UBC Theses and Dissertations

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UBC Theses and Dissertations

Gluconeogenic adaptations in Cancer Magister Guderley, Helga E 1976

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GLUCONEOGENIC ADAPTATIONS IN CANCER MAGISTER by H e l g a E. G u d e r l e y B.Sc. Earlham College, H971 Richmond, Indiana t h e s i s s u b m i t t e d i n p a r t i a l f u l f i l l m e n t t h e r e q u i r e m e n t s f o r t h e d e g r e e o f DOCTOR OF PHILOSOPHY i n t h e D e p a r t m e n t o f Z o o l o g y We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e g u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA A u g u s t 1976 © Helga E l i z a b e t h Guderley, 1976 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i thout my w r i t t e n p e r m i s s i o n . Department o f "ZGOCJJJ rt The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e .v . v Vancouver, Canada V6T 1W5 ' , Date I ABSTRACT The p e r i o d i c r e q u i r e m e n t f o r a new c h i t i n c u s e x c s k e l e t o n i m p o s e s a l a r g e b i o s y n t h e t i c l e a d on t h e m e t a b o l i s m o f c r u s t a c e a n s , w i t h t h e h y p o d e r m i s f a c i n g t h e b r u n t o f t h e l o a d . S i n c e t h e f r e s h l y m o l t e d a n i m a l i s h i g h l y s u s c e p t i b l e t o p r e d a t i o n , t h e mechanisms f o r e f f i c i e n t g l u c c n e c g e n e s i s i n s u p p o r t o f c h i t i n s y n t h e s i s a r e o f d e f i n i t e s u r v i v a l v a l u e t o t h e o r g a n i s m . M e a s u r e m e n t s o f enzyme a c t i v i t i e s i n t h e h y p o d e r m i s , g i l l and m u s c l e o f C a n c e r m a ^ i s r t e r i n d i c a t e t h a t t h e h y p o d e r m i s and m u s c l e u n d e r g o c o n s i d e r a b l e m e t a b o l i c c h a n g e s d u r i n g t h e m e l t c y c l e . F r e s h m o l t h y p o d e r m i s shows e l e v a t e d s p e c i f i c a c t i v i t i e s c f b o t h t h e g l u c o n e o g e n i c and t h e g l y c o l y t i c e n z y m e s , w h i l e f r e s h m o l t m u s c l e shows d e c r e a s e d l e v e l s o f t h e g l y c o l y t i c and i n c r e a s e d l e v e l s o f t h e g l u c o n e o g e n i c e n z y m e s . H y p o d e r m i s m a i n t a i n s a h i g h l y g l u c o n e o g e n i c o r i e n t a t i o n t h r o u g h o u t t h e m e l t c y c l e . P h o s p h o g l y c e r a t e k i n a s e i s c o n s i d e r e d t o be one o f t h e b i f u n c t i c n a l e nzymes i n t h e g l y c o l y t i c p a t h w a y , b u t t h e k i n e t i c c h a r a c t e r i s t i c s c f t h e p r e v i o u s l y s t u d i e d enzymes a r e i l l s u i t e d f o r f u n c t i o n i n a ' g l u c o n e o g e n i c s y s t e m . S i n c e t h e i n t e r m e l t a n d f r e s h m o l t m u s c l e and h y p o d e r n i s p r e s e n t a v a r i e t y c f m e t a b o l i c poises ( i . e. r a n g i n g 'from h i g h l y g l u c o n e o g e n i c t c h i g h l y g l y c o l y t i c ) , I ^ s t u d i e d t h e c o n t r o l o f p h o s | l i d g l y c e r a t e k i n a s e i n t h e s e t i s s u e , s : . I f o u n d t h a t t h e i n t e ^ 0 ^ ^ i u s c 1 e enzyme shows k i n e t i c s much l i k e t h o s e o f t h e mammalian m u s c l e and t h e y e a s t enzyme, w i t h a h i g h s e n s i t i v i t y t o EgADP/ADP i n h i b i t i o n (PgADE K i = 1.3 x 10~ 5M) and a r e l a t i v e l y l e w a f f i n i t y f o r ATP as a i i s u b s t r a t e (Km = 2.03 x 10-*M). By c o n t r a s t , t h e f r e s h m o l t h y p o d e r m a l enzyme shows a c o n s i d e r a b l y d e c r e a s e d s e n s i t i v i t y t c Kg A DP/A DP i n h i b i t i o n (Mg ADP K i = 2 x10-*M) and a c o n s i d e r a b l y i n c r e a s e d a f f i n i t y f c r ATP (Km = 6.8 x 1 0 - 5 M ) . The f r e s h m o l t m u s c l e enzyme a l s o s h a r e s t h e s e c h a n g e d a f f i n i t i e s . The i n t e r m o . l t h y p o d e r n a l p h c s p h o g l y c e r a t e k i n a s e shows t h e d e c r e a s e d s e n s i t i v i t y t o Kg ADP/ADP i n h i b i t i o n b u t s h a r e s t h e ATP a f f i n i t y o f t h e i n t e r m o l t m u s c l e enzyme. The k i n e t i c c h a r a c t e r i s t i c s o f t h e f r e s h m o l t h y p o d e r m a l and m u s c l e enzymes r e d u c e t h e s u s c e p t i b i l i t y o f t h e enzymes t o i n h i b i t i o n by MgADP and f a c i l i t a t e t h e r e v e r s a l c f t h e r e a c t i o n f o r g l u c c n e o g e n e s i s . The c o n t r o l o f p y r u v a t e k i n a s e i s i n t e g r a l t o t h e c c c t r o l o f b o t h g l y c o l y s i s and g l u c o n e o g e n e s i s . I n g l y c o l y s i s , i t f o r m s t h e s e c o n d m a j o r c e n t r o i s i t e ; i n g l u c o n e o g e n e s i s , i t i s one o f t h e p r i m e d e t e r m i n a n t s o f t h e r a t e o f g l u c o n e o g e n e s i s f r o m l a c t a t e and amino a c i d s . M u s c l e and h y p o d e r m a l p y r u v a t e k i n a s e s f r o m C a n c e r j a ^ i s t e r a r e d i s t i n c t p r o t e i n s , cn t h e b a s i s o f i s o e l e c t r i c p o i n t s , k i n e t i c c h a r a c t e r i s t i c s , and t h e r m a l d e n a t u r a t i o n t e h a v i c r . I n c o n t r a s t t o t h e p h c s p h o g l y c e r a t e k i n a s e s y s t e m , t h e r e a r e no p r o n o u n c e d d i f f e r e n c e s b e t w e e n f r e s h m o l t and i n t e r m o l t f o r m s . M u s c l e p y r u v a t e k i n a s e i s a c t i v a t e d by FDP, i n h i b i t e d by KgATP, a r g i n i n e p h o s p h a t e , M g 2 c i t r a t e , t r y p t o p h a n and i s a l s o s e n s i t i v e t o some i n h i b i t i o n by a l a n i n e , oc--giycerolp.hosp.hate, Mg-ma l a t e and oC- ke t o g l u t a r a t e . The m u s c l e enzyme has a h i g h a f f i n i t y f o r PEP (Km = 0.1 mM) and t h e a d d i t i o n c f 0.05 mM FDP d r c p s t h e PEP Km t c 0.05 mM. I n c o m p a r i s o n w i t h o t h e r m u s c l e p y r u v a t e k i n a s e s , t h e enzyme i s q u i t e s e n s i t i v e t o MgATF i n h i b i t i o n ( K i = 1.8 mM) and shews FDP i i i r e v e r s a l o f t h e i n h i b i t i o n . A r g i n i n e p h o s p h a t e i n h i b i t i o n i s • c o m p e t i t i v e w i t h ADP, and i s n o t r e v e r s e d by FDP. The r e v e r s a l o f t h e r e a c t i o n a c c o u n t s f o r o n l y 0.5% o f t h e f o r w a r d r e a c t i o n . A l t h o u g h h i g h l e v e l s o f ATP and a r g i n i n e p h o s p h a t e s t r o n g l y i n h i b i t t h e r e a c t i o n , t h e i n h i b i t i o n i s n o t s u f f i c i e n t t o a l l o w n e t f l u x t h r o u g h t h e l o w l e v e l s o f t h e b y p a s s enzymes p r e s e n t i n t h e m u s c l e o f f r e s h m o l t a n i m a l s . T h u s , m u s c l e p y r u v a t e k i n a s e has k i n e t i c c h a r a c t e r i s t i c s w h i c h s u i t i t f o r f u n c t i o n i n t h e c o n t r o l of g l y c o l y s i s , b u t do n o t a l l o w g l u c o n e o g e n i c f l u x p a s t t h e r e a c t i o n l o c u s . I n c o n t r a s t , h y p o d e r m a l p y r u v a t e k i n a s e i s a c o n s p l e x p r o t e i n c a p a b l e of m a k i n g l a r g e t r a n s i t i o n s b e t w e e n h i g h a c t i v i t y d u r i n g o x i d a t i o n o f c a r b o h y d r a t e s u b s t r a t e s and v i r t u a l l y no a c t i v i t y d u r i n g g l u c o n e o g e n e s i s f r o m l a c t a t e and amino a c i d s . H y p o d e r m a l p y r u v a t e k i n a s e e x i s t s i n twc c o n f o r m a t i o n a l s t a t e s , one a h i g h a f f i n i t y s t a t e (PK I) and t h e e t h e r a l o w a f f i n i t y s t a t e (PK I I ) , PK I has a Km f o r PEP o f 0.1 mM and a Ka f o r FDP o f 1.3 x 10-s.H. PK I I h a s a Km f o r PEP c f 0.55 mM and a Ka f o r FDP o f 9 x 10~ aM, F c r b o t h f o r m s , FDP f a c i l i t a t e s t h e b i n d i n g o f PEP, E o f h f o r m s a r e s e n s i t i v e t c MgATP i n h i b i t i o n and show FDP r e v e r s a l o f t h e i n h i b i t i o n , EK I I i s more s e n s i t i v e t o i n h i b i t i o n by a l a n i n e , s e r i n e , and M g 2 c i t r a t e . F o r PK I I , FDP a l t e r s t h e i n h i b i t i o n due t o t h e s e compounds, c h a n g i n g t h e i n t e r a c t i o n s b e t w e e n t h e s e i n h i b i t o r s and b o t h PEE and ADP. I n c u b a t i o n o f PK I I w i t h 0.05 mK FDP p r o d u c e s PK I . P r o l o n g e d d i a l y s i s o f PK I l e a d s t o an enzyme w i t h t h e c h a r a c t e r i s t i c s o f PK I I . The l e v e l s o f FDP a s s o c i a t e d w i t h PK I a r e h i g h e r t h a n t h e l e v e l s a s s o c i a t e d w i t h PK I I , D u r i n g g l u c o n e o g e n e s i s , t h e FDP l e v e l s i n t h e c e l l a r e l o w . T h i s would s h i f t t h e e q u i l i b r i u m b e t ween t h e tw o f o r m s t o w a r d s PK I I . S i n c e p h y s i o l o g i c a l l e v e l s o f PEP, ADP, ATP, a l a n i n e , and s e r i n e l i m i t PK I I a c t i v i t y t o l e s s t h a n 0.5% o f m a x i m a l , c o n s i d e r a b l e f l u x t h r o u g h t h e p h o s p h c e n o l p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e b y p a s s w o u l d be f e a s i b l e . The r i s e i n FDP l e v e l s d u r i n g i n h i b i t i o n o f g l u c o n e o g e n e s i s w o u l d s h i f t t h e e q u i l i b r i u m i n f a v o r o f PK I . T h i s s h i f t w o u l d i m m e d i a t e l y r a i s e p y r u v a t e k i n a s e a c t i v i t y f r o m l e s s t h a n 0.5% t c a r o u n d 50$ o f m a x i m a l a c t i v i t y . T h i s , c o u p l e d w i t h t h e o t h e r c h a n g e s i n m e t a b o l i t e l e v e l s d u r i n g an i n h i b i t i o n o f g l u c o n e o g e n e s i s , w o u l d l e a d t o a marked a c t i v a t i o n o f p y r u v a t e k i n a s e a c t i v i t y . These c o n f o r m a t i o n a l s t a t e s a l l o w r a p i d c h a n g e s i n f l u x t h r o u g h t h e r e a c t i o n , and t h u s w o u l d a l l o w f l e x i b l e and r e s p o n s i v e r e g u l a t i o n o f t h i s i m p o r t a n t g l y c o l y t i c and g l u c o n e o g e n i c c o n t r o l s i t e . T h u s , b o t h t h e p h c s p h o g l y c e r a t e k i n a s e and p y r u v a t e k i n a s e p r e s e n t i n t h e h y p o d e r m i s c f C a n c e r m a q i s t e r have s p e c i a l c h a r a c t e r i s t i c s w h i c h f a c i l i t a t e e f f i c i e n t g l u c o -n e o g e n e s i s . To e l u c i d a t e t h e p o s s i b l e i m p o r t a n c e o f i o n s i n r e g u l a t i n g t h e a c t i v i t y o f t h e above e n z y m e s , I measured t h e l e v e l s o f s o d i u m , p o t a s s i u m , magnesium, and c a l c i u m i n t h e m u s c l e and h y p o d e r m i s o f i n t e r m c l t and f r e s h m o l t a n i m a l s . I f o u n d t h a t t h e e x t r a c e l l u l a r ' s p a c e o f - t h e h y p o d e r m i s i s c o n s i d e r a b l y h i g h e r t h a n t h a t o f t h e m u s c l e ( 4 5 % v e r s u s 12%) , b u t t h a t t h e r e was no v a r i a t i o n b e t ween i n t e r m o l t and f r e s h m o l t t i s s u e e x t r a c e l l u l a r s p a c e . W h i l e f r e s h m o l t m u s c l e s o d i u m c o n c e n t r a t i o n s were s i g n i f i c a n t l y h i g h e r t h a n i n t e r m c l t s o d i u m c o n c e n t r a t i o n s , none V o f t h e o t h e r i o n s showed s i g n i f i c a n t d i f f e r e n c e s between m o l t c y c l e s t a g e s . However, t h e r e were s i g n i f i c a n t d i f f e r e n c e s between t h e i o n i c c o n c e n t r a t i o n s i n h y p o d e r m i s and m u s c l e . H y p o d e r m i s shewed h i g h e r c a l c i u m l e v e l s a nd l o w e r p o t a s s i u m l e v e l s t h a n m u s c l e i n b o t h f r e s h m o l t and i n t e r m o l t a n i m a l s . A l t h o u g h i c n i c c h a n g e s do n o t p l a y a r o l e i n d i f f e r e n t i a l r e g u l a t i o n o f enzyme a c t i v i t y d u r i n g t h e m o l t c y c l e , t h e i o n i c c o n c e n t r a t i o n s p r e s e n t i n t h e s e t i s s u e s a r e s u c h t h a t t h e i o n s c o u l d s e t g u i d e l i n e s f o r t h e a c t i v i t y o f p h o s p h o g l y c e r a t e k i n a s e , p y r u v a t e 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 f r u c t o s e d i -p h o s p h a t a s e i n t h e s e t i s s u e s . v i T A E I E Of CONTENTS INTBODOCTION . 1 M o l t C y c l e P h y s i o l o g y 6 I n t e r m e d i a r y M e t a b o l i s m ' i n C r u s t a c e a n s 16 C e n t r a l Q u e s t i o n s i n t h i s s t u d y 30 GENEFAL MATERIALS ANC METHODS E x p e r i m e n t a l A n i m a l s 3 8 R e a g e n t s . 3 9 P r e p a r a t i o n o f Enzymes .. 3 9 S t a r c h G e l s and I s c e l e c t r c f c c u s i n g 40 K i n e t i c A n a l y s i s 41 PART I . C h a n g i n g Enzyme A c t i v i t i e s i n t h e H y p o d e r m i s , G i l l , and M u s c l e o f C a n c e r m a ^ i s t e r d u r i n g t h e M o l t C y c l e INTRODUCTION 43 MATERIALS AND METHODS ................................. 43 P r e p a r a t i o n o f t h e Homogenate f c r M e a s u r e m e n t o f Enzyme A c t i v i t i e s k% Enzyme A s s a y s 44 RESULTS AND DISCUSSION 41 PART I I . C o n t r o l o f E y p c d e r m a l P h c s p h o g l y c e r a t e K i n a s e i n I n t e r m o l t a n d F r e s h m o l t C a n c e r m a a i s t e r • INTRODUCTION 5 4 METHODS P r e p a r a t i o n o f P h o s p h o g l y c e r a t e K i n a s e ........... 57 M e t a b o l i t e M e a s u r e m e n t s .......................... 58 v i i Enzyme A s s a y s .................................... 59 RESULTS S p e c i f i c A c t i v i t i e s .... 59 D i f f e r e n t Forms o f P h c s p h o g l y c e r a t e K i n a s e ....... 61 K i n e t i c P r o p e r t i e s 65 Mass A c t i o n R a t i o s 81 DISCUSSION ., 8 5 PART I I I . M u s c l e P y r u v a t e K i n a s e INTRODUCTION 9 3 METHODS P r e p a r a t i o n o f P y r u v a t e K i n a s e ................... 96 Enzyme A c t i v i t i e s 97 RESULTS E l e c t r o p h o r e s i s and I s o e l e c t r o f o c u s i n g 98 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 .............. 99 S u b s t r a t e A f f i n i t i e s 100 A c t i v a t o r s .. 10 2 I n h i b i t o r s 10 2 DISCUSSION 116 PART IV H y p o d e r m a l P y r u v a t e K i n a s e INTRODUCTION 122 METHODS 12 3 Measurement o f Bound F r u c t c s e 1,6 D i p h o s p h a t e .... 12 4 Enzyme A s s a y s 124 v i i i RESULTS E l e c t r o p h o r e s i s and E l e c t r o f o c u s i n g 124 C a t a l y t i c and R e g u l a t o r y C h a r a c t e r i s t i c s ......... 125 S u b s t r a t e A f f i n i t i e s .............................. 128 A c t i v a t o r s 131 I n h i b i t o r s . 133 DISCUSSION , 153 PART V. C a t i c n i c C o m p o s i t i o n o f I n t e r m o l t and F r e s h m o l t H y p o d e r m i s and M u s c l e INTRODUCTION 160 METHODS C o l l e c t i o n and M a i n t e n a n c e o f A n i m a l s 16 2 T i s s u e P r e p a r a t i o n f o r I o n M e a s u r e m e n t s .......... 16 3 D e t e r m i n a t i o n o f I o n C o n c e n t r a t i o n s 16 4 D e t e r m i n a t i o n o f E x t r a c e l l u l a r Space 16 4 RESULTS AND DISCUSSION E x t r a c e l l u l a r Space D e t e r m i n a t i o n 165 I o n i c C o m p o s i t i o n o f t h e Hemolymph 16 7 I o n i c C o m p o s i t i o n o f M u s c l e a n d H y p o d e r m i s ....... 16S GENEEAL DISCUSSION AND SUMMARY 178 H y p o d e r m a l G l u c c n e o g e n e s i s 17 9 M u s c l e G l u c o n e c g e n e s i s 180 M o l t C y c l e V a r i a t i o n s i n Enzyme L e v e l s ................ 182 C o n t r o l o f G l y c o l y s i s and G l u c c n e o g e n e s i s i n t h e H y p o d e r m i s .................................. 183 I n t e g r a t i o n o f G l u c o n e o g e n e s i s and L i p i d O x i d a t i o n .... 186 I n h i b i t i o n c f G l u c o n e o g e n e s i s and A c t i v a t i o n o f G l y c o l y s i s 188 P h o s p h o g l y c e r a t e K i n a s e i n G l u c o n e o g e n i c T i s s u e s ...... 192 R e g u l a t o r y P r o p e r t i e s o f P y r u v a t e K i n a s e f r o m C a n c e r m a g i s t e r 193 EEF EE ENCiS 19 7 X L I S T OF FIGUBES F i g u r e 1. 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 o f ph c s p h o -g l y c e r a t e k i n a s e t h r o u g h o u t t h e m o l t c y c l e i n m u s c l e and h y p o d e r m i s 6 0 F i g u r e 2. A d i a g r a m m a t i c s k e t c h o f a s t a r c h g e l e l e c t r o p h c r e t o g r a m s h e w i n g z e n e s o f p h o s p h o g l y c e r a t e k i n a s e a c t i v i t y e x t r a c t e d f r o m m u s c l e and h y p o d e r m i s o f C a n c e r m a g i s t e r . 