l e s s f requent h e t e r o z y g o t e s was a consequence of t h e i r reduced PGM a c t i v i t y per gram of t i s s u e . No d i f f e r e n c e s were observed between any of these g e n o t y p i c c l a s s e s in the amount of s o l u b l e p r o t e i n e x t r a c t e d from t h e i r adductor m u s c l e s . However i n the mant l e , h e t e r o z y g o t e s for the Pqm-2-100 a l l e l e had s i g n i f i c a n t l y l a r g e r amounts of s o l u b l e p r o t e i n than both homozygotes and h e t e r o z y g o t e s l a c k i n g t h i s a l l e l e . T h e r e f o r e , overdominance for s p e c i f i c a c t i v i t y does not appear to be u b i q u i t o u s at the Pgm-2 l o c u s . He terozygo te s between the 113 T a b l e V I I I . Comparison of enzyme a c t i v i t i e s ( u n i t s / m g p r o t e i n and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) of homozygote and h e t e r o z y g o t e c l a s s e s p o s s e s s i n g or l a c k i n g the Pgm-2-100 a l l e l e . 1 14 M a n t l e A d d u c t o r M u s c l e G e n o t y p l c S p e c i f i c ' PGM* S o l u b l e 3 S p e c i f i c 1 PGM» S o l u b l e ' C l a s s N A c t i v i t y A c t i v i t y P r o t e i n A c t i v i t y A c t i v i t y P r o t e i n Homozygotes 42 0 . 1 O 9 ± . 0 O 5 3 .10+ .163 28 .4+ .856 0 . 2 0 6 ± . 0 O 6 8.83+.281 42.6+.745 f o r 100 a l l e l e Homozygotes 31 0 . 1 1 7 ± . 0 0 5 3 . 0 0 ± . 1 9 3 26 .1+1 .00 0 .211+.007 8 .90+.327 42 .2+ .863 w i t h o u t 100 a l l e l e H e t e r o z y g o t e s 123 0 . 1 2 9 1 . 0 O 3 3 . 7 0 ± . 0 9 5 29 .2+ .500 0 . 2 3 6 1 . 0 0 3 9 . 9 7 ± . 1 6 4 42 .5+ .435 f o r 100 a l l e l e H e t e r o z y g o t e s 33 0 .105+ .005 2 . 6 5 ± . 1 8 7 2 4 . 9 ± . 1 8 7 0 . 1 9 8 ± . 0 0 6 8 . 5 3 ± . 3 2 0 42 .9+ .850 wi t h o u t 100 a l l e l e F ( 3 , 1 9 7 ) = 7 . 7 6 * * * 8 . 8 1 * * * 6 . 7 1 * * * 1 2 . 9 * * * 7 . 7 9 * * * 0 .84 1 u n i t s / m g p r o t e i n * u n i t s / g wet t i s s u e ' mg p r o t e i n / g wet t i s s u e * * * P < .001 115 Pgm-2-92, 96 and 104 a l l e l e s behaved i n a manner analogous to homozygotes and showed a tendency to d i s p l a y underdominance r a t h e r than overdominance . PREDICTED EFFECTS OF A PGM-2 NULL A L L E L E The overdomi'nant s p e c i f i c a c t i v i t i e s of the Pqm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s c o u l d be caused by the unde tec ted presence of a n u l l a l l e l e a t t h i s l o c u s . N u l l h e t e r o z y g o t e s , m i s c l a s s i f i e d as homozygotes and p o o l e d unknowingly i n t o the four homozygote c l a s s e s , would have the e f f e c t of d e p r e s s i n g t h e i r enzyme a c t i v i t i e s r e l a t i v e to h e t e r o z y g o t e s w i t h two f u n c t i o n a l a l l e l e s . T h i s w i l l produce what may be termed " n u l l overdominance". A Pgm-2 n u l l a l l e l e i n homozygous c o n d i t i o n c o u l d be l e t h a l i n C . q i g a s because of the c e n t r a l r o l e p l a y e d by g lycogen i n the energy metabol i sm of marine b i v a l v e s . In accordance w i t h t h i s p r e d i c t i o n , no n u l l homozygotes were observed at the Pgm-2 l o c u s i n the 1442 o y s t e r s examined e l e c t r o p h o r e t i c a l l y . I f n u l l h e t e r o z y g o t e s are f u l l y v i a b l e , an apparent d e f i c i e n c y of h e t e r o z y g o t e s w i l l r e s u l t at t h i s l o c u s because of t h e i r m i s c l a s s i f i c a t i o n as homozygotes. The maximum frequency of t h i s n u l l a l l e l e cannot exceed the magnitude of t h i s h e t e r o z y g o t e d e f i c i e n c y as measured by D = (Ho H e ) / H e , where Ho and He are the observed and expected h e t e r o z y g o s i t i e s , r e s p e c t i v e l y . A l t h o u g h D v a l u e s f l u c t u a t e d between sampl ing dates (see Chapter 2 ) , t h e i r mean l e v e l s at the low and h i g h i n t e r t i d a l s t a t i o n s i n the s tudy p o p u l a t i o n were -116 0.085 and - 0 . 0 6 7 , r e s p e c t i v e l y . The frequency of a n u l l a l l e l e r e q u i r e d to generate these e s t i m a t e s of D are 0.044 f o r the h i g h water s i t e , and 0.034 f o r the low water s i t e . A n u l l a l l e l e i s expected to a f f e c t d i f f e r e n t i a l l y the s p e c i f i c a c t i v i t i e s measured f o r the four homozygote c l a s s e s . These p r e d i c t e d s p e c i f i c a c t i v i t i e s and PGM a c t i v i t i e s / g t i s s u e of these homozygotes are p r e s e n t e d i n T a b l e IX by making the f o l l o w i n g as sumpt ions : 1) the a c t i v i t y of a normal homozygote e q u a l s the mean of the t h r e e overdominant h e t e r o z y g o t e s , 2) n u l l h e t e r o z y g o t e s have 60% of the a c t i v i t y of a genotype w i t h two f u n c t i o n a l a l l e l e s [as shown by Katoh and F o l t z (1987) for Lap n u l l h e t e r o z y g o t e s i n C . v i r q i n i c a ] , and 3) the f requency of the n u l l a l l e l e i s 0 .044. T a b l e IX shows t h a t the magnitude of the p r e d i c t e d r e d u c t i o n s f o r the Pgm-2-92/92, 96/96 and 104/104 homozygote c l a s s e s are remarkably s i m i l a r to t h e i r observed v a l u e s , p a r t i c u l a r l y i n the adduc tor musc le . However, the Pgm-2-100/100 homozygote e x h i b i t s much lower a c t i v i t i e s than expec ted , and once more t h i s i s most apparent i n the adductor muscle t i s s u e . The expected i n e q u a l i t y between these homozygote c l a s s e s i s a consequence of the much h i g h e r frequency of the Pqm-2-100 a l l e l e compared to the three r a r e r a l l e l e s . A c c o r d i n g l y , i t can be e s t i m a t e d tha t on ly 13% of the genotypes s c o r e d as Pqm-2-100/100 homozygotes are expected to be n u l l h e t e r o z y g o t e s , but in the t h r e e o ther homozygote groups the p r o p o r t i o n of n u l l 117 T a b l e I X . P r e d i c t e d r e d u c t i o n s of enzyme a c t i v i t i e s ( u n i t s / m g and u n i t s / g t i s s u e ) and s o l u b l e p r o t e i n l e v e l s (mg/g t i s s u e ) i n Pgm-2 homozygote c l a s s e s assuming a n u l l a l l e l e i s p r e s e n t at a frequency of 0 .044. S o l u b l e p r o t e i n c o n c e n t r a t i o n s were c a l c u l a t e d by assuming PGM enzyme r e p r e s e n t s (a) 1% and (b) 5% of the t o t a l i n t r a c e l l u l a r p r o t e i n p o o l . 118 S p e c i f i c A c t i v i t y 1 PGM A c t i v i t y ' S o l u b l e P r o t e i n ' G e n o t y p e O b s e r v e d E x p e c t e d O b s e r v e d E x p e c t e d O b s e r v e d E x p e c t e d (a) (b) A . M a n t l e H e t e r o z y g o t e s 0 . 1 1 7 0. .117 3 .02 3 .02 ' 25 .8 25 .8 25 .8 100/100 0 .0968 0. .111 2 .47 2 .87 24 .9 25 .8 25 .7 104/104 0 .0849 0. .0998 2 .06 2 .58 22. .6 25 .7 25 .5 9 6 / 9 6 0 . 0 8 3 9 0. .0942 2 .07 2 .43 23. .2 25 .7 25 .4 9 2 / 9 2 0 . 101 0. .0973 2 .46 2 .51 23 .7 25 .7 25 .5 B . A d d u c t o r I M u s c l e H e t e r o z y g o t e s 0 .214 0. .214 a .73 8 .73 41 , .4 41 . .4 41 .4 10O/10O 0 . 179 0 . 203 7 , .31 8 .28 40. . 1 41 . .4 41 . .2 104/104 0 . 173 0 . 183 7 . 01 7. ,47 40. 6 41 . 3 41 . 0 9 6 / 9 6 0.181 0 . 172 7 . ,21 7. .02 39. 9 41 . 3 40. 8 92 /92 0.181 0 . 178 7. 40 7 . 26 40. 5 41 . 3 40. 9 1 u n i t s / m g p r o t e i n * u n i t s / g wet t i s s u e ' mg p r o t e 1 n / g wet t i s s u e 119 h e t e r o z y g o t e s shou l d be s i g n i f i c a n t l y l a r g e r ( a p p r o x i m a t e l y 37%, 42%, and 49% f o r the Pqm-2-104/104, 92/92 , and 96/96 c l a s s e s , r e s p e c t i v e l y ) . T h e r e f o r e , genotypes s c o r e d as Pgm-2-100/100 homozygotes were expected to e x h i b i t s p e c i f i c a c t i v i t i e s and PGM a c t i v i t i e s / g t i s s u e t h a t were s i m i l a r to h e t e r o z y g o t e s f o r the Pqm-2-100 a l l e l e and thus s i g n i f i c a n t l y l a r g e r than the o ther homozygote g r o u p s . The c l e a r absence of t h i s p a t t e r n , which shou ld h o l d i r r e s p e c t i v e of the n u l l a l l e l e ' s a c t u a l f r e q u e n c y , c o n t r a d i c t s the p r e d i c t e d e f f e c t s of a n u l l a l l e l e . T a b l e IX a l s o p r e s e n t s the r e d u c t i o n s i n s o l u b l e p r o t e i n l e v e l s i n Pgm-2 homozygotes expected by a n u l l a l l e l e t h a t produces no p r o t e i n p r o d u c t . These were c a l c u l a t e d by assuming PGM r e p r e s e n t s (a) 1% and (b) 5% of the t o t a l i n t r a c e l l u l a r p r o t e i n p o o l , percentages expected to b r a c k e t the t r u e in_ v i v o p r o p o r t i o n (see Czok and Bucher 1960; Ottaway and Mowbray 1977). V i r t u a l l y no changes i n s o l u b l e p r o t e i n l e v e l s are p r e d i c t e d i f PGM accounts f o r 1% of the t o t a l p o o l and , even at the 5% l e v e l , the expected r e d u c t i o n s are lower than o b s e r v e d . In f a c t , f o r a n u l l a l l e l e p r o d u c i n g no enzyme to e x p l a i n these p a t t e r n s , PGM must r e p r e s e n t a p p r o x i m a t e l y 35% of the p r o t e i n p o o l i n the mantle and 20% of tha t presen t in the adduc tor musc le . The i n c r e a s e d PGM a c t i v i t y / g t i s s u e weight measurements observed i n the overdominant h e t e r o z y g o t e s are f a r too s m a l l to account f o r the h i g h e r p r o t e i n l e v e l s expressed by these genotypes . The mean PGM a c t i v i t y l e v e l e x t r a c t e d per gram t i s s u e i n h e t e r o z y g o t e s exceeded homozygotes by 29% and 24% i n the mantle and adductor 1 20 m u s c l e , r e s p e c t i v e l y . I f PGM r e p r e s e n t s 1% of the i n t r a c e l l u l a r p r o t e i n p o o l , the i n c r e a s e d enzyme l e v e l s expres sed i n h e t e r o z y g o t e s can e x p l a i n o n l y 4% of the d i f f e r e n c e s observed i n the mantle and 7% of tha t i n the adduc tor m u s c l e . The p r o p o r t i o n of t h i s a d d i t i o n a l p r o t e i n a t t r i b u t a b l e to PGM i n c r e a s e s to 21% and 34% f o r the same t i s s u e s i f the enzyme r e p r e s e n t s 5% of the p r o t e i n p o o l , but t h i s s t i l l l eaves a s i z a b l e amount of the d i f f e r e n c e u n e x p l a i n e d . The presence of n u l l h e t e r o z y g o t e s in the f o u r homozygote groups are a l s o expec ted to produce two a d d i t i o n a l e f f e c t s tha t s h o u l d d i s t i n g u i s h them from the overdominant h e t e r o z y g o t e s . F i r s t , the v a r i a n c e of the s p e c i f i c a c t i v i t y and PGM a c t i v i t y / g t i s s u e measurements i n homozygotes s h o u l d be l a r g e r than seen i n h e t e r o z y g o t e s . No ev idence of t h i s p r e d i c t e d i n f l a t i o n of v a r i a n c e was apparent i n the a n a l y s e s of the mantle or adductor muscle d a t a . Second, n o r m a l i z e d frequency d i s t r i b u t i o n s of these a c t i v i t y measurements s h o u l d e x h i b i t a b imodal p a t t e r n i n the homozygotes , c o r r e s p o n d i n g to n u l l h e t e r o z y g o t e s and normal homozygotes , and t h i s i s expected to be most c l e a r l y seen i n homozygotes f o r the t h r e e l e s s f requent Pgm-2 a l l e l e s . V i s u a l i n s p e c t i o n of these d i s t r i b u t i o n s d e t e c t e d no ev idence of t h i s expec ted b i m o d a l i t y . T h e r e f o r e , the overdominance observed for PGM a c t i v i t y and p r o t e i n l e v e l s i n Pgm-2 h e t e r o z y g o t e s does not match any of the p r e d i c t e d e f f e c t s of a n u l l a l l e l e . 121 DISCUSSION Seasona l v a r i a t i o n in the s p e c i f i c a c t i v i t i e s of many enzymes have been documented in a v a r i e t y of marine b i v a l v e s , most n o t a b l y M y t i l u s e d u l i s (rev iewed by L i v i n g s t o n e 1981; Gabbott 1983). These changes are d i r e c t l y r e l a t e d to the seasona l p a t t e r n s of metabol i sm i n these organisms tha t r e f l e c t the a v a i l a b i l i t y of f ood , p r e v a i l i n g a b i o t i c c o n d i t i o n s ( i . e . t emperature and s a l i n i t y ) , and the s t a t e of t h e i r annual r e p r o d u c t i v e c y c l e (Widdows 1978; Zandee et a l . 1980). Phosphoglucomutase a c t i v i t y has not been demonstrated to f l u c t u a t e s e a s o n a l l y i n any s p e c i e s of marine m o l l u s c . However, the g e n e r a l p a t t e r n observed i n t h i s s tudy i s c o n s i s t e n t w i t h that seen f o r a number of o ther enzymes i n the American o y s t e r , C r a s s o s t r e a v i r g i n i c a , i . e . , a r e d u c t i o n i n s p e c i f i c a c t i v i t y d u r i n g the r e p r o d u c t i v e p e r i o d (summer) f o l l o w e d by an i n c r e a s e in the n o n - r e p r o d u c t i v e phase ( f a l l and w i n t e r ) (Chambers et a l . 1975; M a r t i n 1979). The l a c k of a r e l a t i o n s h i p between PGM s p e c i f i c a c t i v i t y and body weight a l s o agrees w i t h the p a t t e r n s e x h i b i t e d f o r 10 out of the 13 enzymes s t u d i e d i n C . v i r g i n i c a by Chambers et a l . (1975) and M a r t i n (1979) . The i n f l u e n c e of t i d a l p o s i t i o n on enzyme a c t i v i t y l e v e l s i n marine b i v a l v e s has r a r e l y been examined. M a r t i n (1979) found the s p e c i f i c a c t i v i t i e s of phosphoglucose i somerase (PGI) and p h o s p h o f r u c t o k i n a s e (PFK) to vary by more than 2 - f o l d between o y s t e r s l o c a t e d i n low and h i g h i n t e r t i d a l a r e a s , but in 122 o p p o s i t e d i r e c t i o n s ; PGI a c t i v i t y i n c r e a s e d but PFK a c t i v i t y d e c r e a s e d as a f u n c t i o n of t i d a l h e i g h t . The e f f e c t of t i d a l p o s i t i o n on PGM a c t i v i t y in the p r e s e n t s tudy was much l e s s pronounced , but showed an i n t e r a c t i o n w i t h season; the s p e c i f i c a c t i v i t i e s of o y s t e r s i n the low i n t e r t i d a l area exceeded those i n the h i g h i n t e r t i d a l zone i n both June and November, but not i n M a r c h . The c a u s a t i v e f a c t o r s r e s p o n s i b l e for the s e a s o n a l v a r i a t i o n i n PGM a c t i v i t y and the observed i n t e r a c t i o n between t i d a l p o s i t i o n and season are d i f f i c u l t to i n t e r p r e t . On one hand, the p a t t e r n s are c o n s i s t e n t w i t h compensatory changes i n enzyme l e v e l s i n response to d i f f e r e n t temperature c o n d i t i o n s as documented f o r a v a r i e t y of g l y c o l y t i c enzymes ( e . g . H a z e l and P r o s s e r 1974). However, the observed changes i n PGM a c t i v i t y i n both mantle and adductor muscle were a l s o i n v e r s e l y r e l a t e d to the amount of g lycogen p r e s e n t (presented i n Chapter 4 ) . S i n c e the a b i l i t y of o y s t e r t i s s u e to s y n t h e s i z e g lycogen i s i n v e r s e l y r e l a t e d to i t s e x i s t i n g g lycogen s t o r e ( L - F a n d o , G a r c i a -Fernandez and R - C a n d e l a 1972; Goromosova 1976), these f l u c t u a t i o n s c o u l d s i m p l y r e f l e c t the annual c y c l e of g lycogen s y n t h e s i s and d e g r a d a t i o n . A f u r t h e r c o m p l i c a t i o n a r i s e s because phosphoglucomutase c a t a l y z e s a f r e e l y r e v e r s i b l e r e a c t i o n and thus f u n c t i o n s i_n v i v o in both g lycogen s y n t h e s i s and g l y c o g e n o l y s i s . I t s r e l a t i v e importance in the l a t t e r p r o c e s s i s s t i l l u n c e r t a i n because an a l t e r n a t i v e d e g r a d a t i v e pathway from g lycogen u t i l i z i n g a m y l o g l u c o s i d a s e has been d e t e c t e d i n M. 123 e d u l i s (Alemany and R o s e l l - P e r e z 1973; Zaba 1981). T h e r e f o r e , i t i s p o s s i b l e that the reduced PGM a c t i v i t y observed i n June , when g lycogen i s be ing degraded f o r gametogenic p u r p o s e s , i s a consequence of i t s d i m i n i s h e d c a t a b o l i c r o l e i n s t e a d of be ing a compensatory response to the warmer p r e v a i l i n g t emperatures at t h i s t ime of y e a r . At p r e s e n t i t does not appear p o s s i b l e to s e p a r a t e the r e l a t i v e importance and i n t e r a c t i o n between these f a c t o r s i n e x p l a i n i n g the observed s e a s o n a l changes in PGM s p e c i f i c a c t i v i t y . A l t h o u g h the e f f e c t s of these e n v i r o n m e n t a l f a c t o r s on PGM a c t i v i t y may be i n t e r p r e t e d w i t h i n the framework of the known s e a s o n a l m e t a b o l i c changes i n C . g i g a s , the overdominance observed i n h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e i s ex tremely u n u s u a l . When homozygotes f o r two d i f f e r e n t e l e c t r o p h o r e t i c a l l e l e s have been found to d i f f e r i n s p e c i f i c a c t i v i t y , h e t e r o z y g o t e i n t e r m e d i a c y i s a lmost i n v a r i a b l y observed ( e . g . G i l l e s p i e and L a n g l e y 1974; H a r r i s 1975), a l t h o u g h dominance has sometimes been r e p o r t e d ( e . g . Gibson et a l . 1986; K i n g and McDonald 1987). Overdominance f o r enzyme a c t i v i t y has r a r e l y been d e s c r i b e d , and then o n l y i n e x c e p t i o n a l c i r c u m s t a n c e s . For example, Whaley (1952) r e p o r t e d an i n c r e a s e d c a t a l a s e a c t i v i t y i n maize m e r i s t e m a t i c t i s s u e i n F1 h y b r i d s r e l a t i v e to t h e i r p a r e n t a l i n b r e d l i n e s . D i c k i n s o n , Rowan and Brennen (1984) a l s o observed a g r e a t e r ADH a c t i v i t y i n i n t e r s p e c i f i c h y b r i d s between D . melanogaster and D. s imulans than e x h i b i t e d by e i t h e r p a r e n t a l s p e c i e s , a l t h o u g h t h i s e f f e c t 124 may have r e s u l t e d s o l e l y from t h e i r i n c r e a s e d s i z e . In n a t u r a l p o p u l a t i o n s a c l e a r - c u t example of overdominance for enzyme a c t i v i t y has not been d e m o n s t r a t e d . H e t e r o z y g o t e s have been shown to d i s p l a y g r e a t e r a c t i v i t i e s than homozygotes a t i n t e r m e d i a t e ranges of temperature at an e s t e r a s e l o c u s i n both the f reshwater s u c k e r , Catastomus c l a r k i i by Koehn (1969) and the sand s h i n e r , N o t r o p i s s t ramineus by Koehn, Perez and M e r r i t t (1971) . Watt (1977, 1983) has r e p o r t e d overdominant Vmax/Km r a t i o s for some phosphoglucose isomerase h e t e r o z y g o t e s in C o l i a s b u t t e r f l i e s . In none of these examples was i t shown t h a t the overdominance r e s u l t e d from i n c r e a s e d enzyme a c t i v i t y l e v e l s i n h e t e r o z y g o t e s . The overdominance f o r s p e c i f i c a c t i v i t y at the Pgm-2 l o c u s i n C . g igas r e p o r t e d i n the p r e s e n t s tudy appears unique i n i t s . c l a r i t y of e x p r e s s i o n , r e p r o d u c i b i l i t y a c r o s s d i f f e r e n t seasons , t i d a l p o s i t i o n s and t i s s u e s , and i t s i n s e n s i t i v i t y to e n v i r o n m e n t a l f a c t o r s . Before examining the causes of t h i s apparent overdominance , i t i s necessary to d i s c o u n t the p o s s i b i l i t y t h a t i t arose through u n c o n t r o l l e d f a c t o r s or by s y s t e m a t i c e r r o r s . One important f a c t o r not examined i n t h i s s tudy was the p o t e n t i a l i n f l u e n c e of an i n d i v i d u a l ' s sex on PGM s p e c i f i c a c t i v i t y . In the mantle t i s s u e of M. e d u l i s , females have been found to possess s i g n i f i c a n t l y g r e a t e r l e v e l s of s o l u b l e p r o t e i n than males at a l l t imes of the year ( L i v i n g s t o n e 1981; L i v i n g s t o n e and C l a r k e 1983) or o n l y i n the p r e - r e p r o d u c t i v e p e r i o d (Koehn and Immerman 1981). T h e r e f o r e , i f both sexes produce e q u a l 125 q u a n t i t i e s of a c e r t a i n enzyme, males w i l l e x h i b i t g r e a t e r s p e c i f i c a c t i v i t i e s than females s o l e l y because of these d i f f e r e n c e s in s o l u b l e p r o t e i n l e v e l s . S i m i l a r p a t t e r n s may a l s o e x i s t between the sexes i n C . g i g a s . However, these s e x - r e l a t e d d i f f e r e n c e s can account f o r the observed overdominance o n l y i f the sex of h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e i s somehow b i a s e d in the d i r e c t i o n of males . From what i s known about the m u l t i p l e - l o c u s sex d e t e r m i n a t i o n mechanisms of o y s t e r s (Haley 1978), and because of t h e i r a b i l i t y to change sex depending upon e n v i r o n m e n t a l c o n d i t i o n s (Hoagland 1978) and the s i z e and the sex of a d j a c e n t i n d i v i d u a l s (Buroker 1983), t h i s requirement appears u n l i k e l y to be met. F u r t h e r m o r e , the overdominance for mantle PGM s p e c i f i c a c t i v i t y was shown not to a r i s e through d i f f e r e n c e s i n t i s s u e p r o t e i n c o n c e n t r a t i o n s , but r a t h e r from g e n o t y p i c d i f f e r e n c e s i n iji v i v o enzyme a c t i v i t y l e v e l s . An i d e n t i c a l p a t t e r n was a l s o noted i n the adductor muscle t i s s u e where p r o t e i n c o n c e n t r a t i o n has not been observed to d i f f e r between the two sexes . Another c o m p l i c a t i n g f a c t o r was the c o n t r i b u t i o n of the more c a t h o d a l Pqm-1 l o c u s to the measured a c t i v i t y of phosphoglucomutase in these crude homogenates. There are s e v e r a l reasons why the Pgm-1 l o c u s c o u l d not have had a s i g n i f i c a n t e f f e c t on these r e s u l t s . F i r s t , t h i s l o c u s remained u n s c o r a b l e i n both t i s s u e s throughout the s tudy due to i t s low l e v e l of a c t i v i t y compared to the Pgm-2 l o c u s . T h e r e f o r e , the vas t m a j o r i t y of enzyme measured in these crude t i s s u e homogenates 1 2 6 must have been produced by the Pgm-2 l o c u s . Second, the Pgm-1 l o c u s c o u l d e x p l a i n these r e s u l t s o n l y i f 1) i t e x i s t e d i n complete l i n k a g e d i s e q u i l i b r i u m w i t h the Pgm-2 l o c u s , 2) both Pgm l o c i were s e g r e g a t i n g for a l l e l i c v a r i a n t s t h a t d i f f e r e d i n s p e c i f i c a c t i v i t y , and 3) the a l l e l e s presen t a t the two l i n k e d l o c i e x h i b i t e d r e v e r s i n g dominance r e l a t i o n s h i p s . As an example, suppose t h a t a h i g h a c t i v i t y a l l e l e e x h i b i t i n g dominance at the Pgm-1 l o c u s was i n d i s e q u i l i b r i u m w i t h the t h r e e l e s s f requent Pgm-2 a l l e l e s , which behaved as low a c t i v i t y r e c e s s i v e s , and the o p p o s i t e p a t t e r n e x i s t e d f o r a Pgm-1 v a r i a n t l i n k e d to the Pgm-2-100 a l l e l e . The p o o l i n g of these dominance e f f e c t s at both l o c i c o u l d produce an a p p a r e n t l y overdominant Pgm-2 h e t e r o z y g o t e . However, to produce the 20-25% l a r g e r enzyme a c t i v i t i e s of Pgm-2 h e t e r o z y g o t e s , s u b s t a n t i a l d i f f e r e n c e s must e x i s t between the s p e c i f i c a c t i v i t i e s of the s e p a r a t e Pgm-1 and Pgm-2 genotypes , and the Pgm-1 l o c u s must account f o r a s i z a b l e (30% or more) p r o p o r t i o n of the t o t a l PGM a c t i v i t y . There was a b s o l u t e l y no ev idence for e i t h e r requirement from the e l e c t r o p h o r e t i c s t a i n i n g p a t t e r n s of o y s t e r PGM. I t i s a l s o d i f f i c u l t to account for t h i s overdominance as a consequence of l ong term s y s t e m a t i c e r r o r s i n e x t r a c t i o n a n d / o r assay t e c h n i q u e s . D a y - t o - d a y e r r o r s were min imized by r a n d o m i z i n g the s e l e c t i o n of Pgm-2 genotypes and the o r d e r i n which they were assayed for both enzyme a c t i v i t y and s o l u b l e p r o t e i n . The spontaneous l o s s of PGM a c t i v i t y shou ld a l s o have been s i m i l a r for a l l genotypes because these measurements were 1 27 completed w i t h i n i d e n t i c a l p e r i o d s of t i m e . I f d i f f e r e n c e s i n the r a t e s of i_n v i v o d e g r a d a t i o n have i n f l u e n c e d these r e s u l t s , h e t e r o z y g o t e s would have been expec ted to d i s p l a y i n t e r m e d i a t e r a t h e r than overdominant s p e c i f i c a c t i v i t i e s , based on the i n v i t r o s t a b i l i t i e s of these a l l o z y m e s observed i n Chapter 2. In a d d i t i o n , because of the marked t h e r m o l a b i l i t i e s of the Pgm-2-92 and 96 a l l e l e s , both homozygotes and h e t e r o z y g o t e s f o r these a l l e l e s may be expected to have l o s t a g r e a t e r p r o p o r t i o n of t h e i r a c t i v i t i e s than genotypes c o n t a i n i n g the more s t a b l e Pgm-2-100 and 104 a l l e l e s . The l a c k of a c l e a r d i s t i n c t i o n i n the s p e c i f i c a c t i v i t i e s of these g e n o t y p i c c l a s s e s suggests t h a t the spontaneous l o s s of enzyme a c t i v i t y has had l i t t l e impact on the r e s u l t s . Hence, the observed overdominance i s u n l i k e l y to have been caused by u n c o n t r o l l e d f a c t o r s or s y s t e m a t i c e r r o r s . The e x i s t e n c e of a n u l l a l l e l e at the Pgm-2 l o c u s has the p o t e n t i a l to p r o v i d e a s imple yet p o w e r f u l e x p l a n a t i o n f o r the overdominant enzyme a c t i v i t i e s of the Pgm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s . Z o u r o s , S i n g h and M i l e s (1980) o r i g i n a l l y d i s c o u n t e d the p o s s i b i l i t y tha t the overdominance f o r growth r a t e observed in C . v i r g i n i c a was caused by n u l l a l l e l e s , e x c l u s i v e l y on t h e o r e t i c a l grounds . However, F o l t z (1986a, 1986b) has r e c e n t l y d e t e c t e d n u l l a l l e l e s s e g r e g a t i n g at s e v e r a l l o c i i n C . v i r g i n i c a , i n c l u d i n g one l o c u s (Lap-2) p r e v i o u s l y s c o r e d by S ingh and Zouros (1978) and Z o u r o s , S ingh and M i l e s (1980) . The p r e d i c t e d e f f e c t s of a n u l l a l l e l e on the enzyme a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s of the four Pgm-2 128 homozygote c l a s s e s shown in T a b l e IX are i n c o m p a t i b l e w i t h the p a t t e r n s e x p r e s s e d by these genotypes . Because of the l a r g e . f requency d i f f e r e n c e s between these a l l o z y m e s in the s tudy p o p u l a t i o n , a marked dichotomy i s expec ted between the r e d u c t i o n s i n enzyme a c t i v i t i e s caused by the n u l l a l l e l e between the Pqm-2-100/100 and the Pqm-2-92/92 , 96/96 and 104/104 homozygote g r o u p s . There was no ev idence f o r t h i s d i s t i n c t i o n between homozygote c l a s s e s . F u r t h e r m o r e , the s l i g h t overdominance f o r s o l u b l e p r o t e i n l e v e l s i n h e t e r o z y g o t e s was f a r g r e a t e r than c o u l d be accounted f o r by 1) the r e d u c t i o n s expec ted i n homozygotes from a n u l l a l l e l e p r o d u c i n g even no p r o t e i n p r o d u c t , or 2) the i n c r e a s e d l e v e l s of PGM a c t i v i t y e x h i b i t e d by the overdominant h e t e r o z y g o t e s . These d i s c r e p a n c i e s d i r e c t l y c o n t r a d i c t the i n f l u e n c e of a n u l l a l l e l e and f u r t h e r suggest tha t the overdominance may i n v o l v e a number of u n i d e n t i f i e d enzyme l o c i i n a d d i t i o n to the Pqm-2 l o c u s . Perhaps the s t r o n g e s t ev idence a g a i n s t the n u l l a l l e l e e x p l a n a t i o n i s p r o v i d e d by the r e l a t i v e performance of the two h e t e r o z y g o t e groups examined i n the f a l l (Tab le V I I I ) . H e r e , h e t e r o z y g o t e s between the Pqm-2-92, 96 and 104 a l l e l e s d i d not e x h i b i t overdominance f o r s p e c i f i c a c t i v i t y , PGM a c t i v i t y expres sed on a t i s s u e weight b a s i s , or s o l u b l e p r o t e i n c o n t e n t s i n e i t h e r the mantle or adductor musc l e . I f the n u l l a l l e l e e x p l a n a t i o n i s c o r r e c t , these h e t e r o z y g o t e s , p o s s e s s i n g two f u n c t i o n a l a l l e l e s , are expected to d i s p l a y PGM a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s tha t were i d e n t i c a l w i th the three 129 h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e . In f a c t , they were i n d i s t i n g u i s h a b l e from homozygotes f o r these t h r e e r a r e r Pgm-2 a l l e l e s . T h e r e f o r e , as w e l l as c o n t r a d i c t i n g a key p r e d i c t i o n of the e f f e c t s of a n u l l a l l e l e , the r e s u l t s show tha t h e t e r o z y g o s i t y per se ( c f . L e r n e r 1954) i s not s u f f i c i e n t f o r the e x p r e s s i o n of overdominance f o r enzyme a c t i v i t y at the Pgm-2 l o c u s i n C . g i g a s . I n s t e a d , a p a r t i c u l a r a l l e l i c c o n f i g u r a t i o n i s r e q u i r e d ; the Pgm-2-100 a l l e l e must be p a i r e d w i t h e i t h e r the Pgm-2-92, 96, or 104 a l l e l e s b e f o r e the overdominant e f f e c t s are m a n i f e s t e d . An a l t e r n a t i v e h y p o t h e s i s , c a p a b l e of p r o v i d i n g a b e t t e r e x p l a n a t i o n for these r e s u l t s , i s t h a t t i g h t l y l i n k e d to or a s s o c i a t e d w i t h the Pgm-2 s t r u c t u r a l l o c u s , i s an overdominant r e g u l a t o r y l o c u s that produces g r e a t e r s t e a d y - s t a t e l e v e l s of enzyme i n h e t e r o z y g o t e s r e l a t i v e to homozygotes . The mode of a c t i o n of t h i s p u t a t i v e r e g u l a t o r y l o c u s i s unknown, but c o n c e i v a b l y i t c o u l d ac t a t any of the known l e v e l s of r e g u l a t o r y c o n t r o l ( i . e . t r a n s c r i p t i o n , mRNA p r o c e s s i n g , t r a n s l a t i o n , or d e g r a d a t i o n ) . I f a r e g u l a t o r y element i s i n v o l v e d , i t s b e h a v i o r i s u n l i k e any p r e v i o u s l y c h a r a c t e r i z e d example. The g e n e r a l p a t t e r n tha t has emerged from s t u d i e s on r e g u l a t o r y polymorphisms in e u k a r y o t e s i s tha t both t r a n s - a c t i n g ( e . g . S c a n d a l i o s et a l . 1980; Doane et a l . 1983; L u s i s et a l . 1983) and c i s - a c t i n g ( e . g . D i c k i n s o n 1975; S h a f f e r and Bewley 1983) v a r i a n t s a f f e c t i n g r a t e s of t r a n s c r i p t i o n produce i n t e r m e d i a t e enzyme a c t i v i t i e s i n h e t e r o z y g o t e s , w h i l e those 130 a c t i n g p o s t - t r a n s l a t i o n a l l y tend to be i n h e r i t e d in a d o m i n a n t / r e c e s s i v e f a s h i o n ( e . g . R e c h c i g l and Heston 1967; L a i and S c a n d a l i o s 1980; K i n g and McDonald 1983, 1987; Gibson et a l . 1986). An overdominant r e g u l a t o r y l o c u s c o u l d t h e o r e t i c a l l y e x e r t i t s e f f e c t s at the l e v e l of enzyme s y n t h e s i s or d e g r a d a t i o n . The i n c r e a s e d s o l u b l e p r o t e i n l e v e l s observed i n the t i s s u e s of ^ h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e , above t h a t e x p l a i n a b l e by phosphoglucomutase enzyme a l o n e , suggests t h a t the l e v e l of r e g u l a t o r y c o n t r o l i s t r a n s c r i p t i o n a l and t h a t p l e i o t r o p i c e f f e c t s are e x e r t e d on o t h e r u n i d e n t i f i e d enzyme l o c i . However, the n e g a t i v e a s s o c i a t i o n between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and p r o t e i n t u r n o v e r r a t e s r e c e n t l y r e p o r t e d by Hawkins , Bayne and Day (1986) o f f e r s the p o s s i b i l i t y that the p u t a t i v e r e g u l a t o r y l o c u s in he terozygous c o n d i t i o n l eads to d e c r e a s e d r a t e s of PGM t u r n o v e r , thus r e s u l t i n g i n g r e a t e r s t e a d y - s t a t e l e v e l s of enzyme i n h e t e r o z y g o t e s r e l a t i v e to homozygotes. S i n c e i t i s u n l i k e l y t h a t such a p r o t e a s e w i l l have ac t s p e c i f i c a l l y on PGM a l o n e , the in. v i v o l e v e l s of i t s o ther enzyme s u b s t r a t e s s h o u l d a l s o i n c r e a s e . T h i s e x p l a n a t i o n i s i n t u i t i v e l y a p p e a l i n g because i t can p r o v i d e a g e n e r a l e x p l a n a t i o n f o r the i n c r e a s e d "metabol ic e f f i c i e n c y " of h e t e r o z y g o t e s ( through the reduced e x p e n d i t u r e of ATP used for p o l y p e p t i d e s y n t h e s i s ) suggested by Berger (1976) a n d • s u p p o r t e d by a number of recent s t u d i e s ( e . g . Koehn and 131 Shumway 1982; G a r t o n 1984; G a r t o n et a l . 1984). E x t r a p o l a t i n g from p r e v i o u s l y c h a r a c t e r i z e d r e g u l a t o r y po lymorphisms , t h i s overdominant l o c u s i n C . g igas must be t r a n s - a c t i n g and somehow d i s p l a y unique p r o p e r t i e s in he terozygous c o n d i t i o n . These r e q u i r e m e n t s c o u l d be met i f the d i f f u s i b l e gene product i s a m u l t i m e r . I f the h e t e r o m u l t i m e r i c gene product produced by a h e t e r o z y g o t e has a l t e r e d f u n c t i o n a l p r o p e r t i e s (produc ing an enhancement of t r a n s c r i p t i o n r a t e s i n the case of a t r a n s c r i p t i o n f a c t o r or d i m i n i s h e d p r o t e o l y t i c f u n c t i o n i f i t i s a p r o t e a s e ) , then overdominance f o r PGM a c t i v i t y would r e s u l t . The e x i s t e n c e of t h i s h y p o t h e t i c a l r e g u l a t o r y l o c u s must await f u r t h e r s t u d y . The i n t e r p r e t a t i o n of these r e s u l t s as a case of genuine overdominance has s e v e r a l advantages over the n u l l a l l e l e e x p l a n a t i o n . F i r s t , i t i s c a p a b l e of p r o v i d i n g an e x p l a n a t i o n f o r the the maintenance of a s t a b l e po lymorph ic e q u i l i b r i u m at the Pgm-2 s t r u c t u r a l l o c u s by c o l l a p s i n g the m u l t i - a l l e l i c system i n t o a two a l l e l e r e g u l a t o r y po lymorphism. Based on the r e s u l t s summarized in T a b l e V I I I i t i s p o s s i b l e to group the 10 Pgm-2 genotypes i n t o t h r e e p h e n o t y p i c c l a s s e s a c c o r d i n g to t h e i r h y p o t h e s i z e d genotype a t t h i s r e g u l a t o r y l o c u s (see T a b l e X ) . A c c o r d i n g to t h i s mode l , r e g u l a t o r y v a r i a n t "A" e x i s t s i n complete d i s e q u i l i b r i u m w i t h the Pqm-2-100 s t r u c t u r a l a l l e l e . A s s o c i a t e d w i t h the Pqm-2-92, 96 and 104 a l l e l e s i s a d i f f e r e n t a l l e l e d e s i g n a t e d as " B " . He terozygo te s f o r the A and B a l l e l e s produce an overdominant phenotype m a n i f e s t e d i n the Pgm-2-132 T a b l e X . C o l l a p s e of the m u l t i - a l l e l i c Pqm-2 s t r u c t u r a l l o c u s polymorphism by a h y p o t h e t i c a l t i g h t l y - l i n k e d r e g u l a t o r y l o c u s s e g r e g a t i n g f o r two a l l e l e s . 133 Phenotyp ic C l a s s R e g u l a t o r y l o c u s Genotype S t r u c t u r a l l o c u s Genotype(s ) 1 " A / A " 100/100 2 " A / B " 92/100 96/100 100/104 3 " B / B " 92/92 96/96 104/104 92/96 92/104 96/104 134 92/100, 96/100 and 100/104 genotypes . Homozygotes f o r the B a l l e l e produce s i x p h e n o t y p i c a l l y e q u i v a l e n t Pgm-2 genotypes c o m p r i s i n g both homozygotes and h e t e r o z y g o t e s f o r the Pgm-2-92, 96 and 104 a l l e l e s . T h i s model can e x p l a i n the s i m i l a r i t i e s observed between the d i f f e r e n t s t r u c t u r a l l o c u s genotypes and e l i m i n a t e s the d i f f i c u l t i e s a s s o c i a t e d w i t h m a i n t a i n i n g s t a b l e m u l t i - a l l e l i c polymorphisms by overdominance ( e . g . L e w o n t i n , G i n z b u r g and T u l j a p u r k a r 1978). For the n u l l a l l e l e e x p l a n a t i o n , i t i s d i f f i c u l t to see how a Pgm-2 n u l l h e t e r o z y g o t e w i th a reduced enzyme a c t i v i t y can enjoy a s e l e c t i v e advantage over genotypes w i t h two f u n c t i o n a l a l l e l e s ( r e q u i r e d to m a i n t a i n the presence of a n u l l a l l e l e in p o p u l a t i o n s of t h i s s p e c i e s ) . The p e r s i s t e n c e of t h i s h y p o t h e s i z e d d i s e q u i l i b r i u m p r e s e n t s a problem i n i n t e r p r e t a t i o n , however, because i t c o u l d be e a s i l y broken down by i n t r a g e n i c " r e c o m b i n a t i o n as d e t e c t e d , for example, between the f a s t and slow a l l e l e s a t the Adh l o c u s in D. melanogaster by Aquadro et a l . (1986) . S i m i l a r c r o s s o v e r s are expected w i t h i n the Pgm-2 l o c u s of C . g i g a s . T h i s becomes even more p r o b a b l e i n l i g h t of the p o t e n t i a l l y g r e a t age of t h i s polymorphism t h a t i s suggested by the s i m i l a r i t y of a l l e l e f requency d i s t r i b u t i o n s between c o n g e n e r i c C r a s s o s t r e a s p e c i e s and c l o s e l y r e l a t e d genera ( c f . B u r o k e r , Hershberger and Chew 1979a, 1979b), some of which are known to have been i s o l a t e d f o r at l e a s t 30 m i l l i o n y e a r s ( S t e n z e l 1971). Perhaps the o n l y way to account for t h i s d i s e q u i l i b r i u m i s f or an i n v e r s i o n to e x i s t w i t h i n the chromosomal r e g i o n encompassing the Pgm-2 s t r u c t u r a l 1 35 l o c u s tha t i n c l u d e s e i t h e r the Pqm-2-100 a l l e l e or the t h r e e l e s s f requent a l l e l e s . The proposed overdominant model a l s o p r e d i c t s tha t the i n d i v i d u a l f r e q u e n c i e s of the Pqm-2-92, 96 and 104 a l l e l e s are of l i t t l e consequence to the e q u i l i b r i u m s t a t e a c h i e v e d . I n s t e a d , i t i s the combined frequency of these l e s s f requent a l l e l e s and t h e i r r e s u l t i n g f i t n e s s e s i n homozygous and he terozygous s t a t e r e l a t i v e to the Pgm-2-100/100 genotype t h a t c o n t r o l the dynamics of the po lymorphism. T h i s i n t e r p r e t a t i o n i s c o n s i s t e n t w i t h the observed f r e q u e n c i e s of these Pgm-2 a l l e l e s i n n a t u r a l and c u l t u r e d p o p u l a t i o n s of the P a c i f i c o y s t e r . For example, i n 23 p o p u l a t i o n samples of C . q i g a s from 5 geographic a r e a s of J a p a n , O z a k i and F u j i o (1985) observed that the f requency of the most common Pgm-2 a l l e l e f e l l w i t h i n a l i m i t e d range , e x h i b i t i n g a mean of 0.596 and a c o e f f i c i e n t of v a r i a t i o n of o n l y 5.4%. In c o n t r a s t , the f r e q u e n c i e s of a l l e l e s i n a d j a c e n t m o b i l i t y c l a s s e s f l u c t u a t e d r a t h e r d r a m a t i c a l l y , each by more than a f a c t o r of 3, g i v i n g r i s e to c o e f f i c i e n t s of v a r i a t i o n r a n g i n g from 29%-49%. The extent of t h i s v a r i a t i o n appears f a r too l a r g e to be caused by sampl ing e r r o r a l o n e . The e r r a t i c p a t t e r n s d i s p l a y e d by the f r e q u e n c i e s of these r a r e r a l l e l e s i s d i f f i c u l t to r e c o n c i l e w i t h the maintenance of a s t a b l e m u l t i - a l l e l i c polymorphism by overdominance , or any o ther type of b a l a n c i n g s e l e c t i o n , u n l e s s they are f u n c t i o n a l l y e q u i v a l e n t as p r e d i c t e d by the mode l . S ince the frequency of the Pgm-2-100 a l l e l e in p o p u l a t i o n s of C . g igas i s always g r e a t e r 136 than 0.5 ( B u r o k e r , Hershberger and Chew 1975, 1979a; O z a k i and F u j i o 1985), the f i t n e s s of the Pqm-2-100/100 genotype must exceed t h a t of homozygotes and h e t e r o z y g o t e s p o s s e s s i n g these l e s s f requent a l l e l e s . I f f i t n e s s i s c o r r e l a t e d w i t h enzyme, a c t i v i t y a t t h i s l o c u s , t h i s p r e d i c t i o n i s s u p p o r t e d i n the mantle t i s s u e where the Pgm-2-100/100 homozygote d i s p l a y s PGM a c t i v i t i e s and s o l u b l e p r o t e i n l e v e l s on a t i s s u e weight b a s i s t h a t are g r e a t e r than the mean of these o t h e r genotypes by 14% and 8%, r e s p e c t i v e l y . The i n t e r p r e t a t i o n of these r e s u l t s by an overdominant model i s a l s o supported by the b i o c h e m i c a l da ta of Chapter 2, i n which i t was c o n c l u d e d t h a t the k i n e t i c and s t r u c t u r a l d i f f e r e n c e s d e t e c t e d between these Pgm-2 a l l o z y m e s were not s u f f i c i e n t to account for- the p h e n o t y p i c e f f e c t s of h e t e r o z y g o s i t y at t h i s l o c u s r e p o r t e d by F u j i o (1982) . The overdominant s p e c i f i c a c t i v i t i e s of h e t e r o z y g o t e s p o s s e s s i n g the Pqm-2-100 a l l e l e s t r o n g l y suggest t h a t i t i s through these d i f f e r e n c e s i n enzyme a c t i v i t y l e v e l s tha t the s e l e c t i v e e f f e c t s of t h i s l o c u s are e x p r e s s e d ; the a l l e l i c d i f f e r e n c e s observed at the s t r u c t u r a l enzyme l o c u s may be l a r g e l y , i f not e n t i r e l y , n e u t r a l . The i n c r e a s e d enzyme a c t i v i t i e s of the Pgm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s a u t o m a t i c a l l y imparts upon these genotypes l a r g e r Vmax/Km r a t i o s and , hence , g r e a t e r f l u x c a p a c i t i e s than the o ther Pgm-2 genotypes l i s t e d i n T a b l e X . T h i s in t u r n may p r o v i d e the b i o c h e m i c a l b a s i s f o r the d e m o n s t r a t i o n of the s e l e c t i v e importance of t h i s enzyme 1 37 polymorphism through i t s i n f l u e n c e on g lycogen metabol i sm and subsequent impact on f i t n e s s r e l a t e d t r a i t s . These r e s u l t s have s e v e r a l important i m p l i c a t i o n s to the growing number of s t u d i e s documenting a s s o c i a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and a v a r i e t y of p h e n o t y p i c - l e v e l t r a i t s (rev iewed by M i t t o n and Grant 1984; Zouros and F o l t z 1987). As r e p o r t e d for the Pg i l o c u s i n C o l i a s b u t t e r f l i e s by Watt (1977, 1983), i t i s one of the few cases i n which overdominance has been d e t e c t e d a t a p o l y m o r p h i c enzyme l o c u s , and i t i s the f i r s t t ime i t has i n v o l v e d one p r e v i o u s l y i m p l i c a t e d w i t h a r e l a t i o n s h i p w i t h m u l t i p l e - l o c u s h e t e r o z y g o s i t y . T h e r e f o r e , f or a t l e a s t one l o c u s , the presen t s tudy has been s u c c e s s f u l in d i s t i n g u i s h i n g between the v a r i o u s a l t e r n a t i v e hypotheses put forward to e x p l a i n these a s s o c i a t i o n s . Both the i n b r e e d i n g and a s s o c i a t i v e overdominance e x p l a n a t i o n s are u n t e n a b l e : overdominance was m a n i f e s t e d at the Pgm-2 l o c u s i n C . g i g a s even though i t may have been caused by a t i g h t l y l i n k e d gene, or b l o c k of genes , in complete d i s e q u i l i b r i u m w i t h the s t r u c t u r a l l o c u s . Another important r a m i f i c a t i o n of these r e s u l t s a r i s e s from the d i s t i n c t i v e p r o p e r t i e s of d i f f e r e n t Pgm-2 h e t e r o z y g o t e s . In most s t u d i e s examining the p h e n o t y p i c e f f e c t s of m u l t i p l e - l o c u s h e t e r o z y g o s i t y , i t has been common p r a c t i s e to p o o l a l l homozygotes and h e t e r o z y g o t e s t o g e t h e r , thus i m p l i c i t l y assuming t h a t a l l genotypes combined w i t h i n these groups are e q u i v a l e n t . 138 The absence of an overdominant e f f e c t i n h e t e r o z y g o t e s between the Pgm-2-92, 96 and 104 a l l e l e s found i n t h i s s tudy suggests tha t a s i z a b l e amount of e r r o r may be i n t r o d u c e d by the assumpt ion of h e t e r o z y g o t e e q u i v a l e n c y , s i n c e these genotypes r e p r e s e n t a p p r o x i m a t e l y 24% of a l l h e t e r o z y g o t e s at t h i s l o c u s . F u r t h e r m o r e , i n s e v e r a l s p e c i e s of marine b i v a l v e s , l a b o r a t o r y c r o s s e s between l i m i t e d numbers of p a r e n t s have o c c a s i o n a l l y r e s u l t e d i n the e l i m i n a t i o n of an a s s o c i a t i o n between m u l t i p l e -l o c u s h e t e r o z y g o s i t y and growth r a t e tha t was observed i n progeny c o l l e c t e d from n a t u r a l p o p u l a t i o n s of the same s p e c i e s ( d i s c u s s e d by Zouros and F o l t z 1987). A l t h o u g h these d i s c r e p a n c i e s have been e x p l a i n e d by the requirement f o r a l a r g e number of d i f f e r i n g p a r e n t a l genomes ( e . g . Gaf fney and S c o t t 1984), the r e s u l t s from the present s tudy suggest t h a t an a d d i t i o n a l e x p l a n a t i o n i s p o s s i b l e : the overdominant e f f e c t s at some l o c i may depend on p a r t i c u l a r he terozygous s t a t e s . The. p o t e n t i a l l y confound ing e f f e c t s of d i s p a r a t e he terozygous groups at an enzyme l o c u s s c o r e d i n these types of s t u d i e s p r o v i d e s an a d d i t i o n a l reason for a d o p t i n g the "adapt ive d i s t a n c e " model of Smouse (1986) in i n t e r p r e t i n g more a c c u r a t e l y the causes of these c o r r e l a t i o n s i n v o l v i n g m u l t i p l e - l o c u s h e t e r o z y g o s i t y . In summary, the p r e s e n t s tudy has demonstrated that overdominance f o r enzyme a c t i v i t y i s expres sed by the t h r e e most common h e t e r o z y g o t e s a t the Pgm-2 l o c u s in C r a s s o s t r e a g i g a s . The magnitude of t h i s overdominant e f f e c t was s i m i l a r in the mantle and adductor muscle t i s s u e s , and was u n a f f e c t e d by 139 e n v i r o n m e n t a l f a c t o r s . Sys t emat i c e r r o r s , unexamined v a r i a b l e s , or an unde tec ted Pqm-2 n u l l a l l e l e are unable to account for these f i n d i n g s . The best e x p l a n a t i o n f o r t h i s overdominance i s v i a a r e g u l a t o r y l o c u s , t i g h t l y l i n k e d to the Pqm-2 s t r u c t u r a l l o c u s , t h a t produces g r e a t e r s t e a d y - s t a t e l e v e l s of PGM a c t i v i t y i n h e t e r o z y g o t e s r e l a t i v e to homozygotes . T h i s i n t e r p r e t a t i o n can e x p l a i n the s i m i l a r i t i e s between an a r r a y of d i f f e r e n t s t r u c t u r a l l o c u s genotypes , account f o r the f r e q u e n c i e s of these a l l e l e s i n n a t u r a l p o p u l a t i o n s , and p r o v i d e an e x p l a n a t i o n f o r the maintenance of the Pqm-2 polymorphism i n a b a l a n c e d s t a t e by c o l l a p s i n g the m u l t i - a l l e l i c system i n t o a two a l l e l e polymorphism at t h i s r e g u l a t o r y l o c u s . D e f i n i t i v e ev idence f o r the s e l e c t i v e importance of the observed overdominance must i n v o l v e a d e m o n s t r a t i o n of i t s impact on g lycogen metabo l i sm, the m e t a b o l i c pathway i n which PGM f u n c t i o n s . E x a m i n a t i o n of the p h y s i o l o g i c a l e f f e c t s of PGM a c t i v i t y v a r i a t i o n on t i s s u e g lycogen c o n c e n t r a t i o n s i s the s u b j e c t of Chapter 4. 140 CHAPTER 4 PHYSIOLOGICAL EFFECTS OF THE PGM-2 LOCUS ON GLYCOGEN METABOLISM INTRODUCTION A l a r g e body of s t u d i e s have documented the e x i s t e n c e of d i f f e r e n c e s i n s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s between a l l o z y m e s at po lymorphic enzyme l o c i . A s i n g l e comprehensive review of t h i s l i t e r a t u r e has not been done, but some of the b e t t e r c h a r a c t e r i z e d polymorphisms have been d i s c u s s e d by McDonald (1983), Koehn, Zera and H a l l (1983), Z e r a , Koehn and H a l l (1985) , and Watt (1985b) . The d e t e c t i o n of b i o c h e m i c a l d i f f e r e n c e s between a l l o z y m e s p r o v i d e s o n l y the f o u n d a t i o n upon which the demonstra t ion of the f u n c t i o n a l , and u l t i m a t e l y the s e l e c t i v e , importance of t h i s type of g e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s must be based . As p o i n t e d out by C l a r k e (1975) and Koehn (1978) , i t i s e s s e n t i a l to show tha t t h i s a l l o z y m i c v a r i a t i o n impar t s s i g n i f i c a n t e f f e c t s on r e l e v a n t p h y s i o l o g i c a l proces se s t h a t d i f f e r e n t i a l l y a f f e c t the f i t n e s s e s of the enzyme genotypes . The vas t m a j o r i t y of b i o c h e m i c a l s t u d i e s on a l l o z y m e s f a l l s h o r t of a c h i e v i n g t h i s end and , as a consequence , have o n l y been a b l e to s p e c u l a t e on the a d a p t i v e s i g n i f i c a n c e of the k i n e t i c data ( e . g . M e r r i t t 1972; Hoffman 1981; H a l l 1985). S t u d i e s that attempt to demonstrate the p h y s i o l o g i c a l 141 consequences of enzyme v a r i a t i o n encounter a number of f o r m i d a b l e d i f f i c u l t i e s . Z e r a , H a l l and Koehn (1985), and Eanes (1984) have addres sed some of the problems t h a t a r i s e from e x t r a p o l a t i n g the s i g n i f i c a n c e of j_n v i t r o k i n e t i c d i f f e r e n c e s to in v i v o f u n c t i o n . Such l i m i t a t i o n s are u n a v o i d a b l e i n these s t u d i e s , but may be m i n i m i z e d through the use of h i g h l y p u r i f i e d a l l o z y m e p r e p a r a t i o n s , p h y s i o l o g i c a l l y r e a l i s t i c assay c o n d i t i o n s , and robus t k i n e t i c e x p e r i m e n t a l d e s i g n s and a n a l y t i c a l m e t h o d o l o g i e s . S u f f i c i e n t background knowledge i s a l s o r e q u i r e d on the c a t a l y t i c p r o p e r t i e s and m e t a b o l i c f u n c t i o n ( s ) of the enzyme s e l e c t e d for s t u d y . T h i s i n f o r m a t i o n i s e s s e n t i a l f o r i d e n t i f y i n g the c a t a l y t i c a n d / o r r e g u l a t o r y parameters t h a t might be of s e l e c t i v e importance (Watt 1983, 1985a) . D y k h u i z e n , Dean and H a r t l (1987) have d i s c u s s e d a d d i t i o n a l o b s t a c l e s faced i n these s t u d i e s tha t a r i s e from the i n h e r e n t c o m p l e x i t y of m e t a b o l i c phenotypes , the s m a l l magnitude of the k i n e t i c d i f f e r e n t i a t i o n u s u a l l y present between a l l o z y m e s , and the unknown e f f e c t s of l o c i t i g h t l y l i n k e d to the one under s t u d y . F i n a l l y , enough must be known about the s tudy o r g a n i s m ' s eco logy to a l l o w r e a s o n a b l e assumptions to be made c o n c e r n i n g the p o t e n t i a l s e l e c t i v e agent ( s ) t h a t c o u l d be a c t i n g on the polymorphism under n a t u r a l c o n d i t i o n s . The a b i l i t y to measure the p h y s i o l o g i c a l e f f e c t s of a l l o z y m i c v a r i a t i o n has a l s o met w i t h some i n t e r e s t i n g t h e o r e t i c a l d i f f i c u l t i e s . A l a r g e p r o p o r t i o n of the enzymes s t u d i e d e l e c t r o p h o r e t i c a l l y do not f u n c t i o n i n d e p e n d e n t l y , but 142 c a t a l y z e r e a c t i o n s t h a t are embedded w i t h i n complex b i o c h e m i c a l pathways . Hence, the p h y s i o l o g i c a l impact of t h i s enzyme v a r i a t i o n must be m a n i f e s t e d at the l e v e l of the e n t i r e pathway in which they f u n c t i o n be fore b e i n g v i s i b l e to s e l e c t i o n . E x t e n d i n g the b a s i c premises of m e t a b o l i c c o n t r o l t h e o r y ( c f . Kacser and Burns 1973, 1979, 1981), H a r t l , Dykhuizen and Dean (1985) have p r e d i c t e d the e x i s t e n c e of a concave r e l a t i o n s h i p between enzymic f l u x and a c t i v i t y for many enzymes of i n t e r m e d i a r y m e t a b o l i s m . They have argued t h a t through the a c t i o n of n a t u r a l s e l e c t i o n , the a c t i v i t i e s of many enzymes have been pushed w e l l onto the p l a t e a u of t h i s r e l a t i o n s h i p , at which p o i n t s u b s t a n t i a l changes i n c a t a l y t i c a c t i v i t y would r e s u l t i n n e g l i g i b l e changes i n f l u x . T h e r e f o r e , t h i s s a t u r a t i o n t h e o r y of H a r t l , Dykhuizen and Dean (1985) p r e d i c t s tha t the m a j o r i t y of e l e c t r o p h o r e t i c v a r i a n t s , which u s u a l l y e x h i b i t minor k i n e t i c d i f f e r e n c e s , are s e l e c t i v e l y n e u t r a l . C o n t r o l theory has s t i m u l a t e d c o n s i d e r a b l e d i s c u s s i o n because of i t s important i m p l i c a t i o n s for the p h y s i o l o g i c a l r e l e v a n c e of a l l o z y m i c v a r i a t i o n i n p a r t i c u l a r , and b i o c h e m i c a l a d a p t a t i o n in g e n e r a l ( e . g . Koehn, Zera and H a l l 1983; Watt 1985b; Burton and P l a c e 1987; Pogson 1988). E x p e r i m e n t a l work has c o n t r a d i c t e d i t s p r e d i c t i o n s i n some cases ( e . g . Cavener and C l e g g 1981; D i M i c h e l e and Powers 1982a, 1982b; H i l b i s h , Deaton and Koehn 1983; Bur ton and Feldman 1983), but not in o t h e r s ( e . g . M i d d l e t o n and Kacser 1983; Barnes and L a u r i e - A h l b e r g 1987). The e l egant s e r i e s of c o m p e t i t i o n exper iments by H a r t l 143 and c o - w o r k e r s u t i l i z i n g E . c o l i s t r a i n s tha t d i f f e r by s i n g l e a l l e l i c s u b s t i t u t i o n s at d i f f e r e n t e l e c t r o p h o r e t i c l o c i have l a r g e l y suppor ted c o n t r o l t h e o r y . No f i t n e s s d i f f e r e n c e s were d e t e c t e d between s t r a i n s p o s s e s s i n g d i f f e r e n t a l l e l e s at the gnd (on s t a n d a r d g e n e t i c backgrounds) (Dykhuizen and H a r t l 1980; H a r t l and Dykhuizen 1981), p g i (Dykhuizen and H a r t l 1983), zwf (Dykhuizen , de Framond and H a r t l 1984), or l a c Z l o c i (Dean, Dykhuizen and H a r t l 1986; D y k h u i z e n , Dean and H a r t l 1987). The o n l y l o c u s at which a l l e l i c v a r i a t i o n e x e r t e d s i g n i f i c a n t e f f e c t s on f i t n e s s was l a c Y (Dykhuizen , Dean and H a r t l 1987). However, a l l of these chemostat exper iments measured the e f f e c t s of these po lymorph ic a l l e l e s on f i t n e s s ( i . e . , growth r a t e s ) , and t h e r e f o r e i t i s not known i n any of these exper iments i f a l l e l i c v a r i a t i o n a f f e c t e d m e t a b o l i c f l u x e s . More e m p i r i c a l s t u d i e s are c l e a r l y needed to a s s e s s both the p h y s i o l o g i c a l consequences of a l l o z y m i c v a r i a t i o n and the g e n e r a l v a l i d i t y of m e t a b o l i c c o n t r o l t h e o r y . The e x p r e s s i o n of overdominant enzyme a c t i v i t i e s by the three most common h e t e r o z y g o t e s a t the Pgm-2 l o c u s i n C . g i g a s has p r o v i d e d the p o t e n t i a l f o r d e m o n s t r a t i n g the a d a p t i v e s i g n i f i c a n c e of t h i s po lymorphism. To be p h y s i o l o g i c a l l y r e l e v a n t , t h i s v a r i a t i o n i n enzyme a c t i v i t y must now be shown to i n f l u e n c e the metabol i sm of g l y c o g e n . S i n c e PGM i s not known to possess any r e g u l a t o r y p r o p e r t i e s (Ray and Peck 1972), i t s f u n c t i o n i s b e l i e v e d to be s t r i c t l y c a t a l y t i c . As argued f o r the phosphoglucose isomerase l o c u s by Watt (1977, 1983), the 1 44 p h y s i o l o g i c a l e f f e c t s of Pgm-2 genotypes are most l i k e l y to be e x p r e s s e d by d i f f e r e n c e s i n t h e i r Vmax/Km r a t i o s . These r a t i o s d i f f e r s i g n i f i c a n t l y between Pgm-2 genotypes f o r r e a c t i o n d i r e c t i o n s because of v a r i a t i o n i n t h e i r s p e c i f i c enzyme a c t i v i t i e s (Chapter 3 ) ; M i c h a e l i s c o n s t a n t s measured for g l u c o s e - 1 - p h o s p h a t e and e s t i m a t e d for g l u c o s e - 6 - p h o s p h a t e e x h i b i t e d o n l y minor d i f f e r e n t i a t i o n between a l l o z y m e s (Chapter 2 ) . A l t h o u g h kcat d i f f e r e n c e s c o u l d e x i s t between a l l o z y m e s tha t may be of s e l e c t i v e impor tance , Vmax (as de termined by s p e c i f i c a c t i v i t y ) i s p r o b a b l y the more important i n v i v o parameter because i t s composi te na ture (Vmax = kcat[enzyme]) ensures tha t d i f f e r e n c e s tha t may e x i s t between genotypes i n s t e a d y - s t a t e enzyme a c t i v i t y l e v e l s are s i m u l t a n e o u s l y i n c o r p o r a t e d (Hoffman 1981). E x a m i n a t i o n of the p h y s i o l o g i c a l e f f e c t s of the Pgm-2 l o c u s on g lycogen metabol i sm i n the P a c i f i c o y s t e r has a number of advantages over p r e v i o u s s t u d i e s of t h i s n a t u r e . The s y n t h e t i c pathway from g l u c o s e to g lycogen i s s h o r t , i n v o l v i n g o n l y four enzymic r e a c t i o n s . Glycogen i s the major energy s torage compound of o y s t e r s , at t imes r e p r e s e n t i n g up to 25% of t h e i r t o t a l body weights (Quayle 1969). Demonstrable e f f e c t s of t h i s l o c u s on g l y c o g e n metabol i sm are thus l i k e l y to be p h y s i o l o g i c a l l y important because of the c e n t r a l r o l e p l a y e d by g lycogen in the energy metabol i sm of o y s t e r s . Because g lycogen i s an e a s i l y q u a n t i f i e d end p r o d u c t , the e f f e c t of v a r i a t i o n in PGM a c t i v i t y on pathway f u n c t i o n may a l s o be more r e a d i l y measurable than i f 1 45 i t c a t a l y z e d a r e a c t i o n i n a more complex e n e r g y - p r o d u c i n g pathway (such as g l y c o l y s i s ) . Seasona l p a t t e r n s of g lycogen s y n t h e s i s and d e g r a d a t i o n are w e l l documented i n marine b i v a l v e s (Gabbott 1975; Bayne 1976). S i n c e g lycogen l e v e l s change s l o w l y (over p e r i o d s of weeks and months) , Pgm-2 genotypes have the c a p a c i t y to e x e r t c u m u l a t i v e e f f e c t s on t i s s u e g lycogen c o n c e n t r a t i o n s which may be more e a s i l y observed than i f the c y c l e o p e r a t e d on a much s h o r t e r t ime p e r i o d . MATERIALS AND METHODS C h e m i c a l s . B u f f e r s , s u b s t r a t e s , c o u p l i n g enzymes and s t a n d a r d s used f o r the g lycogen assays were o b t a i n e d from Sigma. A m y l o g l u c o s i d a s e was p r o v i d e d by B o e h r i n g e r Mannheim and the e l e c t r o s t a r c h for e l e c t r o p h o r e s i s from Connaught L a b o r a t o r i e s . A n i m a l s . The s e a s o n a l c o l l e c t i o n s , t r a n s p o r t a t i o n and s torage of o y s t e r s was i d e n t i c a l to t h a t d e s c r i b e d i n Chapter 3. E l e c t r o p h o r e s i s . Pgm-2 genotypes were i d e n t i f i e d by s t a r c h g e l e l e c t r o p h o r e s i s . D e t a i l s of the e l e c t r o p h o r e t i c procedure can be found i n Chapter 2. Glycogen A s s a y s . Glycogen c o n c e n t r a t i o n s were de termined i n the mantle and adductor muscle t i s s u e s of a l l o y s t e r s s e l e c t e d f o r s p e c i f i c a c t i v i t y measurements d e s c r i b e d i n Chapter 3. The e x p e r i m e n t a l d e s i g n i n v o l v e d the comparison of g lycogen l e v e l s 1 46 in a n i m a l s from four body weight c l a s s e s (12 .0 -23 .9 g; 2 4 . 0 - 3 5 . 9 g; 3 6 . 0 - 4 7 . 9 g; 48.0 g+), from two t i d a l h e i g h t s (low and h i g h ) , i n t h r e e seasons (summer, f a l l and w i n t e r ) c o m p r i s i n g 7 Pgm-2 g e n o t y p i c c l a s s e s . Glycogen c o u l d not be measured in some s m a l l e r i n d i v i d u a l s because of the l i m i t e d amount of t i s s u e p r e s e n t . Hence, the sample s i z e s were s l i g h t l y lower than the s p e c i f i c a c t i v i t y measurements p r e s e n t e d i n Chapter 3. A p p r o x i m a t e l y 0.4 g of t i s s u e was d i s s e c t e d from both the mantle and p o s t e r i o r adduc tor m u s c l e , b l o t t e d , and weighed to the n e a r e s t m i l l i g r a m . The s e c t i o n of mantle removed was d i r e c t l y a d j a c e n t to tha t taken f o r the measurement of PGM a c t i v i t y , thus e n s u r i n g tha t a comparable r e g i o n was s t u d i e d in a l l o y s t e r s . S i m i l a r l y , the adductor muscle d i s s e c t e d r e p r e s e n t e d one h a l f of the t i s s u e chosen for enzymat ic s t u d y . T i s s u e s were added to 2 ml of i c e - c o l d 0.6 N p e r c h l o r i c a c i d (PCA) and homogenized f o r 30 s w i t h an U l t r a - T u r r a x t i s s u e g r i n d e r . A 1 ml a l i q u o t was t r a n s f e r r e d to an Eppendorf m i c r o c e n t r i f u g a t i o n tube and f r o z e n at - 7 0 ° C f o r up to 3 months p r i o r to the d e t e r m i n a t i o n of g l y c o g e n . Glycogen was assayed by the a m y l o g l u c o s i d a s e method m o d i f i e d s l i g h t l y from K e p p l e r and Decker (1974) . D u p l i c a t e 100 M1 samples of the PCA homogenates from both t i s s u e s were added to 1 ml of 200 mM sodium a c e t a t e , 50 mM potas s ium hydrogen c a r b o n a t e b u f f e r , pH 4.8 c o n t a i n i n g a p p r o x i m a t e l y 6 u n i t s of a m y l o g l u c o s i d a s e ( l y o p h i l i z e d from A s p e r g i l l u s n i g e r ) in 10 x 75 147 mm p o l y c a r b o n a t e c u l t u r e t u b e s . The tubes were s e a l e d w i t h p l a s t i c caps and i n c u b a t e d for 2 h in a shak ing water bath at 4 0 ° C . The h y d r o l y s i s of g lycogen was s topped by the a d d i t i o n of 500 Ail 0.6N PCA and the samples were spun for 5 min a t top speed i n a c l i n i c a l c e n t r i f u g e . A 500 M1 sample of the s u p e r n a t a n t s were p i p e t t e d i n t o Eppendorf m i c r o c e n t r i f u g a t i o n tubes and f r o z e n a t - 7 0 ° C . The l i b e r a t e d g lucose was q u a n t i f i e d e n z y m a t i c a l l y at room temperature by the h e x o k i n a s e / g l u c o s e - 6 - p h o s p h a t e dehydrogenase system at 340 nm on a Pye Unicam SP8-400 U V / v i s i b l e s p e c t r o p h o t o m e t e r . The r e a c t i o n medium c o n t a i n e d 300 mM t r i e t h a n o l a m i n e , 4 mM MgS04, 1 mM ATP, 0.5 mM NADP, 0.8 u n i t s g l u c o s e - 6 - p h o s p h a t e dehydrogenase , pH 7.5 i n a f i n a l volume of 1 m l . Assays were performed i n d u p l i c a t e on 20 M1 of the h y d r o s y l a t e s from both t i s s u e s . A f t e r sample a d d i t i o n , the c u v e t t e s were mixed t h o r o u g h l y and a l l o w e d to e q u i l i b r a t e f o r at l e a s t 10 min b e f o r e an i n i t i a l absorbance r e a d i n g was t a k e n . 1.5 u n i t s of hexokinase was then added, the c u v e t t e s were aga in mixed and the r e a c t i o n was a l l o w e d to proceed to c o m p l e t i o n (> 15 min) b e f o r e a second absorbance r e a d i n g t a k e n . The d i f f e r e n c e between these absorbance r e a d i n g s was s t o i c h i o m e t r i c a l l y equa l to the amount of f r e e g l u c o s e presen t i n the h y d r o l y z e d sample. Glycogen c o n c e n t r a t i o n s r e p r e s e n t e d the mean of four assays per i n d i v i d u a l and were expres sed as Mmoles g l u c o s y l u n i t s / g wet t i s s u e weight by assuming an i n t r a c e l l u l a r water c o n c e n t r a t i o n of 75% i n the mantle and 50% in the adduc tor muscle t i s s u e 148 (Walsh, McDonald and Booth 1984). C o r r e c t i o n s were not made f o r the f r e e g l u c o s e presen t i n the o r i g i n a l PCA homogenates because i t s c o n c e n t r a t i o n was found to be too low to a f f e c t the g lycogen e s t i m a t e s . To asses s the e f f i c i e n c y and r e p e a t a b i l i t y of the enzymat ic h y d r o l y s i s of g l y c o g e n , s tandards were p r e p a r e d by d i s s o l v i n g 100 mg o y s t e r g lycogen (Type II ) i n 10 ml of 50 mM i m i d a z o l e - H C l b u f f e r , pH 7.0 ( 2 0 ° C ) . F i f t y nl a l i q u o t s of t h i s s tock s o l u t i o n were h y d r o l y z e d a l o n g s i d e the t i s s u e samples from each season and assayed in d u p l i c a t e f o r g l u c o s e as d e s c r i b e d p r e v i o u s l y . The g lycogen measurements were completed w i t h i n s i x months of each seasona l c o l l e c t i o n . A l t h o u g h the spontaneous d e g r a d a t i o n of g lycogen i n the f r o z e n o y s t e r s was not d e t e r m i n e d , s e v e r a l p r e c a u t i o n s were taken to min imize the i n f l u e n c e of t h i s f a c t o r on the r e s u l t s . As d e s c r i b e d i n Chapter 3, the s e l e c t i o n of genotypes was randomized each day such tha t the t ime taken to p r o c e s s them was s i m i l a r . The s e l e c t i o n of genotypes f o r the enzymatic h y d r o l y s i s of g lycogen was a l s o c o m p l e t e l y randomized , as was the o r d e r in which the g lucose as says were c a r r i e d o u t . T h e r e f o r e , i t was expected tha t d a y - t o -day e r r o r s were kept to a minimum and the spontaneous l o s s of g lycogen was s i m i l a r f o r a l l genotypes . S t a t i s t i c a l A n a l y s e s . The e f f e c t s of season, t i d a l p o s i t i o n , and Pgm-2 genotype on the g lycogen c o n c e n t r a t i o n of the two t i s s u e s s t u d i e d was examined by a n a l y s i s of v a r i a n c e (ANOVA) as o u t l i n e d 149 in S o k a l and Roh l f (1981) . Means were compared by a p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s . RESULTS Comparison of the h y d r o l y s i s of the g lycogen s t a n d a r d s wi th a m y l o g l u c o s i d a s e c o n f i r m e d the c o n s i s t e n c y of the g lycogen assays throughout the s t u d y . The e f f i c i e n c y of h y d r o l y s i s of s tandards i n c l u d e d w i t h the summer, f a l l , and w i n t e r samples averaged 85.9%, 84.8%, and 87.4%, r e s p e c t i v e l y . However, s t a t i s t i c a l comparison of these a n g u l a r t rans formed percentages by one-way ANOVA s t i l l produced a s i g n i f i c a n t e f f e c t of season (F(2 ,95) = 4 .82 , P < . 0 5 ) , a d i f f e r e n c e t h a t m u l t i p l e range t e s t s showed to be a consequence of the h i g h e r e f f i c i e n c y of h y d r o l y s i s i n the w i n t e r r e l a t i v e to the- f a l l . T i s s u e g lycogen c o n c e n t r a t i o n s de termined for the f a l l sample w i l l t h e r e f o r e be u n d e r e s t i m a t e d by 3% compared to the w i n t e r , but t h i s would have had a n e g l i g i b l e e f f e c t on the r e s u l t s . S i m i l a r to the a n a l y s i s of the PGM s p e c i f i c a c t i v i t y data r e p o r t e d i n Chapter 3, the g lycogen d a t a ' f o r both the mantle and adductor muscle t i s s u e s were o r i g i n a l l y a n a l y z e d i n each season by a 3 - f a c t o r ANOVA t r e a t i n g t i d a l p o s i t i o n , body weight c l a s s , and Pgm-2 genotype as independent v a r i a b l e s . Body weight was aga in found to have a minor e f f e c t on t i s s u e g lycogen l e v e l s , e x p l a i n i n g a s i g n i f i c a n t p o r t i o n of the observed v a r i a t i o n o n l y i n the adduc tor muscle t i s s u e from the f a l l sample (F(3 ,144) = 150 5 .99 , P < . 0 0 1 ) . I n t e r e s t i n g l y , t h i s was a l s o the on ly season and t i s s u e in which body weight e x e r t e d a s i g n i f i c a n t e f f e c t on PGM s p e c i f i c a c t i v i t y . An i n v e r s e r e l a t i o n s h i p was observed between these v a r i a b l e s and body s i z e . For the g lycogen d a t a , o y s t e r s i n the s m a l l e s t weight c l a s s ( 1 2 . 0 - 2 3 . 9 g) possessed h i g h e r l e v e l s of t h i s c a r b o h y d r a t e i n t h e i r a d d u c t o r muscles than measured in a l l an imal s above 24.0 g . M u l t i p l e range t e s t s d e t e c t e d a s i g n i f i c a n t d i f f e r e n c e between t h i s group and the two weight c l a s s e s above 36.0 g , but not from those i n the 2 4 . 0 - 3 5 . 9 g r a n g e . S t a t i s t i c a l comparison of the adductor muscle s p e c i f i c a c t i v i t i e s r e v e a l e d s i m i l a r d i f f e r e n c e s between these four weight c l a s s e s . However, the r e l a t i o n s h i p between s p e c i f i c a c t i v i t y and body weight was o p p o s i t e to t h a t seen f o r the g lycogen d a t a ; the s m a l l e s t o y s t e r s e x h i b i t e d lower enzyme a c t i v i t y l e v e l s than the o t h e r three g r o u p s . A s i m i l a r a s s o c i a t i o n between body weight and g lycogen was e v i d e n t in the m a n t l e , but as w i t h the s p e c i f i c a c t i v i t i e s measured i n t h i s t i s s u e the d i f f e r e n c e s between weight c l a s s e s were not s t a t i s t i c a l l y s i g n i f i c a n t . Because of i t s minor importance , body weight was exc luded as a f a c t o r i n the o v e r a l l a n a l y s i s . T h i s i s p r e s e n t e d i n T a b l e XI t o g e t h e r wi th the r e s u l t s from the s p e c i f i c a c t i v i t y da ta from Chapter 3 for c o m p a r a t i v e p u r p o s e s . Season and t i d a l p o s i t i o n e x e r t e d h i g h l y s i g n i f i c a n t e f f e c t s on g lycogen l e v e l s measured i n both the mantle and a d d u c t o r muscle t i s s u e s of C . g i g a s . In c o m b i n a t i o n , these f a c t o r s produced h i g h l y s i g n i f i c a n t s e a s o n - b y - t i d a l h e i g h t 151 T a b l e X I . F - r a t i o s from a n a l y s e s of v a r i a n c e on g lycogen c o n c e n t r a t i o n s and PGM s p e c i f i c a c t i v i t i e s i n the mantle and adductor muscle t i s s u e s . 