6 4 F i g u r e 3, M i c h a e l i s c o n s t a n t s f o r A1P and 3 - P - g l y c e r a t e a t v a r y i n g l e v e l s o f c o s u b s t r a t e and a c t i v a t o r . 66 F i g u r e 4. ADP i n h i b i t i o n o f i n t e r m o l t m u s c l e p h o s p h o -g l y c e r a t e k i n a s e , e f f e c t o f i n c r e a s i n g magnesium c o n c e n t r a t i o n . 7 2 F i g u r e 5. I n t e r m o l t m u s c l e p h o s p h o g l y c e r a t e k i n a s e , ADP and MgADP i n h i b i t i o n p a t t e r n s . 74 F i g u r e 6. M o l t i n g h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e , ADP and MgADP i n h i b i t i o n p a t t e r n s ......................... 78 F i g u r e 7. E f f e c t o f PgATP and FDP on PEP a f f i n i t y o f C a n c e r m a g i s t e r m u s c l e p y r u v a t e k i n a s e . ...................... 103 F i g u r e 8. MgATP i n h i b i t i o n , e f f e c t on t h e a f f i n i t y o f m u s c l e p y r u v a t e k i n a s e f o r PEP. 10 4 F i g u r e 9. MgATP i n h i b i t i o n , e f f e c t on ADP a f f i n i t y o f m u s c l e p y r u v a t e k i n a s e . 105 F i g u r e 10. A r g i n i n e p h o s p h a t e i n h i b i t i o n , e f f e c t on t h e ADP a f f i n i t y c f C a n c e r m a g i s t e r m u s c l e p y r u v a t e k i n a s e . ... 107 F i g u r e 1 1 . A r g i n i n e phosphate, i n h i b i t i o n , e f f e c t on t h e PEP a f f i n i t y o f t h e p y r u v a t e k i n a s e f r c m C a n c e r m a g i s t e r m u s c l e . 10 8 F i g u r e 12. D i x o n p l o t o f t r y p t o p h a n i n h i b i t i o n c f t h e m u s c l e p y r u v a t e k i n a s e f r c m C a n c e r j a g i s f e r . .......... 110 F i g u r e 13. D i x o n p l o t o f Mg-malate i n h i b i t i o n o f t h e m u s c l e p y r u v a t e k i n a s e f r o m C a n c e r m a g i s t e r . ................. 111 F i g u r e 14. D i x o n p l o t o f M g 2 c i t r a t e i n h i b i t i o n o f t h e m u s c l e p y r u v a t e k i n a s e f r o m C a n c e r m a g i s t e r . .......... 113 F i g u r e 15. T h e r m a l d e n a t u r a t i o n o f C a n c e r m a g i s t e r m u s c l e p y r u v a t e k i n a s e . .7 117 F i g u r e 16. P h o s p h o e n c l p y r u v a t e a f f i n i t y o f h y p o d e r m a l PK I . 129 x i F i g u r e 17. P h o s p h o e n c l p y r u v a t e a f f i n i t y o f h y p o d e r m a l PK I I . 13 0 F i g u r e 18. I n f l u e n c e o f FD E cn t h e p h o s p h o e n o l p y r u v a t e a f f i n i t y c f PK I and PK I I f r o m t h e h y p o d e r m i s o f Can£§£ m a g i s t e r . 132 F i g u r e 19. MgATP i n h i b i t i o n o f PK I , i n f l u e n c e c f MgATP on PEP a f f i n i t y and t h e r e v e r s a l o f MgATP i n h i b i t i o n . .... 134 F i g u r e 20. MgATP i n h i b i t i o n o f p h o s p h o e n o l p y r u v a t e b i n d i n g by PK I I and t h e r e v e r s a l c f t h i s i n h i b i t i o n by FDP. ..... 13 5 F i g u r e 2 1 . A l a n i n e i n h i b i t i o n o f PK I a n d PK I I and t h e i n f l u e n c e o f FDP on t h e i n h i b i t i o n p a t t e r n . ........... 13 8 F i g u r e 22. S e r i n e i n h i b i t i o n o f PK I I and t h e i n f l u e n c e o f FDP upon t h e i n h i b i t i o n p a t t e r n . ...................... 139 F i g u r e 2 3 a . M g 2 c i t r a t e i n h i b i t i o n o f PK I I , i n h i b i t i o n v e r s u s ADP and i n f l u e n c e o f FDP upon t h e i n h i b i t i o n p a t t e r n v e r s u s ADP and PEP. 141 F i g u r e 23b. M g 2 c i t r a t e i n h i b i t i o n o f PK I I , i n h i b i t i o n v e r s u s PEE. 142 F i g u r e 24. C o n v e r s i o n o f PK I I i n t o EK I by I n c u b a t i o n w i t h 0.05 mM IDP 147 F i g u r e 25. T h e r m a l d e n a t u r a t i o n o f C a n c e r 1 2 2 i s t e r h y p o d e r m a l p y r u v a t e k i n a s e I . 150 F i g u r e 26. T h e r m a l d e n a t u r a t i o n o f C a n c e r m a g i s t e r h y p o d e r m a l p y r u v a t e k i n a s e I I . 15 1 F i g u r e 27. A summary m e t a b o l i c map o f t h e c o n t r c l o f c a r b o h y d r a t e m e t a b o l i s m i n t h e c r u s t a c e a n h y p o d e r m i s . 156 x i i L I S T OF TABLES T a b l e 1 . S p e c i f i c a c t i v i t i e s o f v a r i o u s enzymes i n t i s s u e s o f f r e s h m o l t and i n t e r m o l t C a n c e r m a g i s t e r . 48 T a b l e 2. ATP and 3 - P - g l y c e r a t e a f f i n i t i e s o f phospho-g l y c e r a t e k i n a s e f r o m f r e s h m o l t a n d i n t e r m c l t t i s s u e s o f C a n c e r m a g i s t e r . 62 T a b l e 3. M i c h a e l i s c o n s t a n t s f o r a c t i v a t i n g c a t i o n s c f f r e s h m o l t and I n t e r m o l t p h c s p h o g l y c e r a t e k i n a s e s . 67 T a b l e 4. M i c h a e l i s c o n s t a n t s f o r ATP and MgATP . ........... 7 0 - T a b l e 5. K i v a l u e s f o r ADP and MgADP i n h i b i t i o n c f phospho-g l y c e r a t e k i n a s e f r o m f r e s h m o l t a n d i n t e r m o l t m u s c l e and h y p o d e r m i s . 73 T a b l e 6. K i v a l u e s f c r AMP, 2,3 DPG and CaC12 i n h i b i t i o n o f p h o s p h o g l y c e r a t e k i n a s e f r o m f r e s h m o l t and i n t e r m o l t m u s c l e and h y p o d e r m i s 82 T a b l e 7. P h o s p h o g l y c e r a t e k i n a s e mass a c t i o n r a t i o s f r c m i n t e r m o l t and f r e s h m o l t m u s c l e and h y p o d e r m i s 83 T a b l e 8. N u c l e o t i d e d i p h o s p h a t e a c c e p t a b i l i t y f o r t h e m u s c l e p y r u v a t e k i n a s e i n C a n c e r m a g i s t e r . . . 101 T a b l e 9. E f f e c t i v e n e s s o f a c t i v a t o r s and i n h i b i t o r s o f m u s c l e p y r u v a t e k i n a s e f r c m C a n c e r m a g i s t e r . 114 T a b l e 10. R e v e r s a l c f t h e m u s c l e p y r u v a t e k i n a s e f r o m C a n c e r m a g i s t e r . 116 T a b l e 11. E f f e c t i v e n e s s o f v a r i o u s i n h i b i t o r s o f h y p o d e r m a l PK I . .. .............. 14 3 T a b l e 12. E f f e c t i v e n e s s o f v a r i o u s i n h i b i t o r s o f h y p o d e r m a l PK I I . 14 4 T a b l e 13. E f f e c t o f FDP, ATE a n d a l a n i n e on t h e i n t e r c o n v e r s i o n s . o f h y p o d e r m a l PK I and PK I I f r o m lS2iSi§£ » * • 14 8 T a b l e 14, E x t r a c e l l u l a r s p a c e i n m u s c l e and h y p o d e r m i s o f S ag i s t e r 166 T a b l e 15. Hemolymph i o n c o n c e n t r a t i o n s i n i n t e r m o l t and f r e s h m o l t C a n c e r m a g i s t e r 166 T a b l e 16. F r e s h m o l t and i n t e r m o l t m u s c l e i o n c o n c e n t r a t i o n s . 17 0 T a b l e 17. F r e s h m o l t and i n t e r m o l t h y p o d e r m a l i o n c o n c e n t r a t i c n s . . 171 T a b l e 18. C o m p a r i s o n o f m u s c l e and h y p c a e r m a l i o n c o n c e n t r a t i o n s 17 3 T a b l e 19. C o m p a r i s o n o f k i n e t i c c h a r a c t e r i s t i c s o f m u s c l e and h y p c d e r m a l p y r u v a t e k i n a s e s f r o m C a n c e r m a g i s t e r . 19 <4 i > XX V ACKNOWLEDGEMENTS F i r s t o f f , I must t h a n k my s u p e r v i s o r , P e t e r H o c h a c h k a f o r i n t z e d u c i n g me t o t h e u n i q u e e x c i t e m e n t c f c o m p a r a t i v e b i o c h e m i s t r y . F u r t h e r m o r e , I must t h a n k P e t e r f o r h i s f r e g u e n t e n c o u r a g e m e n t and c l c s e i n t e r e s t i n t h e p r o g r e s s o f t h i s s t u d y , as w e l l as f o r h i s e n t h u s i a s m , e n e r g y , i n t u i t i o n , and h i g h g u o t i e n t o f s c i e n t i f i c c u r i o s i t y . S p e c i a l t h a n k s go t c Dr. J o h n P h i l l i p s and Dr. S t e v e n Shaw f o r t h e use o f t h e i r e q u i p m e n t d u r i n g t h e measurements o f e x t r a c e l l u l a r s p a c e and i o n i c c o n c e n t r a t i o n s . The a s s i s t a n c e o f J o a n M a r t i n and Tim B r a d l e y d u r i n g t h e s e s t u d i e s was a l s o h i g h l y a p p r e c i a t e d . . The c a r e f u l e v a l u a t i o n o f t h e t h e s i s by D r s . J o h n G o s l i n e , B i l l H o a r , and J o h n P h i l l i p s i s a l s c g r a t e f u l l y a c k n o w l e d g e d . D i s c u s s i o n s w i t h Dr. Dan A t k i n s o n cn p o r t i o n s o f t h e t h e s i s were a l s o g r e a t l y • a p p r e c i a t e d . E s p e c i a l t h a n k s go t c my h u s b a n d , J o h n Himmelman, f o r h i s w i l l i n g n e s s t o d i v e f o r a n i m a l s t h r o u g h o u t t h e y e a r , f o r h i s a s s i s t a n c e i n s t a t i s t i c a l a n a l y s i s , f o r h i s h e l p i n t r a n s f o r m i n g t h i s t h e s i s f r o m a t y p e w r i t t e n r o u g h d r a f t t o a c o m p u t e r i z e d p r o d u c t i o n , and f o r h i s f r e g u e n t h e l p , e n c o u r a g e m e n t , a nd s u p p o r t t h r o u g h o u t t h e w h o l e t h i n g . I must t h a n k t h e c r a b s f o r b e i n g s u c h i n t e r e s t i n g b e a s t s . j My f r i e n d s have been i n v a l u a b l e i n s u s t a i n i n g a n d e n t e r t a i n i n g me t h r o u g h o u t t h e c o u r s e o f my w o r k . I must g i v e s p e c i a l t h a n k s t o P a u l F a l k o w s k i , J o a n M a r t i n , B a r b a r a and D a v i d Moon, M i c h a e l Guppy, B r i a n Murphy, J e r e m y F i e l d s , C a r o l e S t a n l e y , Nancy S c h e r e r , Tim B r a d l e y , S t e v e Ha s w e l l , Owen and Penny B a m f o r d , P o l l y H a s w e l l , L a u r a D o l l i n e r , D i a n e W e s t - B o u r k e , C h r i s F r e n c h , and c f c o u r s e , Tom Mumford f o r m a k i n g l i f e a t t h e u n i v e r s i t y e x c i t i n g and i n t e r e s t i n g . Some o f t h e s e f r i e n d s , p a r t i c u l a r l y , P a u l , J o a n , J e r e m y , B r i a n , D i a n e , and Tom a l s o c o n t r i b u t e d a l o t t o my e n j o y m e n t o f l i f e away f r o m t h e u n i v e r s i t y a s d i d P e t e r N e s r o t h , J o a n n D r u e h l , F r a n k E o a s , G l a d y s B o a s , P e t e r and B r e n d a H o c h a c h k a , M a r t a and S t e w a r t Law, and l a s t b u t n o t l e a s t K a t z l e . 1 INTRODUCTION To g r o w , c r u s t a c e a n s must p e r i o d i c a l l y s h e d t h e i r e x o s k e l e t o n , e x p a n d i n volume w h i l e t h e i r e x o s k e l e t o n i s s o f t , and t h e n q u i c k l y h a r d e n t h e " s o f t s h e l l " . T h i s r a p i d t h i c k e n i n g and h a r d e n i n g r e q u i r e s b o t h e f f i c i e n t g l u c o n e o g e i i e s i s t o s u p p o r t c h i t i n s y n t h e s i s , and r a p i d c a l c i f i c a t i o n o f t h e n e w l y d e p o s i t e d l a m e l l a e o f c h i t i n . H h i l e e c d y s i s i s t h e most d r a m a t i c e v e n t i n t h e p r o c e s s o f c r u s t a c e a n g r o w t h , i t i s o n l y one p h a s e o f a c o m p l e x s e r i e s o f p h y s i o l o g i c a l and b e h a v i o r a l c h a n g e s w h i c h c o n s t i t u t e t h e c r u s t a c e a n m o l t c y c l e . The h y p o d e r m i s , l y i n g d i r e c t l y b e n e a t h t h e e x o s k e l e t o n , i s most d i r e c t l y a s s o c i a t e d w i t h t h i s l a r g e b i o s y n t h e t i c l o a d , b u t o t h e r c r u s t a c e a n t i s s u e s a r e a l s o p r o f o u n d l y a f f e c t e d by t h e m o l t c y c l e . S e v e r a l m a j o r c h a n g e s o c c u r d u r i n g t h e m o l t c y c l e . D u r i n g i n t e r m o l t , t h e a n i m a l i s a c t i v e l y f e e d i n g and d e p o s i t s c o n s i d e r a b l e l i p i d and g l y c o g e n . L i p i d i s s t o r e d p r i m a r i l y i n t h e h e p a t o p a n c r e a s ( P a s s a n o , 1960), w h i l e g l y c o g e n d e p o s i t s o c c u r i n m u s c l e , h y p o d e r m i s , h e p a t o p a n c r e a s , a n d b l o o d c e l l s (Hohnke, 1971; T r a v i s , 1955a; Renaud, 1949; J o h n s t o n & D a v i e s , 1972; D a l l , 1 9 7 5 ) . H o r m o n a l s i g n a l s i n i t i a t e t h e p r e m o l t p e r i o d : one o f t h e e a r l y c h a n g e s i s t h e s e p a r a t i o n o f t h e h y p o d e r m i s ' f r o m t h e e x o s k e l e t o n . A f t e r some i n i t i a l r e s o r p t i o n o f t h e o r g a n i c (and i n f r e s h w a t e r c r u s t a c e a n s , t h e i n o r g a n i c ) c o m p o n e n t s o f t h e e x o s k e l e t o n , t h e new c u t i c l e s t a r t s g r o w i n g u n d e r t h e o l d . New g i l l f i l a m e n t s f o r m i n s i d e t h e o l d , and t h e l e g m u s c l e s d e c r e a s e t o 60% o f t h e i r i n t e r m o l t mass ( S k i n n e r , 1966). D u r i n g l a t e p r e m o l t , w h o l e s a l e r e s o r p t i o n o f c h i t i n f r o m 2 the o l d c u t i c l e o c c u r s (Speck & U r i c h , 1971). J u s t p r i o r t o mo l t , the a n i m a l b e g i n s t o i n c r e a s e i t s volume, r e s o r b s much o f the c h i t i n from t h e m o l t i n g s u t u r e ( T r a v i s , 1955b), s p l i t s t h e s u t u r e and b e g i n s - t o p u l l out o f -the o l d s h e l l . G e n e r a l l y , t h e a n i m a l seeks a s h e l t e r e d p l a c e and ceases f e e d i n g s h o r t l y b e f o r e molt. Once t h e a n i m a l has withdrawn from t h e o l d c u t i c l e , r a p i d t h i c k e n i n g and h a r d e n i n g o f the new c u t i c l e • o c c u r s . T h i s i s c r i t i c a l as t h e s o f t , immobile a n i m a l i s h i g h l y s u s c e p t i b l e t o p r e d a t i o n . C h i t i n s y n t h e s i s i s maximal and r a p i d c a l c i u m d e p o s i t i o n o c c u r s u n t i l t h e c u t i c l e i s hardened ( S t e v e n s o n , ^ 1972; Adelung, 1971; T r a v i s , 1955a; Di g b y , -1966). Once t h e e x o s k e l e t o n i s - r e l a t i v e l y f i r m , t h e a n i m a l resumes f e e d i n g , and t i s s u e growth w i t h i n t h e i n c r e a s e d space b e g i n s (Passano,1960). ) ' . . . . . . . . . . . . The d u r a t i o n o f t h e molt c y c l e i s a f u n c t i o n o f a number o f p h y s i o l o g i c a l and e n v i r o n m e n t a l v a r i a b l e s , and d i f f e r e n t s p e c i e s respond to t h e s e v a r i a b l e s d i f f e r e n t l y ( B l i s s & B o y e r , 1964; Passano, 1960; Adelung, 1971). G e n e r a l l y , t h e c r a b m o l t s when i t has grown a s u f f i c i e n t q u a n t i t y , i . e . a g i v e n percentage o f i t s f r e s h m o l t mass. Thus, a n i m a l s of g r e a t e r s i z e t a k e l o n g e r t o complete t h e c y c l e (Passano, 1960; Adelung, 1971). Loss of l e g s a l s o s e r v e s t o s t i m u l a t e m o l t i n g . I f more than f i v e o f e i g h t w a l k i n g l e g s a r e removed, t h e i n t e r m o l t d u r a t i o n i s reduced by 50% ( S k i n n e r & Graham, 1972; Adelung, 1971). The presence o f l a r g e r members of t h e same s p e c i e s i n the same t a n k w i l l ' c a u s e an i n c r e a s e i n i n t e r m o l t d u r a t i o n / w h i l e t h e presence of an e q u a l s i z e member of t h e s p e c i e s does not a f f e c t t h e i n t e r m o l t d u r a t i o n i n C a r c i n u s l i e n a s (Adelung, 1971). G e c a r c i n u s l a t e r a l i s a l s o shows i n h i b i t i o n of p r o e c d y s i a l growth 3 i n t h e p r e s e n c e o f a n o t h e r c r a b , a s w e l l a s u n d e r h i g h t e m p e r a t u r e and l i g h t c o n d i t i o n s . On t h e o t h e r h a n d , d a r k n e s s , m o d e r a t e t e m p e r a t u r e , s o l i t u d e , and a s o u r c e o f w a t e r a l l - f a v o r p r o e c d y s i a l g r o w t h i n t h i s l a n d c r a b . G e c a r c i n u s lat§£a.lis a l s o h a s t h e c a p a b i l i t y t o d e l a y e c d y s i s o n c e t h e i n i t i a l p o r t i o n s o f p r e m o l t h a v e o c c u r r e d ( B l i s s & B o y e r , 1 9 6 4 ) . The f i r s t e x t e n s i v e f o r m u l a t i o n o f t h e c y c l i c p a t t e r n o f v a r i a t i o n s w h i c h c o n s t i t u t e s t h e m o l t c y c l e was made by D r a c h ( 1 9 3 9 ) , i n h i s s t u d i e s o f C a r e i n u s maenas and o t h e r b r a c h y u r a n s . H i s c l a s s i f i c a t i o n scheme a s s i g n s m o r p h o l o g i c a l c o n d i t i o n s , p r i m a r i l y i n v o l v i n g c h a n g e s i n c u t i c l e s t r u c t u r e , t o e a c h m o l t c y c l e s t a g e . The c r u