152 Glycogen Content S p e c i f i c A c t i v i t y 1 Source of df Mant le Adductor V a r i a t i o n Musc le M a n t l e Adductor Musc le Season T i d a l He ight Genotype Genotype x Season Genotype x T i d a l He ight Season x T i d a l He ight Genotype x T i d a l H e i g h t x Season 2 1 6 2 12 128.2*** 449.7*** 88.2*** 166.2*** 1.13 1.38 3.52*** 1.70 1 .55 1.15 17.4*** 111.9*** 3.06*** 1.44 39.3*** 271.3*** 2.52 5.56* 11.9*** 21.9*** 1.34 1.09 0.54 0.73 0.84 23.8*** 13.7*** 1 .20 E r r o r 451 448 456 456 * P < .05 * * * P < .001 1 from Chapter 3 153 i n t e r a c t i o n terms, as observed f o r t h e i r e f f e c t s on PGM s p e c i f i c a c t i v i t y . D e s p i t e these s i m i l a r i t i e s , s e v e r a l important d i f f e r e n c e s can be seen between the r e s u l t s of these a n a l y s e s . F i r s t , i n t e r t i d a l p o s i t i o n a lone accounted for a h i g h l y s i g n i f i c a n t amount of the v a r i a t i o n i n the g lycogen c o n c e n t r a t i o n s , but not the enzyme a c t i v i t i e s , observed in in these t i s s u e s . Second, the h i g h l y s i g n i f i c a n t e f f e c t of Pqm-2 genotype (as a s eparate f a c t o r ) on s p e c i f i c a c t i v i t y was not repeated i n the g l y c o g e n d a t a . T h i r d , h i g h l y s i g n i f i c a n t g e n o t y p e - b y - s e a s o n , and g e n o t y p e - b y - s e a s o n - b y - t i d a l h e i g h t i n t e r a c t i o n terms were observed in the mantle g lycogen r e s u l t s , but were absent from the s p e c i f i c a c t i v i t y d a t a . F o u r t h , the two t i s s u e s produced n e a r l y i d e n t i c a l r e s u l t s f or the s p e c i f i c a c t i v i t y but not the g lycogen d a t a : the genotype-by-env ironment i n t e r a c t i o n s observed i n the mantle were not presen t i n the adductor muscle t i s s u e . EFFECTS OF SEASON AND INTERTIDAL POSITION The e f f e c t s of season and t i d a l p o s i t i o n on the mantle and adductor muscle g lycogen c o n c e n t r a t i o n s are p r e s e n t e d g r a p h i c a l l y i n F i g u r e 13. In each season , mantle g lycogen l e v e l s were c o n s i s t e n t l y t h r e e - to f o u r - f o l d g r e a t e r than observed in the a d d u c t o r musc le . C o n s e q u e n t l y , the s easona l p a t t e r n s d i s p l a y e d i n these t i s s u e s were s i m i l a r , e x h i b i t i n g rank o r d e r s of summer > f a l l > w i n t e r . O y s t e r s lower i n the i n t e r t i d a l zone possessed s i g n i f i c a n t l y g r e a t e r q u a n t i t i e s of g lycogen than 154 F i g u r e 13. Seasona l v a r i a t i o n i n the mantle and a d d u c t o r muscle g lycogen c o n c e n t r a t i o n s (jumoles g l u c o s y l u n i t s / g t i s s u e ) a t the two i n t e r t i d a l p o s i t i o n s . Open c i r c l e s = l o w water; c l o s e d c i r c l e s = h i g h water . Mant l e sample s i z e s : summer, low=61 and high=57; f a l l , low=96 and h igh= l00 ; w i n t e r , low=88 and h i g h = 9 l . Musc le sample s i z e s : summer, low=58 and high=57; f a l l and w i n t e r are the same as the m a n t l e . Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . A. Mantle B. Adductor Muscle 156 a n i m a l s s i t u a t e d i n the more exposed , h i g h i n t e r t i d a l a r e a . T h i s r e l a t i o n s h i p h e l d in both t i s s u e s i n a l l t h r e e seasons , thus a c c o u n t i n g f o r the s i g n i f i c a n t e f f e c t of t i d a l p o s i t i o n seen i n T a b l e X I . As e v i d e n t from F i g u r e 13, the h i g h l y s i g n i f i c a n t s e a s o n - b y - t i d a l h e i g h t i n t e r a c t i o n terms observed i n T a b l e XI were produced by the more pronounced d i f f e r e n c e i n t i s s u e g lycogen l e v e l s between o y s t e r s at the two i n t e r t i d a l p o s i t i o n s i n the summer compared to the f a l l and w i n t e r . In the summer, o y s t e r s i n the low i n t e r t i d a l had 53% more mantle g lycogen than i n d i v i d u a l s sampled from the h i g h i n t e r t i d a l . zone , whereas comparable d i f f e r e n c e s i n the f a l l and win ter were 25 and 22%, r e s p e c t i v e l y . Even more dramat i c d i f f e r e n c e s were observed i n the adduc tor muscle t i s s u e . H e r e , o y s t e r s low i n the i n t e r t i d a l r e g i o n posses sed 92, 21, and 14% more g lycogen than those in the h i g h i n t e r t i d a l a r e a i n the summer, f a l l , and w i n t e r , r e s p e c t i v e l y . The source of t h i s i n t e r a c t i o n between season and t i d a l h e i g h t on t i s s u e g lycogen c o n c e n t r a t i o n s d i f f e r s from the s i m i l a r i n t e r a c t i o n observed between these f a c t o r s on PGM s p e c i f i c a c t i v i t y shown in T a b l e XI and d i s c u s s e d in Chapter 3. For the s p e c i f i c a c t i v i t y d a t a , the i n t e r a c t i o n was caused by s h i f t s i n the enzyme l e v e l s measured i n o y s t e r s at the two t i d a l h e i g h t s in d i f f e r e n t seasons . In the summer and f a l l , o y s t e r s at the low water s t a t i o n had s i g n i f i c a n t l y g r e a t e r s p e c i f i c a c t i v i t i e s than i n d i v i d u a l s at the h i g h water s i t e , but i n the w i n t e r t h i s p a t t e r n was r e v e r s e d . The na ture of these 157 i n t e r a c t i o n s between season and - i n t e r t i d a l p o s i t i o n i n the g lycogen and s p e c i f i c a c t i v i t y data o f f e r i n s i g h t s i n t o the c a u s a l r e l a t i o n s h i p between these measures . Between the summer and f a l l , g lycogen l e v e l s changed i n a markedly n o n - a d d i t i v e f a s h i o n at the two i n t e r t i d a l p o s i t i o n s . However, over the same p e r i o d PGM s p e c i f i c a c t i v i t y i n c r e a s e d i n a s t r i c t l y a d d i t i v e manner at these t i d a l h e i g h t s . In a d d i t i o n , the r e v e r s a l s of the PGM a c t i v i t i e s e x h i b i t e d by o y s t e r s a t these t i d a l p o s i t i o n s between the f a l l and w i n t e r were not r e f l e c t e d i n the g lycogen c o n c e n t r a t i o n s measured i n these seasons . These d i s c r e p a n c i e s suggest t h a t i t i s not v a r i a t i o n in PGM a c t i v i t y per se t h a t i s r e s p o n s i b l e for the s e a s o n - b y - t i d a l h e i g h t i n t e r a c t i o n s seen i n the g lycogen c o n c e n t r a t i o n s of these t i s s u e s . EFFECT OF PGM-2 GENOTYPE The major f i n d i n g of Chapter 3 was the e x p r e s s i o n of overdominance for enzyme a c t i v i t y at the Pgm-2 l o c u s in C r a s s o s t r e a g i g a s . The Pgm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s possessed g r e a t e r s p e c i f i c a c t i v i t i e s than the Pqm - 2 - 9 2 / 9 2 , 96/96, 100/100, and 104/104 homozygotes . These homozygotes and h e t e r o z y g o t e s behaved as homogeneous g r o u p s , e x h i b i t i n g s i m i l a r p a t t e r n s i n both t i s s u e s s t u d i e d : enzyme a c t i v i t i e s i n h e t e r o z y g o t e s exceeded homozygotes by 24% and 20% i n the mantle and adduc tor musc le , r e s p e c t i v e l y . T h i s knowledge a l l o w e d a p r i o r i comparisons of the a s s o c i a t i o n s between these d i f f e r e n c e s i n PGM a c t i v i t y and t i s s u e g lycogen c o n c e n t r a t i o n s . 158 C o n s e q u e n t l y , a n a l y s e s were performed on 1) the 7 Pqm-2 genotypes s e p a r a t e l y , and 2) the same genotypes p o o l e d i n t o three g e n o t y p i c groups (Pgm-2-100/100 homozygotes, h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e , and homozygotes for the t h r e e l e s s f requent a l l e l e s ) . As w i l l be seen , l i t t l e i n f o r m a t i o n i s l o s t i n these a n a l y s e s by p o o l i n g genotypes i n t o these c l a s s e s . In the o v e r a l l a n a l y s i s , Pgm-2 genotype d i d not e x p l a i n a s i g n i f i c a n t amount of the v a r i a t i o n i n g lycogen l e v e l s observed i n e i t h e r t i s s u e . However i n the m a n t l e , h i g h l y s i g n i f i c a n t i n t e r a c t i o n s were d e t e c t e d between Pgm-2 genotype and season , and for the second o r d e r g e n o t y p e - b y - s e a s o n - b y - t i d a l h e i g h t t e r m . Both i n t e r a c t i o n s were d i r e c t l y a s s o c i a t e d w i t h the d i f f e r e n c e s i n enzyme a c t i v i t i e s expres sed between Pgm-2 homozygote and h e t e r o z y g o t e g r o u p s . The source of the genotype-by-season i n t e r a c t i o n i s i l l u s t r a t e d in F i g u r e 14, which p r e s e n t s the mean g lycogen c o n c e n t r a t i o n s of the Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s , poo l ed a c r o s s t i d a l h e i g h t s , i n each of the t h r e e seasons . In the mantle ( F i g u r e 14A), summer g lycogen l e v e l s d i d not d i f f e r s i g n i f i c a n t l y between a l l e l i c c l a s s e s when averaged over the two t i d a l p o s i t i o n s (F (2 ,95 ) = 1.12, P > . 3 0 ) . (However, a h i g h l y s i g n i f i c a n t a l l e l i c c l a s s - b y - t i d a l h e i g h t i n t e r a c t i o n was p r e s e n t ( F ( 2 , 9 5 ) = 6 .31 , P < .01) tha t w i l l be d i s c u s s e d l a t e r . ) In the f a l l , s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between a l l e l i c groups (F(2 ,172) = 12 .0 , P < . 0 0 1 ) . The Pgm-2 h e t e r o z y g o t e s posses sed h i g h e r g lycogen l e v e l s than the two homozygote groups , 159 F i g u r e 14. Seasona l v a r i a t i o n i n the mantle and adductor muscle g lycogen c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s . Open c i r c l e s = Pqm-2-100/100 homozygotes (summer.n=29, f a l l n=42 f w i n t e r n=38). C l o s e d c i r c l e s = h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e (summer n=7l , f a l l n=123, w i n t e r n= l08) . Open squares=homozygotes f o r the Pgm-2-92, 96, and 104 a l l e l e s (summer n=l8 , f a l l n=3 l , w i n t e r n=33). Bars r e p r e s e n t ±1 s t a n d a r d e r r o r where v i s i b l e or f a l l w i t h i n the p l o t t e d symbol . \ A. Mantle B. Adductor Muscle 161 but were found by m u l t i p l e range t e s t s to d i f f e r s i g n i f i c a n t l y on ly from the Pgm-2-100/100 genotypes . The a l l e l i c c l a s s i f i c a t i o n a l s o e x p l a i n e d a s i g n i f i c a n t amount of the v a r i a t i o n i n mantle g lycogen c o n c e n t r a t i o n s i n the w i n t e r (F(2 ,155) = 10 .2 , P < . 0 0 1 ) , but the s i t u a t i o n was r e v e r s e d from tha t seen i n the f a l l . Both Pgm-2 homozygotes now d i s p l a y e d s i g n i f i c a n t l y more mantle g lycogen than the thr^e Pgm-2 h e t e r o z y g o t e s . Genotypes p o o l e d i n t o these a l l e l i c c l a s s e s behaved s i m i l a r l y i n both the f a l l and w i n t e r . Because g e n o t y p e - b y - t i d a l h e i g h t i n t e r a c t i o n s were absent i n both a n a l y s e s , s i x comparisons of the g lycogen l e v e l s of Pgm-2 h e t e r o z y g o t e s r e l a t i v e to t h e i r r e s p e c t i v e homozygotes were a v a i l a b l e i n each season . In the f a l l , h e t e r o z y g o t e s d i s p l a y e d h i g h e r g lycogen c o n c e n t r a t i o n s in 5 of the 6 c o m p a r i s o n s . In a l l s i x w i n t e r comparisons h e t e r o z y g o t e s posses sed lower q u a n t i t i e s g lycogen than t h e i r r e s p e c t i v e homozygotes. The unusual r e v e r s a l s i n mantle g lycogen l e v e l s observed between homozygotes and h e t e r o z y g o t e s over the f a l l and w i n t e r was r e s p o n s i b l e for the s i g n i f i c a n t genotype -by- season i n t e r a c t i o n term seen in T a b l e X I . Seasona l changes in the adductor muscle g lycogen c o n c e n t r a t i o n s of these a l l e l i c groups are shown in F i g u r e 14B. The s i g n i f i c a n t g e n o t y p e - b y - t i d a l h e i g h t i n t e r a c t i o n i n summer mantle l e v e l s was not r epea ted for the adductor muscle t i s s u e , 162 and , i n both the summer and f a l l , homozygotes and h e t e r o z y g o t e s posses sed i d e n t i c a l q u a n t i t i e s of muscle g l y c o g e n . However, s i g n i f i c a n t d i f f e r e n c e s were p r e s e n t between a l l e l i c c l a s s e s i n the w i n t e r (F(2 ,155) = 8 .13 , P < . 0 0 1 ) . As was seen f o r the mantle t i s s u e i n t h i s season , both homozygote c l a s s e s possessed more g lycogen i n t h e i r adductor muscles than h e t e r o z y g o t e s , but the d i f f e r e n c e was s i g n i f i c a n t o n l y for the Pgm-2-100/100 genotypes . An examinat ion of g e n o t y p i c p a t t e r n s at both t i d a l h e i g h t s in the w i n t e r r e v e a l e d that in 5 of the 6 p o s s i b l e c o m p a r i s o n s , Pgm-2 h e t e r o z y g o t e s d i s p l a y e d lower g lycogen l e v e l s than t h e i r r e s p e c t i v e homozygotes . The d i f f e r e n t i a t i o n between these a l l e l i c c l a s s e s in the w i n t e r was m a r g i n a l l y below t h a t r e q u i r e d to produce a s i g n i f i c a n t genotype -by - season i n t e r a c t i o n f o r the adductor muscle g lycogen da ta (F(6 ,448) = 1.70, P < . 1 0 ) . The mantle g lycogen c o n c e n t r a t i o n s of Pgm-2 homozygotes and h e t e r o z y g o t e s are p r e s e n t e d s e p a r a t e l y f o r both i n t e r t i d a l p o s i t i o n s in T a b l e XII to i l l u s t r a t e the source of the s i g n i f i c a n t g e n o t y p e - b y - s e a s o n - b y - t i d a l h e i g h t i n t e r a c t i o n . S i g n i f i c a n t d i f f e r e n c e s were observed between a l l e l i c c l a s s e s at both t i d a l h e i g h t s i n a l l seasons w i t h the e x c e p t i o n of the h i g h i n t e r t i d a l s i t e in the summer. In t h i s season , homozygotes in the low i n t e r t i d a l zone d i s p l a y e d h i g h e r q u a n t i t i e s of mantle g lycogen than the h e t e r o z y g o t e s . At the h i g h water s i t e however, h e t e r o z y g o t e s possessed l a r g e r g lycogen l e v e l s than both homozygote g r o u p s , but these d i f f e r e n c e s were not s i g n i f i c a n t 163 T a b l e X I I . Combined e f f e c t s of season and i n t e r t i d a l p o s i t i o n on the mantle g lycogen c o n c e n t r a t i o n s (/xmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s . Glycogen Concentration' Season Tidal Homozygotes Heterozygotes Homozygotes Posi t i o n N for 100 N for 100 N without 100 Summer Low High F a l l Low High Winter Low High 15 396.9±19.S 14 189.1±24.3 22 171.4+16.0 20 122.0±12.0 19 179.6+13.0 19 174.2±10.4 38 301.4±12.3 33 236.5115.3 60 220.5+9.7 63 1B3.1+6.7 50 157.7+8.0 58 124.2±5.9 8 375.4+26.8 10 199.3±27.6 14 195.2±20.0 17 148.7+13.1 19 192.8+13.0 14 155.7±12.2 F(2.49) = 8.85*** F(2.47) = 1.62 F(2,84) = 3.76* F(2,88) = 10.9*** F(2.76) = 3.11* F(2,79) = 9.78*** 1 pmoles glucosyl units/g wet tissue * P < .05 *** P < .001 165 ( F ( 2 , 4 7 ) = 1.62, P > .20). In the f a l l and winter the d i f f e r e n c e s expressed between these genotypic c l a s s e s were s i m i l a r at both t i d a l p o s i t i o n s , but i n c o n t r a s t to the summer, the d i f f e r e n c e between a l l e l i c c l a s s e s was more extreme at the high i n t e r t i d a l . Table XII shows that the s e a s o n - b y - t i d a l height component of t h i s second-order i n t e r a c t i o n was a consequence of the gr e a t e r seasonal f l u c t u a t i o n s of glycogen c o n c e n t r a t i o n s i n the four Pgm-2 homozygotes. Taking the average of the two homozygote groups, mantle l e v e l s at the two i n t e r t i d a l p o s i t i o n s were found to d i f f e r by 101, 35, and 12% i n the summer, f a l l , and winter, r e s p e c t i v e l y . In c o n t r a s t , mantle glycogen c o n c e n t r a t i o n s i n heterozygotes at these t i d a l h e i g h t s d e c l i n e d i n a l i n e a r and p a r a l l e l f a s h i o n , d i f f e r i n g by only 27, 20, and 27% i n the summer, f a l l , and winter, r e s p e c t i v e l y . The genotypic component of t h i s second-order i n t e r a c t i o n was caused e n t i r e l y by the d i f f e r e n c e s between these a l l e l i c c l a s s e s . The d i f f e r e n t i a l p a t t e r n s d i s p l a y e d by Pgm-2 homozygotes and heterozygotes between t i d a l p o s i t i o n s i n the summer, combined with the net r e v e r s a l between these groups over the f a l l and winter, was thus r e s p o n s i b l e f o r the s i g n i f i c a n t genotype-by-season-by-tidal height i n t e r a c t i o n . D e s p i t e these d i f f e r e n c e s , the mantle glycogen c o n c e n t r a t i o n s of homozygotes and heterozygotes were n e a r l y i d e n t i c a l when averaged over a l l seasons and t i d a l p o s i t i o n s . 166 T a b l e X I I I p r e s e n t s the s e a s o n a l changes i n adductor muscle g lycogen l e v e l s of Pgm-2 homozygotes and h e t e r o z y g o t e s a t the two i n t e r t i d a l l o c a t i o n s . No s i g n i f i c a n t d i f f e r e n c e s were observed between g e n o t y p i c c l a s s e s at e i t h e r t i d a l p o s i t i o n i n the summer or f a l l . In the w i n t e r , s i g n i f i c a n t d i f f e r e n c e s were p r e s e n t between a l l e l i c groups i n the h i g h i n t e r t i d a l a r e a (F(2 ,79) = 7 .58 , P < . 0 0 1 ) , w i t h the homozygotes p o s s e s s i n g on average 23% more g lycogen than h e t e r o z y g o t e s . A s i m i l a r t r e n d was e v i d e n t at the low water s i t e but the magnitude of t h i s d i f f e r e n c e was d i m i n i s h e d (homozygotes exceed ing h e t e r o z y g o t e s by 8%) and thus was not s t a t i s t i c a l l y s i g n i f i c a n t (F (2 ,76 ) = 1.51, P > . 2 0 ) . Because of these w i n t e r r e s u l t s , homozygotes e x h i b i t e d s l i g h t l y h i g h e r g lycogen l e v e l s than h e t e r o z y g o t e s when averaged over the t h r e e seasons , but s i n c e Pgm-2 genotypes were i n d i s t i n g u i s h a b l e i n the summer and f a l l , these d i f f e r e n c e s were not s i g n i f i c a n t (F(6 ,448) = 1.38, P > 0 . 2 0 ) . In Chapter 3 i t was shown tha t overdominance for enzyme a c t i v i t y was expres sed o n l y in h e t e r o z y g o t e s p o s s e s s i n g the most common Pgm-2-100 a l l e l e . H e t e r o z y g o t e s between the l e s s f requent Pgm-2-92, 96 and 104 a l l e l e s d i s p l a y e d s p e c i f i c a c t i v i t i e s t h a t were s i g n i f i c a n t l y lower than the overdominant h e t e r o z y g o t e s and i n d i s t i n g u i s h a b l e from both homozygote c l a s s e s . In T a b l e XIV the mantle and adductor muscle g lycogen c o n c e n t r a t i o n s of these same g e n o t y p i c c l a s s e s are p r e s e n t e d , toge ther w i t h t h e i r Pgm-2 s p e c i f i c a c t i v i t i e s from Chapter 3. As found f o r the s p e c i f i c a c t i v i t y measurements, a c l e a r d i s t i n c t i o n was observed between 167 T a b l e X I I I . Combined e f f e c t s of season and i n t e r t i d a l p o s i t i o n on the adductor muscle g lycogen c o n c e n t r a t i o n s (nmoles g l u c o s y l u n i t s / g t i s s u e ) of Pgm-2 homozygote and h e t e r o z y g o t e c l a s s e s . Glycogen Concentration' Season Tidal Position N Homozygotes for 100 Heterozygotes for 100 Homozygotes without 100 Summer Low High 15 14 119.0±9.1 60.6+6.1 35 127.6+6.O 33 66.3+3.8 8 10 141 .1±12.5 74.316.9 F(2.46) = 1.16 F(2.47) = 0.95 Fal 1 Low High 22 20 47.7+2.9 39.012.4 60 63 50.6+1.7 40.811.4 14 17 51.013.6 46.412.6 F(2.84) = 0.62 F(2.88) = 2.41 Winter Low High 19 19 44.212.0 43.312.2 50 58 40.411.2 34.0+1.2 19 14 43.412.0 39.912.6 F(2.76) = 1.51 F(2.79) = 7.58*** ' pmoles glucosyl units/g wet tissue *** P < .001 169 T a b l e X I V . Glycogen c o n c e n t r a t i o n s (Mmoles g l u c o s y l u n i t s / g t i s s u e ) and s p e c i f i c a c t i v i t i e s (un i t s /mg p r o t e i n ) of homozygotes and h e t e r o z y g o t e s p o s s e s s i n g or l a c k i n g the Pgm-2-100 a l l e l e . 170 Mant1e Adductor Muscle Genotypic N S p e c i f i c 1 ' Glycogen' Class A c t i v i t y Content S p e c i f i c 1 ' Glycogen' A c t i v i t y Content Homozygotes for 100 a l l e l e 42 0.109±.005 147.9110.0 0.2061.006 43.511.8 Homozygotes wlthout 100 a l l e l e 31 0.1171.005 169.7111.6 0.2111.007 - 48.512.1 Heterozygotes 123 for 100 a l l e l e 0.1291.003 201.315.8 0.2361.003 45.6+1.1 Heterozygotes 33 wlthout 100 a l l e l e 0.1051.005 148.7111.3 0.1981.006 45.112.1 F(3,197) 7.76*** 10.5* 12.9*** 1.21 1 units/mg protein ' from Chapter 3 ' pinoles glucosyl units/g wet tissue *** P < .001 171 these h e t e r o z y g o t e groups in t h e i r mantle g lycogen l e v e l s . The Pgm-2-92/96 , 92/104, and 96/104 h e t e r o z y g o t e s e x h i b i t e d g lycogen c o n c e n t r a t i o n s t h a t were a g a i n s i g n i f i c a n t l y lower than the o ther h e t e r o z y g o t e c l a s s and not d i f f e r e n t from the two homozygote groups . In t h i s t i s s u e a s t r o n g p o s i t i v e r e l a t i o n s h i p e x i s t e d between the s p e c i f i c a c t i v i t i e s of these genotyp ic c l a s s e s and t h e i r g lycogen s t o r e s . Adductor muscle g lycogen c o n c e n t r a t i o n s d i d not d i f f e r between these g e n o t y p i c groups , d e s p i t e showing d i f f e r e n c e s i n enzyme a c t i v i t i e s s i m i l a r to those observed in the m a n t l e . DISCUSSION The p h y s i o l o g i c a l r e l e v a n c e of n a t u r a l l y o c c u r r i n g enzyme polymorphisms can o n l y be a s s e s s e d through an examinat ion of the e f f e c t s of t h i s v a r i a t i o n on the output of the b i o c h e m i c a l pathways i n which these enzymes f u n c t i o n . For the Pgm-2 l o c u s i n C r a s s o s t r e a g i g a s , t h i s requirement t r a n s l a t e s i n t o a d e m o n s t r a t i o n of the impact of t h i s polymorphism on g lycogen m e t a b o l i s m . In marine b i v a l v e s , s e a s o n a l v a r i a t i o n i n the s y n t h e s i s and d e g r a d a t i o n of g lycogen i s f u n c t i o n a l l y c o u p l e d to t h e i r annua l r e p r o d u c t i v e c y c l e (Bayne 1976; Zandee et a l . 1980; Gabbott 1983). One important component of t h i s c y c l e i n v o l v e s long term changes i n the a c t i v i t i e s of enzymes i n v o l v e d in the s t o r a g e and subsequent m o b i l i z a t i o n of these g l y c o g e n r e s e r v e s for gametogenesis ( L i v i n g s t o n e 1981; Zaba 1981; Gabbott and W h i t t l e 1986a). Because changes i n g lycogen l e v e l s take p l a c e 172 r a t h e r s l o w l y , examinat ion of the a s s o c i a t i o n s between the d i f f e r i n g enzyme a c t i v i t i e s of Pgm-2 genotypes and t h e i r e x i s t i n g t i s s u e g lycogen c o n c e n t r a t i o n s enab les an e v a l u a t i o n , a l b e i t i n d i r e c t , of the p h y s i o l o g i c a l e f f e c t s , of t h i s po lymorphism. The h i g h l y s i g n i f i c a n t e f f e c t s of season and i n t e r t i d a l p o s i t i o n on g l y c o g e n l e v e l s and t h e i r i n t e r a c t i o n s w i t h Pgm-2 genotype observed i n t h i s s tudy p r o v i d e some important i n s i g h t s c o n c e r n i n g the i n f l u e n c e of a l l o z y m i c v a r i a t i o n on m e t a b o l i c f l u x . The s e a s o n a l changes i n the g lycogen content of the mantle and adduc tor muscle t i s s u e s of C . g i g a s found i n the p r e s e n t s tudy are i n g e n e r a l agreement w i t h those r e p o r t e d p r e v i o u s l y for B r i t i s h Columbia p o p u l a t i o n s of t h i s s p e c i e s (Quayle 1969; Whyte and E n g l a r 1982). The marked d i f f e r e n c e i n g lycogen l e v e l s observed i n the two t i s s u e s examined was expected s i n c e the mantle i s one of the p r i m a r y s torage s i t e s for t h i s c a r b o h y d r a t e i n marine b i v a l v e s (Eble 1969; Bayne et a l . 1982). I t was unexpected tha t f a l l g lycogen l e v e l s would exceed those measured i n the w i n t e r . However, the w i n t e r sample was taken when n a t u r a l food l e v e l s i n Nanoose Bay were u n u s u a l l y low (P. M a c C l e l l a n d , p e r s o n a l communica t ion) , and thus g lycogen c o n c e n t r a t i o n s c o u l d have been below t h e i r mean l e v e l s for t h i s t ime of y e a r . I n t e r t i d a l p o s i t i o n a l s o e x e r t e d a h i g h l y s i g n i f i c a n t e f f e c t on t i s s u e g lycogen l e v e l s : o y s t e r s from the low i n t e r t i d a l zone c o n s i s t e n t l y d i s p l a y e d s i g n i f i c a n t l y l a r g e r 173 q u a n t i t i e s of g lycogen than a n i m a l s sampled from the more exposed h i g h i n t e r t i d a l a r e a . Two f a c t o r s are p r o b a b l y r e s p o n s i b l e for these o b s e r v a t i o n s . F i r s t , o y s t e r s i n the h i g h i n t e r t i d a l zone e x p e r i e n c e longer p e r i o d s of a e r i a l exposure compared to an imal s i n the low i n t e r t i d a l . T h i s would d i r e c t l y reduce t h e i r o p p o r t u n i t i e s to feed and hence a s s i m i l a t e g lycogen r e s e r v e s . Second, d u r i n g p e r i o d s of a e r i a l exposure o y s t e r s s w i t c h to a n a e r o b i c pathways of energy p r o d u c t i o n (Hochachka and Musta fa 1972; Zandee, Holwerda and de Zwaan 1980). Hypoxic c o n d i t i o n s would occur more f r e q u e n t l y and p e r s i s t f or longer d u r a t i o n s i n i n d i v i d u a l s s i t u a t e d h i g h e r i n the i n t e r t i d a l zone . S i n c e g lycogen i s the p r i m a r y f u e l f or a n a e r o b i c metabo l i sm (de Zwaan 1983), the i n c r e a s e d demands on these energy r e s e r v e s i n the h i g h i n t e r t i d a l area would f u r t h e r l i m i t the c a p a b i l i t y of o y s t e r s i n t h i s a r e a to s t o r e g l y c o g e n . The i n t e r a c t i o n observed between season and t i d a l p o s i t i o n c o u l d in p a r t be e x p l a i n e d by the a c c e n t u a t i o n of these e f f e c t s caused by the i n c r e a s e d a v a i l a b i l i t y of food i n the e a r l y summer c o u p l e d w i t h the more extreme t i d e s at t h i s t ime of y e a r . In Chapter 3, season , i n t e r t i d a l p o s i t i o n , and Pgm-2 genotype were shown to e x e r t h i g h l y s i g n i f i c a n t e f f e c t s on the s p e c i f i c a c t i v i t y of phosphoglucomutase measured i n both the mantle and adductor muscle t i s s u e s . E x a m i n a t i o n of the e f f e c t s of these same f a c t o r s on t i s s u e g lycogen c o n c e n t r a t i o n s r e p o r t e d in the p r e s e n t c h a p t e r showed a number of important d i f f e r e n c e s from the enzyme a c t i v i t y d a t a . F i r s t , g lycogen l e v e l s in o y s t e r s 174 sampled from the two i n t e r t i d a l h e i g h t s s h i f t e d i n a non-a d d i t i v e f a s h i o n between the summer and f a l l when the changes i n PGM a c t i v i t y observed between these seasons was s t r i c t l y a d d i t i v e . Second, PGM s p e c i f i c a c t i v i t y e x h i b i t e d a r e v e r s a l in net r a n k i n g between these t i d a l p o s i t i o n s over the f a l l and win ter tha t had no apparent e f f e c t on t i s s u e g lycogen c o n c e n t r a t i o n s . T h i r d , a n a l y s i s of the mantle g lycogen data produced h i g h l y s i g n i f i c a n t genotype -by-env ironment i n t e r a c t i o n s that were not observed i n the a n a l y s i s of the s p e c i f i c a c t i v i t y d a t a . These i n t e r a c t i o n s were not r epea ted i n the adductor muscle d e s p i t e the g r e a t s i m i l a r i t y of i t s enzyme a c t i v i t y da ta w i t h the m a n t l e . T o g e t h e r , these d i f f e r e n c e s suggest t h a t the observed v a r i a t i o n in PGM a c t i v i t y cannot be d i r e c t l y r e s p o n s i b l e f o r the s i g n i f i c a n t e f f e c t s of these f a c t o r s on t i s s u e g lycogen c o n c e n t r a t i o n s . T h i s c o n c l u s i o n i s not e n t i r e l y unexpec ted . Phosphoglucomutase i s not known to possess any r e g u l a t o r y p r o p e r t i e s and c a t a l y z e s a f r e e l y r e v e r s i b l e r e a c t i o n tha t i n marine b i v a l v e s , as i n o ther organ i sms , appears to operate near e q u i l i b r i u m ^n v i v o ( e . g . E b b e r i n k and de Zwaan 1980). The m e t a b o l i c r o l e a t t r i b u t e d to n e a r - e q u i l i b r i u m r e a c t i o n s l i k e PGM i s to t r a n s m i t the f l u x r a t e s through b i o c h e m i c a l pathways d i c t a t e d by enzymes w i t h c o m p a r a t i v e l y low f l u x c a p a c i t i e s that c a t a l y z e r e a c t i o n s d i s p l a c e d f a r from e q u i l i b r i u m (Newsholme and S t a r t 1973; A t k i n s o n 1977). For the s y n t h e s i s and d e g r a d a t i o n of g l y c o g e n , these p i v o t a l r e a c t i o n s are c a t a l y z e d by g lycogen 175 synthe tase and g lycogen p h o s p h o r y l a s e , r e s p e c t i v e l y . In mammalian t i s s u e s , the a c t i v i t i e s of these enzymes are c o o r d i n a t e l y r e g u l a t e d i n an a n t a g o n i s t i c f a s h i o n by r e v e r s i b l e p h o s p h o r y l a t i o n / d e p h o s p h o r y l a t i o n events c a t a l y z e d by s p e c i f i c k i n a s e s and phosphatases (rev iewed i n Madsen 1986; Roach 1986; Cohen 1986) i n response to n e u r o n a l s t i m u l a t i o n , hormones, and g e n e r a l p h y s i o l o g i c a l c o n d i t i o n through a number of d i f f e r e n t e f f e c t o r s (see rev iews by Cohen 1983; Hems and Whi t ton 1980; Stalmans 1976; Hers 1976). From what i s known about the r e g u l a t i o n of g lycogen metabo l i sm, g lycogen synthe tase and g lycogen p h o s p h o r y l a s e r e p r e s e n t the c l a s s i c " f l u x - g e n e r a t i n g " s teps of g lycogen s y n t h e s i s and g l y c o g e n o l y s i s , r e s p e c t i v e l y . A l t h o u g h the c o n t r o l of g lycogen metabol i sm in marine b i v a l v e s has not been as e x t e n s i v e l y s t u d i e d , g lycogen synthe tase and p h o s p h o r y l a s e undoubtedly serve s i m i l a r r e g u l a t o r y f u n c t i o n s . In the mantle t i s s u e of the b lue mussel M y t i l u s e d u l i s , the a c t i v e d e p h o s p h o r y l a t e d form of g lycogen synthe tase e x h i b i t s marked s easona l v a r i a t i o n (Cook, Gabbott and W h i t t l e 1979; Gabbott and W h i t t l e 1986a), and i t s s teady e l e v a t i o n i n a c t i v i t y throughout the s p r i n g and e a r l y summer d i r e c t l y p a r a l l e l s g lycogen d e p o s i t i o n . For f l u x p r o c e e d i n g i n the g l y c o g e n o l y t i c d i r e c t i o n , Zaba (1981) observed t h a t the a c t i v i t i e s of both g lycogen p h o s p h o r y l a s e and a m y l o g l u c o s i d a s e i n c r e a s e d over a p e r i o d of time when g lycogen was be ing r a p i d l y degraded f o r gametogenes i s . These o b s e r v a t i o n s , toge ther w i t h the n e a r - e q u i l i b r i u m s t a t u s of the phosphoglucomutase r e a c t i o n , 176 would p r e d i c t that the m a r g i n a l l y g r e a t e r enzyme a c t i v i t i e s of Pqm-2 h e t e r o z y g o t e s r e l a t i v e to homozygotes s h o u l d have l i t t l e , i f any , impact on t i s s u e g lycogen c o n c e n t r a t i o n s . S u r p r i s i n g l y , the r e s u l t s of the p r e s e n t s tudy c o n t r a d i c t t h i s p r e d i c t i o n for the mantle but not the a d d u c t o r muscle t i s s u e . C l a s s i f i c a t i o n of Pgm-2 genotypes as e i t h e r homozygotes or h e t e r o z y g o t e s e x p l a i n e d a s i g n i f i c a n t amount of the observed v a r i a t i o n i n mantle g lycogen l e v e l s i n seven of the e i g h t comparisons shown i n T a b l e X I I . In c o n t r a s t , the a s s o c i a t i o n of these s p e c i f i c a c t i v i t y d i f f e r e n c e s and g lycogen c o n c e n t r a t i o n s in the adduc tor muscle was weak, a c c o u n t i n g f o r a s i g n i f i c a n t r e s u l t i n on ly one of the same e i g h t comparisons (Tab le X I I I ) . I t seems r e a s o n a b l e to assume t h a t the o v e r a l l c o n t r o l of the annual c y c l e of s y n t h e s i s and d e g r a d a t i o n i s mediated through the d i f f e r e n t i a l r e g u l a t i o n of the a c t i v i t i e s of g lycogen synthe tase and the combined p h o s p h o r y l a s e / a m y l o g l u c o s i d a s e system. D e s p i t e the c o n t r o l e x e r t e d by these r e g u l a t o r y enzymes, however, PGM s p e c i f i c a c t i v i t y v a r i a t i o n s t i l l appears to have i n f l u e n c e d g lycogen metabo l i sm. T h i s may be a consequence of the d i f f e r e n t i a l a b i l i t i e s of Pgm-2 genotypes to respond to the r a t e s of f l u x d i c t a t e d by these r e g u l a t o r y enzymes. The apparent impact of these a c t i v i t y d i f f e r e n c e s was complex, e x h i b i t i n g r e v e r s a l s between homozygotes and h e t e r o z y g o t e s between seasons ( i . e . f a l l and w i n t e r ) and between i n t e r t i d a l p o s i t i o n s w i t h i n a s easona l season (summer). S i n c e g lycogen was measured at a l i m i t e d number of s tages of i t s annual c y c l e , i t i s not p o s s i b l e 177 to determine from the o u t s e t i f the v a r i a t i o n i n PGM a c t i v i t y d i f f e r e n t i a l l y a f f e c t s g lycogen s y n t h e s i s a n d / o r g l y c o g e n o l y s i s , or r e p r e s e n t s a f o r t u i t o u s a s s o c i a t i o n through a l i n k a g e e f f e c t . Of these a l t e r n a t i v e s , a d i r e c t i n f l u e n c e of t h i s v a r i a t i o n i n enzyme a c t i v i t y on the r a t e of g lycogen s y n t h e s i s i s expected through a p a r t i t i o n i n g e f f e c t a t the g l u c o s e - 6 - p h o s p h a t e branch p o i n t . Net f l u x through the PGM r e a c t i o n i n the d i r e c t i o n of g lycogen f o r m a t i o n i s de termined by the oppos ing r a t e s of the forward and r e v e r s e r e a c t i o n d i r e c t i o n s by the f o l l o w i n g e q u a t i o n (Savageau 1976): Vnet = VmaxCf) /Km(f) [G-6 -P] - VmaxCr) /Km(r) [G-1-P] . . ( 4 ) 1 + [ G - 6 - P ] + [G-1-P] Km(f) Km(r) where Vmax(f) and V m a x ( r ) , and Km(f) and Km(r) are the maximum v e l o c i t i e s and M i c h a e l i s c o n s t a n t s for the forward and r e v e r s e r e a c t i o n d i r e c t i o n s , and [ G - 1 - P ] and [G-6-P] are the i n t r a c e l l u l a r c o n c e n t r a t i o n s of g l u c o s e - 1 - p h o s p h a t e and g l u c o s e -6 -phosphate , r e s p e c t i v e l y . E s t i m a t e s of these k i n e t i c parameters i n the mantle t i s s u e f o r the four homozygotes and the h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e are p r e s e n t e d i n T a b l e X V . Maximum v e l o c i t i e s f or the r e v e r s e r e a c t i o n r e p r e s e n t the mean enzyme a c t i v i t i e s of these g e n o t y p i c c l a s s e s from Chapter T a b l e XV. K i n e t i c parameters i n the mantle t i s s u e s of the four homozygotes and the three h e t e r o z y g o t e s f o r the Pgm-2-100 i 179 Parameter H e t e r o z y g o t e s Homozygotes V m a x ( r ) 1 3.02 2.47 K m ( r ) 2 21 .2 21.5 V m a x ( f ) 1 1.01 0.823 K m ( f ) 3 121.9 123.2 Vmax(r ) /Km(r) 0.142 0.115 Vmax(f ) /Km(f ) 0.00829 0.00668 1 u n i t s / g wet t i s s u e 2 Mmoles glucose- 1-phosphate 3 Mmoles g l u c o s e - 6 - p h o s p h a t e 180 3. S i m i l a r l y , M i c h a e l i s c o n s t a n t s f o r the r e v e r s e r e a c t i o n r e p r e s e n t the means expres sed by these groups over the temperature range of 5 to 3 0 ° p r e s e n t e d i n Chapter 2. K i n e t i c parameters f o r the forward r e a c t i o n d i r e c t i o n were e s t i m a t e d from the Haldane e q u a t i o n , assuming t h a t Vmax(r) exceeds Vmax(f) (as w r i t t e n above) by a f a c t o r of t h r e e (Ray and R o s c e l l i 1964b; Chapter 2 ) . H e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e are thus expected to possess Vmax/Km r a t i o s f o r both r e a c t i o n d i r e c t i o n s tha t are a p p r o x i m a t e l y 24% l a r g e r than the four homozygotes . S i n c e a t e q u i l i b r i u m Vnet i s z e r o , a f l u x towards g lycogen must i n v o l v e a d i s p l a c e m e