s t a c e a n c u t i c l e i s composed o f f o u r m a j o r f l a y e r s : t h e e p i c u t i c l e w h i c h c o n t a i n s a h i g h p r o p o r t i o n o f l i p o p r o t e i n , t h e e x o c u t i c l e w h i c h c o n t a i n s p r o t e i n , c h i t i n , c r o s s l i n k s , a n d c a l c i u m , t h e e n d o c u t i c l e w h i c h c o n t a i n s p r o t e i n , c h i t i n a n d c a l c i u m , a n d t h e u n d e r l y i n g membranous l a y e r composed o f c h i t i n and p r o t e i n a l o n e . I n s t a g e A1, t h e a n i m a l i s f r e s h l y m o l t e d , w h i l e i n s t a g e A2 some m i n e r a l i z a t i o n o f t h e s o f t c u t i c l e has begun; D u r i n g , s t a g e B1 e n d o c u t i c l e s e c r e t i o n b e g i n s ; and i n s t a g e B2 t h e c h e l a e a r e hard> a c t i v e e n d o c u t i c l e f o r m a t i o n i s o c c u r r i n g , and t i s s u e g r o w t h b e g i n s . D u r i n g t h e v a r i o u s "C" s t a g e s t h e a n i m a l i s h a r d ; d u r i n g C1 a n d C2 t h e ma i n t i s s u e g r o w t h o c c u r s , i n C3 t h e c o m p l e t i o n o f t h e e x o s k e l e t o n by t h e f o r m a t i o n o f t h e membranous l a y e r o c c u r s . I n C4 t h e c l a s s i c 1 , i n t e r m o l t n c o n d i t i o n - p r e v a i l s , w i t h c o n s i d e r a b l e - a c c u m u l a t i o n o f o r g a n i c r e s e r v e s . The s t a g e s f r o m D1 t h r o u g h D4 c o n s t i t u t e t h e p r e m o l t p e r i o d ; ' D 0 has no m o r p h o l o g i c a l - c o r r e l a t e s , b u t i t marks t h e i n i t i a t i o n o f p r e m o l t by h o r m o n a l c h a n g e s . D1 i s 4 c h a r a c t e r i z e d by b o t h t h e s e p a r a t i o n o f t h e h y p o d e r m i s f r o m t h e o l d e x o s k e l e t o n and t h e f o r m a t i o n o f an e p i c u t i c l e . D u r i n g D2 t h e f o r m a t i o n o f t h e e x o c u t i c l e b e g i n s , i n D3 t h e m a j o r p o r t i o n o f r e s o r p t i o n o c c u r s , and i n D4 t h e e c d y s i a l s u t u r e s o p e n . S t a g e E i s e c d y s i s > t h e a c t u a l a c t o f m o l t i n g ( O r a c h , 1 9 3 9 ) . H h i l e t h e d e f i n i t i o n o f t h e s e s t a g e s has been m o d i f i e d s l i g h t l y t o f a c i l i t a t e s t a g i n g - i n c e r t a i n o r g a n i s m s ( P a s s a n o > 1960; S t e v e n s o n , 1 9 7 2 ) , t h e b a s i c p a t t e r n has been f o u n d t o a p p l y t o t h e d e v e l o p m e n t o f most c r u s t a c e a n s ( P a s s a n o , 1 9 6 0 ) . D r a c h ' s s t a g i n g c r i t e r i a were u s e d t h r o u g h o u t t h e p r e s e n t s t u d y . \ Numerous s t u d i e s o f t h e m o l t c y c l e h ave f o l l o w e d D r a c h ' s p i o n e e r i n g work. T h e s e s u b s e g u e n t i n v e s t i g a t i o n s have l e d t o a c o n s i d e r a b l e c l a r i f i c a t i o n o f t h e p h y s i o l o g y o f m o l t i n g . , F u r t h e r m o r e , t h e many s t u d i e s o f p a t t e r n s o f c a r b o n f l u x h e l p e l u c i d a t e t h e m e t a b o l i c c o n t r o l mechanisms u t i l i z e d by J c r u s t a c e a n s . I n t h e f o l l o w i n g p a g e s , I • w i l l a t t e m p t t o s u m m a r i z e t h e c u r r e n t p i c t u r e o f c r u s t a c e a n m e t a b o l i s m and i t s v a r y i n g f u n c t i o n s t h r o u g h o u t t h e m o l t c y c l e . The s t u d i e s w h i c h f o r m t h e body o f t h i s t h e s i s c o n c e r n m e t a b o l i c c h a n g e s i n t h e h y p o d e r m i s and m u s c l e o f C a n c e r m a g i s t e r a s t h e a n i m a l p a s s e s t h r o u g h t h e m o l t c y c l e . S p e c i f i c a l l y , t h e work f o c u s e s upon t h e e n z y m a t i c c o n t r o l ' mechanisms w h i c h f a c i l i t a t e g l u c o n e o g e n e s i s , t h e s y n t h e s i s o f s u g a r s f r o m n o n - e a r b o h y d r a t e p r e c u r s o r s , i n t h e h y p o d e r m i s and t h e i r c o m p a r i s o n w i t h c o n t r o l m e c h a n i s m s p r e s e n t i n t h e p r e d o m i n a n t l y g l y c o l y t i c m u s c l e . The two e n z y m e s w h i c h I • e x a m i n e d were p h o s p h o g l y c e r a t e k i n a s e a n d p y r u v a t e k i n a s e . B o t h enzymes p r e s e n t i n t e r e s t i n g 5 l o c i i n t h e c o n t r o l o f g l y c o l y s i s and g l u c o n e o g e n e s i s . P h o s p h o -g l y c e r a t e k i n a s e - i s one o f t h e g l y c o l y t i c enzymes t h o u g h t t o f u n c t i o n d u r i n g t h e r e v e r s a l o f g l y c o l y s i s • f o r g l u c o n e o g e n e s i s . However, t h e k i n e t i c s o f p r e v i o u s l y e x a m i n e d p h o s p h o g l y c e r a t e k i n a s e s s t r o n g l y f a v o r g l y c o l y t i c f u n c t i o n . To e l u c i d a t e how r e v e r s a l o f t h e r e a c t i o n i s f a c i l i t a t e d f o r g l u c o n e o g e n e s i s , I c o m p a r e d t h e r e g u l a t o r y c h a r a c t e r i s t i c s o f m u s c l e and h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s . D u r i n g g l u c o n e o g e n e s i s , p y r u v a t e k i n a s e a c t i v i t y must be c u r t a i l e d t o a l l o w f l u x p a s t t h e r e a c t i o n , w h i l e d u r i n g c a r b o h y d r a t e o x i d a t i o n , p y r u v a t e k i n a s e i s c e n t r a l i n t h e r e g u l a t i o n o f g l y c o l y t i c f l u x . A c o m p a r i s o n o f p y r u v a t e k i n a s e i n h y p o d e r m i s and m u s c l e s h o u l d c l a r i f y how t h e s e d i s t i n c t c o n t r o l r e g u i r e m e n t s a r e f u l f i l l e d . F r u c t o s e d i -p h o s p h a t a s e , t h e o t h e r m a j o r g l u c o n e o g e n i c c o n t r o l s i t e , h a s been s t u d i e d i n t h e h y p o d e r m i s , g i l l , and m u s c l e o f P a r a l i t h o d e s c a m t c h a t i c a ( H o c h a c h k a , 1972; B e h r i s c h , 1971, 1972; B e h r i s c h S J o h n s o n , 1 9 7 4 ) , A c l o s e e x a m i n a t i o n o f t h e r e g u l a t o r y p r o p e r t i e s o f p h o s p h o g l y c e r a t e k i n a s e and p y r u v a t e k i n a s e s h o u l d a l l o w t h e f o r m u l a t i o n o f a m e t a b o l i c c o n t r o l t h e o r y d e s c r i b i n g t h e r e g u l a t i o n o f h y p o d e r m a l g l u c o n e o g e n e s i s . C o m p a r i s o n o f t h e enzymes f r o m m u s c l e - a n d h y p o d e r m i s w i l l i n d i c a t e t o w h a t - e x t e n t s e p a r a t e i s o z y m e s have e v o l v e d t o a n s w e r t h e g l u c o n e o g e n i c r e q u i r e m e n t s o f t h e h y p o d e r m i s , a s w e l l a s c l a r i f y i n g t h e c o n t r o l m e c h anisms o f m u s c l e m e t a b o l i s m . 6 H o l t C y c l e P h y s i o l o g y The m a j o r o r g a n s o f t h e c r u s t a c e a n body u n d e r g o c o n s i d e r a b l e v a r i a t i o n s i n s i z e and c o m p o s i t i o n as t h e a n i m a l g o e s t h r o u g h t h e m o l t c y c l e , P a r t i c u l a r l y marked c h a n g e s a r e shown by t h e h y p o d e r m i s , t h e h e p a t o p a n c r e a s , a n d t h e m u s c l e . The h y p o d e r m i s i s an a c t i v e b i o s y n t h e t i c t i s s u e w h i c h u n d e r g o e s m a j o r a l t e r a t i o n s i n s i z e a c c o r d i n g t o t h e e x t e n t o f i t s b i o s y n t h e t i c a c t i v i t y . The h e p a t o p a n c r e a s i s b o t h a d i g e s t i v e and a s t o r a g e o r g a n , w h i c h shows c o n s i d e r a b l e s h i f t s i n t h e l e v e l s - o f o r g a n i c r e s e r v e s . W h i l e t h e r o l e o f t h e c h a n g e s shown by m u s c l e i s n o t c o m p l e t e l y c l a r i f i e d , t h e y a r e s t i l l r e m a r k a b l e . HypoiLe,£itti§. Upon i n i t i a t i o n o f p r e m o l t , t h e h y p o d e r m i s i n c r e a s e s i n t h i c k n e s s and e x t e n t ( T r a v i s , 1 9 5 5 a ) , shows an i n c r e a s e i n t h e r a t e o f p r o t e i n s y n t h e s i s ( S k i n n e r , 1968; M c S h i n n i e e t a l . , 1972; S t e v e n s o n , 1972; Humphreys & S t e v e n s o n , 1 9 7 3 ) , and shows an i n c r e a s e i n o x y g e n c o n s u m p t i o n ( S k i n n e r , 1958; S k i n n e r , 1 9 6 2 ) . The p r o t e i n s y n t h e s i s i s d i r e c t e d t o w a r d s b o t h t h e e n l a r g e m e n t o f t h e h y p o d e r m i s a n d t h e e v e n t u a l d e p o s i t i o n o f p r o t e i n i n t h e new e x o s k e l e t o n . The i n c r e a s e d o x y g e n c o n s u m p t i o n m a y , a c c o u n t f o r t h e i n c r e a s e i n w h o l e a n i m a l o x y g e n c o n s u m p t i o n f o u n d d u r i n g p r e m o l t ( B u l n h e i m , 1974; L e w i s & H a e f n e r , 1 9 7 6 ) . D u r i n g t h e f i r s t p a r t o f p r e m o l t , t h e h y p o d e r m i s s e c r e t e s f l u i d , e n z y m e s , and p o s s i b l y c e l l s i n t o t h e s p a c e between i t s e l f a n d t h e o l d e x o s k e l e t o n . T h e s e e n z y m e s a r e i n v o l v e d i n t h e r e s o r p t i o n o f c h i t i n and p r o t e i n f r o m t h e o l d e x o s k e l e t o n ( T r a v i s , 1955a; D e n n e l l , 1 9 6 0 ) . The h y p o d e r m i s i s 7 c a p a b l e o f c h i t i n s y n t h e s i s t h r o u g h o u t t h e m o l t c y c l e ( H o r n u n g S S t e v e n s o n , 1 9 7 1 ; S t e v e n s o n , 1 9 7 2 ) ; h o w e v e r , t h e r a t e i n c r e a s e s m a r k e d l y d u r i n g s t a g e D2 o f p r e m o l t , a nd r e a c h e s i t s maximum d u r i n g s t a g e s A a n d B d i r e c t l y f o l l o w i n g e c d y s i s { S t e v e n s o n , 1 9 7 2 ) . D u r i n g p r e m o l t , l a r g e q u a n t i t i e s o f g l y c o g e n and -some l i p i d a r e d e p o s i t e d i n t h e h y p o d e r m i s ( T r a v i s , 1 9 5 5 a ; R e n a u d , 1 9 4 9 ) . T h e s e r e s e r v e s a r e p r e s e n t i m m e d i a t e l y a f t e r t h e m o l t , b u t . t h e n d e c l i n e d u r i n g s e c r e t i o n o f t h e new e x o s k e l e t o n ( R e n a u d , 1949; T r a v i s , 1 9 5 7 ) . The c o n n e c t i v e t i s s u e w h i c h l i e s - d i r e c t l y b e n e a t h t h e h y p o d e r m i s i s s u p p l i e d w i t h numerous r e s e r v e c e l l s , w h i c h p r o b a b l y o r i g i n a t e f r o m b l o o d c e l l s . These c e l l s u n d e r g o c o n s i d e r a b l e v a r i a t i o n s b o t h i n number and i n amount o f s t o r e d m a t e r i a l s , d u r i n g t h e p o s t m o l t p e r i o d ( T r a v i s , 1957; S k i n n e r , 1 9 6 2 ) . T h e s e c e l l s a r e s o m e t i m e s f u l l o f ) g l y c o p r o t e i n , m u c o p o l y s a c c h a r i d e s , o r l i p o p r o t e i n . They may , a l s o c o n t a i n d e p o s i t s o f c a l c i u m and p h o s p h a t e ( S e w e l l , 1 9 5 5 ; T r a v i s , 1957; S k i n n e r , 1 9 6 2 ) . L a r g e q u a n t i t i e s o f i n o r g a n i c m a t e r i a l , p r i m a r i l y c a l c i u m and p h o s p h a t e , a l s o move t h r o u g h t h e ' h y p o d e r m i s t o t h e new e x o s k e l e t o n d u r i n g p o s t m o l t . B o t h t h e p r e e x u v i a l l a y e r s a n d t h e f r e s h l y d e p o s i t e d l a m e l l a e o f c h i t i n a r e c a l c i f i e d a f t e r t h e m o l t . A f t e r t h e s y n t h e s i s a n d c a l c i f i c a t i o n • o f t h e e x o s k e l e t o n i s c o m p l e t e , t h e h y p o d e r m i s d e c r e a s e s i n s i z e a n d o n l y p e r f o r m s a low r a t e o f c h i t i n s y n t h e s i s ( T r a v i s , 1 9 5 5 a ; S k i n n e r , 1962; S t e v e n s o n , 1 9 7 2 ) . S e p a t o p a n c r e a s ^ - The h e p a t o p a n c r e a s o f i n t e r m o l t c r u s t a c e a n s g e n e r a l l y shows h i g h l e v e l s o f g l y c o g e n a n d l i p i d s ( Renaud, 1949; T r a v i s , 1955a; -Heath S B a r n e s , 1 9 7 0 ) . I n C a r c i n u s j a e n a s , t h e l e v e l s o f f a t t y a c i d s a r e t e n f o l d t h o s e o f 8 g l y c o g e n ( H e a t h & B a r n e s , 1 9 7 0 ) . I n C a n c e r £aqurus, l i p i d s a l s o f o r m a m a j o r p a r t o f t h e m e t a b o l i c s t o r e s (Henaud, 1 9 4 9 ) . The h e p a t o p a n c r e a s i s composed o f a v a r i e t y o f c e l l t y p e s , i n c l u d i n g a b s o r p t i v e , s e c r e t o r y , a nd r e s e r v e c e l l s . D u r i n g i n t e r m o l t a n d e a r l y p r e m o l t , f a t a n d g l y c o g e n a r e a b u n d a n t i n b o t h t h e a b s o r p t i v e and t h e s e c r e t o r y c e l l s . I n - l a t e p r e m o l t , t h e r e s e r v e c e l l s i n c r e a s e i n number and a r e f o u n d t o c o n t a i n m u c o p o l y s a c c h a r i d e , ' l i p i d , and s o m e t i m e s d e p o s i t s o f c a l c i u m . I n f r e s h w a t e r f o r m s ; c a l c i u m s t o r a g e b e g i n s - e a r l y i n p r e m o l t w i t h t h e f o r m a t i o n o f g a s t r o l i t h s , w h i l e i n m a r i n e c r u s t a c e a n s c a l c i u m and p h o s p h a t e s t o r a g e o c c u r s o n l y i n l a t e p r e m o l t ( M c W h i n n i e e t a l i , 1972; Chen & L e h n i n g e r , 1 9 7 J ; G l y n n , 1968; T r a v i s , 1955a; R e n a u d , 1 9 4 9 ) . I n P a n u l i r u s a r q u s l a t r i e l l e , g l y c o g e n l e v e l s i n t h e h e p a t o p a n c r e a s a r e a t t h e i r p e a k d u r i n g t h e f i r s t day a f t e r e c d y s i s , and t h e n p r o g r e s s i v e l y d e c l i n e u n t i l t h e s e v e n t h d a y , by w h i c h t i m e t h e y have d i s a p p e a r e d ( T r a v i s , 1 9 5 5 a ) . L i p i d l e v e l s d e c l i n e as w e l l b u t n o t t o t h e same e x t e n t a s t h e g l y c o g e n l e v e l s . The r e s e r v e c e l l s show c y c l i c v a r i a t i o n s , w h i c h , i f t a k e n i n r e l a t i o n t o t h o s e shown by t h e r e s e r v e c e l l s i n t h e h y p o d e r m i s (see above)> c o u l d i n d i c a t e t h a t t h e s e c e l l s a r e moving f r o m t h e h e p a t o p a n c r e a s t o t h e h y p o d e r m i s , ' a n d s u p p l y i n g t h e h y p o d e r m i s w i t h s u b s t r a t e f o r c h i t i n s y n t h e s i s ( T r a v i s , 1955a; T r a v i s , 1 9 5 7 ) : C a l c i u m l e v e l s i n t h e h e p a t o p a n c r e a s d r o p m a r k e d l y s o t h a t a week a f t e r t h e m o l t , no " c a l c o s p h e r i t e s " a r e v i s i b l e i n t h e t i s s u e ( T r a v i s , 1 9 5 7 ) . Renaud (1949 ) has c a l c u l a t e d t h a t 70 % of t h e p r o t e i n n i t r o g e n p r e s e n t ' i n t h e s t a g e D 1 • h e p a t o p a n c r e a s i s u t i l i z e d d u r i n g t h e s u c c e e d i n g m o l t . S i m i l a r c y c l i c v a r i a t i o n s i n 9 h e p a t o p a n c r e a t i c s t o r e s have been f o u n d t o o c c u r i n C a n c e r £ao turus (Kenaud, 1 949) , C a r c i n u s maenas ( H e a t h & B a r n e s , 1 9 7 0 ) , and P a n u l i r u s a r g u s l a t r i e l l e ( T r a v i s , 1955a; T r a v i s , 1 9 5 7 ) . However, s u c h s t o r a g e c y c l e s do n o t n e c e s s a r i l y h o l d f o r a l l c r u s t a c e a n s . ' C a r c i n u s maenas have been o b s e r v e d t o m o l t w i t h v i r t u a l l y no m e t a b o l i c s t o r e s ( H e a t h & B a r n e s , 1 9 7 0 ) . I n O r c o n e c t e s l i m o s u s , t h e m e t a b o l i c s t o r e s p r e s e n t i n t h e a n i m a l do n o t s i g n i f i c a n t l y augment t h e m a i n t e n a n c e m e t a b o l i s m o r t h e a c t i v e b i o s y n t h e t i c o u t p u t d u r i n g e c d y s i s . I n t h e s e a n i m a l s , c a r b o n f r o m r e s o r b e d a c e t y l g l u c o s a m i n e p r o v i d e s t h e s o l e s u p p o r t f o r b o t h c h i t i n s y n t h e s i s and e n e r g y m e t a b o l i s m d u r i n g t h e l a t e p r e m o l t and e a r l y p o s t m o l t p e r i o d ( S p e c k & U r i c h , 1 9 7 1 ) . Muscle.. The c h a n g e s i n c u r r e d by c r u s t a c e a n m u s c l e a s a f u n c t i o n o f t h e m o l t c y c l e may c o n t r i b u t e t o t h e a b o v e d e s c r i b e d c y c l e s o f s t o r e d compounds i n t h e h y p o d e r m i s and h e p a t o p a n c r e a s . However, no i n f o r m a t i o n i s a v a i l a b l e t o e i t h e r s u p p o r t o r r e f u t e t h i s c o n c e p t . The c h a n g e s i n m u s c l e have been t h o r o u g h l y d e s c r i b e d i n o n l y one a n i m a l , G e c a r c i n u s l a t e r a l i s ( S k i n n e r , 1 9 6 6 ) . D u r i n g p r e m o l t , t h e m u s c l e s p r e s