n t of one or both of the two s u b s t r a t e s from t h e i r e q u i l i b r i u m c o n c e n t r a t i o n s (Newsholme and C r a b t r e e 1976). I t might be expec ted t h a t the l a r g e r forward r e a c t i o n r a t e in h e t e r o z y g o t e s would be e x a c t l y c o u n t e r b a l a n c e d by the s i m u l t a n e o u s l y g r e a t e r r e v e r s e r a t e under f l u x - g e n e r a t i n g c o n d i t i o n s , thus n e g a t i n g any c a t a l y t i c advantage . However, numerica l , s o l u t i o n of e q u a t i o n 4 w i t h v a r i o u s c o n c e n t r a t i o n s of G - 1 - P and G - 6 - P p r o d u c i n g a p o s i t i v e Vnet shows tha t the f l u x advantage of h e t e r o z y g o t e s p e r s i s t s under these c o n d i t i o n s i r r e s p e c t i v e of the magnitude of V n e t . For example, i f [G-6 -P] and [G-1 -P] are 300 and 10 MM, r e s p e c t i v e l y , the c a l c u l a t e d Vnet for h e t e r o z y g o t e s (0.271 umoles /min /ml ) i s 24% l a r g e r than e s t i m a t e d f o r homozygotes (0.219 M m o l e s / m i n / m l ) . An i d e n t i c a l r e l a t i o n s h i p between these g e n o t y p i c groups h o l d s f o r f l u x p r o c e e d i n g i n the g l y c o g e n o l y t i c d i r e c t i o n . S u r p r i s i n g l y , these r e s u l t s are u n a f f e c t e d by s u b s t r a t e c o n c e n t r a t i o n s , a l t h o u g h the 181 r e a l i z e d Vnet i s d i r e c t l y de termined by the magnitude of t h e i r d i s p l a c e m e n t from e q u i l i b r i u m . The f l u x advantage of Pgm-2 h e t e r o z y g o t e s might not be expres sed ijn v i v o i f the enzyme c a t a l y z e d a r e a c t i o n o c c u p y i n g an i n t e r m e d i a t e p o s i t i o n i n the pathway. However, PGM i s r e s p o n s i b l e for c a t a l y z i n g the f i r s t s t ep i n the s y n t h e s i s of g lycogen from the g l u c o s e - 6 - p h o s p h a t e branch p o i n t . The s y n t h e s i s of g lycogen o p e r a t e s by a " p u l l " mechanism. A c t i v a t i o n of g lycogen synthe tase i n mammalian l i v e r by the i n j e c t i o n of g l u c o s e has been observed to cause s i g n i f i c a n t r e d u c t i o n s i n the c o n c e n t r a t i o n s of the pathway i n t e r m e d i a t e s g l u c o s e - 6 - p h o s p h a t e and UDP-g lucose (De Wulf and Hers 1967; Hue and Hers 1974). In a d d i t i o n to l o w e r i n g the c o n c e n t r a t i o n s of pathway i n t e r m e d i a t e s , t h i s mechanism must a l s o c r e a t e d i s p l a c e m e n t s from e q u i l i b r i u m at both the PGM and UDP-glucose p y r o p h o s p h o r y l a s e r e a c t i o n s t e p s . When d r i v e n by exogenous g l u c o s e , t h i s p u l l mechanism w i l l c r e a t e c o m p e t i t i o n f o r the a v a i l a b l e G - 6 - P between PGM and o ther enzymes t h a t u t i l i z e t h i s m e t a b o l i t e . The predominant c o m p e t i t o r at the G - 6 - P branch p o i n t (by net a c t i v i t y c r i t e r i a ) i s phosphoglucose i somerase (PGI) which c a t a l y z e s the f o r m a t i o n of f r u c t o s e - 6 - p h o s p h a t e from G - 6 - P f o r e n t r y i n t o g l y c o l y s i s . S i n c e b a s a l metabol i sm must be m a i n t a i n e d d u r i n g p e r i o d s of g lycogen f o r m a t i o n , some p a r t i t i o n i n g of G - 6 - P between g l y c o l y s i s and g lycogen s y n t h e s i s i s u n a v o i d a b l e . An e s t imate of the extent of t h i s p a r t i t i o n i n g i s p r o v i d e d by the f l u x s t u d i e s on mantle t i s s u e s l i c e s from M. 182 e d u l i s by Zaba and D a v i e s (1980, 1981) and Zaba, Gabbott and D a v i e s (1981) . These exper iments found tha t 40-60% of the [14C] -g l u c o s e l a b e l was i n c o r p o r a t e d i n t o g l y c o g e n ; the remainder appeared i n o r g a n i c and amino a c i d s . The p a r t i t i o n i n g of f l u x a t m e t a b o l i c branch p o i n t s has been s t u d i e d t h e o r e t i c a l l y by Kacser (1983) and e m p i r i c a l l y by L a P o r t e , Walsh and Kosh land (1984) . When two enzymes compete for a common s u b s t r a t e under s t e a d y - s t a t e c o n d i t i o n s , an i n c r e a s e d r a t e of f l u x through one w i l l cause an i d e n t i c a l d e c l i n e i n f l u x through the o t h e r . T h i s a n t a g o n i s t i c r e l a t i o n s h i p between compet ing enzymes can produce n e g a t i v e c o n t r o l c o e f f i c i e n t s , i n the language of c o n t r o l t h e o r y (Kacser 1983), or u l t r a - or h y p e r - s e n s i t i v i t y to r e g u l a t i o n depending on the k i n e t i c parameters of the s p e c i f i c enzymes i n v o l v e d ( L a P o r t e , Walsh and Kosh land 1984). The d i v i s i o n of f l u x a t the g l u c o s e - 6 - p h o s p h a t e branch p o i n t may be q u a n t i f i e d by the p a r t i t i o n i n g c o e f f i c i e n t , PC, d e f i n e d by L a P o r t e , Walsh and Kosh land (1984): n -1 PC = Vmax(2) (Km(1) + [ G - 6 - P ] ) + 1 V m a x d ) (Km(2) + [ G - 6 - P ] ) (5) where PC q u a n t i f i e s the p r o p o r t i o n of f l u x d i r e c t e d towards g l y c o g e n , Vmax(l ) and Km(1) r e p r e s e n t the s t a n d a r d k i n e t i c parameters f o r the forward r e a c t i o n (to g lycogen) of PGM, Vmax(2) and Km(2) are the same f o r PGI , and [G-6-P] i s the i n t r a c e l l u l a r c o n c e n t r a t i o n of g l u c o s e - 6 - p h o s p h a t e . I n s p e c t i o n 183 of e q u a t i o n 5 shows t h a t the p a r t i t i o n i n g c o e f f i c i e n t i s u n a f f e c t e d by the c o n c e n t r a t i o n of g l u c o s e - 6 - p h o s p h a t e . F u r t h e r m o r e , s i n c e the Km for G - 6 - P of PGI from s e v e r a l b i v a l v e s p e c i e s i s v e r y s i m i l a r to t h a t e s t i m a t e d for C . q i g a s PGM i n Chapter 2 ( M a r t i n 1979; H a l l 1985), e q u a t i o n 5 may be approx imated a s : PC = PGI S p e c i f i c A c t i v i t y + 1 PGM S p e c i f i c A c t i v i t y . . . (6) T h e r e f o r e , the d i v i s i o n of f l u x at the g l u c o s e - 6 - p h o s p h a t e branch p o i n t when both g l y c o l y s i s and g lycogen s y n t h e s i s a r e o c c u r r i n g i s l a r g e l y de termined by the PGI/PGM s p e c i f i c a c t i v i t y r a t i o . The expected f l u x towards g lycogen and the r e s u l t i n g f l u x d i f f e r e n c e between Pgm-2 h e t e r o z y g o t e s and homozygotes as a f u n c t i o n of the PGI/PGM a c t i v i t y r a t i o i s i l l u s t r a t e d i n F i g u r e 15. At e q u a l a c t i v i t i e s of these compet ing enzymes, 50% of the f l u x i s d i r e c t e d towards g l y c o g e n , however, the f l u x f o r h e t e r o z y g o t e s i s o n l y 11% l a r g e r than homozygotes d e s p i t e t h e i r 24% l a r g e r Vmax/Km r a t i o s . As the PGI/PGM a c t i v i t y r a t i o i n c r e a s e s , the p r o p o r t i o n of f l u x p r o c e e d i n g towards g lycogen d e c l i n e s and the f l u x advantage of h e t e r o z y g o t e s a s y m p t o t i c a l l y approaches t h e i r net c a t a l y t i c advantage . In f r e s h l y ground t i s s u e s from 12 o y s t e r s , the mean r a t i o of the s p e c i f i c a c t i v i t i e s of PGI to PGM was 7.4 and 8.4 i n the mantle and 184 F i g u r e 15. P r e d i c t e d f l u x advantage of Pgm-2 h e t e r o z y g o t e s and p e r c e n t f l u x to g l y c o g e n as a f u n c t i o n of the PGI/PGM a c t i v i t y r a t i o . Open c i r c l e s = p e r c e n t f l u x excess of h e t e r o z y g o t e s over homozygotes; c l o s e d c i r c l e s = p e r c e n t f l u x to g l y c o g e n . Heterozygote/Homozygote Flux, % Percent Flux to Glycogen 186 adduc tor musc l e , r e s p e c t i v e l y . These r a t i o s are s i m i l a r to those r e p o r t e d i n the adduc tor muscle t i s s u e s of the m u s s e l , M y t i l u s  e d u l i s (Ebber ink and de Zwaan 1980) and the bay s c a l l o p , Argopec ten i r r a d i a n s c o n c e n t r i c u s ( C h i h and E l l i n g t o n 1986). T h e r e f o r e , Pgm-2 h e t e r o z y g o t e s a r e expected to e x h i b i t l a r g e r f l u x e s towards g lycogen than homozygotes, s i m i l a r i n magnitude to t h e i r enzyme a c t i v i t y e x c e s s , because of t h i s p a r t i t i o n i n g e f f e c t a t the g l u c o s e - 6 - p h o s p h a t e b r a n c h p o i n t . These p a t t e r n s are expected to h o l d i r r e s p e c t i v e of the o v e r a l l r a t e of g lycogen s y n t h e s i s d i c t a t e d by g lycogen s y n t h e t a s e . When g lycogen i s s y n t h e s i z e d from g l u c o n e o g e n i c p r e c u r s o r s the f l u x advantage of h e t e r o z y g o t e s c o u l d a l s o be m a i n t a i n e d . The o v e r a l l r a t e of s y n t h e s i s would now be de termined by the a v a i l a b i l i t y of the s p e c i f i c p r e c u r s o r i n v o l v e d , the c r u c i a l bypass r e a c t i o n s c a t a l y z e d by p y r u v a t e d e c a r b o x y l a s e and f r u c t o s e b i s p h o s p h a t a s e i n a d d i t i o n to g lycogen s y n t h e t a s e . However, a d i s p l a c e m e n t from e q u i l i b r i u m i s s t i l l r e q u i r e d a t the PGM s tep and under these c o n d i t i o n s the net f l u x in Pqm-2 h e t e r o z y g o t e s i s s t i l l expected to exceed homozygotes . The d i f f e r e n c e s i n mantle g lycogen l e v e l s observed between homozygotes and h e t e r o z y g o t e s c o u l d i n p a r t be e x p l a i n e d by these d i f f e r e n t i a l r a t e s of s y n t h e s i s . The impact of t h i s PGM a c t i v i t y v a r i a t i o n on g l y c o g e n o l y s i s i s l e s s c e r t a i n . Three f a c t o r s s u g g e s t i n g a l i m i t e d e f f e c t of PGM on t h i s c a t a b o l i c p r o c e s s are 1) the r e a c t i o n mechanism of 187 g lycogen p h o s p h o r y l a s e , 2) r e g u l a t o r y a s p e c t s of g lycogen m o b i l i z a t i o n , and 3) the e x i s t e n c e of an a l t e r n a t i v e d e g r a d a t i v e pathway. The r e a c t i o n c a t a l y z e d by g lycogen p h o s p h o r y l a s e d i f f e r s from g lycogen syn the tase i n be ing p o t e n t i a l l y r e v e r s i b l e ( F l e t t e r i c k and Madsen 1980), ye t i s b e l i e v e d to proceed almost e x c l u s i v e l y i n the forward d i r e c t i o n ( i . e . g lycogen breakdown) because of the h i g h molar r a t i o of i n o r g a n i c phosphate to g l u c o s e - 1 - p h o s p h a t e (Stalmans 1976). T h i s h i g h molar : r a t i o and the low r e l a t i v e a c t i v i t y of p h o s p h o r y l a s e compared to PGM would ensure t h a t the g l u c o s e - 1 - p h o s p h a t e m o i e t i e s l i b e r a t e d from g lycogen would be r a p i d l y c o n v e r t e d by PGM i n t o g l u c o s e - 6 -phosphate and fed i n t o g l y c o l y s i s . The l a r g e c a t a l y t i c excess a t the PGM r e a c t i o n s t r o n g l y f a v o r s the u n i - d i r e c t i o n a l f low of g l u c o s y l u n i t s from g lycogen and suggests t h a t the minor v a r i a t i o n s i n a c t i v i t y presen t between genotypes would not a f f e c t the d e g r a d a t i v e r a t e . The e l e g a n t r e g u l a t o r y cascade c o n t r o l l i n g the a c t i v a t i o n of g lycogen p h o s p h o r y l a s e a i s w e l l known, and d i f f e r e n t c e l l types e x h i b i t m o d i f i c a t i o n s of the cascade a r c h i t e c t u r e i n accordance w i t h t h e i r requ irements f o r the r a p i d m o b i l i z a t i o n of g lycogen (Cohen 1978). S i t u a t e d i n an i n t e r m e d i a t e p o s i t i o n between p h o s p h o r y l a s e and a d d i t i o n a l r e g u l a t o r y r e a c t i o n s , PGM i s expec ted to have a l i m i t e d impact on g l y c o g e n o l y t i c r a t e . S e v e r a l exper iments w i t h marine b i v a l v e s have borne out t h i s p r e d i c t i o n . For example, E b b e r i n k and de Zwaan (1980) moni tored mass a c t i o n r a t i o s (MAR) for 14 g l y c o l y t i c enzymes i n the 188 p o s t e r i o r adductor muscle of M. e d u l i s over a 24 h p e r i o d of a n a e r o b i o s i s induced by a e r i a l exposure . They i d e n t i f i e d p h o s p h o f r u c t o k i n a s e as the major c o n t r o l p o i n t d u r i n g the f i r s t few hours of v a l v e c l o s u r e , but noted a s h i f t i n c o n t r o l to p y r u v a t e k inase a f t e r 8 h of p r o l o n g e d a n o x i a . The Keq/MAR r a t i o f o r the PGM r e a c t i o n remained c l o s e to u n i t y throughout the experiment d e s p i t e l a r g e i n c r e a s e s i n the c o n c e n t r a t i o n s of both s u b s t r a t e s . S i n c e the r e g u l a t i o n of g l y c o l y s i s i n o y s t e r s i s p r o b a b l y s i m i l a r to t h a t i n m u s s e l s , the d i f f e r e n t i a l a b i l i t i e s of Pgm-2 genotypes to c a t a l y z e the i n t e r c o n v e r s i o n of G - 1 - P and G - 6 - P would not be expected to a f f e c t g l y c o g e n o l y t i c r a t e s under s i m i l a r anox ic c o n d i t i o n s . In a d d i t i o n to the p h o s p h o r o l y t i c pathway, g lycogen may a l s o be h y d r o l y z e d d i r e c t l y by a m y l o g l u c o s i d a s e to produce f r e e g l u c o s e . T h i s i n t u r n c o u l d be e x p o r t e d from the c e l l or c o n v e r t e d i n t o g l u c o s e - 6 - p h o s p h a t e by h e x o k i n a s e . The e x i s t e n c e of t h i s second d e g r a d a t i v e route i n marine i n v e r t e b r a t e s has been r e c o g n i z e d f o r many y e a r s ( e . g . Alemany and R o s e l l - P e r e z 1973; H i n o , Tazawa and Yasumasu 1978; Zaba 1981). However, the r e l a t i v e c o n t r i b u t i o n s of the p h o s p h o r o l y t i c and h y d r o l y t i c pathways of g lycogen m o b i l i z a t i o n i n marine b i v a l v e s have yet to be d e t e r m i n e d . Knowledge of the p r o p o r t i o n of g l y c o s y l u n i t s c h a n n e l e d through both d e g r a d a t i v e route s i s e s s e n t i a l f o r a s s e s s i n g the p o t e n t i a l e f f e c t of PGM a c t i v i t y v a r i a t i o n on g l y c o g e n o l y s i s . The o b s e r v a t i o n of membrane-enclosed g lycogen p a r t i c l e s in the mantle v e s i c u l a r c e l l s of M. e d u l i s by Bayne et 189 a l . (1982) would suggest an important r o l e for the h y d r o l y t i c mechanism. I f the p h o s p h o r o l y t i c pathway i s used l e s s f r e q u e n t l y , PGM must e x e r t a s i g n i f i c a n t e f f e c t on g l y c o g e n o l y s i s tha t i s too g r e a t to be r e c o n c i l e d w i t h the known f u n c t i o n and r e g u l a t i o n of g lycogen p h o s p h o r y l a s e . A l t h o u g h phosphoglucomutase i s not b e l i e v e d to i n f l u e n c e g l y c o g e n o l y s i s under s t a n d a r d c o n d i t i o n s , s i t u a t i o n s have been d e s c r i b e d where i t c o u l d e x e r t some degree of c o n t r o l . For example, C h i h and E l l i n g t o n (1986) noted tha t a f t e r 80 c o n t r a c t i o n s of the p o s t e r i o r adductor muscle of the bay s c a l l o p , Argopec ten i r r a d i a n s c o n c e n t r i c u s the Keq/MAR r a t i o of g lycogen p h o s p h o r y l a s e f e l l from 175 to 27 w h i l e tha t f o r PGM i n c r e a s e d from 3.5 to 25. Under these c o n d i t i o n s PGM c o u l d e x e r t a s i g n i f i c a n t e f f e c t on the r a t e of g lycogen d e g r a d a t i o n . C i r c u m s t a n t i a l ev idence s u g g e s t i n g an e f f e c t of PGM on g lycogen m o b i l i z a t i o n has a l s o been r e p o r t e d i n the f i e l d mouse, Apodemus  s y l v a t i c u s ( L e i g h Brown 1977) and the rainbow t r o u t , Salmo  g a i r d n e r i ( A l l e n d o r f , L e a r y and Knudsen 1983). The ex tremely h i g h c o n c e n t r a t i o n s of g lycogen s t o r e d i n o y s t e r t i s s u e s suggests a mechanism by which PGM c o u l d a f f e c t g l y c o g e n o l y s i s . In the mantle t i s s u e s of marine b i v a l v e s , g lycogen i s s t o r e d i n v e s i c u l a r c e l l s t h a t l i e i n c l o s e p r o x i m i t y to the d i g e s t i v e g l a n d and d e v e l o p i n g gonad (Gabbott 1983). These c e l l s c o n t a i n l a r g e q u a n t i t i e s of g lycogen c o n c e n t r a t e d i n the c y t o p l a s m as s m a l l g r a n u l e s or l a r g e /3-190 p a r t i c l e s (Eb le 1969; Bayne et a l . 1982). U n f o r t u n a t e l y , the d e g r a d a t i o n of g lycogen i n b i v a l v e v e s i c u l a r c e l l s has not been s t u d i e d . I t i s c o n c e i v a b l e t h a t the h i g h c o n c e n t r a t i o n s of g lycogen p h o s p h o r y l a s e a s s o c i a t e d w i t h these B - p a r t i c l e s (Eb le 1969) c o u l d r e l e a s e l a r g e amounts of G - 1 - P tha t r e s u l t i n s u b s t a n t i a l d i s p l a c e m e n t s from e q u i l i b r i u m at the PGM r e a c t i o n . I f these d i s p l a c e m e n t s o c c u r , d i f f e r e n c e s in enzyme a c t i v i t i e s between Pgm-2 homozygotes and h e t e r o z y g o t e s have the p o t e n t i a l to i n f l u e n c e r a t e s of g lycogen breakdown. From these c o n s i d e r a t i o n s , the genotype -by-env ironment i n t e r a c t i o n s observed f o r the mantle g lycogen da ta c o u l d be caused by the d i f f e r e n t i a l c a p a b i l i t i e s of Pgm-2 genotypes to respond to f l u x r a t e s d i c t a t e d by g lycogen s y n t h e t a s e , and p o s s i b l y g lycogen p h o s p h o r y l a s e . In C r a s s o s t r e a q i g a s , mantle g lycogen i s s y n t h e s i z e d r a p i d l y in the f a l l a f t e r the c o m p l e t i o n of spawning and the neces sary r e o r g a n i z a t i o n of t i s s u e (Quayle 1969). In t h i s season , a s t r o n g a s s o c i a t i o n was observed between PGM a c t i v i t y and mantle g lycogen (Table X I V ) . H e t e r o z y g o t e s p o s s e s s i n g the Pqm-2-100 a l l e l e had h i g h e r l e v e l s of mantle g l y c o g e n than homozygotes or h e t e r o z y g o t e s for the l e s s f requent Pgm-2-92, 96, or 104 a l l e l e s . These r e s u l t s are e x a c t l y as expec ted i f t h i s enzyme a c t i v i t y v a r i a t i o n d i f f e r e n t i a l l y a f f e c t s the r a t e s of g lycogen s y n t h e s i s between these g e n o t y p i c c l a s s e s . When the summer sample was t a k e n , c o n s i d e r a b l e gonadal 191 development was observed a n d , t h e r e f o r e , g lycogen l e v e l s were p r o b a b l y on the d e c l i n e because of the s t r o n g r e l a t i o n s h i p between the d e p l e t i o n of these r e s e r v e s and gametogenesis (Gabbott 1975; Bayne 1976). In t h i s season Pgm-2 homozygotes had h i g h e r c o n c e n t r a t i o n s of mantle g lycogen than h e t e r o z y g o t e s i n the low i n t e r t i d a l a r e a , but lower l e v e l s i n the h i g h i n t e r t i d a l r e g i o n . A c l e a r e x p l a n a t i o n f o r these r e s u l t s i s not p o s s i b l e . These p a t t e r n s c o u l d have a r i s e n from d i f f e r e n c e s i n g lycogen l e v e l s t h a t e x i s t e d b e f o r e the onset of g l y c o g e n o l y s i s , d i f f e r e n t i a l r a t e s of d e g r a d a t i o n , or perhaps to the asynchronous . t i m i n g of gonadal development i n d i f f e r e n t genotypes . N e i t h e r of these p o s s i b i l i t i e s are m u t u a l l y e x c l u s i v e and f u r t h e r s t u d i e s on the metabol i sm of g lycogen i n d i f f e r e n t Pgm-2 genotypes are r e q u i r e d to determine w h i c h , i f any , i s c o r r e c t . In the w i n t e r , Pgm-2 homozygotes had h i g h e r c o n c e n t r a t i o n s of mantle g lycogen than h e t e r o z y g o t e s a t both i n t e r t i d a l l o c a t i o n s . The same r e l a t i o n s h i p between genotypes was e v i d e n t i n the adduc tor muscle t i s s u e , but the d i f f e r e n c e s were s i g n i f i c a n t o n l y in the h i g h i n t e r t i d a l a r e a . As s t a t e d p r e v i o u s l y , t h i s sample was taken when n a t u r a l food c o n d i t i o n s were ex tremely p o o r , a f a c t o r t h a t might e x p l a i n the lower than expected l e v e l s of g lycogen observed at t h i s t i m e . I f the p o p u l a t i o n at t h i s time was e x p e r i e n c i n g n e g a t i v e "scope for growth" ( c f . Bayne and Newel l 1983), g l y c o g e n r e s e r v e s might have been u t i l i z e d to meet b a s a l m e t a b o l i c r e q u i r e m e n t s , g i v e n 192 the dormant s t a t e of the r e p r o d u c t i v e c y c l e . One e x p l a n a t i o n f o r these r e s u l t s i s t h a t Pgm-2 h e t e r o z y g o t e s were d e p l e t i n g these energy r e s e r v e s a t a f a s t e r r a t e than homozygotes. Rodhouse and Gaf fney (1984) s t u d i e d the r e l a t i o n s h i p between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and s t a r v a t i o n - i n d u c e d weight l o s s i n the American o y s t e r , C . v i r g i n i c a , over a 42 day p e r i o d . These a u t h o r s found t h a t the r a t e of weight l o s s was lower in more he terozygous i n d i v i d u a l s , thus s u g g e s t i n g that h e t e r o z y g o t e s are more v i a b l e over p e r i o d s of n u t r i t i v e s t r e s s than homozygotes. I f these c o n c l u s i o n s a r e a p p l i c a b l e to the P a c i f i c o y s t e r , h e t e r o z y g o t e s would have been expec ted to e x h i b i t h i g h e r , r a t h e r than l ower , l e v e l s of mantle g lycogen compared to homozygotes under these adverse e n v i r o n m e n t a l c o n d i t i o n s . Comparison of these r e s u l t s are q u e s t i o n a b l e however, s i n c e Rodhouse and Gaf fney (1984) were a b l e to c o n t r o l food a v a i l a b i l i t y , water t e m p e r a t u r e , and the s t a r v a t i o n p e r i o d . None of these f a c t o r s were known or m a n i p u l a t e d i n t h i s s t u d y . F u r t h e r m o r e , Rodhouse and Gaf fney (1984) d i d not f i n d a s i g n i f i c a n t r e l a t i o n s h i p between c a r b o h y d r a t e l e v e l s and h e t e r o z y g o s i t y e i t h e r be fore or a f t e r the s t a r v a t i o n p e r i o d . I t s h o u l d be p o i n t e d o u t , however, t h a t any e f f e c t c o n t r i b u t e d by the Pqm l o c u s on g lycogen metabol i sm i n t h e i r s tudy may have been obscured by the random ass ignment of Pgm homozygotes and h e t e r o z y g o t e s a c r o s s the d i f f e r e n t h e t e r o z y g o s i t y c l a s s e s examined. Marked d i f f e r e n c e s were observed between the e f f e c t s of Pgm 193 -2 genotype i n the a n a l y s e s of the mantle and adduc tor muscle g lycogen d a t a . These r e s u l t s were s u r p r i s i n g i n l i g h t of the g r e a t s i m i l a r i t y shown by these t i s s u e s i n t h e i r PGM s p e c i f i c a c t i v i t y measurements and the s e a s o n a l p a t t e r n s of t h e i r g lycogen l e v e l s . A p p a r e n t l y , enzyme a c t i v i t y v a r i a t i o n at the Pgm-2 l o c u s e x e r t e d an e f f e c t on g lycogen metabol i sm i n the m a n t l e , but not the adductor musc le . In mammals, the r e g u l a t i o n of g lycogen metabol i sm d i f f e r s between the l i v e r and muscle i n s e v e r a l important a s p e c t s . In c o n t r a s t to the l i v e r , the muscle form of g lycogen syn the tase phosphatase i s i n h i b i t e d d i r e c t l y by g lycogen i t s e l f (Hers , De Wulf and Stalmans 1970). T h i s i n h i b i t i o n p r e v e n t s e x c e s s i v e d e p o s i t i o n of g lycogen i n muscle f i b r e s t h a t c o u l d i n t e r f e r e w i t h c o n t r a c t i l e f u n c t i o n . E x t r a p o l a t i n g these d i f f e r e n c e s to m o l l u s c a n systems i s d i f f i c u l t because the homologous r e g u l a t o r y enzymes have not been c h a r a c t e r i z e d . However, i f a s i m i l a r mechanism e x i s t s i n o y s t e r adductor musc l e , d i f f e r e n c e s between Pqm-2 genotypes i n t h e i r r a t e s of g lycogen s y n t h e s i s may not be e x p r e s s e d . D e p o s i t i o n of g lycogen i n the adduc tor muscle may occur at d i f f e r e n t r a t e s , but no net d i f f e r e n c e s between Pgm-2 genotypes would be expec ted i f f u r t h e r s y n t h e s i s i s p r e v e n t e d beyond t h i s l i m i t . Fewer c o n s t r a i n t s may a p p l y to the a b s o l u t e q u a n t i t y of g lycogen s t o r e d i n the mantle t i s s u e . Upper l i m i t s o b v i o u s l y e x i s t f or the s torage c a p a c i t i e s of i n d i v i d u a l v e s i c u l a r c e l l s , but t h e i r mantle p r o p o r t i o n s are known to v a r y s e a s o n a l l y and 1 94 between d i f f e r e n t p o p u l a t i o n s at the same t ime of year (Lowe, Moore and Bayne 1982). T h i s may a l l o w f o r the l e s s r e s t r i c t i v e a c c u m u l a t i o n of g lycogen i n the mantle i n comparison wi th the adduc tor muscle t i s s u e . Under these c o n d i t i o n s , the e f f e c t of enzyme a c t i v i t y v a r i a t i o n at the Pqm-2 l o c u s on m e t a b o l i c f l u x c o u l d be m a n i f e s t e d , even though the o v e r a l l r a t e s would s t i l l be de termined by r e g u l a t o r y enzymes l i k e g lycogen s y n t h e t a s e . I t shou ld be s t r e s s e d tha t these arguments r e l y h e a v i l y on the assumption t h a t the e f f e c t s of the Pgm-2 l o c u s on g lycogen metabol i sm were randomized w i t h r e s p e c t to the remainder of the g e n e t i c background. Non-random a s s o c i a t i o n s of t h i s PGM a c t i v i t y v a r i a t i o n w i t h a d j a c e n t enzymic r e a c t i o n s of g lycogen metabol i sm c o u l d l e a d to erroneous c o n c l u s i o n s c o n c e r n i n g the e f f e c t s of the Pqm-2 l o c u s . The next c h a p t e r t e s t s the v a l i d i t y of t h i s assumpt ion by examining the a c t i v i t y r e l a t i o n s h i p s between the k i n e t i c a l l y - l i n k e d r e a c t i o n s of the g lycogen s y n t h e s i s pathway. 195 CHAPTER 5 ACTIVITY STRUCTURE OP THE GLYCOGEN SYNTHESIS PATHWAY INTRODUCTION A t t r i b u t i n g a p h y s i o l o g i c a l e f f e c t to a l l o z y m i c v a r i a t i o n at a s i n g l e enzyme l o c u s i n a complex mul t i - enzyme pathway encounters d i f f i c u l t i e s beyond those concerned w i t h i t s d e t e c t i o n ( e . g . D y k h u i z e n , Dean and H a r t l 1987). In s t u d y i n g n a t u r a l p o p u l a t i o n s , a major concern i n v o l v e s e f f e c t s of the g e n e t i c background . Mar ine b i v a l v e s such as C . g i g a s possess s u b s t a n t i a l l e v e l s of p r o t e i n h e t e r o z y g o s i t y ( B u r o k e r , Hershberger and Chew 1979a; O z a k i and F u j i o 1985), and thus the c e n t r a l e n e r g y - p r o d u c i n g pathways are l i k e l y to be s e g r e g a t i n g for g e n e t i c v a r i a t i o n at enzyme l o c i i n a d d i t i o n to the one under s t u d y . A l t h o u g h yet u n q u a n t i f i e d i n marine b i v a l v e s , r e g u l a t o r y gene v a r i a t i o n ( c f . Pa igen 1979) might a l s o be present t h a t has the p o t e n t i a l to e x e r t p l e i o t r o p i c e f f e c t s on a number of d i f f e r e n t pathway enzymes, as suggested by s t u d i e s on D. me lanogas ter ( L a u r i e - A h l b e r g et a l . 1982; W i l t o n et a l . 1982). M o l e c u l a r s t u d i e s of DNA sequence v a r i a t i o n have a l s o r e v e a l e d t h a t c o n s i d e r a b l e l i n k a g e d i s e q u i l i b r i u m may e x i s t between s t r u c t u r a l a l l e l e s and t h e i r 5' and 3' f l a n k i n g r e g i o n s tha t are sometimes a s s o c i a t e d w i t h geno typ ic d i f f e r e n c e s i n i n  v i t r o s p e c i f i c a c t i v i t i e s ( e . g . Aquadro et a l . 1986; L a n g l e y et a l . 1988). Each of these f a c t o r s c o m p l i c a t e s the ass ignment of 196 p h y s i o l o g i c a l e f f e c t s to a l l o z y m i c d i f f e r e n c e s at a s p e c i f i c enzyme l o c u s . S t u d i e s of t h i s na ture i n marine b i v a l v e s are f u r t h e r confounded by the w e l l documented s e a s o n a l i t y of t h e i r metabol i sm of l i p i d , p r o t e i n , and c a r b o h y d r a t e , and i n the a c c u m u l a t i o n of a n a e r o b i c e n d - p r o d u c t s ( e . g . Gabbott 1975; P i e t e r s et a l . 1979; Zandee et a l . 1980). A s s o c i a t e d w i t h these s e a s o n a l c y c l e s are t i s s u e - s p e c i f i c changes in s p e c i f i c a c t i v i t i e s ( e . g . L i v i n g s t o n e 1981; Gabbott 1983), and in some i n s t a n c e s the k i n e t i c p r o p e r t i e s of enzymes ( e . g . L i v i n g s t o n e 1975; L i v i n g s t o n e and C l a r k e 1983). A l t h o u g h PGM s p e c i f i c a c t i v i t y has been shown i n Chapter 3 to v a r y on a s e a s o n a l b a s i s , these changes do not n e c e s s a r i l y have to i n v o l v e the p o l y m o r p h i c l o c u s s t u d i e d to c o m p l i c a t e the d e t e c t i o n of i t s p h y s i o l o g i c a l consequences . A l t e r a t i o n s i n a c t i v i t y l e v e l s a n d / o r k i n e t i c p r o p e r t i e s of pathway enzymes have the p o t e n t i a l to a f f e c t the a r c h i t e c t u r e of the pathway's r e g u l a t o r y c o n t r o l t h a t i n t u r n c o u l d d i f f e r e n t i a l l y a f f e c t the impact of the polymorphism between d i f f e r e n t s easons . In the l a b o r a t o r y , g e n e t i c backgrounds may be s t a n d a r d i z e d through the c r e a t i o n of i s o g e n i c l i n e s ( e . g . L a u r i e - A h l b e r g et a l . 1980, 1981) or by the use of g e n e t i c t r a n s d u c t i o n to i n t r o d u c e s i n g l e a l l e l i c s u b s t i t u t i o n s ( e . g . H a r t l and Dykhuizen 1980; Dykhuizen and H a r t l 1981). N e i t h e r of these t echn iques are amenable to the s tudy of n a t u r a l p o p u l a t i o n s , and thus i t i s 197 n e c e s s a r y to moni tor s i m u l t a n e o u s l y the presence of g e n e t i c v a r i a t i o n i n the e n t i r e m e t a b o l i c pathway s t u d i e d to h e l p a l l e v i a t e problems c o n t r i b u t e d by g e n e t i c background e f f e c t s . F u r t h e r m o r e , due to the annua l c y c l e s of metabol i sm i n marine b i v a l v e s , the p h y s i o l o g i c a l e f f e c t s of enzyme polymorphisms may have to be s t u d i e d on a s eason- and t i s s u e - s p e c i f i c b a s i s i f t h e i r f u l l a d a p t i v e s i g n i f i c a n c e i s to be u n d e r s t o o d . In Chapter 4, mantle g lycogen c o n c e n t r a t i o n s were shown to d i f f e r between Pgm-2 genotypes i n a complex season-dependent f a s h i o n . A l t h o u g h the p a t t e r n s observed were d i r e c t l y a s s o c i a t e d w i t h the d i f f e r e n c e s i n PGM s p e c i f i c a c t i v i t y documented i n Chapter 3, the c o n t r i b u t i o n of a d d i t i o n a l l o c i , p a r t i c u l a r l y those m e t a b o l i c a l l y r e l a t e d w i t h PGM, c o u l d not be d i s c o u n t e d . To examine t h e i r p o t e n t i a l e f f e c t s , the e x i s t e n c e of g e n e t i c v a r i a t i o n and the a c t i v i t y l e v e l s of the remain ing g lycogen s y n t h e s i s pathway enzymes were s t u d i e d in d i f f e r e n t Pgm-2 g e n o t y p i c c l a s s e s i n the f a l l sample . In t h i s season , g lycogen r e s e r v e s a r e r a p i d l y accumulated i n the mantle t i s s u e (Quayle 1969), thus a l l o w i n g a more thorough examinat ion of the e f f e c t s of the Pgm-2 l o c u s on g lycogen s y n t h e s i s wi thout c o m p l i c a t i o n s due to the c o n c u r r e n t o p e r a t i o n of the g l y c o g e n o l y t i c pathway. 198 MATERIALS AND METHODS C h e m i c a l s . B u f f e r s , s u b s t r a t e s , c o f a c t o r s , and c o u p l i n g enzymes used for the e l e c t r o p h o r e s i s and enzyme assays were o b t a i n e d from Sigma. The e l e c t r o s t a r c h f o r e l e c t r o p h o r e s i s was s u p p l i e d by Connaught L a b o r a t o r i e s . A n i m a l s . O y s t e r s were c o l l e c t e d from the Nanoose Bay study p o p u l a t i o n i n the f a l l of 1985. The c o l l e c t i o n , t r a n s p o r t a t i o n and s torage of an imal s has been d e s c r i b e d i n C h a p t e r s 2 and 3. E l e c t r o p h o r e s i s . For the e n t i r e sample, Pgm-2 genotype was de termined by s t a r c h g e l e l e c t r o p h o r e s i s as o u t l i n e d i n Chapter 2. In the subsample of i n d i v i d u a l s s e l e c t e d f o r the enzyme a c t i v i t y and g lycogen measurements, e l e c t r o p h o r e t i c a n a l y s i s was a l s o performed to determine genotypes at the hexokinase and UDP-g lucose p y r o p h o s p h o r y l a s e l o c i . UDP-g lucose p y r o p h o s p h o r y l a s e (UDPGP; E . C . 2 . 7 . 7 . 9 ) a c t i v i t y was r e s o l v e d e l e c t r o p h o r e t i c a l l y by the d i s c o n t i n u o u s T r i s - c i t r a t e , L i - O H b u f f e r system of Ridgway, Sherburne and Lewis (1970) . UDPGP a c t i v i t y was s t a i n e d v i s u a l l y by i n c u b a t i n g a g e l s l i c e for a p p r o x i m a t e l y 30 min i n 100 ml of 100 mM T r i s - H C l b u f f e r , pH 7.5 c o n t a i n i n g 120 mg magnesium c h l o r i d e , 90 mg U D P - g l u c o s e , 65 mg sodium p y r o p h o s p h a t e , 25 mg NADP, 0.7 mg g l u c o s e - 1 , 6 - d i p h o s p h a t e , 80 u n i t s phosphoglucomutase , 40 u n i t s g l u c o s e - 6 - p h o s p h a t e dehydrogenase , 2 mg MTT, and 1 mg PMS. Hexokinase (HK; E . C . 2 . 7 . 1 . 1 ) a c t i v i t y was examined e l e c t r o p h o r e t i c a l l y by the 199 s t a n d a r d T r i s - b o r a t e - E D T A pH 8.3 b u f f e r system d e s c r i b e d i n Chapter 2, and s t a i n e d a c c o r d i n g to Shaw and P r a s a d (1970) . Enzyme A s s a y s . The mantle a c t i v i t i e s of h e x o k i n a s e , phosphoglucomutase , UDP-g lucose p y r o p h o s p h o r y l a s e , and g lycogen s y n t h e t a s e were de termined in a subsample of 229 o y s t e r s (108 from the low i n t e r t i d a l s i t e , 121 from the h i g h i n t e r t i d a l s i t e ) . These i n d i v i d u a l s r e p r e s e n t e d seven Pgm-2; g e n o t y p i c c l a s s e s t h a t were randomly p i c k e d from four a r b i t r a r i l y a s s i g n e d body weight c l a s s e s d e s c r i b e d i n Chapter 3. P r e p a r a t i o n s of the crude t i s s u e homogenates were i d e n t i c a l to t h a t o u t l i n e d i n Chapter 3, except for the i n c l u s i o n of 5 mM d i t h i o t h r e i t o l (DTT) i n the e x t r a c t i o n b u f f e r . A d d i t i o n of DTT s t a b i l i z e d the a c t i v i t i e s of UDP-g lucose p y r o p h o s p h o r y l a s e and g lycogen s y n t h e t a s e , but was found to have no adverse e f f e c t s on e i t h e r hexokinase or phosphoglucomutase . A l l enzyme a c t i v i t i e s were measured in t r i p l i c a t e at 340 nm on a Pye Unicam SP 1800 U V / v i s i b l e s p e c t r o p h o t o m e t e r . Assay temperature was m a i n t a i n e d at 15 C by a Lauda K - 2 / R D c i r c u l a t i n g water b a t h . Phosphoglucomutase a c t i v i t y was de termined by the s t a n d a r d assay on 20 y l a l i q u o t s of the crude homogenates as d e s c r i b e d i n Chapter 2. One u n i t of a c t i v i t y i s d e f i n e d as the q u a n t i t y of enzyme r e q u i r e d to c o n v e r t 1 /xmole of g l u c o s e - 1 -phosphate to g l u c o s e - 6 - p h o s p h a t e per minute under these c o n d i t i o n s . 200 Hexokinase was as sayed a c c o r d i n g to Bergmeyer (1974) on 60 Ml samples of the crude homogenates. The r e a c t i o n medium c o n t a i n e d 50 mM tr . i e thano lamine pH 7 .5 , 200 mM D - g l u c o s e , 5 mM MgC12, 0.6 mM ATP, 0.4 mM NADP, and one u n i t of g l u c o s e - 6 -phosphate dehydrogenase (G6PDH) i n a t o t a l volume of 1 m l . One u n i t of a c t i v i t y c o r r e s p o n d s to the amount of enzyme needed to c o n v e r t 1 Mmole of g l u c o s e to g l u c o s e - 6 - p h o s p h a t e per minute at 15 C . UDP-g lucose p y r o p h o s p h o r y l a s e was a l s o measured as o u t l i n e d by Bergmeyer (1974) on 20 ul a l i q u o t s of the t i s s u e e x t r a c t s . The assay m i x t u r e c o n t a i n e d 50 mM T r i s - H C l pH 7 . 5 , 5 mM MgC12, 1.5 mM sodium p y r o p h o s p h a t e , 1 mM U D P - g l u c o s e , 0.4 mM NADP, 16.nM g l u c o s e - 1 , 6 - d i p h o s p h a t e , 5 u n i t s of PGM, and one u n i t of of G6PDH in a f i n a l volume of 1 m l . The u n i t , d e f i n i t i o n f o r UDPGP i s the amount of enzyme r e q u i r e d to c o n v e r t 1 nmole of UDP-g lucose to g l u c o s e - 1 - p h o s p h a t e per minute a t 15 C . Glycogen synthe tase (GS; B . C . 2 . 4 . 1 . 1 1 ) a c t i v i t y was de termined by the d i r e c t o n e - s t e p assay of Passoneau and R o t t e n b e r g (1973) on 50 jul samples of the crude homogenates. The assay medium c o n t a i n e d 50 mM T r i s - H C l pH 7 .5 , 50 mM KC1, 10 mM h y d r a z i n e - H C l , 10 mM sodium a m o b a r b i t a l , 10 mM U D P - g l u c o s e , 10 mM o y s t e r g lycogen (Type I I ) , 5 mM MgC12, 1 mM Na-EDTA, 1 mM p h o s p h o ( e n o l ) p y r u v a t e , 0.15 mM NADH, 20 u n i t s of p y r u v a t e k i n a s e , 10 u n i t s of l a c t a t e dehydrogenase , and 0.02% (w/v) bov ine serum albumin i n a f i n a l volume of 1 m l . Glycogen syn the tase was not a s sayed i n the presence of g l u c o s e - 6 -phosphate , thus on ly the a c t i v i t y of the d e p h o s p h o r y l a t e d I form 201 was q u a n t i f i e d . One u n i t of GS a c t i v i t y i s d e f i n e d as the q u a n t i t y of enzyme r e q u i r e d to t r a n s f e r the a d d i t i o n 1 Mmole of UDP-g lucose onto the p r i m e r g lycogen per minute under the above c o n d i t i o n s . Because of the s i g n i f i c a n t o x i d a t i o n of NADH by the crude homogenates, even i n the presence of the a m o b a r b i t a l , the a c t i v i t y measurements n e c e s s i t a t e d c o r r e c t i o n s by b lank c u v e t t e s that c o n t a i n e d a l l of the above assay components except UDP-g l u c o s e . S i m i l a r c o r r e c t i o n s were not r e q u i r e d for the o ther a s s a y s . S o l u b l e p r o t e i n l e v e l s i n the crude homogenates were measured i n t r i p l i c a t e as d e s c r i b e d i n Chapters 2 and 3. The enzymes of the g lycogen s y n t h e s i s pathway were assayed c o n c u r r e n t l y w i t h the mantle and a d d u c t o r muscle PGM s p e c i f i c a c t i v i t y measurements f o r the f a l l sample . As d e s c r i b e d i n Chapter 3, the s e l e c t i o n of Pgm-2 genotypes was randomized a c r o s s e x p e r i m e n t a l days thus e n s u r i n g t h a t d a y - t o - d a y v a r i a t i o n i n assay t echn ique and s y s t e m a t i c e r r o r s due to the spontaneous l o s s of enzyme a c t i v i t y would be m i n i m i z e d . Glycogen A s s a y s . D e t e r m i n a t i o n of the mantle g lycogen c o n c e n t r a t i o n s of Pgm-2 genotypes i n t h i s sample have been p r e v i o u s l y d e s c r i b e d i n Chapter 4. S t a t i s t i c a l A n a l y s e s . A c t i v i t y r e l a t i o n s h i p s between pathway enzymes were examined by t e s t i n g the s i g n i f i c a n c e of t h e i r product-moment c o r r e l a t i o n c o e f f i c i e n t s from random e x p e c t a t i o n s . To examine the e f f e c t s of Pgm-2 l o c u s on pathway 202 a c t i v i t y s t r u c t u r e , the sample was p a r t i t i o n e d i n t o the four Pgm -2 a l l e l i c c l a s s e s d e s c r i b e d i n Chapter 3 ( i . e . , homozygotes for the Pgm-2-100 a l l e l e , h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e , homozygotes f o r the l e s s f requent Pgm-2-92, 96, and 104 a l l e l e s , and h e t e r o z y g o t e s between these t h r e e l e s s f requent a l l e l e s ) . Three-way a n a l y s e s of v a r i a n c e were performed on the a c t i v i t i e s of HK, PGM, UDPGP, GS, and mantle g lycogen l e v e l s t r e a t i n g i n t e r t i d a l p o s i t i o n , body weight c l a s s , and Pqm-2 l o c u s genotype as independent v a r i a b l e s . S i g n i f i c a n t e f f e c t s of Pgm-2 genotype c l a s s i n the a n a l y s e s of the HK, UDPGP, or GS da ta thus demonstrate an a s s o c i a t i o n of the Pgm-2 l o c u s w i t h the a c t i v i t i e s of these s e q u e n t i a l enzymic r e a c t i o n s . The e f f e c t s of v a r i a t i o n in the a c t i v i t i e s of HK, PGM, UDPGP, and GS on e x i s t i n g mantle g lycogen l e v e l s was examined by m u l t i p l e l i n e a r r e g r e s s i o n a n a l y s i s . The r e l a t i v e importance of each i n d i v i d u a l enzyme was a s s e s s e d by t e s t i n g the s i g n i f i c a n c e of the t - r a t i o of i t s p a r t i a l r e g r e s s i o n c o e f f i c i e n t to i t s s t a n d a r d e r r o r , a procedure analogous to p a r t i a l F - t e s t s on the e x p l a i n e d sums of squares a t t r i b u t a b l e to each f a c t o r i n the m u l t i p l e r e g r e s s i o n . RESULTS ELECTROPHORESIS The d i s c o n t i n u o u s Ridgway b u f f e r system produced e x c e l l e n t e l e c t r o p h o r e t i c r e s o l u t i o n of UDP-g lucose p y r o p h o s p h o r y l a s e . Two d i s t i n c t zones of a c t i v i t y were v i s i b l e , s u g g e s t i n g the presence 203 of two d i s t i n c t g e n e t i c l o c i . E x a m i n a t i o n of the 229 i n d i v i d u a l s s e l e c t e d f o r the enzyme a c t i v i t y and g lycogen measurements showed t h a t both presumpt ive l o c i were c o m p l e t e l y monomorphic. U s i n g the T r i s - b o r a t e - E D T A pH 8.3 b u f f e r system, hexokinase a l s o appeared to be encoded by two s e p a r a t e l o c i . The more c a t h o d a l l o c u s produced a f a i n t s i n g l e band . The anoda l r e g i o n a l s o s t a i n e d f a i n t l y , but was c h a r a c t e r i z e d by a two-banded p a t t e r n . Because of the ex tremely low a c t i v i t y of HK, d e t e r m i n a t i o n of the e x i s t e n c e of g e n e t i c v a r i a t i o n at e i t h e r l o c u s c o u l d not be de termined f o r the e n t i r e sample . However i n a sample of 62 i n d i v i d u a l s y i e l d i n g good r e s o l u t i o n , no h e t e r o z y g o s i t y was observed a t e i t h e r l o c u s . CORRELATIONS BETWEEN PATHWAY ENZYME ACTIVITIES Product-moment c o r r e l a t i o n c o e f f i c i e n t s between the mantle a c t i v i t i e s of the g lycogen s y n t h e s i s pathway enzymes are p r e s e n t e d i n T a b l e X V I . At both i n t e r t i d a l l o c a t i o n s , h i g h l y s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s were observed between the a c t i v i t i e s of HK and UDPGP, and between PGM and UDPGP. Weak p o s i t i v e c o r r e l a t i o n s were observed between UDPGP and GS, but no r e l a t i o n s h i p s were e v i d e n t between the a c t i v i t i e s of HK and GS. Two n o t a b l e d i f f e r e n c e s were found between the c o r r e l a t i o n m a t r i c e s of the two i n t e r t i d a l samples . F i r s t , HK was s i g n i f i c a n t l y c o r r e l a t e d w i t h PGM i n o y s t e r s from the low i n t e r t i d a l but not in those s i t u a t e d in the h i g h i n t e r t i d a l . Second, a weak p o s i t i v e r e l a t i o n s h i p e x i s t e d between the 204 T a b l e X V I . Product-moment c o r r e l a t i o n c o e f f i c i e n t s between the a c t i v i t i e s of enzymes in the g lycogen s y n t h e s i s pathway. V a l u e s above the d i a g o n a l are f o r the low i n t e r t i d a l sample (n=l08); those below f o r the h i g h i n t e r t i d a l sample (n=121). 205 Enzyme HK Enzyme PGM UDPGP GS HK 1 .000 0.316** 0.463*** 0.084 PGM 0.135 1.000 0.512*** 0.105 UDPGP 0.491*** 0.419*** 1 .000 0.137 GS 0.006 -0 .146 0.134 1 .000 * * P < .01 * * * P < .001 206 a c t i v i t i e s of PGM and GS i n the low i n t e r t i d a l , whereas i n the h i g h i n t e r t i d a l the c o r r e l a t i o n between these enzyme a c t i v i t i e s was n e g a t i v e . S t a t i s t i c a l comparison of the Z - t r a n s f o r m e d c o e f f i c i e n t s of the HK-PGM and PGM-GS enzyme p a i r s between the h i g h and low i n t e r t i d a l zones d i d not d e t e c t any s i g n i f i c a n t d i f f e r e n c e s . PATHWAY ACTIVITIES OF PGM-2 GENOTYPIC GROUPS The r e s u l t s of the t h r e e - f a c t o r ANOVA's on mantle g lycogen l e v e l s and the a c t i v i t i e s of HK, PGM, UDPGP, and GS are summarized i n T a b l e X V I I . I n t e r t i d a l p o s i t i o n e x p l a i n e d a s i g n i f i c a n t p r o p o r t i o n of the v a r i a n c e observed f o r a l l f i v e dependent v a r i a b l e s . A c o n s i s t e n t p a t t e r n was observed i n a l l a n a l y s e s : o y s t e r s i n the low i n t e r t i d a l r e g i o n posses sed s i g n i f i c a n t l y l a r g e r q u a n t i t i e s of g lycogen and s i g n i f i c a n t l y g r e a t e r a c t i v i t i e s of a l l four enzymes compared to those sampled from the more exposed h i g h i n t e r t i d a l a r e a . As e v i d e n t from the magnitude of the F v a l u e s , the most extreme d i f f e r e n c e was observed f o r g lycogen s y n t h e t a s e : i t s mean a c t i v i t y at the low i n t e r t i d a l s i t e exceeded that at the h i g h i n t e r t i d a l s i t e by 32%. In c o n t r a s t , o y s t e r s i n the low i n t e r t i d a l area d i s p l a y e d 22% more HK a c t i v i t y , 15% more PGM a c t i v i t y , 21% more UDPGP a c t i v i t y , and 24% more g lycogen than i n d i v i d u a l s h i g h e r i n the i n t e r t i d a l zone . No s i g n i f i c a n t e f f e c t s of body weight c l a s s as a s e p a r a t e f a c t o r were p r e s e n t i n any of the a n a l y s e s . 207 T a b l e X V I I . F - r a t i o s from the a n a l y s e s of v a r i a n c e on the mantle a c t i v i t i e s of the g lycogen s y n t h e s i s pathway enzymes and mantle g l y c o g e n c o n c e n t r a t i o n s . 2 0 8 Source of Va r i a t i o n df HK Dependent Variable PGM UDPGP GS Glycogen Tidal Height Body Weight A l l e l i c Class Tidal Height x Body Weight Tidal Height x A l l e l i c Class Body Weight x A l l e l i c Class Tidal Height x Body Weight x A l l e i i c Class Error 19.1*** 15.0*** 14.8*** 67.7*** 19.2*** 0.79 2.40 1 .88 1 . 14 1.48 0.75 0.36 1.51 2.06 7.76*** 1.70 3.25* 10.5*** 3.67* 1.21 0.94 0.52 2.27 4.90** 0.61 0.59 0.22 0.31 1.83 0.78 1.95* 1.72 2.14*. 0.67 1.06 197 * P < .05 ** P < .01 *** P < .001 209 H i g h l y s i g n i f i c a n t d i f f e r e n c e s e x i s t e d between the PGM a c t i v i t i e s and g lycogen c o n c e n t r a t i o n s of the four Pgm-2 a l l e l i c c l a s s e s . The s p e c i f i c a c t i v i t i e s and mantle g lycogen l e v e l s of these g e n o t y p i c groups have been p r e s e n t e d p r e v i o u s l y i n T a b l e XIV of Chapter 4. H e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e had s i g n i f i c a n t l y g r e a t e r s p e c i f i c a c t i v i t i e s than the Pgm-2-100/100 homozygotes and the h e t e r o z y g o t e s f o r the Pgm-2-92, 96, and 104 a l l e l e s , but d i d not d i f f e r s i g n i f i c a n t l y from the homozygotes f o r these l e s s f requent a l l e l e s . I d e n t i c a l d i f f e r e n c e s between these g e n o t y p i c c l a s s e s were observed i n t h e i r mantle g lycogen c o n c e n t r a t i o n s . Of p a r t i c u l a r i n t e r e s t i n t h i s s tudy however, were d i f f e r e n c e s tha t might e x i s t between the HK, UDPGP, and GS a c t i v i t i e s of the Pgm-2 a l l e l i c c l a s s e s . T a b l e XVII shows t h a t o n l y g lycogen synthe tase a c t i v i t y d i f f e r e d s i g n i f i c a n t l y between Pgm-2 genotypes in the o v e r a l l a n a l y s i s . Homozygotes for the Pgm - 2 - 9 2 , 96, and 104 a l l e l e s had GS a c t i v i t i e s tha t were 14.4% l a r g e r than the Pgm-2-100/100 homozygotes (7.31 u n i t s / g v s . 6.39 u n i t s / g ) . The two he terozygous groups showed i n t e r m e d i a t e GS a c t i v i t i e s . A p o s t e r i o r i B o n f e r r o n i m u l t i p l e range t e s t s were u n a b l e , however, to d e t e c t any s i g n i f i c a n t d i f f e r e n c e s between the a l l e l i c g r o u p s . The g e n e r a l absence of any r e l a t i o n s h i p s between Pgm-2 g e n o t y p i c c l a s s and the a c t i v i t i e s of these o ther enzymes in the o v e r a l l a n a l y s i s i s somewhat d e c e p t i v e . T a b l e s XVIII and XIX p r e s e n t the r e s u l t s from two-way ANOVA's c a r r i e d out s e p a r a t e l y f o r the low and h i g h i n t e r t i d a l samples , r e s p e c t i v e l y . The 210 T a b l e X V I I I . A c t i v i t i e s ( u n i t s / g s o l u b l e p r o t e i n ) of HK, PGM, UDPGP, and GS of four Pgm-2 g e n o t y p i c c l a s s e s i n the low i n t e r t i d a l zone . Enzyme A c t i v i t y ' Pgm-2 A l l e l i c Class N HK PGM UDPGP GS Glycogen' Homozygotes 22 6.06+.49 116.2+6.2 31.9+2.7 6.75±.31 171.4+16.0 for 100 A l l e l e Homozygotes 14 6.17±.62 124.917.8 35.413.4 7.751.39 195.2120.0 wlthout lOO A l l e l e Heterozygotes 60 6.571.30 139.213.8 35.3+1.6 7.931.19 220.5+9.7 for 100 A l l e l e Heterozygotes 12 6.90+.67 106.2+8.5 29.113.7 7.801.43 154.6+21.8 wlthout 100 A l l e l e F(3.92) = 0.48 5.67** 0.86 3.68* 4.03** * P < .05 ** P < .01 1 un1ts/g soluble protein * ptnoles glucosyl unlts/g wet tissue 212 T a b l e X I X . A c t i v i t i e s ( u n i t s / g s o l u b l e p r o t e i n ) of HK, PGM, UDPGP, and GS of four Pgm-2 g e n o t y p i c c l a s s e s i n the h i g h i n t e r t i d a l zone . 213 Enzyme A c t i v i t y ' Pgm-2 A l l e l i c Class N HK PGM UDPGP GS Glycogen' Homozygotes 20 4.77±.44 101.6+6.4 27.8+2.2 6.00+.40 122.0±12.0 for 100 A l l e l e Homozygotes 17 6.04+.49 106.7+7.0 34.8+2.4 6.94+.44 148.7±13.1 wlthout 100 A l l e l e Heterozygotes 63 4.931.25 118.5+3.6 25.8+1.3 5.30±.23 183.116.8 for 100 A l l e l e Heterozygotes 21 6.19+.44 104.7+6.3 29.7+2.2 6.24+.39 145.3+11.7 W1thout 100 A l l e l e F(3,105) = 3.35* 2.92* 3.25* 4.56** 7.93*** * P < .05 ** P < .01 1 units/g soluble protein ' *,moles glucosyl units/g wet tissue 214 a n a l y s i s of the low i n t e r t i d a l sample produced r e s u l t s tha t were s i m i l a r to tha t seen i n T a b l e X V I I . No s i g n i f i c a n t d i f f e r e n c e s were observed between the HK and UDPGP a c t i v i t i e s of the four Pgm-2 a l l e l i c g r o u p s . S i g n i f i c a n t d i f f e r e n c e s were a g a i n found between t h e i r GS a c t i v i t i e s tha t m u l t i p l e range t e s t s showed to be caused by the g r e a t e r a c t i v i t i e s of h e t e r o z y g o t e s f o r the Pgm -2-100 a l l e l e r e l a t i v e to the Pgm-2-100/100 homozygotes. The overdominance for PGM a c t i v i t y expressed by the Pgm-2-92/100, 96/jlOO and 100/104 h e t e r o z y g o t e s was not m a n i f e s t e d i n the a c t i v i t i e s of these o ther enzymes. These h e t e r o z y g o t e s e x h i b i t e d UDPGP and GS a c t i v i t i e s t h a t were i n d i s t i n g u i s h a b l e from the homozygotes f o r the Pgm-2-92, 96, and 104 a l l e l e s . The HK a c t i v i t i e s of h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e were s l i g h t l y h i g h e r than e i t h e r homozygote g r o u p , but m a r g i n a l l y below tha t measured in the o ther c l a s s of h e t e r o z y g o t e s . Mant l e g lycogen c o n c e n t r a t i o n s d i f f e r e d s i g n i f i c a n t l y between Pqm-2 a l l e l i c groups (F(3 ,92) = 4 .03 , P < . 0 1 ) , i n a p a t t e r n c o n s i s t e n t w i t h t h e i r a c t i v i t i e s of PGM, but not any of the o ther pathway enzymes. In c o n t r a s t to the o v e r a l l a n a l y s i s , s i g n i f i c a n t d i f f e r e n c e s were observed between Pqm-2 g e n o t y p i c groups in t h e i r a c t i v i t i e s of a l l four enzymes i n the h i g h i n t e r t i d a l sample (Tab le X I X ) . However, the l e v e l of s i g n i f i c a n c e i n most a n a l y s e s was low and m u l t i p l e range t e s t s were a b l e to d e t e c t s i g n i f i c a n t d i f f e r e n c e s between a l l e l i c groups o n l y for t h e i r UDPGP and GS a c t i v i t i e s . F o r both enzymes, homozygotes for the 215 Pgm-2-92, 96, and 104 a l l e l e s had s i g n i f i c a n t l y g r e a t e r a c t i v i t i e s than the Pqm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s . Homozygotes and h e t e r o z y g o t e s f o r the three l e s s f r e q u e n t a l l e l e s a l s o e x h i b i t e d l a r g e r HK a c t i v i t i e s than e i t h e r g e n o t y p i c c l a s s p o s s e s s i n g the Pgm-2-100 a l l e l e . In comparison to the o ther g e n o t y p i c g r o u p s , h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e had HK, UDPGP, and GS a c t i v i t i e s t h a t d i f f e r e d s u b s t a n t i a l l y from tha t observed i n the low water sample . A l t h o u g h they s t i l l expressed overdominant PGM a c t i v i t i e s , these genotypes expres sed s l i g h t l y underdominant UDPGP and GS a c t i v i t i e s , and l e v e l s of HK s i m i l a r to the Pgm-2-100/100 homozygotes . In the h i g h i n t e r t i d a l r e g i o n the Pgm-2-92/100, 96/100 and 100/104 h e t e r o z y g o t e s thus d i s p l a y e d HK, UDPGP, and GS a c t i v i t i e s tha t resembled one homozygote (Pgm-2-100/100) , but i n the low i n t e r t i d a l area were more s i m i l a r to the a l t e r n a t e homozygotes (Pqm-2-92/92, 96/96, and 104/104) . C o n s e q u e n t l y , when p o o l e d over the two t i d a l h e i g h t s , h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e expressed overdominant PGM a c t i v i t i e s , but i n t e r m e d i a t e HK, UDPGP, and GS a c t i v i t i e s . As seen i n the low i n t e r t i d a l sample, the g lycogen c o n c e n t r a t i o n s of the g e n o t y p i c groups showed a s t r o n g p o s i t i v e r e l a t i o n s h i p w i t h PGM a c t i v i t y , but not w i th any o t h e r pathway enzymes. A number of s i g n i f i c a n t i n t e r a c t i o n terms were present i n the ANOVA r e s u l t s p r e s e n t e d i n T a b l e X V I I . The a n a l y s e s for both HK and UDPGP produced s i g n i f i c a n t s e c o n d - o r d e r t i d a l h e i g h t - b y -body w e i g h t - b y - a l l e l i c c l a s s i n t e r a c t i o n s . For HK, the two-way 216 ANOVA f o r the h i g h i n t e r t i d a l sample y i e l d e d a s i g n i f i c a n t body w e i g h t - b y - a l l e l i c c l a s s i n t e r a c t i o n term (F(9 ,105) = 2 .15 , P < . 0 5 ) . In t h i s sample, the HK a c t i v i t y i n h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e decreased w i t h i n c r e a s i n g body weight but showed the r e v e r s e t r e n d i n the o ther g e n o t y p i c g r o u p s . In the low i n t e r t i d a l HK a n a l y s i s the i n t e r a c t i o n between body weight and a l l e l i c c l a s s was n o n - s i g n i f i c a n t (F(3 ,92 ) = 0 .59 , P > . 8 0 ) , but a c t i v i t i e s i n h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e tended to i n c r e a s e as a f u n c t i o n of body we ight . No r e l a t i o n s h i p s were observed i n the o ther g e n o t y p i c g r o u p s . The s e c o n d - o r d e r i n t e r a c t i o n observed f o r HK was thus l a r g e l y caused by a r e v e r s a l i n the r e l a t i o n s h i p between body weight and enzyme a c t i v i t y i n h e t e r o z y g o t e s for the Pqm-2-100 a l l e l e between the two t i d a l p o s i t i o n s . The s i m i l a r s e c o n d - o r d e r i n t e r a c t i o n term observed for UDPGP d i f f e r e d i n o r i g i n from tha t seen f o r HK. At both i n t e r t i d a l p o s i t i o n s , the body w e i g h t - b y - a l l e l i c c l a s s i n t e r a c t i o n terms were n o n - s i g n i f i c a n t . In the h i g h i n t e r t i d a l sample, bo th homozygote groups showed a p o s i t i v e a s s o c i a t i o n between UDPGP a c t i v i t y and body we ight . No r e l a t i o n s h i p s were e v i d e n t i n the two h e t e r o z y g o t e g r o u p s . In o y s t e r s from the low i n t e r t i d a l s i t e , h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e d i s p l a y e d a p o s i t i v e a s s o c i a t i o n between UDPGP a c t i v i t y and body we ight , however no r e l a t i o n s h i p s were observed f o r the o ther g e n o t y p i c g r o u p s . The d i f f e r e n t i a l responses of UDPGP a c t i v i t y and body weight in homozygous and heterozygous genotypes between 217 i n t e r t i d a l p o s i t i o n s produced the s i g n i f i c a n t s e c o n d - o r d e r i n t e r a c t i o n observed for t h i s enzyme i n T a b l e X V I I . A n a l y s i s of the g lycogen synthe tase da ta y i e l d e d s i g n i f i c a n t i n t e r a c t i o n s between t i d a l h e i g h t and body weight , and between t i d a l h e i g h t and a l l e l i c c l a s s . In the h i g h i n t e r t i d a l sample , s i g n i f i c a n t d i f f e r e n c e s i n GS a c t i v i t y e x i s t e d between the four body weight groups (F(3 ,105 = 3 .98 , P < . 0 1 ) . M u l t i p l e range t e s t s showed tha t o y s t e r s i n the s m a l l e s t weight c l a s s ( 12 .0 -23 .9 g) had s i g n i f i c a n t l y lower enzyme a c t i v i t i e s than i n d i v i d u a l s above 48.0 g . No s i g n i f i c a n t d i f f e r e n c e s between the GS a c t i v i t i e s of these weight groups were p r e s e n t i n the low i n t e r t i d a l sample . However, a n i m a l s i n the s m a l l e s t weight c l a s s now expres sed g r e a t e r GS a c t i v i t i e s than measured in the l a r g e s t i n d i v i d u a l s . These r e v e r s a l s i n GS a c t i v i t y between the s m a l l e s t and l a r g e s t o y s t e r s over the two t i d a l p o s i t i o n s were r e s p o n s i b l e f o r the s i g n i f i c a n t body w e i g h t - b y - a l l e l i c c l a s s i n t e r a c t i o n t erm. The s i g n i f i c a n t t i d a l h e i g h t - b y - P q m - 2 a l l e l i c c l a s s i n t e r a c t i o n was a consequence of the change i n ranked GS a c t i v i t y l e v e l f i r s t to l a s t by the h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e between the two t i d a l h e i g h t s as seen in T a b l e s XVIII and X I X . 218 MULTIPLE REGRESSION ANALYSES S imul taneous measures of the a c t i v i t i e s of HK, PGM, UDPGP, GS and e x i s t i n g g lycogen l e v e l s a l l o w e d an assessment of the r e l a t i v e e f f e c t s of each enzyme on the s y n t h e t i c pathway by m u l t i p l e r e g r e s s i o n a n a l y s i s . F o r the low i n t e r t i d a l sample (Table X X ) , the o v e r a l l r e g r e s s i o n was h i g h l y s i g n i f i c a n t (F(4 ,103) = 5 .06 , P < . 001 ) , e x p l a i n i n g 16.4% of the observed v a r i a t i o n i n mantle g lycogen l e v e l s . E s t i m a t i o n of the t - r a t i o s for each enzyme i n the pathway showed tha t o n l y PGM accounted for a s i g n i f i c a n t p r o p o r t i o n of the e x p l a i n e d sums of squares of the m u l t i p l e r e g r e s s i o n (t = 2 .35 , P < . 0 5 ) . The m u l t i p l e r e g r e s s i o n on the h i g h i n t e r t i d a l da ta (Tab le XXI) was a l s o s i g n i f i c a n t (F(4 ,116) = 3 .49 , P < . 0 1 ) , but e x p l a i n e d l e s s of the v a r i a t i o n i n g lycogen than found f o r the low water sample ( r 2 = 10.8%). Once a g a i n , o n l y the t - r a t i o f o r PGM was s i g n i f i c a n t (t = 3 .52 , P < . 0 0 1 ) . In c o n t r a s t to the r e g r e s s i o n e q u a t i o n o b t a i n e d from the low water sample , the p a r t i a l r e g r e s s i o n c o e f f i c i e n t s for UDPGP and GS i n the h i g h i n t e r t i d a l sample were n e g a t i v e . The da ta p r e s e n t e d i n T a b l e 4 suggests that t h i s may have been caused by the h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e . These genotypes expres sed the lowest UDPGP and GS a c t i v i t i e s , yet the h i g h e s t g l y c o g e n l e v e l s of the four g e n o t y p i c g r o u p s . 219 T a b l e XX. R e s u l t s from the m u l t i p l e r e g r e s s i o n a n a l y s i s of the g lycogen s y n t h e s i s pathway enzyme a c t i v i t i e s on g lycogen l e v e l s i n the low i n t e r t i d a l sample . 220 R e g r e s s i o n E q u a t i o n : Glycogen = 54.4 + 0.527HK + 0.626PGM + 1.27UDPGP + 2.42GS Enzyme R e g r e s s i o n C o e f f i c i e n t S tandard E r r o r t - r a t i o Hexokinase 0.527 3.61 0.15 Phosphoglucomutase 0.626 0.27 2.35* UDP-Glucose P y r o p h o s p h o r y l a s e 1 .27 0.72 1 .76 Glycogen Synthetase 2.42 4.73 0.51 * P < .05 221 T a b l e X X I . R e s u l t s from the m u l t i p l e r e g r e s s i o n a n a l y s i s of the g lycogen s y n t h e s i s pathway enzyme a c t i v i t i e s on g lycogen l e v e l s i n the h i g h i n t e r t i d a l sample . 222 R e g r e s s i o n E q u a t i o n : Glycogen = 94.1 + 1.76HK + 0.729PGM - 0.673UDPGP - 0.510GS Enzyme R e g r e s s i o n Coe f f i c i e n t S t a n d a r d E r r o r t - r a t i o Hexokinase 1.76 2.90 0.61 Phosphoglucomutase 0.729 0.207 3.52*** UDP-Glucose P y r o p h o s p h o r y l a s e -0 .673 0.656 -1 .03 Glycogen Synthetase -0 .510 2.89 - 0 . 1 8 * * * P < .001 223 DISCUSSION The c o r r e l a t i o n s between the a c t i v i t i e s of the g lycogen s y n t h e s i s pathway enzymes, t h e i r a c t i v i t y l e v e l s i n d i f f e r e n t Pgm-2 g e n o t y p i c g r o u p s , and the r e s u l t s of the m u l t i p l e r e g r e s s i o n a n a l y s e s a l l p r o v i d e c o n s i s t e n t ev idence f a v o r i n g a s i g n i f i c a n t e f f e c t of the Pgm-2 l o c u s on g lycogen metabol i sm in C . g i g a s . In the g lycogen s y n t h e s i s pathway, both hexokinase and g lycogen synthe tase are known to c a t a l y z e n o n - e q u i l i b r i u m r e a c t i o n s (Newsholme and S t a r t 1973), and thus are expected to e x e r t s i g n i f i c a n t e f f e c t s on the o v e r a l l f l u x of D - g l u c o s e u n i t s i n t o g l y c o g e n . S i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s between the a c t i v i t y of PGM w i t h e i t h e r HK or GS c o u l d , t h e r e f o r e , s e r i o u s l y weaken e f f e c t s p r e v i o u s l y a t t r i b u t a b l e to genotype-dependent enzyme a c t i v i t y v a r i a t i o n a t the Pgm-2 l o c u s on s y n t h e t i c r a t e s . An a s s o c i a t i o n between PGM and g lycogen s y n t h e t a s e a c t i v i t y i s of p a r t i c u l a r importance because of the s t r o n g r e l a t i o n s h i p demonstrated between GS a c t i v i t y and r a t e s of g lycogen s y n t h e s i s i n mammalian l i v e r (Stalmans 1976), and between the I form a c t i v i t y and mantle g lycogen l e v e l s i n M. e d u l i s (Gabbot t , Cook and W h i t t l e 1979; Gabbott and W h i t t l e 1986a, 1986b). In comparing the c o r r e l a t i o n m a t r i c e s o b t a i n e d f o r the two i n t e r t i d a l samples shown i n T a b l e X V I , i t i s n o t a b l e that the o n l y observed d i f f e r e n c e s i n v o l v e d the a c t i v i t y r e l a t i o n s h i p s of PGM w i t h both HK and GS. Hexokinase e x h i b i t e d a s i g n i f i c a n t p o s i t i v e a s s o c i a t i o n wi th PGM i n the low but not in the h i g h 224 i n t e r t i d a l sample , and the c o r r e l a t i o n c o e f f i c i e n t s between PGM and GS r e v e r s e d i n s i g n between the two t i d a l p o s i t i o n s . D e s p i t e these d i f f e r e n c e s , the mantle g lycogen c o n c e n t r a t i o n s of the four Pgm-2 g e n o t y p i c groups e x h i b i t e d i d e n t i c a l p a t t e r n s a t both i n t e r t i d a l l o c a t i o n s . T h e r e f o r e , any i n f l u e n c e of the Pgm-2 l o c u s on g lycogen metabol i sm i n t h i s f a l l sample has not appeared to occur through c o r r e l a t i o n s of i t s a c t i v i t y wi th e i t h e r the HK or GS r e a c t i o n s t e p s . In a s tudy of the a c t i v i t y r e l a t i o n s h i p s between 23 enzymes in 48 second and t h i r d chromosome i s o g e n i c s u b s t i t u t i o n l i n e s of D r o s o p h i l a m e l a n o g a s t e r , W i l t o n et a l . (1982) observed a p a t t e r n of s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n s between s e q u e n t i a l enzymic r e a c t i o n s , p a r t i c u l a r l y at the g l u c o s e - 6 - p h o s p h a t e branch p o i n t . The s i g n i f i c a n t r e l a t i o n s h i p s observed in t h i s s tudy between the a c t i v i t i e s of PGM and UDPGP are c o n s i s t e n t w i t h t h i s g e n e r a l t r e n d . In D . melanogaster however, h i g h l y s i g n i f i c a n t c o r r e l a t i o n s were observed between the a c t i v i t i e s of HK and PGM (0.58 i n second chromosome l i n e s , and 0.55 i n t h i r d chromosome l i n e s ) . In the P a c i f i c o y s t e r , hexokinase a c t i v i t y e x h i b i t e d much lower c o r r e l a t i o n s w i th PGM, yet was s t r o n g l y a s s o c i a t e d wi th the a c t i v i t y of UDP-g lucose p y r o p h o s p h o r y l a s e . T h i s d i s c r e p a n c y c o u l d perhaps be e x p l a i n e d by the marked d i f f e r e n c e s in c a r b o h y d r a t e metabol i sm between these groups of organ i sms . In the mant le t i s s u e of M y t i l u s e d u l i s , hexokinase e x h i b i t s s i g n i f i c a n t changes in s p e c i f i c a c t i v i t y , a n d k i n e t i c p r o p e r t i e s on a s e a s o n a l b a s i s tha t c o i n c i d e s w i th the u t i l i z a t i o n of 225 endogenous v e r s u s exogenous s u p p l i e s of g l u c o s e a t d i f f e r e n t t imes of the year ( L i v i n g s t o n e and C l a r k e 1983). A l t h o u g h PGM a c t i v i t y may have been expec ted to show a s t r o n g r e l a t i o n s h i p w i t h hexokinase i n t h i s s e a s o n a l sample (due to the a c t i v e s y n t h e s i s of g lycogen from e x t e r n a l g l u c o s e s u p p l i e s ) , t h e i r a c t i v i t i e s c o u l d a t t imes be e f f e c t i v e l y uncoupled v i a the s e a s o n a l i n d u c t i o n of d i s t i n c t a n a b o l i c or c a t a b o l i c pathways ( c f . L i v i n g s t o n e 1981). The apparent impact of the Pqm-2 l o c u s on g lycogen s y n t h e s i s i s not c o m p l i c a t e d by presence of g e n e t i c v a r i a t i o n at a d j a c e n t enzymic s t e p s . Both presumed UDP-g lucose p y r o p h o s p h o r y l a s e l o c i were monomorphic, and no a l l o z y m i c v a r i a t i o n was observed i n the subsample of i n d i v i d u a l s y i e l d i n g good r e s o l u t i o n of hexokinase a c t i v i t y . Glycogen syn the tase a c t i v i t y c o u l d not be examined e l e c t r o p h o r e t i c a l l y , so the p o s s i b i l i t y of c o n f o u n d i n g e f f e c t s due to g e n e t i c v a r i a n t s of t h i s enzyme cannot be d i s c o u n t e d . The r e s u l t s p r e s e n t e d i n T a b l e s XVIII and XIX demonstrate t h a t enzyme a c t i v i t y v a r i a t i o n at the Pgm-2 l o c u s d i d not occur i n d e p e n d e n t l y of tha t observed for the o ther g lycogen s y n t h e s i s pathway enzymes. S i g n i f i c a n t d i f f e r e n c e s were d e t e c t e d between Pgm-2 g e n o t y p i c c l a s s e s i n t h e i r HK and UDPGP a c t i v i t i e s in the h i g h i n t e r t i d a l sample, and between t h e i r GS a c t i v i t i e s at both i n t e r t i d a l h e i g h t s . An important f i n d i n g was tha t these d i f f e r e n c e s were not caused by the unusua l b e h a v i o r of Pgm-2-226 92/100, 96/100, and 100/104 h e t e r o z y g o t e s . In both i n t e r t i d a l samples , h e t e r o z y g o t e s f o r the Pqm-2-100 a l l e l e expressed overdominance for t h e i r a c t i v i t i e s of PGM, but not i n t h e i r l e v e l s of HK, UDPGP or GS. T h e r e f o r e , the h i g h e r g lycogen c o n c e n t r a t i o n s d i s p l a y e d by these h e t e r o z y g o t e s was not an i n d i r e c t consequence of the c o o r d i n a t e d e l e v a t i o n of the e n t i r e s y n t h e t i c pathway. These non-random a s s o c i a t i o n s do not. appear c a p a b l e of e x p l a i n i n g the d i f f e r e n c e s i n mantle g lycogen c o n c e n t r a t i o n s observed between Pgm-2 genotypes . Homozygotes for the Pgm-2-92, 96 and 104 a l l e l e s had g r e a t e r HK, UDPGP, and GS a c t i v i t i e s than Pgm-2-100/100 homozygotes a t both t i d a l h e i g h t s . E x c l u d i n g t h e i r PGM a c t i v i t i e s , the Pgm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s approx imated the Pgm-2-100/100 homozygotes in the h i g h i n t e r t i d a l r e g i o n , but s h i f t e d to resemble the a l t e r n a t e c l a s s of homozygotes i n the low i n t e r t i d a l zone . D e s p i t e e x h i b i t i n g HK, UDPGP, and GS a c t i v i t i e s i n the low i n t e r t i d a l area t h a t were s i m i l a r to the h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e , the Pgm-2-92/92, 96 /96 , and 104/104 homozygotes d i s p l a y e d lower q u a n t i t i e s of mantle g lycogen than these overdominant h e t e r o z y g o t e s . In the h i g h i n t e r t i d a l zone, these same homozygotes had c o n s i d e r a b l y l a r g e r a c t i v i t i e s of a l l pathway enzymes (wi th e x c e p t i o n of PGM) than the h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e , yet s t i l l possessed s i g n i f i c a n t l y lower mantle g lycogen l e v e l s . These r e s u l t s i n d i c a t e tha t the d i f f e r e n t a c t i v i t y l e v e l s of HK, UDPGP, and GS expres sed between 227 the Pqm-2 g e n o t y p i c groups are poor p r e d i c t o r s of t h e i r d i f f e r i n g mantle g lycogen c o n c e n t r a t i o n s . In Chapter 3 i t was h y p o t h e s i z e d t h a t a t i g h t l y - l i n k e d " r e g u l a t o r y " l o c u s , i n complete d i s e q u i l i b r i u m wi th the Pgm-2 s t r u c t u r a l l o c u s , c o u l d be r e s p o n s i b l e f o r p r o d u c i n g the d i f f e r e n t enzyme a c t i v i t i e s of Pgm-2 genotypes . I t was suggested tha t t h i s r e g u l a t o r y element was s e g r e g a t i n g f o r two a l l e l e s , one of which was a s s o c i a t e d w i t h the Pgm-2-100 a l l e l e , the o ther shared by the Pgm-2-92, 96, and 104 a l l e l e s . The d i f f e r e n t a c t i v i t y l e v e l s of the g lycogen s y n t h e s i s pathway enzymes shown by the two homozygote groups appear to support t h i s e x p l a n a t i o n , but a l s o r e q u i r e that t h i s r e g u l a t o r y element e x e r t p l e i o t r o p i c e f f e c t s on these o ther pathway enzymes. These o b s e r v a t i o n s p r o v i d e f u r t h e r ev idence a g a i n s t a Pgm-2 n u l l a l l e l e b e i n g r e s p o n s i b l e f o r the overdominant PGM a c t i v i t i e s of h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e . I t seems h i g h l y improbable tha t an