e n t i n t h e l e g s o f t h e s e a n i m a l s d e c r e a s e t o 60% o f t h e i r i n t e r m o l t mass. The r a t i o o f p r o t e i n t o wet w e i g h t s t a y s c o n s t a n t , t h u s , t h e c h a n g e i s n o t due t o a c h a n g e i n t i s s u e h y d r a t i o n . The amount o f DNA i n t h e w h o l e m u s c l e s t a y s c o n s t a n t , i n d i c a t i n g t h a t t h e r e i s a d r o p i n t h e amount o f p r o t e i n p r e s e n t p e r c e l l ( S k i n n e r , 1 9 6 6 ) . T h i s d e c r e a s e i n m u s c l e mass may f a c i l i t a t e t h e w i t h d r a w a l o f t h e l e g t h r o u g h t h e n a r r o w j o i n t s i n t h e o l d e x o s k e l e t o n . R e f o r m a t i o n o f t h e m u s c l e d i d n o t o c c u r u n t i l b e t w e e n 6 and 20 d a y s p o s t m o l t ( S k i n n e r , 1 9 6 6 ) . A l t h o u g h S k i n n e r ' s (1966) s t u d y i s t h e o n l y 10 s y s t e m a t i c s t u d y o f t h i s t o p i c , e a r l y r e s e a r c h e r s (Hoet & K e r r i d g e , 1926) f o u n d s i m i l a r c h a n g e s i n t h e o v e r a l l c h a r a c t e r i s t i c s o f m u s c l e f r o m i n t e r m o l t and f r e s h m o l t a n i m a l s . F u r t h e r m o r e , i t i s an e s t a b l i s h e d f a c t among l o b s t e r f i s h e r m e n t h a t t h e m u s c l e o f f r e s h l y m o l t e d a n i m a l s i s q u i t e d i f f e r e n t f r o m t h a t i n i n t e r m o l t a n i m a l s . The l i p o p r o t e i n c e l l s , w h i c h a r e known t o i n v a d e t h e m u s c l e d u r i n g p r e m o l t ( S e w e l l , 1 9 5 5 ) , may a i d e i n t h e d e s t r u c t i o n o f m u s c l e p r o t e i n . A c c o r d i n g t o a r e c e n t s t u d y , t h e p r o t e i n l o s t f r o m t h e m u s c l e a c c o u n t s f o r t h e i n c r e a s e d l e v e l s o f hemolymph p r o t e i n w h i c h o c c u r j u s t b e f o r e m o l t ( M a n t e l e t a l . , 1 9 7 5 ) . The m u s c l e p r o t e i n c o u l d a l s o c o n t r i b u t e t o t h e i n c r e a s e d l e v e l s o f t i s s u e f r e e a m i n o a c i d s f o u n d d u r i n g t h e l a t e p r e m o l t p e r i o d ( D u c h a t e a u e t a , l . , 1959; McWh i n n i e e t a l . , 1 9 72; Speck & U r i c h , 1 9 7 1 ) . Hemolyj^)h.._ The o t h e r t i s s u e w h i c h u n d e r g o e s a f a i r number o f c h a n g e s i s t h e hemolymph. D u r i n g p r e m o l t , t h e wh o l e b l o o d i s c h a r a c t e r i z e d by h i g h l e v e l s o f a n i n o a c i d s , p r o t e i n , c a l c i u m ' , p h o s p h a t e , g l u c o s e , and f a t s ( T r a v i s , 1955b; F l o r k i n , 1960; A d e l u n g , 1 9 7 1 ) . I n G e e a r e i n u s l a t e r a l i s , the b l o o d o s m o t i c c o n c e n t r a t i o n d r o p s f r o m 1000 mOsm b e f o r e m o l t t o 875 mOsm a t e c d y s i s ( M a n t e l e t a l . , 1 9 7 5 ) . P r e s u m a b l y , t h e p r e m o l t i n c r e a s e i n b l o o d o s m o l a r i t y f a c i l i t a t e s t h e u p t a k e o f w a t e r d u r i n g e c d y s i s and l a t e p r e m o l t ( A d e l u n g , 1 9 7 1 ) . Changes i n t h e i o n i c c o m p o s i t i o n o f t h e serum a l s o o c c u r . S p e c i f i c a l l y , p o s t m o l t s o d i u m , p o t a s s i u m , magnesium, and c h l o r i d e c o n c e n t r a t i o n s a r e c o n s i d e r a b l y l o w e r t h a n t h e p r e m o l t v a l u e s , f o r C a r c i n u s maenas ( S o b e r t s o n , 1 9 6 0 ) . C h a n g e s i n t h e magnesium and c a l c i u m c o n c e n t r a t i o n were f o u n d f o r Homarus v u l g a r i s b y G l y n n (196 8) 11 and C a r e i n u s maenas by A d e l u n g ( 1 9 7 1 ) . I n t h e l a t t e r s t u d y , p r o n o u n c e d v a r i a t i o n s i n t h e p o t a s s i u m c o n c e n t r a t i o n were n o t f o u n d , a n d s o d i u m c o n c e n t r a t i o n s were f o u n d t o v a r y i n a l e s s s y s t e m a t i c f a s h i o n t h a n i n B o b e r t s o n ' s (1960) s t u d y . The a b s o l u t e number o f b l o o d c e l l s c i r c u l a t i n g a t m o l t i s 2 t o 5 t i m e s h i g h e r t h a n d u r i n g i n t e r m o l t ( M a y n a r d , 1 9 6 0 ) . T h e r e a r e two t y p e s o f b l o o d c e l l s i n c r u s t a c e a n s , one o f w n i c h h a s been f o u n d t o c o n t a i n a l a r g e g l y c o g e n and a c i d p o l y s a c c h a r i d e s t o r e , a s w e l l a s t h e enzyme g l u c o s e - 6 - p h o s p h a t a s e ( J o h n s t o n e t a l , , 1 9 7 3 ) . The c a r b o h y d r a t e s c o n t a i n e d by t h e s e c a l l s r e p r e s e n t a p p r o x i m a t e l y 5 0 % o f t h e t o t a l a v a i l a b l e c a r b o h y d r a t e i n C a r e i n u s maenas ( J o h n s t o n e t a l . , 1 9 7 3 ) , By c o n t r a s t , D a l l ( 1 9 7 5 ) , f o u n d o n l y 5.5% o f a v a i l a b l e c a r b o h y d r a t e i n t h e b l o o d c e l l s o f P a n u l i r u s I g n g i p e s . The w h o l e b l o o d c o n t a i n s a v a r i e t y o f c i r c u l a t i n g c a r b o h y d r a t e s , i n c l u d i n g g l u c o s e , t r e h a l o s e , m a l t o s e , f r u c t o s e , f u c o s e , g l u c o s a m i n e , g l u c o s e s -p h o s p h a t e , g a l a c t o s e , mannose, m a l t o t r i o s e , v a r i o u s o l i g o -s a c c h a r i d e s , and i n P a n u l i r u s I o n g i p e s , two d i s t i n c t g l y c o p r o t e i n s ( D a l l , 1975; T e l f o r d , 1968; S c h w o c h , 1972; P a r v a r t h y , 1970; J o h n s t o n S D a v i a s , 1 9 7 2 ) . W i t h t h e e x c e p t i o n o f g l u c o s e , no c l e a r v a r i a t i o n o f any o f t h e s e c o m p o n e n t s w i t h / t h e m o l t c y c l e h a s been f o u n d ( J e n i a u x , 1 9 7 1 ) . E v e n t h e v a r i a t i o n due t o g l u c o s e i s somewhat e r r a t i c ( A d e l u n g , 1 9 7 1 ) . However, g l u c o s e c o n c e n t r a t i o n s g e n e r a l l y d e c r e a s e a f t e r m o l t ( A d e l u n g , 1971; D a l l , 1 9 7 5 ) . M 2 £ 1 2 M i C o n t r o l M e c h a n i s m s . H o r m o n a l c o n t r o l m e c h a n i s m s a r e u n d o u b t e d l y o f u n d e r l y i n g i m p o r t a n c e i n t h e c o n t r o l o f t h e m o l t c y c l e . C o n s i d e r a b l e work has been done on h o r m o n a l c o n t r o l 12 mechanisms i n C r u s t a c e a , and a r e l a t i v e l y c l e a r p i c t u r e o f t h e c o n t r o l o f m o l t i n g h a s now emerged. Two m a j o r e n d o c r i n e g l a n d s a r e i n v o l v e d i n t h e c o n t r o l o f t h e m o l t c y c l e , t h e s i n u s g l a n d -m e d u l l a t e r m i n a l i s X o r g a n c o m p l e x and t h e Y o r g a n . The s i n u s g l a n d a n d t h e m e d u l l a t e r m i n a l i s X o r g a n a r e l o c a t e d i n t h e e y e s t a l k ; t h e y c o n t a i n and r e l e a s e , among o t h e r h o r m o n e s , a m o l t i n h i b i t i n g hormone. T h u s , e y e s t a l k a b l a t i o n u s u a l l y r e s u l t s i n p r e c o c i o u s e c d y s i s . The m a j o r t a r g e t o r g a n o f t h i s m o l t i n h i b i t i n g hormone i s p r o b a b l y t h e Y o r g a n . T h i s o r g a n shows h i s t o l o g i c a l c h a n g e s w h i c h a r e w e l l c o r r e l a t e d w i t h t h e m o l t c y c l e (Gabe, 1 9 5 3 ) . f u r t h e r m o r e , r e m o v a l o f t h e s e g l a n d s l e a d s t o a p e r m a n e n t i n h i b i t i o n o f m e l t i n g i n i n t e r m o l t a n i m a l s ( E c h a l i e r , 1 9 5 4 ) . The I o r g a n r e l e a s e s a m o l t a c c e l e r a t i n g hormone o r hormones ( F i n g e r m a n , 1 9 7 3 ) . The Y o r g a n may s e c r e t e a p r o h o r m o n e w h i c h i s t h e n c o n v e r t e d t o an a c t i v e f o r m i n v a r i o u s t i s s u e s t h r o u g h o u t t h e body ( F i n g e r m a n , 1 9 7 4 ) . The hormones i n v o l v e d a r e s t e r o i d s s i m i l a r t o e c d y s o n e : c r u s t e c d y s o n e i s 2 0 - h y d r o x y e c d y s o n e and c a l l i n e c d y s o n e A and B a r e i n o k s t e r o n e and m a k i s t e r o n e r e s p e c t i v e l y ( F i n g e r m a n , 1 9 7 3 ) . The h o r m o n a l c o n t e n t o f C a r e i n u s maenas and C a l l r n e c t e s s a j i d u s h a s been measured a t i n t e r v a l s t h r o u g h o u t t h e m o l t c y c l e ( A d e l u n g , 1 9 7 1 ; F a u x e t a l . , 1 9 6 9 ) . I n C a r e i n u s m a e nas, t h e t i t r e o f 2 0 - h y d r o x y e c d y s o n e p a s s e s t h r o u g h s e v e r a l d i s t i n c t maxima d u r i n g p r e m o l t , s r i t h t h e m a j o r peak and f a l l o c c u r r i n g s h o r t l y b e f o r e m o l t ( A d e l u n g , 1 9 7 1 ) . O£conectes l i m o s u s a l s o showed a s t e e p d r o p i n 2 0 - h y d r o x y e c d y s o n e j u s t b e f o r e m o l t ( K e l l e r & A d e l u n g , 1 9 7 0 ) . I n C a l l i n e c t e s s a p i d u s . a r i s i n g t i t r e o f hormones was f o u n d a s w e l l , b u t i n t h i s o r g a n i s m 13 s e v e r a l hormones were m e a s u r e d , c a l l i n e c d y s o n e A, 20-h y d r o x y e c d y s o n e , and c a l l i n e c d y s o n e B. E a r l y i n p r e m o l t , o n l y c a l l i n e c d y s o n e A was p r e s e n t . I t c o n t i n u e d t o i n c r e a s e i n c o n c e n t r a t i o n and l a t e r i n p r e m o l t was j o i n e d by l o w l e v e l s o f 2 0 - h y d r o x y e c d y s o n e . A f t e r e c d y s i s , t h e l a t t e r hormone was p r e s e n t i n 70 f o l d h i g h e r l e v e l s t h a n b e f o r e m o l t , i n c o n t r a s t t o t h e s i t u a t i o n i n C a r c i n u s maenas. C a l l i n e c d y s o n e B was a l s o p r e s e n t i n l o w l e v e l s d u r i n g p o s t m o l t (Faux e t a l . , 1 9 6 9 ) . T h u s , a c o m p l e x i n t e r p l a y o f d i f f e r e n t e c d y s o n e s may o c c u r , w i t h p e a k s o f hormone c o n t e n t i n i t i a t i n g c e r t a i n a s p e c t s o f d e v e l o p m e n t . A d e l u n g (1971) c o r r e l a t e d t h a p e a k s i n 20-h y d r o x y e c d y s o n e w i t h p e a k s i n hemolymph p r o t e i n and a m i n o a c i d c o n c e n t r a t i o n , s u g g e s t i n g t h a t t h e s t e r o i d s t i m u l a t e d p r o t e i n s y n t h e s i s . E c d y s o n e s t i m u l a t i o n o f p r o t e i n s y n t h e s i s i s i n d i c a t e d i n some i n s e c t s y s t e m s ( K a r l s o n S S e k e r i s , 1 9 6 2 ) . The e v i d e n c e i s n o t a s p e r f e c t e d f o r c r u s t a c e a n s y s t e m s , b u t t h e c i r c u m s t a n t i a l e v i d e n c e i s g u i t e g o o d . I n c r e a s e s i n rRNA p a r a l l e l t h e i n c r e a s e i n p r o t e i n s y n t h e s i s i n G e c a r c i n u s l a t e r a l i s ( S k i n n e r , 1 9 6 8 ) . A c t i n o m y c i n D i n h i b i t i o n o f RNA s y n t h e s i s p r e v e n t s t h e p r e m o l t i n i t i a t i o n o f c h i t i n s y n t h e s i s i n t h e c r a y f i s h ( S t e v e n s o n S T ung, 1 9 7 1 ) . S l i g h t i n c r e a s e s i n t h e r a t e o f a m ino a c i d i n c o r p o r a t i o n w e r e -found upon e c d y s o n e s t i m u l a t i o n o f t h e h y p o d e r m i s o f O r c o n e c t e s y j . r i . l i s - ( M c W h i n n i e a t a l . , 1 9 7 2 ) . I n O r c o n e c t e s o b s c u r u s and O r c o n e c t e s s a n b o r n i t h e r a t e ' o f p r o t e i n s y n t h e s i s i n c r e a s e s d u r i n g p r e m o l t s h o w i n g two maxima ( S t e v e n s o n , 1972) w h i c h c o r r e s p o n d w i t h t h e maxima o f 2 0 - h y d r o x y e c d y s o n e shown d u r i n g p r e m o l t i n C a r c i n u s maenas { A d e l u n g , 1 9 7 1 ) . I n O r c o n e c t e s l i m o s u s , p e a k s o f RNA s y n t h e s i s 14 f o l l o w t h e p e a k s i n 2 0 - h y d r o x y e c d y s o n e w i t h a c e r t a i n d e l a y ( K e l l e r & A d e l u n g , 1 9 7 0 ) . I n summary, t h e i n i t i a t i o n o f p r e m o l t comes a b o u t t h r o u g h a r e d u c t i o n i n t h e l e v e l s o f a m o l t i n h i b i t i n g hormone s e c r e t e d by t h e m e d u l l a t e r m i n a l i s X o r g a n . T h i s t h e n r e l e a s e s t h e I o r g a n f r o m i n h i b i t i o n and a l l o w s i t t o r e l e a s e t h e a p p r o p r i a t e m o l t a c c e l e r a t i n g hormone o r p r o h o r m o n e . T h i s hormone t h e n a c t i v a t e s t h e s y s t e m s o f p r o t e i n s y n t h e s i s , p r i m a r i l y i n t h e t e g u m e n t a l g l a n d s and t h e h y p o d e r m i s ( K e l l e r & A d e l u n g , 1 9 7 0 ) . The i n c r e a s i n g t i t r e s o f hormone a s t h e a n i m a l p a s s e s t h r o u g h p r e m o l t p r o b a b l y p l a y an i m p o r t a n t r o l e i n i n i t i a t i n g c e r t a i n p h y s i o l o g i c a l a c t i v i t i e s . H o wever, t h e g u e s t i o n a s t o what c a u s e s t h e i n i t i a l r e d u c t i o n o f m o l t i n h i b i t i n g hormone needs c o n s i d e r a t i o n . I n some c a r e f u l s t u d i e s o f C a r c i n u s maenas, A d e l u n g (1971) showed t h a t r e d u c t i o n o f m o l t i n h i b i t i n g hormone was p r o b a b l y d e p e n d e n t upon t h e a n i m a l r e a c h i n g a c e r t a i n d e g r e e o f g r o w t h . A s h o r t e n i n g o f t h e i n t e r m o l t p e r i o d , i . e . a p r e c o c i o u s r e d u c t i o n i n t h e l e v e l s o f m o l t i n h i b i t i n g hormone, o c c u r r e d o n l y a f t e r r e m o v a l o f more t h a n f i v e o f t h e e i g h t w a l k i n g l e g s . A l l t h e f a c t o r s w h i c h i n h i b i t p r e m o l t i n i t i a t i o n , seem t o do s o v i a an i n h i b i t i o n o f g r o w t h . The a n i m a l s consume l e s s f o o d d u r i n g u n f a v o r a b l e e n v i r o n m e n t a l c o n d i t i o n s , i n t h e p r e s e n c e o f a l a r g e r member o f t h e same s p e c i e s , o r i n c o l d t e m p e r a t u r e s ( A d e l u n g , 1 9 7 1 ) . F u r t h e r p r o o f o f t h e s e i n t e r a c t i n g c o n t r o l mechanisms comes f r o m t h e f a c t t h a t d i f f e r e n t s i z e a n i m a l s , w h i c h w o u l d o r d i n a r i l y t a k e d i f f e r e n t l e n g t h s o f t i m e t o m o l t , m o l t w i t h e g u a l r a p i d i t y a f t e r e y e s t a l k a b l a t i o n . F u r t h e r m o r e , a n i m a l s f r o m d i f f e r e n t n u t r i t i o n a l 15 r e g i m e s a l s o m o l t w i t h t h e same f r e g u e n c y a f t e r e y e s t a l k a b l a t i o n (Adelung> 1971) . However, t h e e x a c t p h y s i c a l mechanism whereby a d e g u a t e g r o w t h i s t r a n s l a t e d i n t o t h e c e s s a t i o n o f s e c r e t i o n o f m o l t i n h i b i t i n g hormone i s n o t known. The m o l t c y c l e i s n o t t h e o n l y s y s t e m w h i c h i s u n d e r h o r m o n a l c o n t r o l i n c r u s t a c e a n s . - B e s i d e s d i r e c t i n g t h e - p a t t e r n c f r e p r o d u c t i v e e v e n t s ( F i n g e r m a n , 1 9 7 3 ) , hormones a r e i n v o l v e d w i t h t h e c o n t r o l o f s a l t a n d w a t e r b a l a n c e ( H e i t 6 F i n g e r m a n , 1975) and w i t h c o n t r o l o f r e s p i r a t i o n d u r i n g t e m p e r a t u r e a c c l i m a t i o n ( S i l v e r t h o r n , 1975)* F u r t h e r m o r e , hormones a l s o c o n t r o l c e r t a i n • a s p e c t s o f c a r b o h y d r a t e m e t a b o l i s m . The s i n u s g l a n d c o n t a i n s a h y p e r g l y c e m i c p e p t i d e hormone. I n j e c t i o n o f e y e s t a l k e x t r a c t i n C a n c e r m a q i s t e r and HemijgraDSus n u d u s c a u s e d an i n c r e a s e i n b l o o d m a l t o s e l e v e l s ( R a m a m u r t h i e t a l . , 1968). D e s t a l k i n g t h e s e a n i m a l s l e a d s t o an i n c r e a s e i n m u s c l e g l y c o g e n s y n t h e t a s e l e v e l s and a c o n c o m i t a n t d e c r e a s e i n t o t a l p h o s p h o r y l a s e . The e x p e c t e d i n c r e a s e i n g l y c o g e n d e p o s i t i o n was n o t f o u n d , i n s t e a d , an i n c r e a s e i n t u r n o v e r was f o u n d ( R a m a m u r t h i e t a l . , 1968). K e l l e r (1969) f o u n d t h a t t h e h y p e r g l y c e m i c p e p t i d e hormone o f Q r c o n e c t e s l i m o s u s was c r o s s r e a c t i v e w i t h c l o s e l y r e l a t e d s p e c i e s b u t n o t w i t h a v a r i e t y o f o t h e r c r u s t a c e a n s . T e l f o r d (1975) f o u n d t h a t t h i s p e p t i d e i n d u c e d i n c r e a s e d m u s c l e g l y c o g e n p h o s p h o r y l a s e a c t i v i t y a n d t h a t m u s c l e c o n t r i b u t e d t h e b u l k o f t h e g l u c o s e t o h y p e r g l y c e m i a . 