unde tec ted Pqm-2 n u l l a l l e l e would have any impact on the pathway a c t i v i t y s t r u c t u r e o ther than d e p r e s s i n g the PGM a c t i v i t i e s of the two homozygous g r o u p s . M o d i f i e r l o c i hav ing c o o r d i n a t e e f f e c t s on d i f f e r e n t enzymes have been observed i n Mus musculus (Womack, Yan and P o t i e r 1980) and D. melanogaster ( B e n t l e y and W i l l i a m s o n 1979; B e l o t e and L u c c h e s i 1980). I f t h i s p u t a t i v e r e g u l a t o r y element i s r e s p o n s i b l e f o r these r e s u l t s i n C . g i g a s , i t d i s p l a y s t h r e e ex tremely unusua l p r o p e r t i e s . F i r s t , i n he terozygous c o n d i t i o n 228 i t g i v e s r i s e to overdominant PGM a c t i v i t y l e v e l s . Second, the a l l e l e s e g r e g a t i n g w i t h the t h r e e l e s s f requent s t r u c t u r a l a l l e l e s produces g r e a t e r HK, UDPGP, and GS a c t i v i t i e s when homozygous than the v a r i a n t l i n k e d to the Pgm-2-100 a l l e l e . T h i r d , i t appears to cause r e v e r s a l s i n the dominance r e l a t i o n s h i p s of r e g u l a t o r y genotypes between the two i n t e r t i d a l p o s i t i o n s such tha t h e t e r o z y g o t e s resemble the Pgm-2-100/100 homozygote i n the h i g h i n t e r t i d a l , but the Pgm-2-92/92 , 96/96, and 104/104 homozygotes in the low i n t e r t i d a l . The r e s u l t s p r e s e n t e d i n Chapter 3 have shown t h a t the f i r s t p r o p e r t y i s c o n s i s t e n t l y observed i n the mantle and adductor muscle t i s s u e s at both i n t e r t i d a l p o s i t i o n s i n t h r e e d i f f e r e n t s easons . The second and t h i r d e f f e c t s suggested by the data p r e s e n t e d in t h i s s e c t i o n r e q u i r e f u r t h e r study to determine t h e i r r e p e a t a b i l i t y i n d i f f e r e n t seasons and t i s s u e s . A s p e c i f i c p r e d i c t i o n of the h y p o t h e s i s of an overdominant r e g u l a t o r y l o c u s i s the e q u i v a l e n c e of homozygotes and h e t e r o z y g o t e s formed between the three l e s s f requent Pgm-2 s t r u c t u r a l a l l e l e s , s i n c e both would be homozygous f o r the same r e g u l a t o r y e lement . Homozygotes and h e t e r o z y g o t e s l a c k i n g the Pgm-2-100 a l l e l e posses sed very s i m i l a r HK a c t i v i t i e s i n the h i g h i n t e r t i d a l r e g i o n , and GS a c t i v i t i e s in the low i n t e r t i d a l a r e a . However, the UDPGP a c t i v i t i e s of the Pgm-2-92/96 , 92/104, and 96/104 h e t e r o z y g o t e s were c o n s i d e r a b l y lower than measured i n the homozygotes f o r these a l l e l e s . F u r t h e r d i s c r e p a n c i e s from the p r e d i c t e d p a t t e r n s were observed between t h e i r low water HK 229 a c t i v i t i e s and h i g h water GS a c t i v i t i e s . T h e r e f o r e , a p a r t from t h e i r PGM a c t i v i t i e s , homozygotes and h e t e r o z y g o t e s f o r these l e s s f requent Pgm-2 a l l e l e s d i d not e x h i b i t the s t r o n g s i m i l a r i t i e s expected by the r e g u l a t o r y mode l . One f e a t u r e of the model s u p p o r t e d by these r e s u l t s however, was the d i s p a r i t y of the two he terozygous g r o u p s . These two a l l e l i c c l a s s e s expres sed s u b s t a n t i a l l y d i f f e r e n t PGM a c t i v i t i e s i n the low i n t e r t i d a l sample , a l t e r e d l e v e l s of a l l four enzymes i n the h i g h i n t e r t i d a l sample, and d i s s i m i l a r g lycogen c o n c e n t r a t i o n s a t both t i d a l h e i g h t s . An a l t e r n a t i v e e x p l a n a t i o n for the d i f f e r e n t enzyme a c t i v i t i e s of Pgm-2 g e n o t y p i c c l a s s e s c o u l d be t h a t they arose as an i n d i r e c t consequence of the amount of g lycogen p r e s e n t i n t h e i r mantle t i s s u e s . An i n v e r s e r e l a t i o n s h i p i s p r e d i c t e d between g lycogen c o n c e n t r a t i o n and enzyme a c t i v i t y l e v e l s s i m p l y because of the l i m i t e d s torage c a p a c i t i e s of mantle v e s i c u l a r c e l l s . Some ev idence s u p p o r t i n g t h i s r e l a t i o n s h i p was seen i n the h i g h i n t e r t i d a l zone , where h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e e x h i b i t e d the h i g h e s t g lycogen l e v e l s and depres sed HK, UDPGP, and GS a c t i v i t i e s . C o n v e r s e l y , the two g e n o t y p i c groups l a c k i n g the Pgm-2-100 a l l e l e possessed lower g lycogen c o n c e n t r a t i o n s and h i g h e r a c t i v i t i e s of these three enzymes. The Pgm-2-100/100 homozygotes s t r o n g l y c o n t r a d i c t e d t h i s p a t t e r n however, by e x p r e s s i n g low a c t i v i t i e s throughout the e n t i r e pathway, but g lycogen l e v e l s tha t were 50% lower than the overdominant h e t e r o z y g o t e s . V i r t u a l l y no ev idence f a v o r i n g t h i s 230 e x p l a n a t i o n was observed i n the low i n t e r t i d a l sample . T h e r e f o r e , even i f g lycogen c o n c e n t r a t i o n s had some e f f e c t on the pathway enzyme a c t i v i t i e s , some o ther f a c t o r ( s ) must a l s o be i n v o l v e d . The m u l t i p l e r e g r e s s i o n a n a l y s e s s e r v e d to c o n f i r m what was a l r e a d y e v i d e n t from T a b l e s XVIII and X I X . Of the four pathway enzymes, o n l y PGM e x p l a i n e d a s i g n i f i c a n t amount of the observed v a r i a t i o n i n mant le g lycogen l e v e l s . S i m i l a r r e s u l t s were observed for both i n t e r t i d a l samples , but the t - r a t i o of PGM was more h i g h l y s i g n i f i c a n t in the h i g h i n t e r t i d a l a r e a . The complete absence of e f f e c t s by e i t h e r hexokinase or g lycogen syn the tase i n these a n a l y s e s i s r a t h e r s u r p r i s i n g . The c a t a l y t i c and r e g u l a t o r y p r o p e r t i e s of hexokinase have i d e n t i f i e d t h i s enzyme as a major r e g u l a t o r y s i t e i n c a r b o h y d r a t e metabol i sm (Newsholme and S t a r t 1973). R e c e n t l y , T o r r e s et a l . (1986) e s t i m a t e d the g l y c o l y t i c c o n t r o l c o e f f i c i e n t of hexokinase in r a t l i v e r p r e p a r a t i o n s to be 0 .77 , s u g g e s t i n g t h a t t h i s r e a c t i o n may a l s o e x e r t a dominant r o l e i n s u p p l y i n g g l u c o s e - 6 - p h o s p h a t e f o r the s y n t h e s i s of g l y c o g e n . The n o n - s i g n i f i c a n t e f f e c t s of hexokinase a c t i v i t y v a r i a t i o n on mantle g lycogen l e v e l s in C . g i g a s c o u l d perhaps be a consequence of 1) the r e a c t i o n o p e r a t i n g below i t s c a t a l y t i c p o t e n t i a l because of the low haemolymph g l u c o s e c o n c e n t r a t i o n s i n marine b i v a l v e s ( e . g . Zaba 1981; L i v i n g s t o n e and C l a r k e 1983), 2) i t s c o n t r o l of f l u x be ing o v e r r i d d e n by the g lycogen syn the tase r e a c t i o n , or 3) tha t a l a r g e p r o p o r t i o n of g lycogen was s y n t h e s i z e d i n the f a l l from 231 g luconeogen ic p r e c u r s o r s . The a c t i v i t y of g lycogen synthe tase i n mammalian l i v e r has been found to d i r e c t l y p a r a l l e l g lycogen s y n t h e s i s r a t e s (Hue and Hers 1974, Stalmans 1976). E v i d e n c e i s a c c u m u l a t i n g tha t t h i s enzyme e x e r t s a s i m i l a r degree of c o n t r o l i n m o l l u s c a n mantle t i s s u e (see F i g u r e 13 i n Gabbott 1983). The s i g n i f i c a n t e f f e c t of PGM a c t i v i t y v a r i a t i o n on mantle g l y c o g e n , and the c o r r e s p o n d i n g absence of any r e l a t i o n s h i p w i t h GS a c t i v i t y , may at f i r s t appear to c o n t r a d i c t these p r e v i o u s s t u d i e s . However, an important d i s t i n c t i o n must be made between a r e g u l a t o r y enzyme's d i c t a t i o n of o v e r a l l pathway f l u x , and m o d u l a t i o n of these f l u x r a t e s by n o n - r e g u l a t o r y enzymes l i k e PGM. Glycogen synthe tase may w e l l be l a r g e l y r e s p o n s i b l e f o r c o n t r o l l i n g the net r a t e of g lycogen s y n t h e s i s . The h i g h e r g lycogen c o n c e n t r a t i o n s measured i n o y s t e r s from the low compared to the h i g h i n t e r t i d a l area i s most l i k e l y due to a c o m b i n a t i o n of t h e i r g r e a t e r "scope f o r growth" and h i g h e r GS, not PGM, a c t i v i t i e s . As shown i n Chapter 4, PGM a c t i v i t y v a r i a t i o n i s expected to i n f l u e n c e these f l u x r a t e s through a p a r t i t i o n i n g e f f e c t at the g l u c o s e - 6 - p h o s p h a t e branch p o i n t ( c f . L a P o r t e , Walsh and K o s h l a n d 1984). In re sponding to f l u x r a t e s de termined by GS, the overdominant PGM a c t i v i t i e s of the Pgm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s s h o u l d r e s u l t i n h i g h e r r a t e s of s y n t h e s i s than observed i n the o ther g e n o t y p i c c l a s s e s . In a s i n g l e p o p u l a t i o n sample , the i n f l u e n c e of g lycogen synthe tase would be obscured by e f f e c t s of the Pgm-2 l o c u s . However i f Pgm-232 2 genotype i s i g n o r e d , a s e r i e s of s i m i l a r measurements taken over a p e r i o d of a c t i v e s y n t h e s i s would demonstrate a s t r o n g c o r r e l a t i o n between GS a c t i v i t y and mantle g lycogen l e v e l s , as r e p o r t e d i n M. e d u l i s by G a b b o t t , Cook and W h i t t l e (1979), and Gabbott and W h i t t l e (1986a) . The impact of the Pgm-2 l o c u s on g lycogen s y n t h e s i s would be s i m i l a r f o r ' d i f f e r e n t genotypes o n l y i f t h e i r a c t i v i t i e s of g l y c o g e n syn the tase were e q u a l . However in both i n t e r t i d a l samples , s i g n i f i c a n t d i f f e r e n c e s i n these a c t i v i t i e s were d e t e c t e d between the four g e n o t y p i c c l a s s e s . In the low i n t e r t i d a l sample," the two heterozygous groups and the homozygotes l a c k i n g the Pgm-2-100 a l l e l e posses sed on average 17% h i g h e r GS a c t i v i t i e s than the Pgm-2-100/100 homozygotes . The mean g lycogen c o n c e n t r a t i o n s of these three g e n o t y p i c c l a s s e s were i n t u r n 21% l a r g e r than measured in the Pqm-2-100/100 homozygotes . A l t h o u g h e x h i b i t i n g s i m i l a r l e v e l s of g lycogen s y n t h e t a s e a c t i v i t y , s u b s t a n t i a l d i f f e r e n c e s i n g lycogen l e v e l s were observed between these a l l e l i c c l a s s e s t h a t were d i r e c t l y c o r r e l a t e d w i t h t h e i r PGM a c t i v i t i e s . The overdominant c l a s s of h e t e r o z y g o t e s had 11% h i g h e r PGM a c t i v i t i e s and 13% more g l y c o g e n than the Pgm-2-92/92, 96 /96 , and 104/104 homozygotes. Compared to the o ther h e t e r o z y g o t e group , the overdominant h e t e r o z y g o t e s expressed 31% more PGM a c t i v i t y and 43% h i g h e r g lycogen l e v e l s . These p a t t e r n s demonstrate tha t GS a c t i v i t y i s a major f a c t o r in d e t e r m i n i n g g lycogen l e v e l s , but the r e a l i z e d f l u x r a t e s appear s t r o n g l y a f f e c t e d by Pgm-2 genotype-dependent 233 enzyme a c t i v i t y v a r i a t i o n . An i n t e r a c t i o n between PGM and GS a c t i v i t y i n a f f e c t i n g mantle g lycogen was a l s o e v i d e n t i n the h i g h i n t e r t i d a l sample , but was c o m p l i c a t e d by the unusual b e h a v i o r of the Pgm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s . These genotypes e x h i b i t e d the h i g h e s t c o n c e n t r a t i o n s of g lycogen and the lowest GS a c t i v i t i e s of the four a l l e l i c c l a s s e s . T h i s d i s c r e p a n c y b r i n g s to l i g h t an important assumption t h a t s i g n i f i c a n t l y a f f e c t s the c o n c l u s i o n s of the m u l t i p l e r e g r e s s i o n a n a l y s e s , namely, that the a c t i v i t y r e l a t i o n s h i p s of the pathway enzymes have remained unchanged over the t ime p e r i o d p r e c e d i n g the sampl ing d a t e . G lycogen has been i m p l i c a t e d as a n e g a t i v e r e g u l a t o r of i t s own s y n t h e s i s by i n h i b i t i n g g lycogen s y n t h e t a s e phosphatase and thus l o w e r i n g the p r o p o r t i o n of the enzyme i n the a c t i v e ( I ) form ( D a n f o r t h 1965; Watts and Mal thus 1980). T h e r e f o r e , i t i s p o s s i b l e t h a t the 'GS l e v e l s i n h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e were l a r g e r be fore the sample was c o l l e c t e d but have been s u b s e q u e n t l y depressed through the i n h i b i t o r y e f f e c t of t h e i r h i g h e r g lycogen c o n c e n t r a t i o n s . E s t i m a t e s of the t o t a l (I p l u s D) g lycogen synthe tase a c t i v i t y of Pgm-2 g e n o t y p i c c l a s s e s c o u l d have p r o v i d e d a t e s t of t h i s p r e d i c t i o n , but these assays were not p e r f o r m e d . The e x p r e s s i o n of overdominant GS a c t i v i t i e s by the h e t e r o z y g o t e s for the Pgm-2-100 a l l e l e i n the h i g h i n t e r t i d a l area p r i o r to the sampl ing date cannot be d i s c o u n t e d . However i n 234 the h i g h i n t e r t i d a l , the magnitude of the i n h i b i t i o n of GS a c t i v i t y by g lycogen appears f a r too grea t to be r e c t i f i e d w i t h i t s observed l e v e l s , s i n c e these were below those present a t o ther t i d a l h e i g h t . Another f a c t o r d i s c o u n t i n g the importance of t h i s e x p l a n a t i o n i s the f a c t t h a t mantle g lycogen c o n c e n t r a t i o n s can r e a c h c o n s i d e r a b l y h i g h e r l e v e l s than measured i n t h i s season (Chapter 4 ) . T h e r e f o r e , the extent of t h i s p o t e n t i a l i n h i b i t i o n by g lycogen on GS a c t i v i t y i n the f a l l i s expected to have been n e g l i g i b l e . The comparable HK and UDPGP a c t i v i t i e s of the h e t e r o z y g o t e s p o s s e s s i n g the Pgm-2-100 a l l e l e and the Pgm-2-100/100 homozygotes i n the h i g h i n t e r t i d a l sample suggest t h a t the GS a c t i v i t i e s of these groups may have p r e v i o u s l y been at s i m i l a r l e v e l s . The h i g h e r g lycogen c o n c e n t r a t i o n s of the Pqm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s i n the h i g h i n t e r t i d a l sample c o u l d i n d i c a t e tha t t h e i r pathway a c t i v i t y l e v e l s are more o p t i m a l l y s u i t e d to the s y n t h e s i s of g lycogen than expres sed i n the homozygotes and h e t e r o z y g o t e s l a c k i n g the Pgm-2-100 a l l e l e . In summary, the a c t i v i t y r e l a t i o n s h i p s between the g lycogen s y n t h e s i s pathway enzymes p r o v i d e s f u r t h e r ev idence f a v o r i n g an e f f e c t of the Pgm-2 l o c u s on g lycogen metabo l i sm. There i s some ev idence s u g g e s t i n g a d d i t i o n a l e f f e c t s of the p u t a t i v e r e g u l a t o r y element p r o d u c i n g the overdominant a c t i v i t y l e v e l s of h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e on the a c t i v i t i e s of these o ther enzymes, but none t h a t can account f o r the d i f f e r i n g g lycogen l e v e l s of these g e n o t y p i c g r o u p s . These r e s u l t s 235 demonstrate a s u r p r i s i n g l y s t r o n g e f f e c t of a l l o z y m i c v a r i a t i o n at the Pgm-2 l o c u s on m e t a b o l i c f l u x , c o n s i s t e n t w i t h tha t expected from a p a r t i t i o n i n g e f f e c t a t the g l u c o s e - 6 - p h o s p h a t e branch p o i n t d i s c u s s e d i n Chapter 4. 236 CHAPTER 6 GENERAL DISCUSSION H e t e r o z y g o t e s at f i v e enzyme l o c i ( i n c l u d i n g Pgm-2) have been shown by F u j i o (1982) to e x h i b i t g r e a t e r a d u l t body weights than homozygotes i n 20 Japanese p o p u l a t i o n s of C r a s s o s t r e a  g i g a s . A l t h o u g h s i m i l a r r e l a t i o n s h i p s have not been demonstrated i n the p r e s e n t s t u d y , there are h i s t o r i c a l reasons for b e l i e v i n g tha t the same p a t t e r n s would be presen t i n B r i t i s h Columbia p o p u l a t i o n s of t h i s s p e c i e s . The c o l o n i z a t i o n of B r i t i s h Columbia by C . g igas has not been accompanied by founder e f f e c t s s i g n i f i c a n t l y r e d u c i n g l e v e l s of g e n e t i c v a r i a b i l i t y : h e t e r o z y g o s i t y e s t i m a t e s from both geographic r e g i o n s are n e a r l y i d e n t i c a l ( e . g . B u r o k e r , Hershberger and Chew 1975, 1979a; O z a k i and F u j i o 1985). F u r t h e r m o r e , a l l e l i c f requency d i s t r i b u t i o n s at these po lymorph ic enzyme l o c i have not d i v e r g e d to any a p p r e c i a b l e ex tent between the western and e a s t e r n P a c i f i c . For example at the Pgm-2 l o c u s , the frequency of the most common a l l e l e i n the Nanoose Bay study p o p u l a t i o n (0.595) was a lmost i d e n t i c a l t o i t s mean frequency i n the 23 Japanese p o p u l a t i o n s (0.596) s t u d i e d by Ozak i and F u j i o (1985) . The c l o s e s i m i l a r i t y of C . g i g a s p o p u l a t i o n s between both P a c i f i c c o a s t s suggests tha t i f a g e n e t i c mechanism(s) i s r e s p o n s i b l e for these m u l t i p l e - l o c u s h e t e r o z y g o s i t y r e l a t i o n s h i p s , i t sh ou ld a l s o e x i s t i n the B . C . p o p u l a t i o n s t u d i e d . 237 F u j i o (1982) proposed tha t i n b r e e d i n g was the most l i k e l y e x p l a n a t i o n for the g r e a t e r s i z e s of h e t e r o z y g o t e s at a l l f i v e l o c i because a s i g n i f i c a n t n e g a t i v e r e l a t i o n s h i p was observed between the mean body weights of the p o p u l a t i o n samples and the magnitude of t h e i r i n b r e e d i n g c o e f f i c i e n t s . However, h i s data showed tha t the 20 p o p u l a t i o n s c o u l d be e q u a l l y p a r t i t i o n e d i n t o two d i s t i n c t groups ; ones y i e l d i n g h i g h F v a l u e s , and o thers w i t h i n b r e e d i n g c o e f f i c i e n t s c l o s e to u n i t y . In n e a r l y a l l samples , he terozygous i n d i v i d u a l s were l a r g e r than homozygotes, i r r e s p e c t i v e of the extent of the apparent i n b r e e d i n g . I f the i n b r e e d i n g h y p o t h e s i s i s c o r r e c t , t h i s p a t t e r n s h o u l d have been observed o n l y i n the p o p u l a t i o n s e x h i b i t i n g h i g h F v a l u e s . The f a c t t h a t i t was p r e s e n t i n a l l samples shows t h a t i n b r e e d i n g per se c o u l d not have been r e s p o n s i b l e f o r the l a r g e r body weights of he terozygous o y s t e r s . My study has demonstrated t h a t overdominance f o r enzyme a c t i v i t y i s expres sed by the t h r e e most common h e t e r o z y g o t e s at the Pqm-2 l o c u s i n C r a s s o s t r e a g i g a s . The magnitude of t h i s overdominance was n e a r l y i d e n t i c a l i n both the mantle and a d d u c t o r muscle t i s s u e s , and was c o n s i s t e n t l y observed at both i n t e r t i d a l l o c a t i o n s sampled i n three d i f f e r e n t s easons . Since, a l l e l e f r e q u e n c i e s at t h i s l o c u s show o n l y l i m i t e d i n t e r -p o p u l a t i o n a l d i f f e r e n t i a t i o n ( c o n f i n e d to the three l e s s f requent a l l e l e s ) , these same genotypes must have been r e s p o n s i b l e for the i n c r e a s e d s i z e of Pgm-2 h e t e r o z y g o t e s r e p o r t e d in F u j i o ' s s t u d y . These r e s u l t s are unique i n p r o v i d i n g 238 ev idence f a v o r i n g the overdominance e x p l a n a t i o n f o r one l o c u s i n v o l v e d i n a p o s i t i v e a s s o c i a t i o n between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and growth r a t e . The enzyme a c t i v i t y l e v e l s expressed by d i f f e r e n t Pgm-2 homozygotes and h e t e r o z y g o t e s were i n c o m p a t i b l e w i t h the p r e d i c t e d e f f e c t s of an undetec ted n u l l a l l e l e , and the a s s o c i a t i v e overdominance h y p o t h e s i s , i n v o k i n g u n i d e n t i f i e d t i g h t l y - l i n k e d l o c i s e g r e g a t i n g for d e l e t e r i o u s r e c e s s i v e s , may t h e r e f o r e be deemed u n n e c e s s a r y . The u n d e r l y i n g mechanism p r o d u c i n g the overdominant PGM a c t i v i t i e s of h e t e r o z y g o t e s f o r the Pqm-2-100 a l l e l e i s unknown. A l t h o u g h e n t i r e l y s p e c u l a t i v e , the overdominant po lymorphic " r e g u l a t o r y " l o c u s model d i s c u s s e d i n Chapter 3 i s i n t u i t i v e l y a p p e a l i n g because i t can s i m u l t a n e o u s l y e x p l a i n the enzyme a c t i v i t y l e v e l s of ten Pgm-2 genotypes , a l l e l i c f requency d i s t r i b u t i o n s i n n a t u r a l p o p u l a t i o n s , and the maintenance of t h i s g e n e t i c v a r i a t i o n in a b a l a n c e d s t a t e , by d i s t i l l i n g the m u l t i - a l l e l i c system i n t o a two a l l e l e polymorphism e x h i b i t i n g overdominance . However, a s s o c i a t e d wi th the overdominance observed at the Pgm-2 l o c u s were p l e i o t r o p i c e f f e c t s on the s o l u b l e p r o t e i n l e v e l s measured i n both t i s s u e s and the mantle a c t i v i t i e s of the a d j a c e n t enzymes of the g lycogen s y n t h e s i s pathway. These r e s u l t s suggest tha t h e t e r o z y g o s i t y i n the v i c i n i t y of l o c i s cored i n m u l t i p l e - l o c u s s t u d i e s c o u l d e x e r t a number of m e t a b o l i c e f f e c t s tha t may ac t to c o m p l i c a t e the e x p l a n a t i o n ( s ) f o r the c o r r e l a t i o n s o b s e r v e d . 239 Genotype-dependent enzyme a c t i v i t y v a r i a t i o n at the Pqm-2 l o c u s was shown i n Chapter 4 to be d i r e c t l y a s s o c i a t e d w i t h d i f f e r i n g g lycogen c o n c e n t r a t i o n s i n the m a n t l e , but not the adduc tor muscle t i s s u e . Complex Pgm-2 genotype by environment i n t e r a c t i o n s were observed i n the a n a l y s i s of the mantle g lycogen d a t a : the r e l a t i v e l e v e l s measured i n homozygotes and h e t e r o z y g o t e s r e v e r s e d between the f a l l and w i n t e r , and over the two i n t e r t i d a l p o s i t i o n s w i t h i n the summer sample . In a d d i t i o n to d e m o n s t r a t i n g a p h y s i o l o g i c a l impact of t h i s po lymorphism, a compar i son of the mantle g lycogen c o n c e n t r a t i o n s of Pgm-2 genotypes a l l o w s an assessment of the r e l a t i v e importance of k i n e t i c v e r s u s enzyme a c t i v i t y v a r i a t i o n a t t h i s l o c u s . For the s p e c i f i c a c t i v i t y d a t a , h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e behaved as a homogeneous group i n a l l p o p u l a t i o n samples; the four homozygotes a c t e d as another homogeneous s p e c i f i c a c t i v i t y g r o u p . W i t h i n both of these g r o u p s , genotypes p o s s e s s i n g the Pgm -2-92 a l l o z y m e may have been expected to enjoy a c a t a l y t i c advantage over the o t h e r s due to the s u p e r i o r k i n e t i c p r o p e r t i e s of t h i s v a r i a n t shown i n Chapter 2. However, the g lycogen c o n c e n t r a t i o n s of homozygotes and h e t e r o z y g o t e s d i f f e r e d i n a manner c o n s i s t e n t o n l y w i t h t h e i r enzyme a c t i v i t y d i f f e r e n c e s : genotypes hav ing the Pgm-2-92 a l l e l e showed a b s o l u t e l y no tendency to ou tper form o t h e r s as expected from t h e i r advantageous k i n e t i c p r o p e r t i e s . These r e s u l t s , combined w i t h low o b s e r v e d frequency of the Pgm-2-92 a l l e l e , argue tha t the a l l o z y m e s themselves c o u l d be n e u t r a l markers of the g e n e t i c d i f f e r e n c e s that g i v e r i s e to the d i f f e r e n c e s i n s p e c i f i c 240 a c t i v i t y . The a d a p t i v e - s i g n i f i c a n c e of t h i s overdominance would be r e a l i z e d o n l y i f i t , in t u r n , a f f e c t e d the r e l a t i v e f i t n e s s e s of Pgm-2 genotypes . Through i t s e f f e c t s on mantle g lycogen l e v e l s , t h i s polymorphism has the p o t e n t i a l to i n f l u e n c e two important f i t n e s s components: v i a b i l i t y and f e c u n d i t y . The d i f f e r e n t i a l a b i l i t i e s of Pqm-2 genotypes to s y n t h e s i z e g lycogen r e s e r v e s c o u l d d i r e c t l y a f f e c t v i a b i l i t y , p a r t i c u l a r l y under adverse e n v i r o n m e n t a l c o n d i t i o n s when t h i s c a r b o h y d r a t e i s important as a f u e l f o r s u p p o r t i n g b a s a l metabol i sm (de Zwaan 1983). T h i s e f f e c t on v i a b i l i t y s h o u l d be more pronounced i n immature o y s t e r s , which u t i l i z e a much h i g h e r p r o p o r t i o n of t h e i r g lycogen s t o r e s d u r i n g the w i n t e r months ( H o l l a n d and Hannant 1976). S i n c e g lycogen has been s t r o n g l y i m p l i c a t e d as the major energy source f o r gametogenesis (Gabbott 1983), h e t e r o z y g o t e s f o r the Pqm-2-100 a l l e l e c o u l d a l s o enjoy a net f e c u n d i t y advantage . The a l l o c a t i o n of energy between somatic growth and gamete p r o d u c t i o n has been shown by Rodhouse (1978) to s h i f t towards the l a t t e r as marine b i v a l v e s i n c r e a s e in age . The impact of t h i s polymorphism on f e c u n d i t y i s thus expected to be more s i g n i f i c a n t in o l d e r i n d i v i d u a l s . I n t e r e s t i n g l y , an e f f e c t of h e t e r o z y g o s i t y at the Pgm l o c u s has been d e s c r i b e d for both f i t n e s s components; on v i a b i l i t y to 2 and 3 y e a r s of age i n C r a s s o s t r e a v i r g i n i c a (Zouros et a l . 1983), and on the f e c u n d i t y of the l a r g e s t s i z e c l a s s i n M y t i l u s e d u l i s (Rodhouse et a l . 1986). 241 In C r a s s o s t r e a q i g a s , m u l t i p l e - l o c u s h e t e r o z y g o s i t y has been s i g n i f i c a n t l y c o r r e l a t e d o n l y w i t h a d u l t body w e i g h t s . S i n c e these r e s u l t s were o b t a i n e d by p o o l i n g 20 p o p u l a t i o n s of d i f f e r i n g age s t r u c t u r e s , i t cannot be de termined i f h e t e r o z y g o s i t y was a s s o c i a t e d w i t h d i f f e r e n t i a l growth r a t e s or v i a b i l i t i e s . In o ther s p e c i e s of marine b i v a l v e s , m u l t i p l e - l o c u s h e t e r o z y g o s i t y has been c o r r e l a t e d w i t h a number of p h y s i o l o g i c a l parameters which suggest t h a t he terozygous i n d i v i d u a l s are m e t a b o l i c a l l y more e f f i c i e n t than homozygotes . T h i s e n e r g e t i c advantage i s presumably r e f l e c t e d i n the n e g a t i v e r e l a t i o n s h i p s between h e t e r o z y g o s i t y and r a t e s of oxygen consumption (Koehn and Shumway 1982; G a r t o n , Koehn and S c o t t 1984; D i e h l et a l . 1985) and p r o t e i n t u r n o v e r (Hawkins, Bayne and Day 1986), lower r a t e s of weight l o s s under n u t r i t i v e s t r e s s (Rodhouse and Gaffney 1984), and the o b s e r v a t i o n t h a t i n some i n s t a n c e s growth r a t e s are s i g n i f i c a n t l y c o r r e l a t e d w i t h h e t e r o z y g o s i t y o n l y under more s t r e s s f u l e n v i r o n m e n t a l c o n d i t i o n s (Green et a l . 1983; G e n t i l i and Beaumont 1988). U n d e r s t a n d i n g the b i o c h e m i c a l mechanism(s) r e s p o n s i b l e for the i n c r e a s e d m e t a b o l i c e f f i c i e n c y of enzyme h e t e r o z y g o t e s may thus p r o v i d e a f o u n d a t i o n f o r the e x p l a n a t i o n of these r e l a t i o n s h i p s in b i v a l v e s and o ther s p e c i e s . R e c e n t l y , Koehn, D i e h l and S c o t t (1988) examined the r e l a t i v e c o n t r i b u t i o n of 15 l o c i to a p o s i t i v e c o r r e l a t i o n between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and growth r a t e i n a 242 s i n g l e c o h o r t of the coot c l a m , M u l i n i a l a t e r a l i s . T h i s s tudy was unique i n d e m o n s t r a t i n g a s i g n i f i c a n t c o n t r i b u t i o n by enzymes t h a t f u n c t i o n e d i n g l y c o l y s i s and p r o t e i n c a t a b o l i s m , but not f o r those i n v o l v e d i n m a i n t a i n i n g c e l l u l a r redox b a l a n c e or the g e n e r a t i o n of r e d u c i n g e q u i v a l e n t s . To account f o r these r e s u l t s and to p r o v i d e a g e n e r a l e x p l a n a t i o n f o r the e f f e c t s of enzyme h e t e r o z y g o s i t y , these a u t h o r s proposed tha t (p . 128): " . . . the c o s t of pathway maintenance ( i . e . , b i o s y n t h e s i s and d e g r a d a t i o n of c o n s t i t u e n t enzymes) i s i n v e r s e l y r e l a t e d to the magnitude of c a t a l y t i c v a r i a t i o n among the sequence of r e a c t i o n s i n a pathway." T h i s e x p l a n a t i o n p r e d i c t s t h a t more he terozygous b i o c h e m i c a l pathways are m a i n t a i n e d by lower e x p e n d i t u r e s of energy , thus r e s u l t i n g i n a more e f f i c i e n t p h y s i o l o g i c a l phenotype . T h i s m e t a b o l i c advantage of a l l e l i c d i v e r s i t y i s an e x t e n s i o n of ideas f i r s t deve loped by E a s t (1936) , L e r n e r (1954) and Berger (1976) . The r e s u l t s from the presen t s tudy may be i n t e r p r e t e d i n a manner c o n s i s t e n t w i t h the above p r e m i s e s , but d i f f e r i n the e x p r e s s i o n of t h i s overdominance . Koehn, D i e h l and S c o t t (1988), a c t i n g on the w e l l - f o u n d e d b a s i s of h e t e r o z y g o t e i n t e r m e d i a c y , suggested tha t the "advantage" of h e t e r o z y g o s i s a r i s e s from the "averag ing out" of the d i f f e r i n g c a t a l y t i c performances of the two homozygotes ( i . e . m a r g i n a l overdominance ) . The minor k i n e t i c d i f f e r e n t i a t i o n e x h i b i t e d by the four most common Pgm-2 a l l o z y m e s shown in Chapter 2 appears to p r e c l u d e t h i s mechanism 243 from o p e r a t i n g at t h i s l o c u s . I n s t e a d , the g r e a t e r s p e c i f i c a c t i v i t i e s of the Pgm-2-92/100, 96/100, and 100/104 h e t e r o z y g o t e s has p r o v i d e d d i r e c t ev idence f o r the e x p r e s s i o n of u n c o n d i t i o n a l b i o c h e m i c a l overdominance . Because t h i s enzyme i s a monomer, the l a r g e r enzyme a c t i v i t i e s of these h e t e r o z y g o t e s are most e a s i l y e n v i s a g e d as a r i s i n g through i n c r e a s e d l e v e l s of PGM enzyme. S t e a d y - s t a t e enzyme c o n c e n t r a t i o n s are a c h i e v e d through a b a l a n c e between the oppos ing r a t e s of s y n t h e s i s and d e g r a d a t i o n (Goldberg and S t . John 1976). I f the overdominant enzyme a c t i v i t i e s r e s u l t from i n c r e a s e d s y n t h e t i c r a t e s , h e t e r o z y g o t e s f o r the Pqm-2-100 a l l e l e would be expected to e x p e r i e n c e h i g h e r , r a t h e r than l ower , e n e r g e t i c r e q u i r e m e n t s . However, i f s y n t h e t i c r a t e s a r e the same f o r a l l genotypes , but the he terozygous enzyme ensembles are degraded at lower r a t e s , overdominant enzyme a c t i v i t y l e v e l s would r e s u l t through the reduced e x p e n d i t u r e of c e l l u l a r energy r e s e r v e s . The i n c r e a s e d m e t a b o l i c e f f i c i e n c y of these genotypes would in t u r n be r e f l e c t e d i n t h e i r i n c r e a s e d r a t e s of growth and a l s o i n d e c r e a s e d r a t e s of oxygen consumpt ion . These arguments suggest t h a t the overdominance observed a t the Pgm-2 l o c u s may not be the cause of the i n c r e a s e d " e f f i c i e n c y " of h e t e r o z y g o t e s , but r a t h e r an e f f e c t of an a l t e r e d f e a t u r e of r o u t i n e m e t a b o l i s m , such as p r o t e i n t u r n o v e r . The h i g h e r Vmax/Km r a t i o s of the overdominant Pgm-2 h e t e r o z y g o t e s c o u l d produce a more e f f i c i e n t phenotype o n l y through 1) re sponding more e f f e c t i v e l y to the f l u x r a t e s 244 d i c t a t e d by r e g u l a t o r y enzymes l i k e g lycogen s y n t h e t a s e , or 2) s imply c a t a l y z i n g the i n t e r c o n v e r s i o n of G1P and G6P more e f f i c i e n t l y as suggested by the t h e o r e t i c a l a n a l y s i s of Watt (1986) . The e n e r g e t i c advantage t h a t would r e s u l t from e i t h e r mechanism i s unknown, but p r o b a b l y much lower than reduced b i o s y n t h e t i c c o s t s p r e d i c t e d by the a l t e r n a t i v e e x p l a n a t i o n . I f the m e t a b o l i c advantage of h e t e r o z y g o s i t y at the Pgm-2 l o c u s r e s u l t e d s o l e l y from a d e c r e a s e d r a t e of p r o t e i n t u r n o v e r , no d i r e c t e f f e c t of t h i s polymorphism i s n e c e s s a r i l y p r e d i c t e d on g lycogen metabo l i sm. A l t h o u g h the i n c r e a s e d e f f i c i e n c y of these h e t e r o z y g o t e s c o u l d i n d i r e c t l y r e s u l t i n h i g h e r mantle g lycogen c o n c e n t r a t i o n s , t h i s was not a c o n s i s t e n t o b s e r v a t i o n of Chapter 4. The s t r o n g r e l a t i o n s h i p between PGM a c t i v i t y v a r i a t i o n and g lycogen l e v e l s i n the f a l l argues for a major impact of t h i s polymorphism on m e t a b o l i c f l u x , as expected from the p a r t i t i o n i n g of s u b s t r a t e at the g l u c o s e - 6 - p h o s p h a t e branch p o i n t . T h e r e f o r e , a d u a l advantage might r e s u l t from h e t e r o z y g o s i t y in the r e g i o n of the Pgm-2 l o c u s i n C r a s s o s t r e a  g i g a s . F i r s t , an i n d i r e c t e f f e c t from reduced r a t e s of p r o t e i n d e g r a d a t i o n ( r e s u l t i n g i n the e x p r e s s i o n of overdominant enzyme a c t i v i t i e s ) , and second, a d i r e c t impact on v i a b i l i t y or f e c u n d i t y through the e f f e c t of t h i s v a r i a t i o n i n enzyme a c t i v i t y on g lycogen m e t a b o l i s m . The e x p r e s s i o n of overdominant enzyme a c t i v i t i e s by h e t e r o z y g o t e s f o r the Pgm-2-100 a l l e l e i s ex tremely u n u s u a l , and 245 thus u n l i k e l y to p r o v i d e a g e n e r a l e x p l a n a t i o n for the a s s o c i a t i o n s between m u l t i p l e - l o c u s h e t e r o z y g o s i t y and f i t n e s s -r e l a t e d t r a i t s i n marine b i v a l v e s . H e t e r o z y g o s i t y at the PGI l o c u s i n C r a s s o s t r e a v i r g i n i c a has been i n v o l v e d i n p o s i t i v e c o r r e l a t i o n s w i t h both growth r a t e (S ingh and Zouros 1978; Z o u r o s , S ingh and M i l e s 1980) and v i a b i l i t y (Zouros et a l . 