16 I n t e r m e d i a r y M e t a b o l i s m i n C r u s t a c e a n s W i t h i n t h i s f r a m e w o r k o f m a j o r p h y s i o l o g i c a l a l t e r a t i o n s d u r i n g t h e m o l t c y c l e , t h e c o n t r o l mechanisms and b a s i c p a t t e r n s o f i n t e r m e d i a r y m e t a b o l i s m i n c r u s t a c e a n s assume a d d i t i o n a l i n t e r e s t . Not o n l y w o u l d t h e g r o w i n g p r e m o l t h y p o d e r m i s s e r v e a s a g o o d s y s t e m f o r t h e s t u d y o f h o r m o n a l i n d u c t i o n o f enzyme s y n t h e s i s , b u t t h e m u s c l e w o u l d s e r v e a s a g ood s y s t e m w i t h i n w h i c h t o c o n s i d e r t h e c h a n g e s w h i c h o c c u r d u r i n g t i s s u e b r e a k d o w n . F u r t h e r m o r e , and o f c e n t r a l i n t e r e s t t o t h i s s t u d y , t h e g l u c o n e o g e n i c a n d b i o s y n t h e t i c m e c hanisms u t i l i z e d by t h e h y p o d e r m i s d u r i n g t h e i n i t i a l p o s t m o l t p e r i o d a r e c r i t i c a l f o r t h e s u r v i v a l o f t h e s o f t , h i g h l y v u l n e r a b l e a n i m a l . S i n c e t h e s e m e chanisms h a v e b e e n p l a c e d u n d e r e v o l u t i o n a r y s e l e c t i v e p r e s s u r e s , t h e y s h o u l d s e r v e a s a good s y s t e m f o r t h e e x a m i n a t i o n o f e f f i c i e n t s y s t e m s f o r g l u c o n e o g e n e s i s a n d c h i t i n b i o s y n t h e s i s . R e a c t i o n P a t h w a y s . Most p r e v i o u s s t u d i e s o f c r u s t a c e a n m e t a b o l i s m have c e n t e r e d a r o u n d i d e n t i f i c a t i o n o f m ajor r e a c t i o n p a t h w a y s . D e s p i t e e a r l y d o u b t s a b o u t t h e p r e s e n c e o f t h e g l y c o l y t i c p a thway i n c r u s t a c e a n t i s s u e s ( S c h e e r & S c h e e r , 1 9 5 1 ) , t h e p r e s e n c e o f g l y c o l y t i c and K r e b s c y c l e enzymes i s now w e l l e s t a b l i s h e d ( V a t s i s S S c h a t z l e i n , 1972; K e l l e r , 1 9 6 5 ; B o u l t o n & H u g g i n s , 1 9 7 0 ) . T h e r e i s c o n s i d e r a b l e v a r i a t i o n i n t h e l e v e l s o f g l y c o l y t i c a nd K r e b s c y c l e enzymes i n d i f f e r e n t t i s s u e s . M u s c l e i s p r i m a r i l y g l y c o l y t i c , w h i l e h e a r t , g i l l s , and a n t e n n a l g l a n d show h i g h l e v e l s o f K r e b s c y c l e e n z y m e s , h e x o k i n a s e , and m a l i c enzyme ( K e l l e r , 1 9 6 5 ) . H y p o d e r m i s showed 17 r e l a t i v e l y l o w enzyme a c t i v i t i e s i n t h e s e s t a r v e d i n t e r m o l t a n i m a l s , h o w e v e r , i t e v i d e n c e d an ample c a p a c i t y f o r o x i d a t i o n o f c a r b o h y d r a t e s u b s t r a t e s ( K e l l e r , 1 9 6 5 ) . K e l l e r ' s (1965) s t u d y o f Cambarus a f f i n i s m e t a b o l i s m i s t h e o n l y s t u d y i n w h i c h enzyme l e v e l s i n a v a r i e t y o f o r g a n s w i t h i n t h e same o r g a n i s m a r e c o m p a r e d . He a l s o c o m p a r e d t h e - o x i d a t i o n o f a v a r i e t y o f s u b s t r a t e s by t h e s e o r g a n s . I n c o r r e s p o n d e n c e w i t h s t u d i e s o f l o b s t e r m u s c l e m e t a b o l i s m (Kermack e t a l . , 1 9 5 4 ) , K e l l e r (1965) f o u n d o n l y m i n i m a l o x y g e n u p t a k e by m u s c l e . • However, s u c c i n a t e , g l u t a m a t e , o t — g l y c e r o p h o s p h a t e , g l u c o s e , and g l u c o s e - 6 -p h o s p h a t e were o x i d i z e d by homogenates o f h e a r t , a n t e n n a l g l a n d , g o n a d s , g i l l s , i n t e s t i n e , a n d h e p a t o p a n c r e a s . S u c c i n a t e was t h e most e f f i c i e n t l y o x i d i z e d s u b s t r a t e ( K e l l e r , 1 9 6 5 ) . S u c c i n a t e d e h y d r o g e n a s e f r o m b o t h t h e m u s c l e and g i l l o f £asllI9£§P§ u§ c r a s s i p e s d i f f e r s f r o m t h e enzyme i n o t h e r s y s t e m s i n t h a t i t u t i l i z e s NAD and -NADP a s e l e c t r o n a c c e p t o r s ( V a t s i s & S c h a t z l e i n , 1 9 7 2 ) . A c o n i t a s e , f u m a r a s e , i s o c i t r a t e d e h y d r o g -g e n a s e , and s u c c i n a t e d e h y d r o g e n a s e were f o u n d b o t h i n t h e m i t o c h o n d r i a and i n t h e c y t o s o l by t h e s e w o r k e r s . O n l y t h e NADP l i n k e d i s o c i t r a t e d e h y d r o g e n a s e was f o u n d i n m u s c l e and g i l l ( V a t s i s & S c h a t z l e i n , 1 9 7 2 ) . W h i l e c i t r a t e s y n t h a s e and t h e N A D P ' l i n k e d i s o c i t r a t e d e h y d r o g e n a s e were f o u n d i n a l l t h e c r u s t a c e a n m u s c l e s e x a m i n e d by A l p and c o w o r k e r s ( 1 9 7 6 ) / t h e NAD l i n k e d i s o c i t r a t e d e h y d r o g e n a s e was f o u n d o n l y i n P o r t u n u s p u b e r and L i m u l u s p o l y p h e m u s . These two a n i m a l s showed c o n s i d e r a b l y h i g h e r l e v e l s o f m u s c l e c i t r a t e s y n t h a s e and t h e NADP l i n k e d i s o c i t r a t e d e h y d r o g e n a s e t h a n t h e o t h e r c r u s t a c e a n s e x a m i n e d ( A l p e t a l . , 1 9 7 6 ) . O n l y t h e NADP l i n k e d i s o c i t r a t e d e h y d r o -18 g e n a s e was f o u n d i n t h e t h e n e r v o u s t i s s u e o f Hojaarus v u l g a r i s (Sugden S Newsholme, 1975a)., M u s c l e M e t a b o l i s m , . C r u s t a c e a n m u s c l e m e t a b o l i s m h a s r e c e i v e d c o n s i d e r a b l e a t t e n t i o n , w i t h t h e r e s u l t o f c o n s i d e r a b l e c l a r i f i c a t i o n o f i t s m e t a b o l i c c o n t r o l m e c h a n i s m s . B o u l t o n and H u g g i n s (1970) measured t h e m a j o r i t y o f t h e g l y c o l y t i c enzymes i n t h e m u s c l e o f C a r e i n us maenas, C a n c e r fiagurus, a n d s e v e r a l o t h e r c r u s t a c e a n s . As was f o u n d by K e l l e r ( 1 9 6 5 ) , h e x o k i n a s e had t h e l o w e s t s p e c i f i c a c t i v i t y i n a l l t h e m u s c l e s where i t c o u l d be m e a s u r e d . G l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e l e v e l s were t h r e e f o l d h i g h e r t h a n t h o s e o f l a c t a t e d e h y d r o -g e n a s e , i n c o n t r a s t t o t h e s i t u a t i o n i n most mammalian t i s s u e s i n w h i c h t h e two a r e p r e s e n t i n e g u a l l e v e l s ( B o u l t o n & H u g g i n s , 1 9 7 0 ) . T h i s f i n d i n g , i n c o m b i n a t i o n w i t h m e a s u r e m e n t s o f r e l a t i v e l y h i g h l e v e l s o f o<— g l y c e r o p h o s p h a t e d e h y d r o g e n a s e i n Cambarus a f f i n i s a n d Cambarus b a r t o n i m u s c l e ( K e l l e r , 1965; Popham & Dandy, 1 9 7 6 ) , i m p l i e s t h a t r e d o x r e g u l a t i o n i n t h e s e m u s c l e s may p r o c e e d a c c o r d i n g t o a d i f f e r e n t p a t t e r n t h a n i n mammalian m u s c l e . The d e h y d r o g e n a s e s may a l s o be d i r e c t l y a f f e c t e d by h o r m o n a l c o n t r o l s , s i n c e T i l g n e r a nd L e s i c k i (1976) have f o u n d t h a t e y e s t a l k r e m o v a l l e a d s t o c h a n g e s i n t h e X i n e t i c s o f m u s c l e l a c t a t e d e h y d r o g e n a s e . These c h a n g e s were n o t due t o t h e s y n t h e s i s o f a new i s o z y m e o f l a c t a t e d e h y d r o -g e n a s e . B o t h g l y c o g e n 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 a r e p r e s e n t i n f i v e f o l d h i g h e r l e v e l s t h a n h e x o k i n a s e i n t h e m u s c l e o f C a r c i n u s maenas ( C r a b t r e e & Newsholme, 1 9 7 2 ) . M u s c l e 19 p h o s p f a o r y l a s e and g l y c o g e n s y n t h e t a s e l e v e l s v a r y w i t h t h e m o l t c y c l e (HohnJce, 1 9 7 1 ) . O p i e and Newsholme (1967) m e a s u r e d f r u c t o s e d i p h o s p h a t a s e i n t h e p i n c e r m u s c l e o f Ma,ia s g u i n a d o and f o u n d 0.48 u n i t s / g r a m / m i n u t e a t 25 °C. I f s i m i l a r l e v e l s a r e f o u n d I n C a r c i n u s maenas, t h e r a t i o o f p h o s p h o f r u c t o k i n a s e t o f r u c t o s e d i p h o s p h a t a s e w o u l d l i e a r o u n d 10. A t s u c h l o w v a l u e s o f t h i s r a t i o , c o n s i d e r a b l e c y c l i n g c a n o c c u r a r o u n d t h i s r e a c t i o n l o c u s . w h i l e t h i s c y c l i n g i s w a s t e f u l , i t p r o v i d e s t h e p o t e n t i a l o f c o n s i d e r a b l e a m p l i f i c a t i o n o f n e t g l y c o l y t i c o r g l u c o n e o g e n i c f l u x . AMP i s t h e most e f f i c i e n t r e g u l a t o r y s i g n a l a t t h i s r e a c t i o n l o c u s s i n c e i t l e a d s t o n e t c h a n g e s i n f l u x f a r g r e a t e r t h a n i t s a c t u a l c h a n g e s i n c o n c e n t r a t i o n s . T h i s o c c u r s s i n c e i t a c t i v a t e s p h o s p h o r r u c t o k i n a s e w h i l e i n h i b i t i n g f r u c t o s e d i p h o s p h a t a s e (Newsholme & S t a r t , 1 9 7 3 ) . C hanges i n t h e l e v e l s o f i n o r g a n i c p h o s p h a t e and ammonium i o n (Sugden S Newsholme, 1975b) a s w e l l as c h a n g e s i n t h e l e v e l s o f i n h i b i t o r s o f f r u c t o s e d i p h o s p h a t a s e w o u l d a l s o l e a d t o n e t c h a n g e s i n g l y c o l y t i c f l u x . H i g h d e g r e e s of c y c l i n g a r e c h a r a c t e r i s t i c o f p r e d o m i n a n t l y g l y c o l y t i c t i s s u e s (Newsholme & S t a r t , 1 9 7 3 ) . S t u d i e s o f g l y c o l y t i c c o n t r o l i n P a r a l i t h o d e s c a m t c h a t i c a m u s c l e i n d i c a t e d 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 p y r u v a t e k i n a s e a s t h e i m p o r t a n t c o n t r o l s i t e s ( H o c h a c h k a e t a l . , 1 9 7 0 ) . : T h u s , i n t h e s e c h a r a c t e r i s t i c s , t h i s h i g h l y g l y c o l y t i c c r u s t a c e a n m u s c l e i s s i m i l a r t o v e r t e b r a t e w h i t e m u s c l e . P h o s p h o f r u c t o k i n a s e has been e x a m i n e d i n m u s c l e - o f - P a r a -l i t h o d e s c a m t c h a t i c a ( F r e e d , 1971) and o f Homarus v u l g a r i s (Sugden S Newsholme, 1 9 7 5 b ) . The k i n g c r a b m u s c l e enzyme shows 20 k i n e t i c s s i m i l a r t o p r e v i o u s l y e x a m i n e d p h o s p h o f r u c t o k i n a s e s , i n c l u d i n g i n h i b i t i o n by h i g h l e v e l s o f ATP, a c t i v a t i o n by AMP, and a c c e n t u a t i o n o f ATP i n h i b i t i o n by c i t r a t e ( F r e e d , 1 9 7 1 ) . The l o b s t e r m u s c l e enzyme showed a c t i v a t i o n by ammonium i o n w h i c h was i n c r e a s e d s y n e r g i s t i c a l l y by t h e p r e s e n c e o f i n o r g a n i c p h o s p h a t e . C o n c e n t r a t i o n s o f K + o v e r 5 0 mM c a u s e d i n h i b i t i o n o f t h e l o b s t e r m u s c l e enzyme (Sugden & Newsholme, 1 9 7 5 b ) . A p a r t f r o m ' a s t u d y o f t e m p e r a t u r e e f f e c t s upon k i n g c r a b p y r u v a t e k i n a s e (Somero, 1 9 6 9 ) , t h e r e g u l a t o r y p r o p e r t i e s o f t h i s enzyme h a v e n o t been e x a m i n e d i n c r u s t a c e a n t i s s u e s . B e h r i s c h (1972; 1976) i n s t u d i e s o n P a r a l i t h o d e s c a m t c h a t i c a m u s c l e , h a s f o u n d h i g h l e v e l s o f t h e g l u c o n e o g e n i c enzymes> f r u c t o s e d i p h o s p h a -t a s e , p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e , and p y r u v a t e c a r b o x y l a s e . He has a l s o f o u n d i s o t o p i c e v i d e n c e f o r g l u c o s e s y n t h e s i s f r o m a l a n i n e , l a c t a t e , and p y r u v a t e ( B e h r i s c h , 1 9 7 6 ) . The p r e s e n c e o f ' t h i s g l u c o n e o g e n i c c a p a c i t y m a r k e d l y d i f f e r e n t i a t e s P a r a l i t h o d e s c a m t c h a t i c a m u s c l e f r o m o t h e r m u s c l e s . P a t h w a y s o f C a r b o h y d r a t e M e t a b c l J . s n u The c o n t r i b u t i o n s o f t h e d i f f e r e n t p a t h w a y s o f c a r b o h y d r a t e m e t a b o l i s m h a v e b e e n c o n s i d e r e d i n t h e c r u s t a c e a n g i l l ( H o c h a c h k a e t a l . , 1970; Thabrew e t a l . , 1 9 7 1 ) , i n t h e h e p a t o p a n c r a a s ( H o c h a c h k a e t a l . , 1962; P u y e a r , 1 9 6 7 ) , i n t h e i n t a c t i n t e r m o l t c r a y f i s h - ( P u y e a r e t a l . , 1 9 6 5 ) , and i n v a r i o u s t i s s u e o f O r c o n e c t e s l i m o s u s ( L a n g , 1 9 7 1 ) . P u y e a r a n d c o w o r k e r s (1965) f o u n d t h a t g l y c o l y s i s , t h e p e n t o s e p h o s p h a t e s h u n t , t h e g l u c u r o n i c a c i d p a t h w a y , and t h e K r e b s c y c l e a l l p l a y i m p o r t a n t r o l e s i n t h e m e t a b o l i s m o f i n t e r m o l t P a c i f a s t a c u s l e n i u s c u l u s . S t u d i e s by H o c h a c h k a e t a l . 21 (1962) i n d i c a t e t h e p r e s e n c e o f t h e p e n t o s e p h o s p h a t e s h u n t , g l u c o n e o g e n e s i s , g l y c o l y s i s , a nd K r e b s c y c l e a c t i v i t y i n l o b s t e r h e p a t o p a n c r e a s . E v i d e n c e f o r g l u c o n e o g e n e s i s comes f r o m i n c o r p o r a t i o n o f r a d i o a c t i v e c a r b o n f r o m a c e t a t e i n t o g l y c o g e n . P u y e a r (1967) d e m o n s t r a t e d t h e p r e s e n c e o f t h e g l u c u r o n i c a c i d p a t h w a y i n t h e h e p a t o p a n c r e a s o f C a l l i n e c t e s s a f i i d u s . • • • • I n g i l l s o f b o t h P a r a l i t h o d e s c a m t c h a t i c a and C a r e i n u s iae£as^ t h e c o n t r i b u t i o n o f t h e p e n t o s e p h o s p h a t e s h u n t i s h i g h r e l a t i v e t o g l y c o l y s i s ( H o c h a c h k a e t a l . , 1970; Thabrew e t a l . , 1 9 7 1 ) . B o t h g r o u p s a l s o f o u n d t h a t t h e K r e b s c y c l e made a m a j o r c o n t r i b u t i o n t o r e s p i r a t i o n , i n J a g r e e m e n t w i t h K e l l e r (1965) and Thabrew and c o w o r k e r s ( 1 9 7 3 ) . I n P a r a l i t h o d e s c a m t c h a t i c a g i l l , t h e l e v e l s o f p h o s p h o f r u c t o k i n a s e a r e c o n s i d e r a b l y l o w e r t h a n t h o s e o f h e x o k i n a s e ( H o c h a c h k a e t a l . , 1 9 7 0 ) , w h i l e i n Q a r e i n u s maenas g i l l t h e two a r e p r e s e n t i n e g u a l l e v e l s (Thabrew e t a l . , 1 9 7 1 ) . I n C a r c i n u s maenas g i l l , f r u c t o s e d i p h o s p h a t a s e and p h o s p h o -f r u c t o k i n a s e a r e p r e s e n t i n e g u a l l e v e l s a s w e l l . S u c h h i q h l e v e l s o f f r u c t o s e d i p h o s p h a t a s e b r i n g a h i g h g l u c o n e o g e n i c p o t e n t i a l . I n a c c o r d a n c e w i t h t h i s , i s o l a t e d g i l l s p e r f o r m h i g h r a t e s o f - g l u c o n e o g e n e s i s , p a r t i c u l a r l y i n s o d i u m d e p l e t e d s e a w a t e r . The r a t e s u n d e r t h e s e c o n d i t i o n s a p p r o a c h t h o s e o f r a t l i v e r s l i c e s (Thabrew e t a l . , 1 9 7 1 ) . D u r i n g p r e m o l t , t h e g i l l s h a v e t o s y n t h e s i z e new c h i t i n o u s c o v e r i n g s f o r t h e i n d i v i d u a l g i l l f i l a m e n t s . G l u c o n e o g e n e s i s w i l l t h u s p r o v i d e t h e h e x o s e u n i t s n e eded f o r c h i t i n s y n t h e s i s . M i t o c h o n d r i a l M e t a b o l i s m . M o s t s t u d i e s o f c r a b m i t o c h o n d r i a •have b e e n done w i t h h e p a t o p a n c r e a s m i t o c h o n d r i a . The i n i t i a l w o rk done by B e e c h e y ( 1 9 6 1 ) , Munday and Thompson 22 ( 1 9 6 2 ) , and Munday and Munn (1962) showed t h a t t h e m i t o c h o n d r i a were s i m i l a r t o mammalian m i t o c h o n d r i a . B e e c h e y (1961) f o u n d f l a v o p r o t e i n s and c y t o c h r o m e s i n t h e c h a r a c t e r i s t i c r a t i o s p r e s e n t i n o t h e r a n i m a l s , and f o u n d r e d o x c h a n g e s w h i c h a c c o m p a n i e d t h e p h o s p h o r y l a t i o n o f ADP t o ATP. Munday and Thompson (1962) f o u n d o x i d a t i o n o f t h e v a r i o u s K r e b s c y c l e i n t e r m e d i a t e s and s p a r k i n g o f p y r u v a t e o x i d a t i o n by a d d i t i o n o f t r a c e l e v e l s o f • f u m a r a t e . F u r t h e r m o r e , t h e s e m i t o c h o n d r i a c o n t a i n e d v a r i o u s K r e b s c y c l e enzymes a s w e l l as a s p a r t a t e and a l a n i n e a m i n o t r a n s f e r a s e s . Munday and Munn (1962) f o u n d t h a t h e p a t o p a n c r e a s m i t o c h o n d r i a were a b l e t o o x i d i z e o c t a n o a t e t o c a r b o n d i o x i d e w i t h o u t t h e a d d i t i o n o f K r e b s c y c l e i n t e r m e d i a t e s o r c a r b o h y d r a t e p r e c u r s o r s . However, t h e a d d i t i o n o f t r a c e amounts o f s u c c i n a t e l e d t o a s p a r k i n g • o f f a t o x i d t i o n . Chen and L e h n i n g e r (1973) p r e p a r e d h e p a t o p a n c r e a s m i t o c h o n d r i a i n t h e p r e s e n c e o f b o v i n e serum a l b u m i n , and were a b l e t o o b t a i n a h i g h d e g r e e o f r e s p i r a t o r y c o n t r o l . The a d d i t i o n o f b o t h p y r u v a t e and p r o l i n e s t i m u l a t e d r e s p i r a t i o n , w h i l e t h e a d d i t i o n o f e i t h e r compound a l o n e • d i d n o t . As w i t h mammalian m i t o c h o n d r i a , d i c a r b o x y l a t e , t r i c a r b o x y l a t e , o t — k e t o g l u t a r a t e , and p y r u v a t e c a r r i e r s a r e p r e s e n t . E x t e r n a l NADH, ^ - O H b u t y r a t e , and o t - g l y c e r o l p h o s p h a t e a r e n o t o x i d i z e d by t h e s e m i t o c h o n d r i a (Chen S L e h n i n g e r , 1 9 7 3 ) . By c o n t r a s t , K e l l e r (1965) was a b l e t o d e m o n s t r a t e some o < - g l y c e r o l p h o s p h a t e s t i m u l a t i o n o f o x y g e n u p t a k e by Cambarus a f f i n i s h e p a t o p a n c r e a s h o m o g e n a t e s . B o t h Chen a n d L e h n i n g e r (1973) and Munday and Thompson (1962) f o u n d t h a t h i g h l e v e l s o f p h o s p h a t e and magnesium were n e e d e d f o r f u l l s t i m u l a t i o n o f o x y g e n u p t a k e by t h e s e m i t o c h o n d r i a . Chen and 23 L e h n i n g e r (1973) a l s o n o t e d t h e h i g h p h o s p h a t e and c a l c i u m c o n t e n t o f t h e s e m i t o c h o n d r i a . I n f u r t h e r s t u d i e s , Chen e t a l . (1974) were a b l e t o d e m o n s t r a t e a marked s t i m u l a t i o n o f r e s p i r a t i o n by c a l c i u m a s w e l l a s t h e c o n c o m i t a n t a c c u m u l a t i o n o f h i g h l e v e l s o f c a l c i u m and p h o s p h a t e . C a l c i u m u p t a k e t o o k p r e c e d e n c e o v e r p h o s p h o r y l a t i o n o f ADP f o r r e s p i r a t o r y e n e r g y . A n o t h e r n o v e l c h a r a c t e r i s t i c o f t h e s e C a l l i n e c f e s s a p i d u s h e p a t o p a n c r e a s m i t o c h o n d r i a i s a marked s t i m u l a t i o n o f ATP f o r m a t i o n by t h e a d d i t i o n o f a r g i n i n e . The a r g i n i n e k i n a s e t h r o u g h w h i c h t h i s e f f e c t i s m e d i a t e d i s l o c a t e d o u t s i d e t h e a t r a c t y l o s i d e b a r r i e r . T h u s , t h e t r a n s f e r o f t h e t e r m i n a l p h o s p h a t e g r o u p o f ATP o n t o a r g i n i n e d o e s n o t o c c u r d i r e c t l y i n t h e m a t r i x . A r g i n i n e k i n a s e i s u s u a l l y t h o u g h t t o be r e s t r i c t e d t o m u s c l e and n e r v o u s t i s s u e where i t f u n c t i o n s i n t h e a c c u m u l a t i o n and b r e a k d o w n o f a r g i n i n e p h o s p h a t e . L i p i d M e t a b o l i s m . L i p i d m e t a b o l i s m i n c r u s t a c e a n s has n o t been t h e o b j e c t o f much s t u d y , a l t h o u g h l i p i d s r e p r e s e n t a m a j o r p o r t i o n o f t h e m e t a b o l i c s t o r e s t h r o u g h o u t t h e m o l t c y c l e . D u r i n g t h e b e g i n n i n g o f p r e m o l t t h e l i p i d c o n t e n t o f t h e h e p a t o p a n c r e a s i n c r e a s e s (Henaud, 1949; B o l l e n b a c h e r e t a l . , 1 9 7 2 ) . I n some c r u s t a c e a n s , s u c h a s t h e i s o p o d P o r c e l l i o l a e y i s , t h e l i p i d s r e p r e s e n t t h e m a j o r m o l t c y c l e r e s e r v e ( A l i k h a n , 1 9 7 2 ) . The d i g e s t i v e j u i c e o f t h e h e p a t o p a n c r e a s c o n t a i n s a h i g h l e v e l o f s u r f a c t a n t s w h i c h f a c i l i t a t e l i p i d a b s o r p t i o n (Vonk, 1 9 6 0 ) . L i p i d s a r e a l s o i n v o l v e d w i t h v i t e l l o g e n e s i s ; a new hemolymph l i p o p r o t e i n a p p e a r s i n f e m a l e s d u r i n g t h i s p e r i o d o f r a p i d i n c o r p o r a t i o n o f l i p i d i n t o t h e o v a r y ( A l l e n , 1 9 7 2 ) . F r e e f a t t y a c i d s a r e t h e m a j o r f o r m i n 24 w h i c h l i p i d i s t r a n s p o r t e d t h r o u g h t h e hemolymph (Speck. & U r i c h , 1 9 7 0 ; A l l e n , 1 9 7 2 ) . The o x i d a t i o n o f l i p i d s was d e m o n s t r a t e d by Munday and Munn (1962) i n t h e i r s t u d i e s o f h e p a t o p a n c r e a s m i t o c h o n d r i a . L a b e l l e d c a r b o n f r o m a c e t a t e i s q u i c k l y c o n v e r t e d t o amino a c i d s , K r e b s c y c l e i n t e r m e d i a t e s , a n d e v e n t u a l l y t o s u g a r p h o s p h a t e s i n C a r c i n u s maenas ( H u g g i n s , 1966),. i n A s t a c u s a s t a c u s a n d A s t a c u s l e p t o d a c t y l u s ( Z a n d e e , 1 9 6 6 a ; Z a n d e e , 1966b; van Marrewyk & Z a n d e e , • 1 9 7 5 ) v i n P a l a e m o n s e j r a t u s (Cowey & F o r s t e r , 1971) and i n A r t e m i a s a l i n a ( H u g g i n s , 1969) . C a r b o n f r o m p a l m i t a t e i s ' p r e f e r e n t i a l l y c o n v e r t e d i n t o - a m i n o a c i d s i n 9££SBg£t§§ l i m o s u s ( S p e c k & U r i c h , 1 9 6 9 a ) . T h e r e i s some e v i d e n c e t h a t f a t s a r e t h e p r e f e r e n t i a l s u b s t r a t e s d u r i n g s t a r v a t i o n i n b o t h O r c o n e c t e s l i m o s u s and G a r c i n u s maenas ( S p e c k & U r i c h , 1969b; H e a t h & B a r n e s , 1 9 7 0 ) . However, a n o t h e r s t u d y o f t h e e f f e c t o f s t a r v a t i o n upon t h e m e t a b o l i c r e s e r v e s o f C a r c i n u s maenas i n d i c a t e s t h a t s t a r v a t i o n l e a d s t o no c h a n g e i n t h e l e v e l o f c a r b o h y d r a t e s o r l i p i d s i n t h e h e p a t o p a n c r e a s o r g i l l ( B a r s d e n e t a l . , 1 9 7 3 ) . . The c o n t r o l o f t h e m e t a b o l i c p a t h w a y s i n v o l v e d w i t h l i p i d s y n t h e s i s and d e g r a d a t i o n h a s n o t been s t u d i e d i n c r u s t a c e a n s . Amino A c i d M e t a b o l i s m ; . Amino a c i d s p l a y an i m p o r t a n t r o l e i n o s m o r e g u l a t i o n , a s e n e r g y s o u r c e s , a s w e l l a s a c t i n g a s t h e s u b s t r a t e s f o r p r o t e i n s y n t h e s i s . The e n z y m a t i c r e a c t i o n s i n v o l v e d i n t h e i r b i o s y n t h e s i s and u t i l i z a t i o n h ave r e c e i v e d c o n s i d e r a b l e ' a t t e n t i o n . Zandee (1966a>- 1966b) has p u b l i s h e d some s t u d i e s o n A s t a c u s a s t a c u s i n d i c a t i n g t h a t t h e f o l l o w i n g amino a c i d s were n o n - e s s e n t i a l ; g l y c i n e , a l a n i n e , s e r i n e , a s p a r t a t e , g l u t a m a t e ( g l u t a m i n e ) , t y r o s i n e , p r o l i n e , O H - p r o l i n e , 25 and a r g i n i n e . No l a b e l f r o m U l*G g l u c o s e o r 1 l*C a c e t a t e was i n c o r p o r a t e d i n t o v a l i n e , l e u c i n e , i s o l e u c i n e , t h r e o n i n e , l y s i n e , h i s t i d i n e , p h e n y l a l a n i n e , c y s t e i n e , o r t r y p t o p h a n . S i m i l a r ' r e s u l t s "-were f o u n d i n more r e c e n t e x p e r i m e n t s w i t h flstacus l e p t o d a c t y l u s (van Marrewyk & Z a n d e e , 1975) w i t h A r t e m i a s a l i n a ( f l u g g i n s , * 1969) , w i t h P a l a e m o j i s e r r a t us (Cowey & F o r s t e r , 1 9 7 1 ) , w i t h C a r c i n u s maenas ( H u g g i n s , 1 9 6 6 ) , w i t h P a n a e u s a z t e c us ( S h e w b a r t e t a l . , 1 9 7 2 ) , and w i t h ficap.uaila.tor ( C l a y b r o o k , 1 9 7 6 ) . Amino a c i d s seem t o e n j o y a c e n t r a l r o l e i n e n e r g y m e t a b o l i s m s i n c e t h e y a r e h i g h l y l a b e l l e d by c a r b o n f r o m g l u c o s e and f r o m a c e t a t e . The s y n t h e s i s o f t h e n o n - e s s e n t i a l amino a c i d s c a n be e x p l a i n e d on t h e b a s i s o f s t a n d a r d e n z y m a t i c r e a c t i o n s as b o t h a s p a r t a t e a m i n o t r a n s f e r a s e and a l a n i n e a m i n o t r a n s f e r a s e a r e p r e s e n t i n h i g h l e v e l s i n v a r i o u s c r u s t a c e a n t i s s u e s . A v a r i e t y o f o t h e r t r a n s a m i n a t i o n s h a v e a l s o b een d e m o n s t r a t e d i n - g i l l , m u s c l e and h e p a t o p a n c r e a s o f C a r c i n u s maenas ( C h a p l i n e t a l . , 1 S 6 7 ) . G i l l e s and S c h o f f e n i e l s (1964) f o u n d a n o m a l o u s l a b e l l i n g p a t t e r n s i n t h e i r e x p e r i m e n t s on amino a c i d f o r m a t i o n by l o b s t e r n e r v e c o r d . They c o n c l u d e d t h a t some p y r u v a t e c a r b o n must e n t e r t h e K r e b s c y c l e by way o f o x a l o a c e t a t e o r ' m a l a t e , a n d a l s o t h a t some p y r u v a t e i s e v e n t u a l l y g e n e r a t e d f r o m K r e b s c y c l e i n t e r m e d i a t e s s u c h a s m a l a t e . M a l i c enzyme i s p r e s e n t i n v a r i o u s c r a b t i s s u e s ( K e l l e r , 1 9 6 5 ) , t h u s , t h e s u g g e s t i o n s made by G i l l e s a n d S c h o f f e n i e l s ' (1964) may i n f a c t be c o r r e c t ; The marked c h a n g e s i n t h e l e v e l s o f t i s s u e f r e e amino a c i d s d u r i n g t h e m o l t c y c l e o f - C a r c i n u s •maenas and C a l l i n e c t e s s a p i d u s n a u p l i i a r e l a r g e l y due t o c h a n g e s i n t h e c o n c e n t r a t i o n s o f t h e n o n - e s s e n t i a l amino 26 a c i d s / g l u t a m a t e , a l a n i n e , g l y c i n e , and p r o l i n e ( D u c h a t e a u e t a l . , 1959; T u c k e r S C o s t l o w , 1 9 7 5 ) . M e t a b o l i c F l u x P a t t e r n s d u r i n g t h e M o l t C j r o l e T h e most c o m p r e h e n s i v e s t u d i e s o f c r u s t a c e a n m e t a b o l i s m and p h y s i o l o g y h a v e been c a r r i e d o u t on t h e c r a y f i s h O r c o n e c t e s l i m o s u s by U r i c h a nd c o w o r k e r s . These s t u d i e s c e n t e r upon t h e c h a n g i n g p a t t e r n s o f c a r b o n f l u x d u r i n g t h e m o l t c y c l e and t h e m e t a b o l i c f a t e o f c a r b o n f r o m d i f f e r e n t f o o d s t u f f s and r e s o r b e d c h i t i n . P a r t i c u l a r e m p h a s i s was p l a c e d upon c a r b o n f l u x d u r i n g t h e p a s s a g e o f compounds f r o m one t i s s u e t o a n o t h e r * T h e s e s t u d i e s show t h a t i n g e s t e d o r r e s o r b e d compounds a r e s u b j e c t t o e x t e n s i v e i n t e r c o n v e r s i o n s , a n d t h a t a m i n o a c i d s a n d l a c t a t e a r e o f c e n t r a l i m p o r t a n c e i n t h e e n e r g y m e t a b o l i s m o f c r u s t a c e a n s . The p i c t u r e o f c a r b o n f l u x w h i c h e m e r g e s f r o m t h e h i s t o l o g i c a l s t u d i e s o f Renaud (1949) and T r a v i s ( 1 9 5 5 a ; 1957) i m p l i e s t h a t g l y c o g e n and f a t p r o v i d e t h e b u l k o f t h e c a r b o n f o r c h i t i n s y n t h e s i s . I n a n i m a l s , t h e n e t s y n t h e s i s o f c a r b o h y d r a t e f r o m f a t i s i m p o s s i b l e * By c o n t r a s t , g l u c o s e u n i t s f r o m g l y c o g e n c o u l d w e l l be u s e d t o s u p p o r t c h i t i n s y n t h e s i s . The e a r l y h i s t o l o g i c a l s t u d i e s a l s o i m p l y t h a t c a r b o n f r o m t h e r e s o r b e d c h i t i n i s q u i c k l y c o n v e r t e d t o g l y c o g e n s t o r e d w i t h i n t h e h y p o d e r m i s a n d - t h e h e p a t o p a n c r e a s . The s t u d i e s o f U r i c h a nd c o w o r k e r s show t h a t t h i s p i c t u r e o f c a r b o n f l u x i s a c o n s i d e r a b l e o v e r - s i m p l i f i c a t i o n . C o n s i d e r a b l e r e d i s t r i b u t i o n o f - c a r b o n f r o m b l o o d g l u c o s e o r r e s o r b e d a c e t y l g i u c o s a m i n e i n t o s u g a r s , o r g a n i c a c i d s , amino a c i d s , p o l y s a c c h a r i d e s * p r o t e i n s , and l i p i d s o c c u r s ( H e r z - H u b n e r S U r i c h , 1 9 7 3 ) * B e t w e e n 20 and 40% o f c a r b o n f r o m hemclymph g l u c o s e a n d p a l m i t a t e i s i n i t i a l l y 27 c o n v e r t e d i n t o amino a c i d s . I n f r e s h m o l t a n i m a l s , c a r b o n f r o m amino a c i d s i s s u b s e q u e n t l y f o u n d b o t h i n c h i t i n and i n r e s p i r a t o r y c a r b o n d i o x i d e . I n b o t h i n t e r m o l t and f r e s h m o l t a n i m a l s , a m ino a c i d s a r e o x i d i z e d more r e a d i l y t h a n g l u c o s e o r p a l m i t a t e ( S p e c k 6 U r i c h , * 1 9 6 9 a ) . T h u s , amino a c i d s p l a y a c e n t r a l r o l e i n ' e n e r g y m e t a b o l i s m , and g l u c o n e o g e n e s i s f r o m a m i no a c i d s and o r g a n i c a c i d s p l a y s a c r i t i c a l r o l e a s a s o u r c e o f h e x o s e u n i t s f o r c h i t i n s y n t h e s i s . O r c o n e c t e s l i m o s u s l a c k s a p p r e c i a b l e m e t a b o l i c s t o r e s , a n d d u r i n g t h e m o l t c o v e r s a l l o f i t s e n e r g e t i c and b i o s y n t h e t i c n e e d s w i t h r e s o r b e d a c e t y l g l u c o s a m i n e (Speck & U r i c h , 1 9 7 1 ) . I n t h i s r e s p e c t i t d i f f e r s f r o m c r u s t a c e a n s w h i c h u t i l i z e s t o r e d g l y c o g e n , l i p i d s and p r o t e i n s as w e l l a s r e s o r b e d a c e t y l g l u c o s a m i n e t o s u p p o r t t h e i r a c t i v i t y d u r i n g m o l t ( R e n a u d , 1949; T r a v i s , 1 9 5 5 a ; T r a v i s , 1957; H e a t h & B a r n e s , 1 9 7 0 ) . H o w e v e r , t h e d e g r e e t o w h i c h d e c a p o d c r u s t a c e a n s a c c u m u l a t e s t o r e s v a r i e s c o n s i d e r a b l y ( H e a t h & B a r n e s , 1 9 7 0 ) . T h e r e f o r e , t h e u t i l i z a t i o n o f r e s o r b e d a c e t y l g l u c o s a m i n e r e p r e s e n t s a b a s i c f e a t u r e o f m o l t i n g p h y s i o l o g y w h i c h i s u t i l i z e d t o d i f f e r i n g d e g r e e s d e p e n d i n g - u p o n t h e e l a b o r a t i o n o f m e t a b o l i c s t o r e s . The p a t h w a y t o and f r o m c h i t i n j o i n s g l y c o l y s i s a t t h e l e v e l o f f r u c t o s e - 6 - p h o s p h a t e . A c e t y l g l u c o s a m i n e r e s o r b e d f r o m t h e o l d e x o s k e l e t o n must be d e a c e t y l a t e d b e f o r e i t c a n be r e c o n v e r t e d i n t o c h i t i n («olter, 1 9 6 8 ) , b u t t h e n i t f o r m s t h e p r e f e r e n t i a l s u b s t r a t e f o r p r e m o l t c h i t i n s y n t h e s i s ( S p e c k e_t a 1. , 1972; S t e v e n s o n , 1 9 7 2 ) w O n l y 20% o f t h e r e s o r b e d a c e t y l g l u c o s a m i n e r e m a i n s u n c h a n g e d t e n m i n u t e s a f t e r r e s o r p t i o n by t h e h y p o d e r m i s . The r e m a i n d e r i s c o n v e r t e d l a r g e l y i n t o v a r i o u s 28 o r g a n i c and a mino a c i d s a s w e l l as t o c h i t i n , p r o t e i n , and o l i g o s a c c h a r i d e ( S p e c k 6 U r i c h , 1 9 7 2 ) . Some c a r b o n f r o m r e s o r b e d a c e t y l g l u c o s a m i n e a c c u m u l a t e s i n t h e a b d o m i n a l m u s c l e and i s n o t r e l e a s e d o r r e d i s t r i b u t e d u n t i l a c o n s i d e r a b l e p e r i o d a f t e r t h e m o l t ( S p e c k & U r i c h , 1 9 7 2 ) . C a r b o n f r o m a c e t y l g l u c o s a m i n e a c c u m u l a t e s i n t h e h y p o d e r m i s b e f o r e t h e m o l t . A f t e r t h e m o l t t h e o r g a n i c a c i d s , s u g a r s , p r o t e i n s - and p o l y s a c c h a r i d e s i n t o w h i c h i t was c o n v e r t e d a r e p r e f e r e n t i a l l y u t i l i z e d f o r c h i t i n s y n t h e s