1983). M a r t i n (1979) examined the b i o c h e m i c a l p r o p e r t i e s of these PGI a l l o z y m e s and found s i g n i f i c a n t d i f f e r e n c e s between the t h r e e most common genotypes i n t h e i r s p e c i f i c enzyme a c t i v i t i e s . However, h e t e r o z y g o t e s e x h i b i t e d s t r i c t i n t e r m e d i a c y in t h e i r a c t i v i t y l e v e l s tha t were i n t e r p r e t e d as be ing advantageous i n the face of f l u c t u a t i n g e n v i r o n m e n t a l c o n d i t i o n s . Given these g e n o t y p i c r e l a t i o n s h i p s , h e t e r o z y g o s i t y at the PGI l o c u s i n C . v i r g i n i c a c o u l d f i t i n t o the g e n e r a l scheme proposed by Koehn, D i e h l and S c o t t (1988) . A p a r t i c u l a r l y w e l l - s t u d i e d polymorphism i n M. e d u l i s , a t which h e t e r o z y g o s i t y has been i m p l i c a t e d i n r e l a t i o n s h i p s w i t h growth r a t e (Koehn and Gaf fney 1984), m o r p h o l o g i c a l v a r i a t i o n ( M i t t o n and Koehn 1985), v i a b i l i t y ( D i e h l and Koehn 1985) and oxygen consumption ( D i e h l et a l . 1985), i n v o l v e s the aminopept idase -1 (Lap) l o c u s . Genotypes p o s s e s s i n g the Lap-94 a l l e l e e x h i b i t the g r e a t e s t enzyme a c t i v i t i e s i n h i g h s a l i n i t y environments (Koehn and Immerman 1981) caused by the g r e a t e r kcat of t h i s a l l e l i c v a r i a n t (Koehn and S i e b e n a l l e r 1981). A p h y s i o l o g i c a l e f f e c t on o s m o r e g u l a t i o n of t h i s genotype-dependent v a r i a t i o n i n a c t i v i t y has been documented ( H i l b i s h , 246 Deaton and Koehn 1982) which can e x p l a i n the a d a p t i v e s i g n i f i c a n c e of t h i s polymorphism (rev iewed by Koehn and H i l b i s h 1987). H i l b i s h and Koehn (1985) have demonstrated t h a t dominance i s expres sed a t t h i s l o c u s a t bo th the b i o c h e m i c a l and p h y s i o l o g i c a l l e v e l s : Lap genotypes form two homogeneous groups depending on the presence or absence of the 94 a l l e l e . T h i s i s a p a r t i c u l a r l y important r e s u l t , s i n c e the p o o l i n g of the L a p -94/94 genotype wi th the two low a c t i v i t y homozygotes in m u l t i p l e - l o c u s s t u d i e s , would r e s u l t in the apparent e x p r e s s i o n of overdominance by h e t e r o z y g o t e s f o r the Lap-94 a l l e l e a l t h o u g h the u n d e r l y i n g mechanism i s s t r i c t dominance . The p r e v a l e n c e of s i m i l a r dominance r e l a t i o n s h i p s at o t h e r p o l y m o r p h i c l o c i i n v o l v e d i n these c o r r e l a t i o n s i s unknown. F o l t z (1986a, 1986b) has r e c e n t l y d e t e c t e d n u l l a l l e l e s s e g r e g a t i n g at the Lap l o c u s in C r a s s o s t r e a v i r g i n i c a . The r e s u l t i n g m i s c l a s s i f i c a t i o n of n u l l h e t e r o z y g o t e s as homozygotes has the p o t e n t i a l to s i m u l t a n e o u s l y e x p l a i n the h e t e r o z y g o t e d e f i c i e n c i e s and s u p e r i o r performance of h e t e r o z y g o t e s for two f u n c t i o n a l a l l e l e s r e p o r t e d i n e a r l i e r s t u d i e s (S ingh and Zouros 1978; Z o u r o s , S ingh and M i l e s 1980; F o l t z et a l . 1983). The r e s u l t s of the present s tudy and the g e n o t y p i c r e l a t i o n s h i p s at these three o ther l o c i s t r o n g l y suggest t h a t a s i n g l e g e n e t i c mechanism may not be r e s p o n s i b l e f o r these c o r r e l a t i o n s i n v o l v i n g m u l t i p l e - l o c u s h e t e r o z y g o s i t y . R a t h e r , a f u l l e x p l a n a t i o n for these p a t t e r n s may have to depend on separate l o c u s - s p e c i f i c s t u d i e s . A l t h o u g h d e l a y i n g the u l t i m a t e g o a l of 247 e x p l a i n i n g these s t i m u l a t i n g o b s e r v a t i o n s , t h i s approach w i l l undoubted ly i n c r e a s e our u n d e r s t a n d i n g of the f u n c t i o n a l r e l e v a n c e of enzyme po lymorphisms , the i n t r i c a c i e s of the s e l e c t i v e p r o c e s s , and the e v o l u t i o n of complex b i o c h e m i c a l sys tems. 248 LITERATURE CITED Alemany, M. , and M. R o s e l l - P e r e z . 1973. Two d i f f e r e n t amylase a c t i v i t i e s i n the sea mussel M y t i l u s e d u l i s L . Rev. E x p . F i s i o l . 29:217-222. A l l e n d o r f , F . W . , R .F . . L e a r y , and K . L . Knudsen. 1983. S t r u c t u r a l and r e g u l a t o r y v a r i a t i o n of phosphoglucomutase in Rainbow t r o u t . Isozymes C u r r . T o p . B i o l . Med. Res . 9:123-142. A t k i n s o n , D . E . 1977. C e l l u l a r Energy M e t a b o l i s m and i t s R e g u l a t i o n . Academic P r e s s , N . Y . Aquadro , C . F . , S . F . Desse , M . M . B l a n d , C H . L a n g l e y , and C . C . L a u r i e - A h l b e r g . 1986. M o l e c u l a r p o p u l a t i o n g e n e t i c s of the a l c o h o l dehydrogenase gene r e g i o n of D r o s o p h i l a  m e l a n o g a s t e r . G e n e t i c s 114:1165-1190. A y a l a , F . J . , M . L . T r a c e y , L . G . B a r r , J . F . McDonald , and S. P e r e z - S a l a s . 1974. G e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s of f i v e D r o s o p h i l a s p e c i e s and the h y p o t h e s i s of the s e l e c t i v e n e u t r a l i t y of p r o t e i n po lymorphisms . G e n e t i c s 77:343-384. B a r n e s , P . T . , and C . C . L a u r i e - A h l b e r g . .1986. G e n e t i c v a r i a b i l i t y of f l i g h t metabol i sm i n D r o s o p h i l a m e l a n o g a s t e r . I I I . E f f e c t s of Gpdh a l l o z y m e s and e n v i r o n m e n t a l temperature on power o u t p u t . G e n e t i c s 112:267-294. Bayne, B . L . 1976. A s p e c t s of r e p r o d u c t i o n i n marine b i v a l v e s . I_n : E s t u a r i n e P r o c e s s e s , V o l I . U s e s , S t r e s s e s , and A d a p t a t i o n to the E s t u a r y . E d i t e d by M. W i l e y . Academic P r e s s , N . Y . pp . 432-448. Bayne, B . L . , A . B u b e l , P . A . G a b b o t t , D . R . L i v i n g s t o n e , D . M . Lowe, and M . N . Moore . 1982. Glycogen u t i l i s a t i o n and gametogenesis i n M y t i l u s e d u l i s L . Mar . B i o l . L e t t . 2:£39~ 105. Bayne, B . L . , and R . C . N e w e l l . 1983. P h y s i o l o g i c a l e n e r g e t i c s of marine m o l l u s c s . I_n: The M o l l u s c a , V o l . 4. P h y s i o l o g y , P a r t 1. E d i t e d by A . S . M . S a l e u d d i n and K . M . W i l b u r . Academic P r e s s , N . Y . p p . 407-515. 249 B e a t t i e , J . H . , J . Perdue , W. H e r s h b e r g e r , and K . Chew. 1987. E f f e c t s of i n b r e e d i n g on growth i n the P a c i f i c o y s t e r ( C r a s s o s t r e a g i g a s ) . J . S h e l l f i s h Res . £ : 2 5 - 2 8 . Beaumont, A . R . , and C M . B e v e r i d g e . 1983. R e s o l u t i o n of phosphoglucomutase isozymes i n M y t i l u s e d u l i s L . Mar . B i o l . L e t t . 4:97-103. B e i t n e r , R. 1984. C o n t r o l of l e v e l s of g l u c o s e 1 , 6 - b i s p h o s p h a t e . I n t . J . Biochem. 16:579-585. B e i t n e r , R . , S. Haberman, and J . Nordenberg . 1978. The e f f e c t of e p i n e p h r i n e and d i b u t y r y l c y c l i c AMP on g l u c o s e 1,6-b i sphosphate l e v e l s and the a c t i v i t i e s of h e x o k i n a s e , p h o s p h o f r u c t o k i n a s e and phosphoglucomutase i n the i s o l a t e d r a t d iaphragm. M o l . C e l l . E n d o c r i n . 10:135-147. B e l o t e , J . , and J . C . L u c c h e s i . 1980. C o n t r o l of X chromosome t r a n s c r i p t i o n by the m a l e l e s s gene i n D r o s o p h i l a . Nature 285:573-575. B e n t l e y , M . M . , and J . H . W i l l i a m s o n . 1979. The c o n t r o l of a ldehyde ox idase and x a n t h i n e dehydrogenase a c t i v i t i e s by the cinnamon gene i n D r o s o p h i l a m e l a n o g a s t e r . C a n . J . Genet . C y t o l . 2±: 457-471. B e r g e r , E . 1976. H e t e r o s i s and the maintenance of enzyme po lymorphism. Am. N a t . 110:823-839. Bergmeyer, H . U . . 1974a. H e x o k i n a s e . I_n: Methods of Enzymat ic A n a l y s i s , 2nd E d i t i o n . E d i t e d by H . U . Bergmeyer. Academic P r e s s , N . Y . pp . 473-474. Bergmeyer, H . U . 1974b. U r i d i n e d i p h o s p h o g l u c o s e p y r o p h o s p h o r y l a s e . I_n: Methods of Enzymat ic A n a l y s i s , 2nd E d i t i o n . E d i t e d by H . U . Bergmeyer. Academic P r e s s , N . Y . p p . 519-520. Bergmeyer, H . U . , and G . M i c h a l . 1974. D - g l u c o s e - 1 - p h o s p h a t e . I n : Methods of Enzymat ic A n a l y s i s , 2nd E d i t i o n . E d i t e d by H . U . Bergmeyer. Academic P r e s s , N . Y . pp . 1233-1237. 250 B r a d f o r d , M.M. 1976. A r a p i d and s e n s i t i v e method f o r the q u a n t i t a t i o n of microgram q u a n t i t i e s of p r o t e i n u t i l i z i n g the p r i n c i p l e of p r o t e i n - d y e b i n d i n g . A n a l . Biochem. 72: 248-254. Buroker, N.E. 1975. A survey of p r o t e i n v a r i a t i o n i n p o p u l a t i o n s of the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s . M.Sc. T h e s i s , U n i v e r s i t y of Washington. Buroker, N.E. 1979. Overdominance of a muscle p r o t e i n (Mp-1) l o c u s i n the Japanese o y s t e r , C r a s s o s t r e a gigas ( O s t r e i d a e ) . J . F i s h . Res. Board Can. 36:1313-1318. Buroker, N.E. 1983. S e x u a l i t y with r e s p e c t to s h e l l l e n g t h and group s i z e i n the Japanese o y s t e r C r a s s o s t r e a g i g a s . M a l a c o l o g i a 23:271-279. Buroker, N.E., W.K. Hershberger, and K.K. Chew. 1979a. P o p u l a t i o n g e n e t i c s of the f a m i l y O s t r e i d a e . I. I n t r a s p e c i f i c s t u d i e s of C r a s s o s t r e a gigas and S a c c o s t r e a  commercialis. Mar. B i o l . 54:157-169. Buroker, N.E., W.K. Hershberger, and K.K. Chew. 1979b. P o p u l a t i o n g e n e t i c s of the f a m i l y O s t r e i d a e . I I . I n t e r s p e c i f i c s t u d i e s of the genera C r a s s o s t r e a and S a c c o s t r e a . Mar. B i o l . 54:171-184. Burton, R.S., and M.W. Feldman. 1983. P h y s i o l o g i c a l e f f e c t s of an allozyme polymorphism: glutamate-pyruvate transaminase and response to hyperosmotic s t r e s s i n the copepod T i g r i o p u s c a l i f o r n i c u s . Biochem. Genet. 21:239-251. Burton, R.S., and A.R. P l a c e . 1986. E v o l u t i o n of s e l e c t i v e n e u t r a l i t y : f u r t h e r c o n s i d e r a t i o n s . G e n e t i c s 114:1033-1 036. Bush, R.M., P.E. Smouse, and F.T. L e d i g . 1987. The f i t n e s s consequences of m u l t i p l e - l o c u s h e t e r o z y g o s i t y : the r e l a t i o n s h i p between h e t e r o z y g o s i t y and growth r a t e i n P i t c h pine (Pinus r i g i d a M i l l . ) . E v o l u t i o n 4J_:787-798. Cavener, D.R., and M..T. Clegg. 1981. Evidence f o r b i o c h e m i c a l and p h y s i o l o g i c a l d i f f e r e n c e s between enzyme genotypes i n D r o s o p h i l a melanogaster. Proc. N a t l . Acad. S c i . U.S.A. 78: 4444-4447. 251 C h a k r a b o r t y , R. 1981. The d i s t r i b u t i o n of the number of he terozygous l o c i i n an i n d i v i d u a l i n n a t u r a l p o p u l a t i o n s . G e n e t i c s 98:461-466. Chambers, J . E . , F . M . M c C o r k l e , J . W . C a r r o l l , J . R . H e i t z , L . L e w i s , and J . D . Y a r b r o u g h . 1975. V a r i a t i o n i n enzyme a c t i v i t i e s of the American o y s t e r ( C r a s s o s t r e a v i r g i n i c a ) r e l a t i v e to s i z e and season . Comp. Biochem. P h y s i o l . 51B: 145-150. C h i h , C P . , and W.R. E l l i n g t o n . 1986. C o n t r o l of g l y c o l y s i s d u r i n g c o n t r a c t i l e a c t i v i t y i n the p h a s i c adductor muscle of the bay s c a l l o p , Argopec ten i r r a d i a n s c o n c e n t r i c u s : i d e n t i f i c a t i o n of p o t e n t i a l s i t e s of r e g u l a t i o n and a c o n s i d e r a t i o n of the r o l e of o c t o p i n e dehydrogenase a c t i v i t y . P h y s i o l . Z o o l . 59:563-573. C l a r k , R . L . , E . B o e r w i n k l e , G . J . Brewer, and C . F . S i n g . 1983. S t u d i e s of enzyme polymorphism i n the Kamuela p o p u l a t i o n of D r o s o p h i l a mercatorum. I I I . E f f e c t s of v a r i a t i o n a t the a lpha-GPD l o c u s and s u b f l i g h t s t r e s s on the energy charge and g l y c o l y t i c i n t e r m e d i a t e c o n c e n t r a t i o n s . G e n e t i c s 104: 661-675. C l a r k e , B . 1975. The c o n t r i b u t i o n of e c o l o g i c a l g e n e t i c s to e v o l u t i o n a r y t h e o r y : d e t e c t i n g the d i r e c t e f f e c t s of n a t u r a l s e l e c t i o n on p a r t i c u l a r po lymorphic l o c i . G e n e t i c s 79:101-113. C l a r k e , B.. 1979. The e v o l u t i o n of g e n e t i c d i v e r s i t y . P r o c . R. S o c . L o n d . B 205:453-474. C l e l a n d , W.W. 1963. The k i n e t i c s of e n z y m e - c a t a l y z e d r e a c t i o n s w i t h two or more s u b s t r a t e s or p r o d u c t s . B i o c h i m . B i o p h y s . A c t a 67:104-137. Cohen, P . 1978. The r o l e of c y c l i c - A M P - d e p e n d e n t p r o t e i n k inase i n the r e g u l a t i o n of g lycogen metabol i sm i n mammalian s k e l e t a l m usc l e . C u r r . T o p . C e l l . Reg. 14:117—196. Cohen, P . 1983. P r o t e i n p h o s p h o r y l a t i o n and the c o n t r o l of g lycogen metabol i sm i n s k e l e t a l musc l e . P h i l . T r a n s . R. Soc . L o n d . B 302:13-25. 252 Cohen, P . 1986. Musc le g lycogen s y n t h a s e . I_n: The Enzymes, V o l X V I I . E d i t e d by P . D . Boyer and E . G . K r e b s . Academic P r e s s , O r l a n d o , p p . 461-497. C o l o w i c k , S . P . , and E . W . S u t h e r l a n d . 1942. P o l y s a c c h a r i d e s y n t h e s i s from g l u c o s e by means of p u r i f i e d enzymes. J . B i o l . Chem. 144:423-437. Cook, P . A . , and P . A . G a b b o t t . 1978. Glycogen s y n t h e t a s e i n the sea mussel M y t i l u s e d u l i s L . I . P u r i f i c a t i o n , i n t e r c o n v e r s i o n and k i n e t i c p r o p e r t i e s of the I and D forms . Comp. Biochem. P h y s i o l . 60B:419-421 . C o r n i s h - B o w d e n , A . 1985. A f o r t r a n program for robus t r e g r e s s i o n of enzyme k i n e t i c d a t a . Techn iques in the L i f e S c i e n c e s . B I / I I Supplement. P r o t e i n and Enzyme B i o c h e m i s t r y BS115:1 -22. Coyne, J . 1976. Lack of genie s i m i l a r i t y between two s i b l i n g s p e c i e s of D r o s o p h i l a as r e v e a l e d by v a r i e d t e c h n i q u e s . G e n e t i c s 84:593-607. C u r r i e , D . J . 1982. E s t i m a t i n g M i c h a e l i s - M e n t e n p a r a m e t e r s : b i a s , v a r i a n c e and e x p e r i m e n t a l d e s i g n . B i o m e t r i c s 38:907-919. Czok , R . , and T h . B u c h e r . 1960. C r y s t a l l i z e d enzymes from the myogen of r a b b i t s k e l e t a l m u s c l e . Adv . P r o t e i n Chem. 15:. 315-415. . D a n f o r t h , N . D . , and J . A . Beardmore. 1979. B i o c h e m i c a l p r o p e r t i e s of e s t e r a s e 6 i n D r o s o p h i l a m e l a n o g a s t e r . B iochem. Genet . 17:1-22. D a n f o r t h , W.H. 1965. Glycogen synthe tase a c t i v i t y i n s k e l e t a l m u s c l e . I n t e r c o n v e r s i o n of two forms and c o n t r o l of g lycogen s y n t h e s i s . J . B i o l . Chem. 240:588-593. D a r w i n , C . 1859. On the O r i g i n of S p e c i e s by Means of N a t u r a l S e l e c t i o n or the P r e s e r v a t i o n of Favored Races i n the S t r u g g l e for L i f e . M u r r a y , London . 253 Dean, A . M . , D . E . D y k h u i z e n , and D . L . H a r t l . 1986. F i t n e s s as a f u n c t i o n of 0 - g a l a c t o s i d a s e a c t i v i t y i n E s c h e r i c h i a c o l i . Genet . R e s . , Camb. 4 8 : 1 - 8 . De Wulf , H . , and H . G . H e r s . 1967. The s t i m u l a t i o n of g lycogen s y n t h e s i s and of g lycogen syn the tase i n the l i v e r by the a d m i n i s t r a t i o n of g l u c o s e . E u r . J . Biochem. 2:50-56 . D i c k i n s o n , W . J . 1975. A g e n e t i c l o c u s a f f e c t i n g the deve lopmenta l e x p r e s s i o n of an enzyme i n D r o s o p h i l a  m e l a n o g a s t e r . Dev. B i o l . 42:131 -140 . D i c k i n s o n , W . J . , R . G . Rowan, and M . D . Brennan . 1984. R e g u l a t o r y gene e v o l u t i o n : a d a p t i v e d i f f e r e n c e s i n e x p r e s s i o n of a l c o h o l dehydrogenases i n D r o s o p h i l a melanogaster and D r o s o p h i l a s i m u l a n s . H e r e d i t y 52:215-225. D i e h l , W . J . , P . M . G a f f n e y , J . H . McDonald , and R . K . Koehn. 1985. R e l a t i o n s h i p between w e i g h t - s t a n d a r d i z e d oxygen consumption and m u l t i p l e - l o c u s h e t e r o z y g o s i t y i n the m u s s e l , M y t i l u s e d u l i s . I n : P r o c . 19th E u r . mar. b i o l . Symp. E d i t e d by P . G i b b s . Cambridge U n i v e r s i t y P r e s s , Cambridge , pp . 529-534. D i e h l , W . J . , and R . K . Koehn. 1985. M u l t i p l e - l o c u s h e t e r o z y g o s i t y , m o r t a l i t y , and growth i n a c o h o r t of M y t i l u s e d u l i s . Mar . B i o l . 88:265-271. D i M i c h e l e , L . , and D . A . Powers, ,1982a. P h y s i o l o g i c a l b a s i s f o r swimming endurance d i f f e r e n c e s between LDH-B genotypes of Fundulus h e t e r o c l i t u s . S c i e n c e 216:1014-1016. D i M i c h e l e , L . , and D . A . Powers. 1982b. LDH-B g e n o t y p e - s p e c i f i c h a t c h i n g t imes of Fundulus h e t e r o c l i t u s embryos. Nature 296:563-564. Doane, W.W. , L . G . T r e a t - C l e m o n s , R . M . G e m m i l l , J . N . L e v y , S . A . Hawley, A . M . Buchberg , and K. P a i g e n . 1983. G e n e t i c mechanism f o r the t i s s u e - s p e c i f i c c o n t r o l of a l p h a - a m y l a s e e x p r e s s i o n i n D r o s o p h i l a m e l a n o g a s t e r . Isozymes C u r r . T o p . B i o l . Med. Res . 9 :63-90 . 254 Dobzhansky, T h . 1937. G e n e t i c s and the O r i g i n of S p e c i e s . Columbia U n i v e r s i t y P r e s s , N . Y . Dobzhansky, T h . 1955. A review of some fundamental concept s and problems of p o p u l a t i o n g e n e t i c s . C o l d S p r i n g Harbor Symp. Quant . B i o l . 20 :1 -15 . Dobzhansky, T h . , and H . Levene . 1948. G e n e t i c s of n a t u r a l p o p u l a t i o n s . X V I I . Proof of o p e r a t i o n of n a t u r a l s e l e c t i o n i n w i l d p o p u l a t i o n s of D r o s o p h i l a p s e u d o o b s c u r a . G e n e t i c s 33:537-547. Dobzhansky, T h . , and H . Levene . 1951. Development of h e t e r o s i s through n a t u r a l s e l e c t i o n i n e x p e r i m e n t a l p o p u l a t i o n s of D r o s o p h i l a p s e u d o o b s c u r a . Am. N a t . 85:247-264. Dugg leby , R . G . 1979. E x p e r i m e n t a l d e s i g n s for e s t i m a t i n g the k i n e t i c parameters f o r e n z y m e - c a t a l y s e d r e a c t i o n s . J . t h e o r . B i o l . 8J_: 671 -684 . D y k h u i z e n , D . E . , A . M . Dean, and D . L . H a r t l . 1987. M e t a b o l i c f l u x and f i t n e s s . G e n e t i c s 115:25-31 . D y k h u i z e n , D . E . , de Framond, and D . L . H a r t l . 1984. S e l e c t i v e n e u t r a l i t y of g l u c o s e - 6 - p h o s p h a t e dehydrogenase a l l o z y m e s i n E s c h e r i c h i a c o l i . M o l . B i o l . E v o l . 162-170. D y k h u i z e n , D . E . , and D . L . H a r t l . 1980. S e l e c t i v e n e u t r a l i t y of 6PGDH a l l o z y m e s i n E . c o l i and the e f f e c t s of the g e n e t i c background . G e n e t i c s 96:801-817. D y k h u i z e n , D . E . , and D . L . H a r t l . 1983. F u n c t i o n a l e f f e c t s of PGI a l l o z y m e s i n E s c h e r i c h i a c o l i . G e n e t i c s 105:1-18. E a n e s , W . F . 1984. V i a b i l i t y i n t e r a c t i o n s , in v i v o a c t i v i t y and the G6PD polymorphism i n D r o s o p h i l a m e l a n o g a s t e r . G e n e t i c s 106:95-107. E a s t , E . M . 1936. H e t e r o s i s . G e n e t i c s 21:375-397. 255 E b b e r i n k , R . H . M . , and A . de Zwaan. 1980. C o n t r o l of g l y c o l y s i s i n the p o s t e r i o r adduc tor muscle of the sea mussel M y t i l u s  e d u l i s . J . Comp. P h y s i o l . 137:165-171. E b b e r i n k , R . H . M . , and M. S a l i m a n s . 1982. C o n t r o l of g lycogen p h o s p h o r y l a s e a c t i v i t y i n the p o s t e r i o r adduc tor muscle of the sea mussel M y t i l u s e d u l i s . J . Comp. P h y s i o l . 148:27-33. E b l e , A . F . 1969. A h i s t o c h e m i c a l d e m o n s t r a t i o n of g l y c o g e n , g lycogen p h o s p h o r y l a s e and b r a n c h i n g enzyme i n the American o y s t e r . P r o c . N a t l . S h e l l f i s h . A s s o c . 59:13-25 . E i s e n t h a l , R . , and A . C o r n i s h - B o w d e n . 1974. The d i r e c t l i n e a r p l o t . A new g r a p h i c a l proced u re for e s t i m a t i n g enzyme k i n e t i c p a r a m e t e r s . Biochem. J . 139:715-720. E n d r e n y i , L . , and F . - Y . Chan . 1981. O p t i m a l d e s i g n s of exper iments for the e s t i m a t i o n of p r e c i s e h y p e r b o l i c k i n e t i c and b i n d i n g p a r a m e t e r s . J . t h e o r . B i o l . 90:241-263. E s t e l l e , M . A . , and R . B . H o d g e t t s . 1984. G e n e t i c e lements near the s t r u c t u r a l gene modulate the l e v e l of dopa d e c a r b o x y l a s e d u r i n g D r o s o p h i l a deve lopment . M o l . Gen. Genet . 195:434-441. Ewens, W . J . 1977. P o p u l a t i o n g e n e t i c s theory i n r e l a t i o n to the n e u t r a l i s t - s e l e c t i o n i s t c o n t r o v e r s y . Adv . Hum. Genet . 8: 67-134. F e l s e n s t e i . n , J . 1976. The t h e o r e t i c a l p o p u l a t i o n g e n e t i c s of v a r i a b l e s e l e c t i o n and m i g r a t i o n . Ann . Rev. Genet . 10:253-280. F i s h e r , R . A . 1922. On the dominance r a t i o . P r o c . Roy. S o c . E d i n . 42:321-341 . F l e t t e r i c k , R . J . , and N . B . Madsen. 1980. The s t r u c t u r e s and r e l a t e d f u n c t i o n s of p h o s p h o r y l a s e a . A n n . Rev. Biochem. 49:31-61 . 256 F o l t z , D.W. 1986a. N u l l a l l e l e s as a p o s s i b l e cause of h e t e r o z y g o t e d e f i c i e n c i e s i n the o y s t e r C r a s s o s t r e a  v i r g i n i c a and o t h e r b i v a l v e s . E v o l u t i o n 40:869-870. F o l t z , D.W. 1986b. S e g r e g a t i o n and l i n k a g e s t u d i e s of a l lozyme l o c i i n p a i r c r o s s e s of the o y s t e r C r a s s o s t r e a v i r g i n i c a . Biochem. Genet . 24:941-956. F o r d , E . B . 1965. E c o l o g i c a l G e n e t i c s , 3rd E d i t i o n . John Wi l ey and Sons , N . Y . F u c c i , L . , L . G a u d i o , R. Rao, A . Spano, and M. C a r f a g n a . 1979. P r o p e r t i e s of the two common e l e c t r o p h o r e t i c v a r i a n t s of phosphoglucomutase i n D r o s o p h i l a m e l a n o g a s t e r . Biochem. G e n e t . 17:825-836. F u j i o , Y . 1982. A c o r r e l a t i o n of h e t e r o z y g o s i t y w i t h growth r a t e i n the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s . Tohoku J . A g r i c . Res . 33:66-75 . F u j i o , Y . , Y . Nakamura, and M. S u g i t a . 1979. S e l e c t i v e advantage of h e t e r o z y g o t e s at c a t a l a s e l o c u s i n the P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s . J a p a n . J . G e n e t . 54:359-366. G a b b o t t , P . A . 1975. S torage c y c l e s i n marine b i v a l v e m o l l u s c s : a h y p o t h e s i s c o n c e r n i n g the r e l a t i o n s h i p between g lycogen metabo l i sm and gametogenes i s . I_n: P r o c . 9th E u r . mar. b i o l . Symp. E d i t e d by H . B a r n e s . Aberdeen U n i v e r s i t y P r e s s , Aberdeen , p p . 191-211. G a b b o t t , P . A . 1976. Energy M e t a b o l i s m . I n : Mar ine M u s s e l s : T h e i r E c o l o g y and P h y s i o l o g y . E d i t e d by B . L . Bayne, Cambridge U n i v e r s i t y P r e s s , Cambridge , p p . 293-356. G a b b o t t , P . A . 1983. Deve lopmenta l and seasona l m e t a b o l i c a c t i v i t i e s i n marine m o l l u s c s . I_n: The M o l l u s c a , V o l . 2. E n v i r o n m e n t a l B i o c h e m i s t r y and P h y s i o l o g y . E d i t e d by P.W. Hochachka . Academic P r e s s , N . Y . pp . 165-217. G a b b o t t , P . A . , P . A . Cook, and A . M . W h i t t l e . 1979. Seasona l changes in g lycogen synthase a c t i v i t y i n the mantle t i s s u e of the mussel M y t i l u s e d u l i s L . : r e g u l a t i o n by t i s s u e g l u c o s e . Biochem. Soc . T r a n s . 7:895-896. 257 G a b b o t t , P . A . , and M . A . W h i t t l e . 1986a. G lycogen syn the tase i n the sea mussel M y t i l u s e d u l i s L . - I I . Seasona l changes in g l y c o g e n content and g lycogen synthe tase a c t i v i t y i n the mant le t i s s u e . Comp. Biochem. P h y s i o l . 83B:197-207. G a b b o t t , P . A . , and M . A . W h i t t l e . 1986b. Glycogen syn the tase i n the sea mussel M y t i l u s e d u l i s L . - I I I . ' R e g u l a t i o n by g l u c o s e i n a mantle t i s s u e s l i c e p r e p a r a t i o n . Comp. Biochem. P h y s i o l . 8_3B: 209-2 1 4. G a f f n e y , P . M . , and T . M . S c o t t . 1984.. G e n e t i c h e t e r o z y g o s i t y and p r o d u c t i o n t r a i t s i n n a t u r a l and h a t c h e r y p o p u l a t i o n s of b i v a l v e s . A q u a c u l t u r e 42:289-302. G a l t s o f f , P . S . 1964. The American O y s t e r C r a s s o s t r e a v i r g i n i c a G m e l i n . F i s h e r y B u l l e t i n of the F i s h and W i l d l i f e S e r v i c e . V o l . 64, Washington D . C . G a r t o n , D.W. 1984. R e l a t i o n s h i p between m u l t i p l e l o c u s h e t e r o z y g o s i t y and p h y s i o l o g i c a l e n e r g e t i c s of growth in the e s t u a r i n e g a s t r o p o d T h a i s haemastoma. P h y s i o l . Z o o l . 57:530-543. G a r t o n , D . W . , R . K . Koehn, and T . M . S c o t t . 1984. M u l t i p l e - l o c u s h e t e r o z y g o s i t y and p h y s i o l o g i c a l e n e r g e t i c s of growth i n the coo t c l a m , M u l i n i a l a t e r a l i s , from a n a t u r a l p o p u l a t i o n . G e n e t i c s 108:445-455. G a u l d i e , R.W. 1984. A r e c i p r o c a l r e l a t i o n s h i p between h e t e r o z y g o s i t i e s of the phosphoglucomutase and g l u c o s e phosphate isomerase l o c i . G e n e t i c a 63:93-103 . G e n t i l i , M . R . , and A . R . Beaumont. 1988. E n v i r o n m e n t a l s t r e s s , h e t e r o z y g o s i t y , and growth r a t e i n M y t i l u s e d u l i s L . J . exp . mar. B i o l . E c o l . 120:145-153. G i b s o n , J . B . , A . V . W i l k s , A . Cao , and A . L . F r e e t h . 1986. Dominance for s n - g l y c e r o l - 3 - p h o s p h a t e dehydrogenase a c t i v i t y i n D r o s o p h i l a me lanogas ter : ev idence f o r d i f f e r e n t i a l a l l e l i c e x p r e s s i o n mediated v i a a t r a n s -a c t i n g e f f e c t . H e r e d i t y 56:227-235. 258 G i l l e s p i e , J . H . 1977. A g e n e r a l model to account for enzyme v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . I I I . M u l t i p l e a l l e l e s . E v o l u t i o n 31:85-90 . G i l l e s p i e , J . H . 1978. A g e n e r a l model to account for enzyme v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . V . The SAS-CFF model . T h e o r . Pop. B i o l . 14:1-45. G i l l e s p i e , J . H . , and C H . L a n g l e y . 1974. A g e n e r a l model to account f o r enzyme v a r i a t i o n i n n a t u r a l p o p u l a t i o n s . G e n e t i c s 76:837-848. G o l d b e r g , A . L . , and A . C S t . J o h n . 1976. I n t r a c e l l u l a r p r o t e i n d e g r a d a t i o n in mammalian and b a c t e r i a l c e l l s : p a r t 2. Ann . Rev. Biochem. 45:747-803. Goromosova, S . A . 1976. Glycogen syn the tase and f r u c t o s e d i p h o s p h a t a s e a c t i v i t y in the t i s s u e s of M u l l u s k s ( s i c ) ( M y t i l u s q a l l o p r o v i n c i a l i s ) and C r u s t a c e a n s (Balanus  i m p r o v i s u s , C a r e i n u s maenas) . J . E v o l . Biochem. P h y s i o l . 12:331-334. G o u l d , S . J . 1983. The h a r d e n i n g of the modern s y n t h e s i s . I n : Dimensions of Darwin i sm. E d i t e d by M. G r e n e . Cambridge U n i v e r s i t y P r e s s , Cambridge , pp . 71-93. G o u l d , S . J . , and R . C L e w o n t i n . 1979. The s p a n d r e l s of San Marco and the P a n g l o s s i a n parad igm: a c r i t i q u e of the a d a p t a t i o n i s t programme. P r o c . Roy. Soc . London B 205:581-598. Gowen, J . W . ( e d . ) . 1952. H e t e r o s i s . H a f n e r , N . Y . G r e e n , R . H . , S . M . S i n g h , B . H i c k s , and J . M . M c C u a i g . 1983. An a r c t i c i n t e r t i d a l p o p u l a t i o n of Macoma b a l t h i c a ( M o l l u s c a , P e l e c y p o d a ) : g e n o t y p i c and p h e n o t y p i c components of p o p u l a t i o n s t r u c t u r e . C a n . J . F i s h . A q u a t . S c i . 40:1360-1371 . H a l d a n e , J . B . S . 1930. Enzymes. Longmans G r e e n , London. 259 H a l e y , L . E . 1978. Sex d e t e r m i n a t i o n in the American o y s t e r . J . H e r e d i t y 68:114-116. H a l l , J . G . 1985. T e m p e r a t u r e - r e l a t e d k i n e t i c d i f f e r e n t i a t i o n of g lucosephosphate i somerase a l l e l o e n z y m e s i s o l a t e d from the b l u e m u s s e l , M y t i l u s e d u l i s . Biochem. G e n e t . 23:705-728. Hanabusa, K . , H.W. Dougher ty , C . d e l R i o , T . Hashimoto , and P. H a n d l e r . 1966. Phosphoglucomutase . I I . P r e p a r a t i o n and p r o p e r t i e s of phosphoglucomutases from M i c r o c o c c u s  l y s o d e i k t i c u s and B a c i l l u s c e r e u s . J . B i o l . Chem. 241: 3930-3939. H a r r i s , H . 1975. P r i n c i p l e s of Human B i o c h e m i c a l G e n e t i c s . N o r t h H o l l a n d , Amsterdam. H a r t l , D . L , and D . E . D y k h u i z e n . 1981. P o t e n t i a l f or s e l e c t i o n among n e a r l y n e u t r a l a l l o z y m e s of 6 -phosphogluconate dehydrogenase i n E s c h e r i c h i a c o l i . P r o c . N a t l . A c a d . S c i . U . S . A . 78:6344-6348. H a r t l , D . L . , D . E . D y k h u i z e n , and A . M . Dean. 1985. L i m i t s of a d a p t a t i o n : the e v o l u t i o n of s e l e c t i v e n e u t r a l i t y . G e n e t i c s 111:655-674. Hashimoto , T . , : - C . d e l R i o , and P . H a n d l e r . 1966. Comparat ive s t r u c t u r e and f u n c t i o n of phosphoglucomutases . F e d . P r o c . 25:408. Hashimoto , T . , and P . H a n d l e r . 1966. Phosphoglucomutase . I I I . P u r i f i c a t i o n and p r o p e r t i e s of phosphoglucomutases from f l o u n d e r and shark m usc l e . J . B i o l . Chem. 241:3940-3948. Hawkins , A . J . S . , B . L . Bayne, and A . J . Day. 1986. P r o t e i n t u r n o v e r , p h y s i o l o g i c a l e n e r g e t i c s and h e t e r o z y g o s i t y i n the b lue m u s s e l , M y t i l u s e d u l i s : the b a s i s of v a r i a b l e a g e - s p e c i f i c growth . P r o c . R. Soc . L o n d . B 229:161-176. Hay, R . E . , and F . B . A r m s t r o n g . 1976. B i o c h e m i c a l c h a r a c t e r i z a t i o n of a l l e l i c forms of s o l u b l e malate dehydrogenase of D r o s o p h i l a m e l a n o g a s t e r . Insec t Biochem. 6:367-376. 260 H a z e l , J . R . , and C . L . P r o s s e r . 1974. M o l e c u l a r mechanisms of temperature compensat ion i n p o i k i l o t h e r m s . P h y s i o l . Rev. 54:620-677. H e d r i c k , P.W. 1986. G e n e t i c polymorphism i n heterogeneous env ironments : a decade l a t e r . Ann . Rev. E c o l . S y s t . 17: 535-566. H e d r i c k , P . W . , M . E . G i n e v a n , and E . P . Ewing . 1976. G e n e t i c polymorphism i n heterogeneous env i ronments . Ann . Rev. E c o l . S y s t . 9 :1 -32 . H e i n s t r a , P . W . H . , W. S c h a r l o o , and G . E . W . T h o r i g . 1987. P h y s i o l o g i c a l s i g n i f i c a n c e of the a l c o h o l dehydrogenase polymorphism i n l a r v a e of D r o s o p h i l a . G e n e t i c s 117:75-84. Hems, D . A . , and P . D . W h i t t o n . 1980. C o n t r o l of h e p a t i c g l y c o g e n o l y s i s . P h y s i o l . Rev . 60:1-50 . H e r s , H . G . 1976. The c o n t r o l of g lycogen metabol i sm i n the l i v e r . A n n . Rev. Biochem. 45:167-189. H e r s , H . G . , H . De W u l f , and W. S ta lmans . 1970. The c o n t r o l of g lycogen metabol i sm i n the l i v e r . FEBS L e t t . 12:73-82. H i c k e y , D . A . 1977. S e l e c t i o n f o r amylase a l l o z y m e s i n D r o s o p h i l a  m e l a n o g a s t e r . E v o l u t i o n 31:800-804. H i l b i s h , T . J . , Deaton , T . E . , and R . K . Koehn. 1982. E f f e c t of an a l l o z y m e polymorphism on r e g u l a t i o n of c e l l volume. Nature 298:688-689. H i l b i s h , T . J . , and R . K . Koehn. 1985. Dominance i n p h y s i o l o g i c a l phenotypes and f i t n e s s at an enzyme l o c u s . S c i e n c e 229:52-54. H i n o , A . , E . Tazawa, and I . Yasumasu. 1978. Two pathways from g lycogen to g i u c o s e - 6 - p h o s p h a t e in sea u r c h i n eggs w i t h s p e c i a l r e f e r e n c e to the d i f f e r e n c e between the s p e c i e s ( s i c ) i n the c o n t r i b u t i o n r a t i o of each pathway. Gamete Res . 1:117-128. 261 Hoag land , K . E . 1978. P r o t a n d r y and the e v o l u t i o n of e n v i r o n m e n t a l l y - m e d i a t e d sex change: a s tudy of the M o l l u s c a . M a l a c o l o g i a 17:365-391. Hochachka, P . W . , and T . M u s t a f a . 1972. I n v e r t e b r a t e f a c u l t a t i v e a n a e r o b i o s i s . S c i e n c e 178:1056-1060. Hoffman, R . J . 1981. E v o l u t i o n a r y g e n e t i c s of M e t r i d i u m s e n i l e . I . K i n e t i c d i f f e r e n c e s in phosphoglucose i somerase a l l o z y m e s . Biochem. Genet . 19:129-144• Hoffman, R . J . 1985. P r o p e r t i e s of a l l e l i c v a r i a n t s of phosphoglucomutase from the sea anemone Metr id iurn s e n i l e . Biochem. Genet . 23:859-876. H o l l a n d , D . L . , and P . J . Hannant . 1976. The g lycogen c o n t e n t i n w i n t e r and summer of o y s t e r s O s t r e a e d u l i s L . , of d i f f e r e n t ages . J . C o n s . i n t . E x p l o r . Mer 36:240-242. . H o o r n , A . J . W . , and W. S c h a r l o o . 1978. The f u n c t i o n a l s i g n i f i c a n c e of amylase polymorphism in D r o s o p h i l a  m e l a n o g a s t e r . I . P r o p e r t i e s of two amylase v a r i a n t s . G e n e t i c a 49:173-180. Hue, L . , and H . G . H e r s . 1974. U t i l e and f u t i l e c y c l e s i n the l i v e r . Biochem. B i o p h y s . Res . Commun. 58:540-548• J o s h i , J . G . , and P. H a n d l e r . 1964. Phosphoglucomutase . I . P u r i f i c a t i o n and p r o p e r t i e s of phosphoglucomutase from E s c h e r i c h i a c o l i . J . B i o l . Chem. 239:2741-2751. J o s h i , J . G . , and P. H a n d l e r . 1969. Phosphoglucomutase . V I . P u r i f i c a t i o n and p r o p e r t i e s of phosphoglucomutases from human musc l e . J . B i o l . Chem. 244:3343-3351. J o s h i , J . G . , J . Hooper , T . Kuwaki , T . S a k u r a d a , J . R . Swanson, and P . H a n d l e r . 1967. Phosphoglucomutase , V . M u l t i p l e forms of phosphoglucomutase . P r o c . N a t l . A c a d . S c i . U . S . A . 57:1482-1489. K a c s e r , H . 1983. The c o n t r o l of enzyme systems _in v i v o : e l a s t i c i t y a n a l y s i s of the s teady s t a t e . Biochem. Soc . T r a n s . 