i s ( S p e c k & U r i c h , 1972; H a r z - H u b n e r & U r i c h , 1 9 7 3 ) . The means by w h i c h t h i s p r e f e r e n t i a l u t i l i z a t i o n i s a c h i e v e d a r e n o t c l e a r . G l u c o s e i s n o t u s e d f o r c h i t i n s y n t h e s i s u n t i l a f t e r m o l t , p r e s u m a b l y due t o an i n h i b i t i o n a t t h e l e v e l o f g l u t a m i n e ; f r u c t o s e - 6 - p h o s p h a t e a m i d o t r a n s f e r a s e (E.C.2.6.1.16) ( S p e c k e t a l . , 1 9 7 2 ) . T h i s i n h i b i t i o n may f a c i l i t a t e e f f i c i e n t r e s o r p t i o n and d i s t r i b u t i o n o f c a r b o n f r o m t h e o l d e x o s k e l e t o n i n t o t h e g e n e r a l m e t a b o l i c p o o l s . A f t e r t h e m o l t , h o w e v e r , c h i t i n s y n t h e s i s f r o m g l u c o s e i s r a p i d (Hornung & S t e v e n s o n , 1 9 7 1 ; S t e v e n s o n , 1972; S p e c k e t a l . , 1 9 7 2 ) . C a r b o n f r o m r e s o r b e d a c e t y l g l u c o s a m i n e a n d hemolymph g l u c o s e i s r a p i d l y d i s t r i b u t e d a mongst amino a c i d s , g l y c o l y t i c and K r e b s c y c l e i n t e r m e d i a t e s , c h i t i n , p r o t e i n , p o l y s a c c h a r i d e s , and C02 ( S p e c k & U r i c h , 1972; H e r z - H u b n e r & U r i c h , 1 9 7 3 ) . T h r e e h o u r s a f t e r i n j e c t i o n o f l a b e l , 3 3 % o f hemolymph g l u c o s e c a r b o n i s i n c o r p o r a t e d i n t o p r o t e i n s , w h i l e 5 5 % i s p r e s e n t i n t h e f o r m o f i n t e r m e d i a r y compounds. I n c o m p a r i s o n , 18% o f t h e a c e t y l g l u c o s a m i n e c a r b o n i s i n p r o t e i n , -44% i n i n t e r m e d i a r y compounds ( o f • w h i c h amino a c i d s c o n s t i t u t e b e t w e e n 15 and 30%) and 24% i s i n c h i t i n ( H e r z -29 Hubner & U r i c h , 1 9 7 3 ) . C u r i n g a b s o r p t i o n o f g l u c o s e f r o m f o o d s t u f f s , o n l y 9% o f t h e g l u c o s e l e a v e s t h e d i g e s t i v e s y s t e m u n c h a n g e d . * O t h e r s u g a r s , o r g a n i c a c i d s , and a m i n o a c i d s c o n s t i t u t e one t h i r d e a c h o f t h e t o t a l m a t e r i a l t r a n s f e r r e d f r o m t h e h e p a t o p a n c r e a s ' a n d g u t t o t h e hemolymph ( U r i c h e t a l . , 1973) ; A c o m p a r i s o n o f t h e u t i l i z a t i o n o f g l u c o s e , g l u t a m a t e , and p a I m i t a t e by O r c o n e c t e s l i m o s u s shows t h a t b o t h g l u c o s e a n d g l u t a m a t e a r e q u i c k l y m e t a b o l i z e d . G l u c o s e c a r b o n i s g u i c k l y i n c o r p o r a t e d i n t o amino a c i d s , w h i l e g l u t a m a t e c a r b o n i s more g u i c k l y o x i d i z e d t o C02 t h a n c a r b o n f r o m e i t h e r g l u c o s e o r p a l m i t a t e ( S p e c k 5 U r i c h , 1 9 6 9 ) . L a c t a t e a c c o u n t s f o r 57% o f t h e s u b s t a n c e t r a n s f e r r e d b e t w e e n hemolymph and b o d y t i s s u e s ( H e r z - H l i b n e r e t a l . , 1 9 7 3 ) . I n Cambarus r o b u s t u s , b l o o d l a c t a t e l e v e l s e q u a l b l o o d g l u c o s e l e v e l s ( T e l f o r d , 1 9 7 5 ) , i n O r c o n e c t e s l i m o s u s t h e y e x c e e d t h e b l o o d g l u c o s e l e v e l s ( A n d r e w s , 1 9 6 7 ) , w h i l e i n Homarus a m e r i c a n u s t h e y a r e somewhat l o w e r ( S t e w a r t e t a l . , 1 9 6 6 ) . T h u s , a mino a c i d s and l a c t a t e a r e o f c e n t r a l i m p o r t a n c e i n c r u s t a c e a n e n e r g y m e t a b o l i s m . S i n c e hemolymph a n d h e p a t o p a n c r e a t i c g l u c o s e a s w e l l a s r e s o r b e d a c e t y l g l u c o s a m i n e show p r e f e r e n t i a l c o n v e r s i o n t o amino a c i d s and l a c t a t e ; c h i t i n s y n t h e s i s f r o m b o t h s t o r e d g l u c o s e and r e s o r b e d a c e t y l g l u c o s a m i n e w i l l i n v o l v e g l u c o n e o g e n e s i s . C e n t r a l Q u e s t i o n s i n t h i s S t u d y G l u c o n e o g e n e s i s i n c r u s t a c e a n s n o r m a l l y s u p p o r t s g l y c o g e n s y n t h e s i s , s y n t h e s i s o f b l o o d g l u c o s e , t r e h a l o s e , m a l t o s e , a nd 30 mannose a s w e l l a s c h i t i n s y n t h e s i s . T h i s d i v e r s i t y o f e n d p r o d u c t s i n c r e a s e s t h e c o m p l e x i t y o f t h e m e t a b o l i c c o n t r o l r e q u i r e m e n t s . S i n c e amino a c i d s and l a c t a t e a r e o f c e n t r a l i m p o r t a n c e i n c r u s t a c e a n e n e r g y m e t a b o l i s m ( H e r z - H u n n e r e t a l . , 1973) t h e s e compounds l o g i c a l l y f o r m m a j o r s u b s t r a t e s f o r g l u c o n e o g e n e s i s . G l u c o n e o g e n e s i s o c c u r s i n a v a r i e t y o f c r u s t a c e a n t i s s u e s i n c l u d i n g g i l l (Thabrew e t a l . > 1 9 7 1 ) , h y p o d e r m i s "(Herz-Hubner 6 U r i c h , 1 9 7 3 ) , h e p a t o p a n c r e a s ( H o c h a c h k a e t a l . , 1962) and p o s s i b l y b l o o d ( J o h n s t o n e t a l . , 1972) a n d m u s c l e ( B e h r i s c h , 1 9 7 6 ) . S i n c e h y p o d e r m a l g l u c o n e o -g e n e s i s s u p p o r t s c h i t i n s y n t h e s i s d u r i n g t h e c r i t i c a l h a r d e n i n g o f t h e f r e s h l y m o l t e d a n i m a l , t h e h y p o d e r m i s i s the- t i s s u e o f c h o i c e w i t h i n w h i c h t o s t u d y g l u c o n e o g e n i c c o n t r o l m e c h a n i s m s . G l u c o n e o g e n e s i s h a s two w e l l d e f i n e d c o n t r o l s i t e s i n v e r t e b r a t e t i s s u e s . One l i e s a t t h e r e v e r s a l o f t h e p y r u v a t e k i n a s e r e a c t i o n , i . e . t h e p h o s p h o e n o l p y r u v a t e c a r b o x y K i n a s e a n d p y r u v a t e c a r b o x y l a s e r e a c t i o n s , and t h e o t h e r a t t h e r e v e r s a l o f t h e p h o s p h o f r u c t o k i n a s e r e a c t i o n by f r u c t o s e d i p h o s p h a t a s e ( S c r u t t o n 5 U t t e r , 1 9 6 8 ) . B o t h o f t h e s e c o n t r o l s i t e s o c c u r i n a p p o s i t i o n t o g l y c o l y t i c r e a c t i o n s w h i c h a r e c h a r a c t e r i z e d by l a r g e f r e e e n e r g y c h a n g e s , a r e f u n c t i o n a l l y i r r e v e r s i b l e i n t h e c e l l , a n d c o n s t i t u t e m a j o r g l y c o l y t i c c o n t r o l s i t e s ( S c r u t t o n S U t t e r , 1968; Newsholme & S t a r t , 1973; S o l l e s t o n , 1 9 7 2 ) . A n o t h e r g l y c o l y t i c r e a c t i o n w h i c h i s c h a r a c t e r i z e d by a l a r g e f r e e e n e r g y c h a n g e i s t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n . The e q u i l i b r i u m o f t h e r e a c t i o n s t r o n g l y f a v o r s ATP and 3 - p h o s p h o g l y c e r a t e f o r m a t i o n ( K r i e t s c h 6 B t i c h e r , 1 9 7 0 ; Rose & Harms, 1 9 7 0 ) . I n t h e c e l l , t h e r e a c t i o n i s c o n s i d e r e d t o be 31 f r e e l y r e v e r s i b l e f o r g l u c o n e o g e n e s i s , and t h u s t o c a t a l y z e a n e a r e q u i l i b r i u m r e a c t i o n . E v i d e n c e o f a c r o s s o v e r b e t w e e n g l y c e r a l d e h y d e - 3 - p h o s p h a t e a n d 3 - p h o s p h o g l y c e r a t e i s u s u a l l y i n t e r p r e t e d a s r a t e l i m i t a t i o n a t t h e d e h y d r o g e n a s e r e a c t i o n ( W i l l i a m s o n , 1965; Rose & Warms, 1970: B o l i e s t o n , 1 9 7 2 ) . P r e c i s e e l u c i d a t i o n o f t h e i n v i v o r o l e o f p h o s p h o g l y c e r a t e k i n a s e a n d g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e h a s b e e n hampered by t h e d i f f i c u l t y i n m e a s u r i n g t h e i r - common s u b s t r a t e 1,3 d i p h o s p h o g l y c e r a t e . The k i n e t i c p r o p e r t i e s o f t h e enzyme ha v e been e x a m i n e d i n y e a s t and r a b b i t m u s c l e . The enzyme f r o m t h e s e s o u r c e s shows a h i g h a f f i n i t y f o r ' 1 , 3 d i p h o s p h o g l y c e r a t e (Km = 2 juh) and a 1000 f o l d l o w e r a f f i n i t y f o r 3 - p h o s p h o g l y -c e r a t e (Km = a p p r o x i m a t e l y 2 mM) ( K r i e t s c h S B u c h e r , 1 9 7 0 ) . F o r b o t h t h e y e a s t and t h e r a b b i t m u s c l e enzyme, t h e K i f o r MgADP a s an i n h i b i t o r i s l o w e r t h a n i t s Km v a l u e a s t h e g l y c o l y t i c s u b s t r a t e ( L a r s s o n - R a z n i k i e w i c z & A r v i d s s o n , 1970; S c o p e s , 1 9 7 3 ) . T h u s , b o t h k i n e t i c and t h e r m o d y n a m i c b a r r i e r s h a v e t o be o v e r c o m e t o a c h i e v e n e t g l u c o n e o g e n i c f l u x . I n c o n t r a s t t o t h e s i t u a t i o n i n g l y c o l y t i c t i s s u e s where t h e e x a c t r o l e o f p h o s p h o -g l y c e r a t e k i n a s e and g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e i s n o t c l e a r ( W i l l i a m s o n , 1965; P a r k e r 6 H o f f m a n , 1967; Newsholme S S t a r t , 1973; T o r n h e i m & L o w e n s t e i n , 1 9 7 5 ) , t h e p n o s p h o g l y c e r a t e k i n a s e , g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e s y s t e m i s a t e q u i l i b r i u m u n d e r ' a - v a r i e t y o f m e t a b o l i c - c o n d i t i o n s i n t h e r a t l i v e r ( V e e c h e t a l , , 1970; S t u b b s e t a l . , 1 9 7 2 ) . ; W h i l e some c a r e f u l k i n e t i c s t u d i e s o f t h e l i v e r g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ' h a v e e l u c i d a t e d t h e means w h e r e b y t h i s r e a c t i o n i s r e v e r s e d u n d e r g l u c o n e o g e n i c c o n d i t i o n s ( S m i t h & V e l i c k , 1 9 7 2 ) , 32 no i n v e s t i g a t i o n s o f k i n e t i c m e c h a n i s m s w h i c h m i g h t f a c i l i t a t e t h e r e v e r s a l o f t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n f o r g l u c o -n e o g e n e s i s h a v e been made. S i n c e c r u s t a c e a n h y p o d e r m i s a n d m u s c l e show a v a r i e t y o f m e t a b o l i c p o i s e s d u r i n g t h e c o u r s e o f t h e m o l t c y c l e , t h e y p r o v i d e an e x c e l l e n t b i o l o g i c a l s i t u a t i o n w i t h i n w h i c h t h e c o n t r o l m e c h a n i s m s o f t h i s r e a c t i o n c a n be e x a m i n e d , u n d e r b o t h g l u c o n e o g e n i c and g l y c o l y t i c c o n d i t i o n s . Of t h e two w e l l d e f i n e d c o n t r c l s i t e s i n g l u c o n e o g e n e s i s , t h e f r u c t o s e d i p h o s p h a t a s e r e a c t i o n (FDPase) has been w e l l c h a r a c t e r i z e d i n h y p o d e r m i s , g i l l , and m u s c l e o f £aralithodes c a m t c h a t i c a ( H o c h a c h k a , 1972; B e h r i s c h , 1972; B e h r i s c h S J o h n s o n , 1974; B e h r i s c h , 1 9 7 5 ) . The h y p o d e r m a l FDPase i s p r e s e n t i n two f o r m s ( I and I I ) . FDPase I I i s t h e p r e d o m i n a n t f o r m p r e s e n t i n t h e g i l l . T h i s f o r m i s h i g h l y s e n s i t i v e t o f e e d b a c k i n h i b i t i o n by r i b o s e - 5 - p h o s p h a t e , UDP-a c e t y l g l u c o s a m i n e , and D D P - g l u c o s e . Low l e v e l s o f UDP-a c e t y l g l u c o s a m i n e a c t i v a t e t h e b i n d i n g o f f r u c t o s e - 1 , 6 d i p h o s p h a t e by FDPase I I . B o t h f o r m s a r e s e n s i t i v e t o AMP i n h i b i t i o n ( H o c h a c h k a , 1 9 7 2 ) . B o t h t i s s u e s h a v e a r e l a t i v e l y c o m p l e x c a r b o h y d r a t e : m e t a b o l i s m . The h y p o d e r m i s s y n t h e s i z e s l a r g e q u a n t i t i e s - - o f b o t h c h i t i n and g l y c o g e n , w h i l e t h e g i l l ' s p r i m a r y p o l y s a c c h a r i d e p r o d u c t i s c h i t i n . The p e n t o s e s h u n t i s a c t i v e i n b o t h g i l l and h y p o d e r m i s (Thabrew e t a l , 1971; H o c h a c h k a e t a l . , 1 9 70; K e l l e r , 1 9 6 5 ) . W h i l e g l u c o n e o g e n i c f l u x h a s n o t been m e a s u r e d i n t h e h y p o d e r m i s , g l u c o s e p r o d u c t i o n by t h e g i l l r e a c h e s r a t e s e q u i v a l e n t t o t h o s e a t t a i n e d by r a t l i v e r s l i c e s (Thabrew e t a l . , 1 9 7 1 ) . , I n t h e g i l l o f G a r c i n u s maenas, p h o s p h o f r u c t o k i n a s e and f r u c t o s e d i p h o s p h a t a s e a r e i n a 1:1 33 r a t i o (Thabrew e t a l . , 1971)••,--•••A s i m i l a r s i t u a t i o n seems t o p r e v a i l i n t h e g i l l o f P a r a l i t h o d e s c a m t c h a t i c a ( H o c h a c h k a , 1972; H o c h a c h k a e t a l . , 1 9 7 0 ) . The p r e s e n c e o f h i g h l e v e l s o f t h e c l o s e l y r e g u l a t e d FDPase I I , i n c o m b i n a t i o n w i t h l o w l e v e l s o f FDPase I , " w i l l c o n t r o l f u t i l e c y c l i n g a nd c h a n n e l c a r b o n f l u x i n t o t h e a p p r o p r i a t e s y n t h e t i c p a t h w a y . A s i m i l a r * b u t more f l e x i b l e s i t u a t i o n • o c c u r s i n t h e h y p o d e r m i s w h i c h h a s h i g h e r l e v e l s o f FDPase I . The r e l a t i v e l e v e l s o f p h o s p h o f r u c t o k i n a s e a n d f r u c t o s e d i p h o s p h a t a s e i n t h e h y p o d e r m i s a r e n o t known; t h u s t h i s a s p e c t o f c o n t r o l c a n n o t be d i s c u s s e d . One f o r m o f f r u c t o s e d i p h o s p h a t a s e o c c u r s i n m u s c l e ; and w h i l e i t s k i n e t i c s a r e s i m i l a r t o t h o s e o f FDPase I I , i t i s n o t s e n s i t i v e t o i n h i b i t i o n by O D P - g l u c o s e , and i s a c t i v a t e d by p h o s p h o e n o l -p y r u v a t e ( B e h r i s c h , 1S72; B e h r i s c h S J o h n s o n , 1 9 7 4 ) . A c o m p a r i s o n o f t h e r e g u l a t o r y p r o p e r t i e s o f t h e s e c r u s t a c e a n F D P a s e s w i t h t h o s e o f mammalian F D P a s e s r e v e a l s a c o n s i d e r a b l y g r e a t e r c o m p l e x i t y o f t h e r e g u l a t i o n o f t h i s g l u c o n e o g e n i c enzyme i n c r u s t a c e a n t i s s u e s . The s e c o n d m a j o r g l u c o n e o g e n i c c o n t r o l s i t e i s c e n t r a l t o t h e m e t a b o l i s m o f t h e s e a n i m a l s , s i n c e i t r e p r e s e n t s b o t h a c r i t i c a l s t e p i n t h e c o n v e r s i o n o f a mino a c i d s and s m a l l o r g a n i c a c i d s i n t o s u g a r s , and an i m p o r t a n t r a t e l i m i t i n g s t e p i n g l y c o l y s i s . The t w o " s i d e s " o f t h i s c o n t r o l s i t e c o n s i s t o f p y r u v a t e k i n a s e i n t h e g l y c o l y t i c s p a n and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e i n t h e g l u c o n e o g e n i c s p a n ( S c r u t t o n & U t t e r , 1 9 6 8 ) . W h i l e t h e p r e s e n c e o f p y r u v a t e k i n a s e i n c r u s t a c e a n t i s s u e s i s w e l l * d o c u m e n t e d , t h e p r e s e n c e o f t h e g l u c o n e o g e n i c enzymes h a s n o t been c l e a r l y e s t a b l i s h e d (Munday & 34 P o a t , 1 9 7 0 ) . F u r t h e r m o r e , t h e o v e r a l l c o n t r o l o f t h i s r e a c t i o n l o c u s has not* been e x a m i n e d . I n v e r t e b r a t e t i s s u e s , p h o s p h o -e n o l p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e l e v e l s a r e l o w i n c o m p a r i s o n w i t h t h o s e o f p y r u v a t e k i n a s e ( S c r u t t o n & O t t e r , 1968; Newsholme S S t a r t , 1 9 7 3 ) . T h u s , a c r i t i c a l e l e m e n t i n t h e c o n t r o l o f t h i s r e a c t i o n l o c u s i s t h e c o n t r o l o f p y r u v a t e k i n a s e . The p r e m o l t a n d p o s t m o l t h y p o d e r m i s shows h i g h r a t e s o f c h i t i n s y n t h e s i s and a marked i n c r e a s e i n o x y g e n c o n s u m p t i o n ( S k i n n e r , 1962; M c H h i n n i e e t a l . , 1 9 7 2 ) , and c a r b o h y d r a t e o x i d a t i o n ( H e r z - H u b n e r S O r i c h , 1 9 7 3 ) . T h u s , f l u x t h r o u g h t h e p y r u v a t e k i n a s e r e a c t i o n must be s u b j e c t t o f l e x i b l e y e t c l o s e c o n t r o l . S t u d i e s o f t h e c o n t r o l o f m u s c l e m e t a b o l i s m i n P a r a -iiiiL2^S§ c a m t c h a t i c a i n d i c a t e t h a t p y r u v a t e k i n a s e i s an i m p o r t a n t c o n t r o l s i t e i n g l y c o l y s i s ( H o c h a c h k a e t a l . , 1 9 7 1 ) . However no s t u d i e s o f