11:35-40. 262 K a c s e r , H . , and J . A . B u r n s . 1973. The c o n t r o l of f l u x . I n : Rate C o n t r o l of B i o l o g i c a l P r o c e s s e s . E d i t e d by D . D . D a v i e s . Symp. S o c . E x p . B i o l . 27:65-104. K a c s e r , H . , and J . A . B u r n s . 1979. M o l e c u l a r democracy: who shares the c o n t r o l s ? Biochem. S o c . T r a n s . 7:1149-1160. K a c s e r , H . , and J . A . B u r n s . 1981. The m o l e c u l a r b a s i s of dominance. G e n e t i c s 97:639-666. K a t o h , M . , and D.W. F o l t z . 1987. L e u c i n e aminopept idase s p e c i f i c a c t i v i t y i s s i g n i f i c a n t l y reduced i n Lap n u l l he terozygous o y s t e r s ( C r a s s o s t r e a v i r g i n i c a ) . G e n e t i c s 1 1 6 ( S u p p l ) : s 4 4 . K e p p l e r , D . , and K. D e c k e r . 1974. Glycogen d e t e r m i n a t i o n w i t h a m y l o g l u c o s i d a s e . Ijn: Methods of Enzymat ic A n a l y s i s , 2nd E d i t i o n . E d i t e d by H . U . Bergmeyer. Academic P r e s s , N . Y . p p . 1127-1131. K i m u r a , M. 1983. The N e u t r a l Theory of M o l e c u l a r E v o l u t i o n . Cambridge U n i v e r s i t y P r e s s , Cambridge . K i n g , J . J . , and J . F . McDonald . 1983. G e n e t i c l o c a l i z a t i o n and b i o c h e m i c a l c h a r a c t e r i z a t i o n of a t r a n s - a c t i n g r e g u l a t o r y e f f e c t i n D r o s o p h i l a . G e n e t i c s 105:55-69. K i n g , J . J . , and J . F . . M c D o n a l d . 1987. P o s t - t r a n s l a t i o n a l c o n t r o l of a l c o h o l dehydrogenase l e v e l s i n D r o s o p h i l a melanogaster . G e n e t i c s 115:693-699. Koehn, R . K . 1969. E s t e r a s e h e t e r o g e n e i t y : dynamics of a po lymorphism. S c i e n c e 163:943-944 . Koehn, R . K . 1978. P h y s i o l o g y and b i o c h e m i s t r y of enzyme v a r i a t i o n : the i n t e r f a c e of eco logy and p o p u l a t i o n g e n e t i c s . I_n: E c o l o g i c a l G e n e t i c s : The I n t e r f a c e . E d i t e d by P . F . B r u s s a r d , S p r i n g e r - V e r l a g , N . Y . p p . 51-72. Koehn, R . K . , W . J . D i e h l , and T . M . S c o t t . 1988. The d i f f e r e n t i a l c o n t r i b u t i o n by i n d i v i d u a l enzymes of g l y c o l y s i s and p r o t e i n c a t a b o l i s m to the r e l a t i o n s h i p between h e t e r o z y g o s i t y and growth r a t e in the coot c l a m , M u l i n i a l a t e r a l i s . G e n e t i c s 118:121-130. 263 Koehn, R . K . , and P . M . G a f f n e y . 1984. G e n e t i c h e t e r o z y g o s i t y and growth r a t e i n M y t i l u s e d u l i s . M a r . B i o l . 8 2 : 1 - 7 . Koehn, R . K . , and T . J . H i l b i s h . 1987. The a d a p t i v e s i g n i f i c a n c e of g e n e t i c v a r i a t i o n . Am. S c i . 75:134-141. Koehn, R . K . , and F . W . Immerman. 1981. B i o c h e m i c a l s t u d i e s of aminopept idase polymorphism i n M y t i l u s e d u l i s . I . Dependence of enzyme a c t i v i t y on season, t i s s u e and genotype . Biochem. Genet . 19:1115-1142. Koehn, R . K . , R . I . E . N e w e l l , and F . W . Immerman. 1980. Maintenance of an aminopept idase a l l e l e f requency c l i n e by n a t u r a l s e l e c t i o n . P r o c . N a t l . A c a d . S c i . U . S . A . 77:5385-5389. Koehn, R . K . , J . E . P e r e z , and R . B . M e r r i t t . 1971. E s t e r a s e enzyme f u n c t i o n and g e n e t i c a l s t r u c t u r e of p o p u l a t i o n s of the f re shwater f i s h , N o t r o p i s s t r a m i n e u s . Am. N a t . 105:51-69. Koehn, R . K . , and S . E . Shumway. 1982. A g e n e t i c / p h y s i o l o g i c a l e x p l a n a t i o n f o r d i f f e r e n t i a l growth r a t e among i n d i v i d u a l s of the American o y s t e r , C r a s s o s t r e a v i r g i n i c a ( G m e l i n ) . M a r . B i o l . L e t t . 3 :35-42 . Koehn, R . K . , and J . F . S i e b e n a l l e r . 1981. B i o c h e m i c a l s t u d i e s on aminopept idase polymorphism i n M y t i l u s e d u l i s . I I . Dependence of r e a c t i o n r a t e on p h y s i c a l f a c t o r s and enzyme c o n c e n t r a t i o n . Biochem. Genet . 19:1143-1162. Koehn, R . K . , A . J . Z e r a , and J . G . H a l l . 1983. Enzyme polymorphism and n a t u r a l s e l e c t i o n . I n : E v o l u t i o n of Genes and P r o t e i n s . E d i t e d by M. Nei and R . K . Koehn. S i n a u e r , S u n d e r l a n d , Mass . pp . 115-136. K r i s t j a n s s o n , F . D . 1963. G e n e t i c c o n t r o l of two p r e - a l b u m i n s i n p i g s . G e n e t i c s 48:1059-1063. L - F a n d o , J . J . , M . C . G a r c i a - F e r n a n d e z , and J . L . R - C a n d e l a . 1972. Glycogen metabol i sm i n O s t r e a e d u l i s ( L . ) - f a c t o r s a f f e c t i n g g lycogen s y n t h e s i s . Comp. Biochem. P h y s i o l . 43B: 807-814. 264 L a i , Y . - K . , and J . G . S c a n d a l i o s . 1980. G e n e t i c d e t e r m i n a t i o n of the deve lopmenta l program f o r maize s c u t e l l a r a l c o h o l dehydrogenase: involvement of a r e c e s s i v e , t r a n s - a c t i n g , t empora l r e g u l a t o r y gene. Dev. Genet . _1_:31 1-324. L a n g , G . , and G . M i c h a l . 1974. D - g l u c o s e - 6 - p h o s p h a t e and D-f r u c t o s e - 6 - p h o s p h a t e . I_n: Methods of Enzymat ic A n a l y s i s , 2nd E d i t i o n . E d i t e d by H . U . Bergmeyer. Academic P r e s s , N . Y . p p . 1238-1242. L a n g l e y , C . H . , A . E . S h r i m p t o n , T . Yamazaki , N . M i y a s h i t a , Y . Matsuo , and C F . Aquadro . 1988. N a t u r a l l y o c c u r r i n g v a r i a t i o n i n the r e s t r i c t i o n map of the Amy r e g i o n of D r o s o p h i l a m e l a n o g a s t e r . G e n e t i c s 119:619-629. L a P o r t e , D . C , K . Walsh , and D . E . K o s h l a n d , J r . 1984. The branch p o i n t e f f e c t . U l t r a s e n s i t i v i t y and s u b s e n s i t i v i t y to m e t a b o l i c c o n t r o l . J . B i o l . Chem. 269:14068-14075. L a t t e r , B . D . H . 1975. Enzyme polymorphisms: gene frequency d i s t r i b u t i o n s w i t h muta t ion and s e l e c t i o n for o p t i m a l a c t i v i t y . G e n e t i c s 79:325-331 . L a u r i e - A h l b e r g , C . C 1985. G e n e t i c v a r i a t i o n a f f e c t i n g the e x p r e s s i o n of enzyme c o d i n g genes in D r o s o p h i l a : an e v o l u t i o n a r y p e r s p e c t i v e . Isozymes C u r r . T o p . B i o l . Med. Res . 12:33-88. L a u r i e - A h l b e r g , C . C , G . M a r o n i , G . C Bewley, J . C L u c c h e s i , and B . S . W e i r . 1980. Q u a n t i t i v e g e n e t i c v a r i a t i o n of enzyme a c t i v i t i e s i n n a t u r a l p o p u l a t i o n s of D r o s o p h i l a  m e l a n o g a s t e r . P r o c . N a t l . A c a d . S c i . . U . S . A . 77:1 073-1 077. L a u r i e - A h l b e r g , C . C , J . H . W i l l i a m s o n , B . J . Cochrane , A . N . W i l t o n , and F . I . Chasalow. 1981. Autosomal f a c t o r s w i t h c o r r e l a t e d e f f e c t s on the a c t i v i t i e s of the g l u c o s e - 6 -phosphate and 6-phosphogluconate dehydrogenases i n D r o s o p h i l a me lanogas ter . G e n e t i c s 99:127-150. L a u r i e - A h l b e r g , C . C . , A . N . W i l t o n , J . W . K u r t s i n g e r , and T . M . Emigh. 1982. N a t u r a l l y o c c u r r i n g enzyme a c t i v i t y v a r i a t i o n i n . D r o s o p h i l a m e l a n o g a s t e r . I . Sources of v a r i a t i o n f o r 23 enzymes. G e n e t i c s 102:191-206. 265 L e i g h Brown, A . J . 1977. P h y s i o l o g i c a l c o r r e l a t e s of an enzyme po lymorphi sm. Nature 269:803-804. L e r n e r , I . M . 1 954. G e n e t i c Homeos tas i s . O l i v e r and Boyd, E d i n b u r g h . L e w o n t i n , R . C . 1972. T e s t i n g the t h e o r y of n a t u r a l s e l e c t i o n . Nature 236:181-182. L e w o n t i n , R . C . 1974. The G e n e t i c B a s i s of E v o l u t i o n a r y Change. Columbia U n i v e r s i t y P r e s s , N . Y . L e w o n t i n , R . C , L . R . G i n z b u r g , and S . D . T u l j a p u r k a r . 1978. H e t e r o s i s as an e x p l a n a t i o n f o r l a r g e amounts of genie po lymorphism. G e n e t i c s 88:149-170. L i v i n g s t o n e , D . R . 1975. A comparison of the k i n e t i c p r o p e r t i e s of p y r u v a t e k inase i n three p o p u l a t i o n s of M y t i l u s e d u l i s L . from d i f f e r e n t e n v i r o n m e n t s . I_n: P r o c . 9th E u r o p . mar. b i o l . Symp. E d i t e d by H . B a r n e s . Aberdeen U n i v e r s i t y P r e s s , Aberdeen , p p . 151-164. L i v i n g s t o n e , D . R . 1976. Some k i n e t i c and r e g u l a t o r y p r o p e r t i e s of L - m a l a t e dehydrogenases from the p o s t e r i o r adduc tor muscle and mantle t i s s u e s of the common mussel M y t i l u s e d u l i s . Biochem. S o c . T r a n s . 4:447-451. L i v i n g s t o n e , D . R . 1981. I n d u c t i o n of enzymes as a mechanism for the s e a s o n a l c o n t r o l of metabol i sm i n marine i n v e r t e b r a t e s : g l u c o s e - 6 - p h o s p h a t e dehydrogenases from the mantle and hepatopancreas of the common mussel M y t i l u s  e d u l i s . Comp. Biochem. P h y s i o l . 69B:147-156. L i v i n g s t o n e , D . R . , and K . R . C l a r k e . 1983. Seasona l changes in hexokinase from the mantle t i s s u e of the common mussel M y t i l u s . e d u l i s L . Comp. Biochem. P h y s i o l . 74B:69 l -702 . L o n g w e l l , A . C , and S . S . S t i l e s . 1973. Gamete c r o s s i n c o m p a t i b i l i t y and i n b r e e d i n g i n the commerc ia l American o y s t e r , C r a s s o s t r e a v i r g i n i c a G m e l i n . C y t o l o g i a 38:521-533. 2 6 6 Lowry, O.H., and J.V. Passoneau. 1 9 6 9 . Phosphoglucomutase k i n e t i c s w i t h the phosphates of f r u c t o s e , g l u c o s e , mannose, r i b o s e , and g a l a c t o s e . J . B i o l . Chem. 2 4 4 ; 9 1 0 -9 1 6 . L u s i s , A . J . , V.M. Chapman, R.W. Wangenstein, and K. P a i g e n . 1 9 8 3 . T r a n s - a c t i n g t e m p o r a l l o c u s w i t h i n the 0-g l u c u r o n i d a s e gene complex. P r o c . N a t l . Acad. S c i . U.S.A. 8 0 : 4 3 9 8 - 4 4 0 2 . Madsen, N.B. 1 9 8 6 . Glycogen p h o s p h o r y l a s e . I n : The Enzymes, V o l . X V I I . E d i t e d by P.D. Boyer and E.G. Kre b s . Academic P r e s s , O r l a n d o , pp. 3 6 5 - 3 9 4 . Mandel, S.P.H. 1 9 5 9 . The s t a b i l i t y of a m u l t i p l e a l l e l i c system. H e r e d i t y 2 1 : 2 8 9 - 3 0 2 . M a r t i n , J.P. 1 9 7 9 . B i o c h e m i c a l , e c o l o g i c a l and g e n e t i c s t u d i e s on g l u c o s e - 6 - p h o s p h a t e isomerase a l l o z y m e s and a c c l i m a t i o n s t u d i e s on a d d u c t o r muscle p h o s p h o f r u c t o k i n a s e of the American o y s t e r , C r a s s o s t r e a v i r g i n i c a ( G m e l i n ) . Ph.D. D i s s e r t a t i o n , Duke U n i v e r s i t y . Mayr, E. 1 9 8 2 . The Growth of B i o l o g i c a l Thought. B e l k n a p , Cambridge, Mass. McDonald, J.F. 1 9 8 3 . The m o l e c u l a r b a s i s of a d a p t a t i o n : a c r i t i c a l r e v i e w of r e l e v a n t i d e a s and o b s e r v a t i o n s . Ann. Rev. E c o l . S y s t . J _ 4 : 7 7 - 1 0 2 . McDonald, J.F., S.M. Anderson, and M. San t o s . 1 9 8 0 . B i o c h e m i c a l d i f f e r e n c e s between p r o d u c t s of the Adh l o c u s i n D r o s o p h i l a . G e n e t i c s 9 5 : 1 0 1 3 - 1 0 2 2 . M e r r i t t , R.B. 1 9 7 2 . Geographic d i s t r i b u t i o n and en z y m a t i c p r o p e r t i e s of l a c t a t e dehydrogenase a l l o z y m e s i n the f a t h e a d minnow Pimephales promelas . Am. Nat. 1 0 6 : 1 7 3 - 1 8 4 . M i d d l e t o n , R.J., and H. K a c s e r . 1 9 8 3 . Enzyme v a r i a t i o n , m e t a b o l i c f l u x and f i t n e s s : a l c o h o l dehydrogenase i n D r o s o p h i l a m e l a n o g a s t e r . G e n e t i c s 1 0 5 : 6 3 3 - 6 5 0 . 267 M i l s t e i n , C , and F . Sanger . 1961. An amino a c i d sequence i n the a c t i v e c e n t r e of phosphoglucomutase . Biochem. J . 79;456-469. M i t t o n , J . B . , C . C a r e y , and T . D . K o c h e r . 1986. The r e l a t i o n of enzyme h e t e r o z y g o s i t y to s t a n d a r d and a c t i v e oxygen consumption and body s i z e of t i g e r sa lamanders , Ambystoma  t i g r i n u m . P h y s i o l . Z o o l . 59:574-582. M i t t o n , J . B . , and R . K . Koehn. 1985. S h e l l shape v a r i a t i o n i n the b l u e m u s s e l , M y t i l u s e d u l i s L . , and i t s a s s o c i a t i o n w i t h enzyme h e t e r o z y g o s i t y . J . exp . mar. B i o l . E c o l . 90:73-80. M i t t o n , J . B . , and M . C . G r a n t . 1984. A s s o c i a t i o n s among p r o t e i n h e t e r o z y g o s i t y , growth r a t e , and deve lopmenta l h o m e o s t a s i s . A n n . Rev. E c o l . S y s t . 15:479-499. M i t t o n , J . B . , and B . A . P i e r c e . 1980. The d i s t r i b u t i o n of i n d i v i d u a l h e t e r o z y g o s i t y i n n a t u r a l p o p u l a t i o n s . G e n e t i c s 95:1043-1054. M u k a i , T . , R . A . C a r d e l l i n o , T . K . Watanabe, and J . F . Crow. 1974. The g e n e t i c v a r i a n c e for v i a b i l i t y and i t s components i n a l o c a l p o p u l a t i o n of D r o s o p h i l a me lanogas ter . G e n e t i c s 78: 1195-1208. Nagaya, N . , K . S a s a k i , and K. F u j i n o . 1978. B i o c h e m i c a l polymorphism i n the P a c i f i c o y s t e r - I I I . L o c a l p o p u l a t i o n s i n Hokkaido and the N o r t h e a s t d i s t r i c t s of J a p a n . B u l l . J a p . Soc . S c i . F i s h . 44:1041-1045. N a j j a r , V . A . , and M . E . P u l l m a n . 1954. The o c c u r r e n c e of a group t r a n s f e r i n v o l v i n g enzyme (phosphoglucomutase) and s u b s t r a t e . S c i e n c e 119:631-634. N e i , M. 1975. M o l e c u l a r P o p u l a t i o n G e n e t i c s and E v o l u t i o n . N o r t h - H o l l a n d , Amsterdam. N e i , M . , and R . K . Koehn ( e d s . ) . 1983. E v o l u t i o n of Genes and P r o t e i n s . S i n a u e r , S u n d e r l a n d , Mass . 268 Nevo, E . 1978. G e n e t i c v a r i a t i o n i n n a t u r a l p o p u l a t i o n s : p a t t e r n s and t h e o r y . T h e o r . Pop. B i o l . 13:121 -177 . Newsholme, E . A . , and B . C r a b t r e e . 1976. S u b s t r a t e c y c l e s i n m e t a b o l i c r e g u l a t i o n and i n heat g e n e r a t i o n . Biochem. Soc . Symp. 41;61 -109 . Newsholme, E . A . , and C . S t a r t . 1973. R e g u l a t i o n i n M e t a b o l i s m . W i l e y , N . Y . O a k e s h o t t , J . G . , G . K . Chambers, J . B . G i b s o n , and D . A . W i l l c o c k s . 1981. L a t i t u d i n a l r e l a t i o n s h i p s of e s t e r a s e - 6 and phosphoglucomutase gene f r e q u e n c i e s i n D r o s o p h i l a  m e l a n o g a s t e r . H e r e d i t y 47:385-396. O a k e s h o t t , J . G . , J . B . G i b s o n , P . R . A n d e r s o n , W.R. K n i b b , D . G . A n d e r s o n , and G . K . Chambers. 1982. A l c o h o l dehydrogenase and g l y c e r o l - 3 - p h o s p h a t e dehydrogenase c l i n e s i n D r o s o p h i l a melanogaster on d i f f e r e n t c o n t i n e n t s . E v o l u t i o n 36:86-96 . O h t a , T . 1971. A s s o c i a t i v e overdominance caused by l i n k e d d e t r i m e n t a l m u t a t i o n s . Genet . R e s . , Camb. 18:277-286. O h t a , T . , and K. Aok i ( e d s . ) . 1985. P o p u l a t i o n G e n e t i c s and M o l e c u l a r E v o l u t i o n . S p r i n g e r - V e r l a g , T o k y o . Ottaway, J . H . , and J . Mowbray. 1977. The r o l e of c o m p a r t m e n t a l i z a t i o n i n the c o n t r o l of g l y c o l y s i s . C u r r . T o p . C e l l . Reg. j_2: 1 07-208. O z a k i , H . , and Y . F u j i o . . 1985. G e n e t i c d i f f e r e n t i a t i o n in g e o g r a p h i c a l p o p u l a t i o n s of the P a c i f i c o y s t e r ( C r a s s o s t r e a g igas ) around J a p a n . Tohoku J . A g r i c . Res . 36:49-61 . P a i g e n , K. 1979. G e n e t i c f a c t o r s i n deve lopmenta l r e g u l a t i o n . In : P h y s i o l o g i c a l G e n e t i c s . E d i t e d by J . G . S c a n d a l i o s . Academic P r e s s , N . Y . p p . 1-49. P a i g e n , K. 1986. Gene r e g u l a t i o n and i t s r o l e i n e v o l u t i o n a r y p r o c e s s e s . I_n: E v o l u t i o n a r y Proces se s and T h e o r y . E d i t e d by S. K a r l i n and E . Nevo. Academic P r e s s , N . Y . p p . 3-36 . 269 Passonneau, J . V . , O . H . Lowry, D.W. S c h u l z , and J . G . Brown. 1969. G l u c o s e 1 ,6 -d iphosphate f o r m a t i o n by phosphoglucomutase i n mammalian t i s s u e s . J . B i o l . Chem. 244:902-909. Passonneau, J . V . , and D . A . R o t t e n b e r g . 1973. An assessment of methods for measurement of g l y c o g e n syn the tase a c t i v i t y i n c l u d i n g a new d i r e c t o n e - s t e p a s s a y . A n a l . Biochem. 51: 528-541. P i e t e r s , H . , J . H . Kluytmans , W. Z u r b u r g , and D . I . Zandee. 1979. The i n f l u e n c e of s easona l changes on energy metabol i sm i n M y t i l u s e d u l i s ( L . ) . I . Growth r a t e and b i o c h e m i c a l c o m p o s i t i o n i n r e l a t i o n to e n v i r o n m e n t a l parameters and spawning. I_n: C y c l i c Phenomena i n Marine P l a n t s and A n i m a l s . E d i t e d by E . N a y l o r and R . G . H a r t n o l l . Pergamon, O x f o r d , p p . 285-292. P l a c e , A . R . , and D . A . Powers. 1979. G e n e t i c v a r i a t i o n and r e l a t i v e c a t a l y t i c e f f i c i e n c i e s : l a c t a t e dehydrogenase B a l l o z y m e s of Fundulus h e t e r o c l i t u s . P r o c . N a t l . A c a d . S c i . U . S . A . 76:2354-2358. P l a c e , A . R . , and D . A . Powers. 1984a. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of the l a c t a t e dehydrogenase (LDH-B4) a l l o z y m e s of Fundulus h e t e r o c l i t u s . J . B i o l . Chem. 259: 1299-1308. P l a c e , A . R . , and D . A . Powers. 1984b. K i n e t i c c h a r a c t e r i z a t i o n of the l a c t a t e dehydrogenase (LDH-B4) a l l o z y m e s of Fundulus  h e t e r o c l i t u s . J . B i o l . Chem. 259:1309-1318. Pogson, G . H . 1988. C o n s t r a i n t s on the g e n e t i c p r o c e s s of b i o c h e m i c a l a d a p t a t i o n . C a n . J . Z o o l . 66:1139-1145. Powers, D . A . , L . D i M i c h e l e , and A . R . P l a c e . 1983. The use of enzyme k i n e t i c s to p r e d i c t d i f f e r e n c e s i n c e l l u l a r m e t a b o l i s m , deve lopmenta l r a t e , and swimming performance between LDH-B genotypes of the f i s h , Fundulus  h e t e r o c l i t u s . Isozymes C u r r . T o p . B i o l . Med. Res . 10:147-170. P r o v i n e , W.B . 1986. Sewa l l Wright and E v o l u t i o n a r y B i o l o g y . U n i v e r s i t y of Ch icago P r e s s , C h i c a g o . 270 Q u a y l e , D . B . 1969. P a c i f i c o y s t e r c u l t u r e i n B r i t i s h C o l u m b i a . B u l l . F i s h . Res . Board C a n . No. 169. Q u i c k , C . B . , R . A . F i s h e r , and H . H a r r i s . 1974. A k i n e t i c s tudy of the isozymes de termined by the three human phosphoglucomutase l o c i PGM1, PGM2 and PGM3. E u r . J . Biochem. 42:511-517. Ray, W . J . J r . , M . A . Hermodson, J . M . P u v a t h i n g a l , and W . C . Mahoney. 1983. The complete amino a c i d sequence of r a b b i t muscle phosphoglucomutase . J . B i o l . Chem. 258:9166-9174. Ray, W . J . J r . , and E . J . Peck J r . 1972. Phosphomutases. I_n: The Enzymes, V o l . V I , 3rd E d i t i o n . E d i t e d by P . D . B o y e r , Academic P r e s s , N . Y . p p . 407-477. Ray, W . J . J r . , and G . A . R o s c e l l i . 1964a. A k i n e t i c s tudy of the phosphoglucomutase pathway. J . B i o l . Chem. 239:1228-1236. Ray, W . J . J r . , and G . A . R o s c e l l i . 1964b. The phosphoglucomutase pathway. An i n v e s t i g a t i o n of phospho-enzyme i s o m e r i z a t i o n . J . B i o l . Chem. 239:3935-3941. R e c h c i g l , M . , and W . E . H e s t o n . 1967. G e n e t i c r e g u l a t i o n of enzyme a c t i v i t y in mammalian ( s i c ) by the a l t e r a t i o n of the r a t e s of enzyme d e g r a d a t i o n . Biochem. B i o p h y s . Res . Commun.27:119-124 . Reeves , R . B . 1972. An i m i d a z o l e a l p h a s t a t h y p o t h e s i s f or v e r t e b r a t e a c i d - b a s e r e g u l a t i o n : t i s s u e carbon d i o x i d e c o n t e n t and body temperature in b u l l f r o g s . Resp. P h y s i o l . 14:219-236. Ridgway, G . J . , S.W. S h e r b u r n e , and R . D . L e w i s . 1970. Polymorphism i n the e s t e r a s e s of A t l a n t i c h e r r i n g . T r a n s . Am. F i s h . Soc . 99:147-151. Roach, P . J . 1986. L i v e r g lycogen s y n t h a s e . I_n: The Enzymes, V o l . X V I I . E d i t e d by P . D . Boyer and E . G . K r e b s . Academic P r e s s , O r l a n d o , pp . 499-539. 271 Robson, G . C . , and O.W. R i c h a r d s . 1936. The V a r i a t i o n of Animals i n N a t u r e . Longmans G r e e n , London . Rodhouse, P . G . 1978. Energy t r a n s f o r m a t i o n s by the o y s t e r O s t r e a e d u l i s L . i n a temperate e s t u a r y . J . exp . mar. B i o l . E c o l . 37 :1-22 . Rodhouse, P . G . , and P . M . G a f f n e y . 1984. E f f e c t of h e t e r o z y g o s i t y on metabol i sm d u r i n g s t a r v a t i o n i n the American o y s t e r C r a s s o s t r e a v i r g i n i c a . M a r . B i o l . 80:179-187. Rodhouse, P . G . , J . H . McDonald , R . I . E . N e w e l l , and R . K . Koehn. 1986. Gamete p r o d u c t i o n , somatic growth and m u l t i p l e - l o c u s h e t e r o z y g o s i t y i n M y t i l u s e d u l i s . Mar . B i o l . 90:209-214. Savageau, M . A . 1976. B i o c h e m i c a l Systems A n a l y s i s . A d d i s o n -Wes ley , R e a d i n g , Mass . S c a n d a l i o s , J . G . , and J . A . Baum. 1982. R e g u l a t o r y gene v a r i a t i o n i n h i g h e r p l a n t s . A d v . G e n e t . 21:347-370. S c a n d a l i o s , J . G . , D . - Y . Chang, D . E . M c M i l l i n , A . T s a f t a r i s , and R . H . M o l l . 1980. G e n e t i c r e g u l a t i o n of the c a t a l a s e deve lopmenta l program i n maize s c u t e l l u m : i d e n t i f i c a t i o n of a temporal r e g u l a t o r y gene. P r o c . N a t l . A c a d . S c i . U . S . A . 77:5360-5364. Schwartz , D . , and W . J . Laughner . 1969. A m o l e c u l a r b a s i s for h e t e r o s i s . S c i e n c e 166:626-627. S e d c o l e , J . R . 1981. A review of the t h e o r i e s of h e t e r o s i s . E g y p t . J . Genet . C y t o l . 10:117-146. S e l a n d e r , R . K . , and B . R . L e w i n . 1980. G e n e t i c d i v e r s i t y and s t r u c t u r e in E s c h e r i c h i a c o l i p o p u l a t i o n s . S c i e n c e 210: 545-547. S h a f f e r , J . B . , and G . C . Bewley. 1983. G e n e t i c d e t e r m i n a t i o n of s n - g l y c e r o l - 3 - p h o s p h a t e dehydrogenase s y n t h e s i s in D r o s o p h i l a m e l a n o g a s t e r . J . B i o l . Chem. 258:10027-10033. 272 Shaw, C . R . , and R. P r a s a d . 1970. S t a r c h g e l e l e c t r o p h o r e s i s of enzymes - a c o m p i l a t i o n of r e c i p e s . Biochem. Genet . 4:297-320. Simpson, G . G . 1944. Tempo and Mode i n E v o l u t i o n . Columbia U n i v e r s i t y P r e s s , N . Y . S i n g h , R . S . , J . L . Hubby, and R . C . L e w o n t i n . 1974. M o l e c u l a r h e t e r o s i s for h e a t - s e n s i t i v e enzyme a l l e l e s . P r o c . N a t l . A c a d . S c i . U . S . A . 71:1808-1810. S i n g h , . S . M . 1982. Enzyme h e t e r o z y g o s i t y a s s o c i a t e d w i t h growth a t d i f f e r e n t deve lopmenta l s tages i n o y s t e r s . C a n . J . G e n e t . C y t o l . 24:451-458. S i n g h , S . M . , and R . H . G r e e n . 1984. Excess of a l l o z y m e homozygos i ty i n marine m o l l u s c s and i t s p o s s i b l e b i o l o g i c a l s i g n i f i c a n c e . M a l a c o l o g i a 25:569-581. S i n g h , S . M . , and E . Z o u r o s . 1978. G e n e t i c v a r i a t i o n a s s o c i a t e d w i t h growth r a t e i n the American o y s t e r ( C r a s s o s t r e a  v i r g i n i c a ) . E v o l u t i o n 32:342-353. Smouse, P . E . 1986. The f i t n e s s consequences of m u l t i p l e - l o c u s h e t e r o z y g o s i t y under the m u l t i p l i c a t i v e overdominance and i n b r e e d i n g d e p r e s s i o n mode l s . E v o l u t i o n 40:946-957 . S o k a l , R . R . , and F . J . R o h l f . 1981. B i o m e t r y . W . H . Freeman, San F r a n c i s c o . Somero, G . N . 1981. pH-temperature i n t e r a c t i o n s on p r o t e i n s : p r i n c i p l e s of o p t i m a l pH and b u f f e r system d e s i g n . M a r . B i o l . L e t t . 2:163-178. S ta lmans , W. 1976. The r o l e of the l i v e r on the homeostas i s of b l o o d g l u c o s e . C u r r . T o p . C e l l . Reg . 11:51-97. S t e n z e l , H . B . 1971. O y s t e r s . I_n: T r e a t i s e on I n v e r t e b r a t e P a l e o n t o l o g y . P a r t N . V o l . 3. M o l l u s c a 6. E d i t e d by K . C . Moore . G e o l o g i c a l S o c i e t y of Amer ica and U n i v e r s i t y of Kansas P r e s s , B o u l d e r . 273 S t r a u s s , S . H . 1986. H e t e r o s i s a t a l l o z y m e l o c i under i n b r e e d i n g and c r o s s b r e e d i n g i n P i n u s a t t e n u a t a . G e n e t i c s 113:115-1 34. S t r a u s s , S . H . 1987. H e t e r o z y g o s i t y and deve lopmenta l s t a b i l i t y under i n b r e e d i n g and c r o s s b r e e d i n g i n P i n u s a t t e n u a t a . E v o l u t i o n 41:331~339. S u g i t a , M . , and Y . F u j i o . 1982. E f f e c t s of genotypes at the A a t -1 l o c u s on the s u r v i v a l and growth r a t e s i n the c u l t u r e d o y s t e r . Tohoku J . A g r i c . Res . 33:42-49 . T r i p p a , G . , A . L o v e r r e , and A . Catamo. 1976. T h e r m o s t a b i l i t y s t u d i e s for i n v e s t i g a t i n g n o n - e l e c t r o p h o r e t i c po lymorphic a l l e l e s i n D r o s o p h i l a m e l a n o g a s t e r . Nature 260:42-44. T r i p p a , G . , A . Catamo, A . L o m b a r d o z z i , and R. C i c c h e t t i . 1978. A s imple approach for d i s c o v e r i n g common n o n - e l e c t r o p h o r e t i c enzyme v a r i a b i l i t y : a heat d e n a t u r a t i o n s tudy i n D r o s o p h i l a m e l a n o g a s t e r . Biochem. Genet . 16:299-305. T o r r e s , N . V . , F . Mateo , E . M e l e n d e z - H e v i a , and H . K a c s e r . 1986. K i n e t i c s of m e t a b o l i c pathways . A system i_n v i t r o to s tudy the c o n t r o l of f l u x . Biochem. J . 234:169-174. T u r e l l i , M . , and L . R . G i n z b u r g . 1 9 8 3 . . S h o u l d i n d i v i d u a l f i t n e s s i n c r e a s e w i t h h e t e r o z y g o s i t y ? G e n e t i c s 104:191-209. V i g u e , C . L . , and F . M . Johnson . 1973. Isozyme v a r i a b i l i t y i n the genus D r o s o p h i l a . V I . F r e q u e n c y - p r o p e r t y - e n v i r o n m e n t r e l a t i o n s h i p s of a l l e l i c a l c o h o l dehydrogenase . Biochem. Genet . 9:213-227. W a l l a c e , B . 1959. The r o l e of h e t e r o z y g o s i t y i n D r o s o p h i l a p o p u l a t i o n s . P r o c . 10th I n t e r n . Cong. G e n e t . U 4 0 8 - 4 1 9 . W a l s h , P . J . 1981. P u r i f i c a t i o n and c h a r a c t e r i z a t i o n of two a l l o z y m i c forms of o c t o p i n e dehydrogenase from C a l i f o r n i a p o p u l a t i o n s of M e t r i d i u m s e n i l e . J . Comp. P h y s i o l . 143: 213-222. 274 Walsh , P . J . , D . G . McDonald , and C E . B o o t h . 1984. A c i d - b a s e ba lance in the sea m u s s e l , M y t i l u s e d u l i s . I I . E f f e c t s of hypox ia and a i r exposure on i n t r a c e l l u l a r a c i d - b a s e s t a t u s . Mar . B i o l . L e t t . 5:359-369. Ward, R . D . , and J . A . Beardmore. 1977. P r o t e i n v a r i a t i o n in the p l a i c e , P l e u r o n e c t e s p l a t e s s a L . G e n e t . R e s . , Camb. 30:45-62. Watt , W.B. 1977. A d a p t a t i o n at s p e c i f i c l o c i . I . N a t u r a l s e l e c t i o n on phosphoglucose isomerase of C o l i a s b u t t e r f l i e s : b i o c h e m i c a l and p o p u l a t i o n a s p e c t s . G e n e t i c s 87:177-194. Watt , W.B. 1983. A d a p t a t i o n at s p e c i f i c l o c i . I I . Demographic and b i o c h e m i c a l e lements i n the maintenance of the C o l i a s PGI po lymorphism. G e n e t i c s 103:691-724. Wat t , W.B . 1985a. B i o e n e r g e t i c s and e v o l u t i o n a r y g e n e t i c s : o p p o r t u n i t i e s for new s y n t h e s i s . Am. N a t . 125:118-143. Watt , W.B . 1985b. A l l e l i c isozymes and the m e c h a n i s t i c s tudy of e v o l u t i o n . Isozymes C u r r . T o p . B i o l . Med. Res . 12:89-132. Watt , W.B . 1986. Power and e f f i c i e n c y as indexes of f i t n e s s i n m e t a b o l i c o r g a n i z a t i o n . Am. Nat . 127:629-653. Watt , W . B . , P . A . C a r t e r , and S . M . B l o w e r . 1985. A d a p t a t i o n a t s p e c i f i c l o c i . I V . D i f f e r e n t i a l mat ing success among g l y c o l y t i c a l l o z y m e genotypes of C o l i a s b u t t e r f l i e s . G e n e t i c s 109:157-175. Watt , W . B . , R . C . C a s s i n , and M . S . Swan. 1983. A d a p t a t i o n at s p e c i f i c l o c i . I I I . F i e l d b e h a v i o r and s u r v i v o r s h i p d i f f e r e n c e s among C o l i a s PGI genotypes are p r e d i c t a b l e from in_ v i t r o b i o c h e m i s t r y . G e n e t i c s 103:725-739. W a t t s , C , and R . S . M a l t h u s . 1980. L i v e r g lycogen syn the tase in r a t s w i t h a g l y c o g e n - s t o r a g e d i s o r d e r . The r o l e of g lycogen in the r e g u l a t i o n of g lycogen s y n t h e t a s e . E u r . J . Biochem. 240:588-593. 275 Whaley, W . G . 1952. P h y s i o l o g y of gene a c t i o n i n h y b r i d s . I n : H e t e r o s i s . E d i t e d by J . W . Gowen. H a f n e r , N . Y . p p . 98-113. Whyte, J . N . C . , and J . R . E n g l a r . 1982. Seasona l v a r i a t i o n in the c h e m i c a l c o m p o s i t i o n and c o n d i t i o n i n d i c e s of P a c i f i c o y s t e r , C r a s s o s t r e a g i g a s , grown i n t r a y s or on the sea bed . C a n . J . F i s h . A q u a t . S c i . 39:1084-1094. Widdows, J . 1978. Combined e f f e c t s of body s i z e , food c o n c e n t r a t i o n and season on the p h y s i o l o g y of M y t i l u s e d u l i s . J . M a r . B i o l . A s s o c . U . K . 58:109-124. Wijsman, T . C . M . 1975. pH f l u c t u a t i o n s i n M y t i l u s e d u l i s L . i n r e l a t i o n to s h e l l movements under a e r o b i c and a n a e r o b i c c o n d i t i o n s . I_n: P r o c . 9th E u r . mar. b i o l . Symp. E d i t e d by H . B a r n e s . Aberdeen U n i v e r s i t y P r e s s , Aberdeen , pp . 139-149. W i l k i n s , N . P . 1976. Genie v a r i a b i l i t y i n marine B i v a l v i a : i m p l i c a t i o n s and a p p l i c a t i o n s i n m o l l u s c a n a q u a c u l t u r e . 10th E u r . Sym. Mar . B i o l . J_:549-563. W i l l s , C . 1981. G e n e t i c V a r i a b i l i t y . C l a r e n d o n , N . Y . W i l s o n , A . C . , S . S . C a r l s o n , and T . J . W h i t e . 1977. B i o c h e m i c a l e v o l u t i o n . A n n . Rev. Biochem. 46:573-639. W i l t o n , A . N . , C . C . L a u r i e - A h l b e r g , T . M . Emigh, and J . W . C u r t s i n g e r . 1982. N a t u r a l l y o c c u r r i n g enzyme a c t i v i t y v a r i a t i o n i n D r o s o p h i l a m e l a n o g a s t e r . I I . R e l a t i o n s h i p s among enzymes. G e n e t i c s 102:207-221. Womack, J . E . , L . S . Yan , and M. P o t i e r . 1980. Gene f o r neuramin idase a c t i v i t y on mouse chromosome 17 near H - 2 : p l e i o t r o p i c e f f e c t s on m u l t i p l e h y d r o l a s e s . S c i e n c e 212: 63-64. W r i g h t , S. 1977. E v o l u t i o n and the G e n e t i c s of P o p u l a t i o n s , V o l . 3. E x p e r i m e n t a l R e s u l t s and E v o l u t i o n a r y D e d u c t i o n s . U n i v e r s i t y of Ch icago P r e s s , C h i c a g o . Yamazaki , T . , and T . Maruyama. 1972. E v i d e n c e for the n e u t r a l h y p o t h e s i s of p r o t e i n po lymorphism. S c i e n c e 178:56-58. Yancey , P . H . , and G . N . Somero. 1978. Temperature dependence of i n t r a c e l l u l a r pH: i t s r o l e in the c o n s e r v a t i o n of p y r u v a t e apparent Km v a l u e s of v e r t e b r a t e l a c t a t e dehydrogenases . J . Comp. P h y s i o l . 125:129-134. Yonge, C M . 1960. O y s t e r s . C o l l i n s , London. Zaba, B . N . 1981. G l y c o g e n o l y t i c pathways in the mantle t i s s u e of M y t i l u s e d u l i s L . Mar . B i o l . L e t t . 2:67-74. Zaba, B . N . , and J . I . D a v i e s . 1980. G l u c o s e metabol i sm i n an in v i t r o p r e p a r a t i o n of the mantle t i s s u e from M y t i l u s e d u l i s L . Mar . B i o l . L e t t . 1:235-243. Zaba, B . N . , and J . I . D a v i e s . 1981. C a r b o h y d r a t e metabol i sm i n i s o l a t e d mantle t i s s u e of M y t i l u s e d u l i s L . I s o t o p i c s t u d i e s on the a c t i v i t i e s of the Embden-Meyerhof and pentose phosphate pathways. M o l . P h y s i o l . J_:97—1 12. Zaba, B . N . , P . A . G a b b o t t , and J . I . D a v i e s . 1981. Seasona l changes in the u t i l i s a t i o n of 14C- and 3 H - l a b e l l e d g l u c o s e i n a mantle t i s s u e s l i c e p r e p a r a t i o n of M y t i l u s e d u l i s L . Comp. Biochem. P h y s i o l . 70B:689-695. Zandee, D . I . , D . A . Holwerda , and A . de Zwaan. 1980. Energy metabol i sm i n b i v a l v e s and c e p h a l o p o d s . Ij i : Animals and E n v i r o n m e n t a l F i t n e s s . E d i t e d by R. G i l l e s . Pergamon, O x f o r d , pp . 185-206. Zandee, D . I . , J . H . Kluytmans , W. Z u r b u r g , and K. P i e t e r s . 1980. Seasona l v a r i a t i o n s i n b i o c h e m i c a l c o m p o s i t i o n of M y t i l u s  e d u l i s wi th r e f e r e n c e to energy metabol i sm and. gametogenes i s . N e t h . J . Sea Res . 14:1-29 . Z e r a , A . J . 1987. Temperature-dependent k i n e t i c v a r i a t i o n among phosphoglucose isomerase a l l o z y m e s from the wing-po lymorphic water s t r i d e r , Limnoporus c a n a l i c u l a t u s . M o l . B i o l . E v o l . 4:266-285. 277 Z e r a , A . J . , R . K . Koehn, and J . G . H a l l . 1985. A l l o z y m e s and b i o c h e m i c a l a d a p t a t i o n . I_n: Comprehensive I n s e c t P h y s i o l o g y , B i o c h e m i s t r y , and Pharmacology , V o l . 10. E d i t e d by G . A . Kerkut and L . I . G i l b e r t . Pergamon, N . Y . pp . 633-674. Z o u r o s , E . , and D.W. F o l t z . 1984. P o s s i b l e e x p l a n a t i o n s of h e t e r o z y g o t e d e f i c i e n c y in b i v a l v e m o l l u s c s . M a l a c o l o g i a 25:583-591. Z o u r o s , E . , and D.W. F o l t z . 1987. The use of a l l e l i c isozyme v a r i a t i o n f o r the s tudy of h e t e r o s i s . Isozymes C u r r . T o p . B i o l . Med. Res . 13:1-59. Z o u r o s , E . , S . M . S i n g h , D.W. F o l t z , and A . L . M a l l e t . 1983. P o s t -s e t t l e m e n t v i a b i l i t y i n the American o y s t e r ( C r a s s o s t r e a  v i r g i n i c a ) : an overdominant phenotype . G e n e t . R e s . , Camb. ' 41:259-270. Z o u r o s , E . , S . M . S i n g h , and H . E . M i l e s . 1980. Growth r a t e i n o y s t e r s : an overdominant phenotype and i t s p o s s i b l e e x p l a n a t i o n s . E v o l u t i o n 34:856-867. 

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