t h e r e g u l a t o r y p r o p e r t i e s o f t h i s enzyme i n m u s c l e o r h y p o d e r m i s h a v e been p e r f o r m e d . Somero (1969) f o u n d t e m p e r a t u r e d e p e n d e n t i n t e r c o n v e r s i o n s o f t h e m u s c l e enzyme f r o m P a r a l i t h o d e s c a m t c h a t i c a , b u t d i d n o t e x a m i n e t h e f u r t h e r r e g u l a t i o n o f t h e enzyme i n h i s s t u d y . T h u s , t o e l u c i d a t e h o w - t h i s i m p o r t a n t c o n t r o l s i t e i s r e g u l a t e d i n b o t h g l u c o n e o g e n i c - and g l y c o l y t i c t i s s u e s , a c o m p a r i s o n o f p y r u v a t e k i n a s e i n m u s c l e and h y p o d e r m i s was u n d e r t a k e n . M e t a b o l i c c o n t r o l c a n be e x e r t e d on v a r i o u s l e v e l s , one i s v i a c o n t r o l o f enzyme l e v e l s , a n o t h e r i s v i a k i n e t i c m e c h a n i s m s w h i c h c o n t r o l t h e a c t i v i t y o f t h e enzyme i n t h e c e l l , T h e s e two means o b v i o u s l y i n t e r a c t i n d e t e r m i n i n g t h e m e t a b o l i c p o t e n t i a l o f a t i s s u e . C r u s t a c e a n t i s s u e s u n d e r g o c o n s i d e r a b l e c h a n g e s a s t h e o r g a n i s m p a s s e s t h r o u g h t h e m o l t c y c l e . The h i g h l e v e l s o f p r o t e i n s y n t h e s i s 1 o c c u r r i n g d u r i n g e a r l y p r e m o l t i n t h e h y p o d e r m i s ( S t e v e n s o n , 1972; M c W h i n n i e e t a l . ; 1 9 7 2 ) , a n d t h e c o n s i d e r a b l e b r e a k d o w n o f p r o t e i n i n p r e m o l t m u s c l e ( S k i n n e r , 1966) c e r t a i n l y r a i s e t h e p o s s i b i l i t y o f c h a n g e s i n enzyme l e v e l s i n t h e s e two t i s s u e s d u r i n g t h e m o l t c y c l e . •  F u r t h e r m o r e , t h e n a t u r e o f m e t a b o l i c c o n t r o l i n t h e t i s s u e s m i g h t c h a n g e a s t h e l e v e l s o f enzymes a r e a l t e r e d . To e l u c i d a t e t h e d e g r e e t o w h i c h s u c h m e t a b o l i c • c h a n g e s o c c u r i n t h e m u s c l e a n d h y p o d e r m i s °f C a n c e r m a g i s t e r , t h e l e v e l s o f s e v e r a l k e y g l y c o l y t i c a n d g l u c o n e o g e n i c enzymes were m e a s u r e d i n i n t e r m o l t and f r e s h m o l t a n i m a l s . H y p o d e r m a l enzymes g e n e r a l l y show i n c r e a s e d s p e c i f i c a c t i v i t y i n f r e s h m o l t a n i m a l s . The o v e r a l l p o i s e r e m a i n s s t r o n g l y g l u c o n e o g e n i c i n b o t h f r e s h m o l t a n d i n t e r m o l t a n i m a l s . I n t e r m o l t m u s c l e shows a c o n s i d e r a b l y s t r o n g e r g l y c o l y t i c o r i e n t a t i o n t h a n f r e s h m o l t m u s c l e . To a s s e s s t h e i m p o r t a n c e and n a t u r e o f k i n e t i c c o n t r o l s , I c h o s e t o e x a m i n e t h e 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 o f t h e p h o s p h o g l y c e r a t e k i n a s e and p y r u v a t e k i n a s e r e a c t i o n i n t h e m u s c l e and h y p o d e r m i s o f i n t e r m o l t and f r e s h m o l t a n i m a l s . W h i l e t h e mechanisms f o r t h e r e v e r s a l o f p h o s p h o g l y c e r a t e k i n a s e a n d t h e i n h i b i t i o n o f p y r u v a t e k i n a s e a r e o f c r i t i c a l i m p o r t a n c e f o r e f f e c t i v e g l u c o n e o g e n e s i s i n t h e h y p o d e r m i s , t h e a c t i v a t i o n o f b o t h o f t h e s e r e a c t i o n s i n t h e g l y c o l y t i c d i r e c t i o n i s e s s e n t i a l f o r b o t h t h e h y p o d e r m i s and t h e h i g h l y g l y c o l y t i c m u s c l e . An e x a m i n a t i o n and c o m p a r i s o n o f t h e c o n t r o l m echanisms shown by t h e s e enzymes i n f r e s h m o l t and i n t e r m o l t m u s c l e a n d h y p o d e r m i s s h o u l d e l u c i d a t e t h e means by w h i c h t h e s e c o n t r o l r e q u i r e m e n t s h ave b e e n f u l f i l l e d i n C a n c e r m a g i s t e r . S e v e r a l s o l u t i o n s 36 seemed p o s s i b l e : 1) t h e e v o l u t i o n o f d i f f e r e n t t i s s u e f o r m s o f t h e enzymes e a c h 1 w i t h r e g u l a t o r y c h a r a c t e r i s t i c s s u i t e d f o r t h e t i s s u e ' s m e t a b o l i s m , 2) t h e e v o l u t i o n o f f o r m s w h i c h a r e s y n t h e s i z e d d u r i n g d i f f e r e n t p o r t i o n s o f t h e m o l t c y c l e i n r e s p o n s e t o t h e c h a n g i n g n e e d s o f t h e t i s s u e s , o r 3) t h e e v o l u t i o n o f a s i n g l e h i g h l y s o p h i s t i c a t e d p r o t e i n s p e c i e s whose k i n e t i c r e g u l a t i o n makes i t s f u n c t i o n u n d e r a v a r i e t y o f m e t a b o l i c c i r c u m s t a n c e s f e a s i b l e . I n e x a m i n i n g t h e p r o p e r t i e s o f p y r u v a t e k i n a s e s and p h o s -p h o g l y c e r a t e k i n a s e s f r o m t h e s e a n i m a l s , I f o u n d t i s s u e s p e c i f i c f o r m s o f p y r u v a t e k i n a s e and m o l t c y c l e r e l a t e d f o r m s o f p h o s -p h o g l y c e r a t e k i n a s e . The f r e s h m o l t f o r m s o f p n o s p h o g l y c e r a t e k i n a s e show an i n c r e a s e d a f f i n i t y f o r ATP* and a d e c r e a s e d s e n s i t i v i t y t o MgADP i n h i b i t i o n c o m p a r e d t o t h e i n t e r m o l t m u s c l e enzyme. Under • p h y s i o l o g i c a l c o n c e n t r a t i o n s o f s u b s t r a t e s a n d i n h i b i t o r s , t h e s e k i n e t i c a l t e r a t i o n s w o u l d s e r v e t o f a c i l i t a t e t h e r e v e r s a l o f t h e r e a c t i o n f o r g l u c o n e o g e n e s i s . M u s c l e and h y p o d e r m a l p y r u v a t e k i n a s e s show no m o l t c y c l e c o r r e l a t e d c h a n g e s , b u t t h e two t i s s u e s h ave d i s t i n c t i s o z y m e s . The m u s c l e enzyme i s i n h i b i t e d by ATP, a r g i n i n e p h o s p h a t e a n d M g 2 c i t r a t e , and i s a c t i v a t e d by FDP. The h y p o d e r m a l enzyme e x i s t s i n t w o i n t e r c o n v e r t i b l e s t a t e s , PK I and PK I I . PK I shows a h i g h a f f i n i t y f o r PEP and a low a f f i n i t y f o r FDP. By c o n t r a s t , PK I I shows a l o w a f f i n i t y f o r PEP, a h i g h a f f i n i t y f o r FDP and an i n c r e a s e d s e n s i t i v i t y t o i n h i b i t i o n by amino a c i d s and M g 2 c i t r a t e . I n t e r c o n v e r s i o n o f t h e two f o r m s c a n be a c h i e v e d by t h e a d d i t i o n o r r e m o v a l o f FDP. S h i f t i n g t h e enzyme i n t o t h e l o w a f f i n i t y c o n f o r m a t i o n w o u l d l e a d t o a d r a s t i c l i m i t a t i o n o f 37 enzyme a c t i v i t y u n d e r p h y s i o l o g i c a l c o n c e n t r a t i o n s o f s u b s t r a t e s and i n h i b i t o r s , t h u s a l l o w i n g g l u c o n e o g e n i c f l u x t h r o u g h t h e e n z y m a t i c b y p a s s . • T h e s e two c o n f o r m a t i o n a l s t a t e s a l l o w s e n s i t i v e r e g u l a t i o n o f f l u x t h r o u g h t h i s m a j o r c o n t r o l s i t e i n b o t h g l u c o n e o g e n e s i s and g l y c o l y s i s . • T h u s , t h e c r u s t a c e a n h y p o d e r m i s h a s e v o l v e d e n z y m a t i c a d a p t a t i o n s w h i c h f a c i l i t a t e g l u c o n e o g e n i c c a r b o n f l u x . 38 GENERAL MATERIALS AND METHODS E x p e r i m e n t a l A n i m a l s F r e s h m o l t and i n t e r m o l t C a n c e r m a g i s t e r were c o l l e c t e d by SCUBA d i v i n g c o u r t e s y • o f J o h n Himmelman, a t J e r i c h o P i e r , V a n c o u v e r , B.C. F r e s h m o l t a n i m a l s were u s e d w i t h i n a day o f c o l l e c t i o n . i n t e r m o l t a n i m a l s were e i t h e r u s e d i m m e d i a t e l y o r m a i n t a i n e d f o r a t l e a s t one week i n t h e r e f r i g e r a t e d , r e c i r c u l a t i n g s e a w a t e r s y s t e m t o a l l o w a c c l i m a t i o n t o t h e new c o n d i t i o n s . B o t h m ale a n d f e m a l e a n i m a l s were u s e d i n t h i s s t u d y . M a t u r e f e m a l e C a n c e r m a g i s t e r m o l t - b e f o r e m a t i n g , a t w h i c h t i m e t h e y a r e b e i n g h e l d and p r o t e c t e d by a h a r d s h e l l e d male.' M a t i n g o c c u r s m o s t - o f t e n b e t w e e n A p r i l a n d S e p t e m b e r , so f e m a l e m o l t i n g o c c u r s i n t h i s p e r i o d . M a l e s m o l t a s y n c h r o n o u s l y w i t h t h e f e m a l e s . T h u s , i t i s p o s s i b l e t o f i n d m o l t i n g a n i m a l s t h r o u g h o u t t h e y e a r . M o l t i n g m a l e s a r e p a r t i c u l a r l y common i n t h e l a t e f a l l and e a r l y w i n t e r , w h i l e m o l t i n g f e m a l e s a r e common i n t h e s p r i n g a n d summer months. M o l t c y c l e s t a g e s w e re d e t e r m i n e d a c c o r d i n g t o t h e c r i t e r i a o f D r a c h ( 1 9 3 9 ) . A n i m a l s were f e d c a n n e d s a l m c n o r f r e s h l y c o l l e c t e d m u s s e l s t w i c e a week. I n p r e l i m i n a r y e x p e r i m e n t s , I t r i e d t o s t i m u l a t e m o l t i n g by a' v a r i e t y o f m e t h o d s . The v a r i o u s t r e a t m e n t s i n c l u d e d : m a i n t a i n i n g t h e ' . a n i m a l s i n warmer w a t e r (10 °C) i n i n d i v i d u a l c o n t a i n e r s , m a i n t a i n i n g t h e a n i m a l s i n t h e d a r k u n d e r b o t h c o l d and warm c o n d i t i o n s i n p r i v a t e c o n t a i n e r s , e y e s t a l k a b l a t i o n , and c r u s t e c d y s o n e i n j e c t i o n (3 jig/gm body w e i g h t ) . None o f 39 t h e s e t r e a t m e n t s p r o v e d s u c c e s s f u l . E y e s t a l k a b l a t i o n l e d p r i m a r i l y t o d e a t h , w h i l e m o l t i n g i n t h e o t h e r c o n d i t i o n s was n o t i n c r e a s e d o v e r m o l t i n g i n t h e "communal" t a n k s . T h u s , i t was d e c i d e d t o r e l y upon f i e l d c o l l e c t i o n s o f m o l t i n g a n i m a l s . R e a g e n t s A l l r e a g e n t s were t h e h i g h e s t g r a d e a v a i l a b l e , most were f r o m .Sigma C h e m i c a l s ( S t . L o u i s , M i s s o u r i ) . C o u p l i n g enzymes s u c h a s b e e f h e a r t l a c t a t e d e h y d r o g e n a s e and m a l a t e - d e h y d r o -g e n a s e , r a b b i t m u s c l e g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , t r i o s e — p h o s p h a t e i s o m e r a s e , and ••• c * — g l y c e r o p h o s p h a t e : d e h y d r o -g e n a s e , a s w e l l as c i t r a t e s y n t h a s e , p h o s p h o g l y c e r a t e k i n a s e , g l u t a m a t e d e h y d r o g e n a s e , a l d o l a s e , p h o s p h o g l u c o s i s o m e r a s e , a n d g l u c o s e - 6 - p h o s p h a t e d e h y d r o g e n a s e were f r o m Sigma a s w e l l . P e a n u t p h o s p h o e n o l p y r u v a t e c a r b o x y l a s e was p u r i f i e d a c c o r d i n g t o Dyson e t a l . , (1975) a nd L a n e e t a l . , ( 1 9 6 7 ) . H y d r o x y l a p a t i t e , c e l l u l o s e p h o s p h a t e a n d DEAE-Sephadex i o n e x c h a n g e r s were f r o m S igma a nd P h a r m a c i a r e s p e c t i v e l y . I o n e x c h a n g e r s were p r e t r e a t e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n s t r u c t i o n s . S t a r c h was f r o m C o n n a u g h t L a b o r a t o r i e s ( T o r o n t o ) and a m p h o l i n e s were f r o m 1KB ( U p s a l a , S w e d e n ) . ££S£aration o f Enzymes S i n c e t h e • p u r i f i c a t i o n p r o c e d u r e s a r e o f d i r e c t i m p o r t a n c e t o t h e d i s c u s s i o n o f e a c h enzyme, t h e method s u t i l i z e d i n p r e p a r i n g e a c h enzyme - w i l l be p r e s e n t e d t o g e t h e r w i t h t h e r e s u l t s . G e n e r a l l y a l l c r a b s were " a n a e s t h e t i z e d " by c o o l i n g i n 40 a - f r e e z e r u n t i l o n l y s l o w movements came i n r e s p o n s e t o a s t i m u l u s . The a n i m a l s were t h e n s a c r i f i c e d by c u t t i n g away t h e d o r s a l s u r f a c e o f t h e c a r a p a c e . C a r e was t a k e n t o d e s t r o y t h e c e r e b r a l g a n g l i a i n t h e p r o c e s s . The d i s s e c t i o n o f t h e t i s s u e s f r o m t h e a n i m a l t o o k p l a c e on i c e . The h y p o d e r m i s , t h e e p i t h e l i u m d i r e c t l y u n d e r l y i n g t h e e x o s k e l e t o n , was s i m p l y p e e l e d o f f o f t h e d o r s a l c a r a p a c e a n d t h e o u t e r c o v e r s o f t h e b r a n c h i a l c h a m b e r s . M u s c l e was r e m o v e d f r o m t h e w a l k i n g l e g s a nd t h e c h e l i p e d s a nd f r e e d o f a d h e r i n g h y p o d e r m i s . A l l s u b s e q u e n t o p e r a t i o n s were a t 4 °C o r on i c e . S p e c t r o -p h o t o m e t r i c a s s a y s o f enzyme a c t i v i t y t o o k p l a c e on a U n i c a m SP180Q r e c o r d i n g u l t r a v i o l e t s p e c t r o p h o t o m e t e r , most o f t e n by m o n i t o r i n g t h e c h a n g e i n a b s o r b a n c e due t o t h e o x i d a t i o n o r r e d u c t i o n o f NAD (H) a t 340 a n g s t r o m s . S t a r c h G e l s and I s p e l e c t r o f o c u s i n g T h i r t e e n p e r c e n t s t a r c h g e l s were p r e p a r e d u s i n g T r i s - c i t r a t e o r T r i s — c i t r a t e - K C l b u f f e r s y s t e m s w i t h v a r i o u s c o m b i n a t i o n s o f g e l and t a n k b u f f e r pH and i o n i c s t r e n g t h . The most e f f e c t i v e s y s t e m f o r s e p a r a t i o n o f p h o s p h o g l y c e r a t e k i n a s e was 35.6 gm T r i s and 9.5 gm c i t r a t e p e r l i t e r (pH = 7 . 5 ) , w h i l e t h e most e f f e c t i v e s y s t e m f o r e l e c t r o p h o r e s i s o f p y r u v a t e k i n a s e was 37.8 gm T r i s , 9.5 gm c i t r a t e , and 7.45 g m K C l p e r l i t e r (pH — 8.5) a s t a n k b u f f e r . I n b o t h c a s e s , t h e g e l b u f f e r was a 1:20 d i l u t i o n o f t h e t a n k b u f f e r . A f t e r t h e g e l s had c o o l e d t o 4 °C c e n t r i f u g e d , d i a l y z e d t i s s u e e x t r a c t s were c o a t e d o n t o f i l t e r p a p e r w i c k s a n d i n s e r t e d i n t o t h e g e l . To e n s u r e s u f f i c i e n t a c t i v i t y , t w o o r more w i c k s were o f t e n i n s e r t e d i n t o 41 t h e g e l . A p r e c h i l l e d p l e x i g l a s s b l o c k was l a i d a c r o s s t h e g e l . G e l s were r u n a t 150—200 v o l t s ( c u r r e n t 25-45 mAmps) f o r v a r y i n g l e n g t h s o f t i m e * (6—18 h o u r s ) . B o t h p h o s p h o g l y c e r a t e k i n a s e and p y r u v a t e k i n a s e were s t a i n e d by t h e n e g a t i v e method d e s c r i b e d by S c o p e s ( 1 9 6 8 ) . D e v e l o p i n g t h e s t a i n w i t h p h e n a z i n e m e t h a s u l p h a t e and n i t r o b l u e t e t r a z o l i u m , g a v e 1 o n l y p a r t i a l l y s a t i s f a c t o r y ' r e s u l t s . I n s t e a d , t h e s t a i n was m o n i t o r e d • w i t h an u l t r a v i o l e t l i g h t . NADH a p p e a r s y e l l o w u n d e r u l t r a v i o l e t l i g h t , w h i l e NAD a p p e a r s d a r k p u r p l e . T h u s , t h e a p p e a r a n c e o f enzyme a c t i v i t y c a n be v i s u a l i z e d . The r e l a t i v e m i g r a t i o n v e l o c i t i e s were m e a s u r e d a nd t h e p o s i t i o n s r e c o r d e d a s t h e s t a i n s became v i s i b l e . I s o e l e c t r o f o c u s i n g was c a r r i e d o u t a c c o r d i n g t o t h e method o f H a g l u n d ( 1 9 6 7 ) . Enzyme p r e p a r a t i o n s were d i a l y z e d a g a i n s t 1% g l y c i n e , pH 7.0 i n p r e p a r a t i o n f o r e l e c t r o f o c u s i n g . As t h e s e c r u s t a c e a n e n z y m e s , p a r t i c u l a r l y t h e h y p o d e r m a l f o r m s , were r e l a t i v e l y u n s t a b l e o n l y 500 m v o l t s were u s e d d u r i n g e l e c t r o f o c u s i n g * The enzymes were e l e c t r o f o c u s e d o n l y u n t i l t h e c u r r e n t s t a b i l i z e d a t a l o w l e v e l , s i n c e l o n g e r e l e c t r o f o c u s i n g t i m e s l e d t o c o n s i d e r a b l e l o s s o f enzyme a c t i v i t y . K i n e t i c A n a l y s e s " K i n e t i c p a r a m e t e r s were d e t e r m i n e d by s e v e r a l means. The p r i m a r y method u t i l i z e d • f o r t h e d e t e r m i n a t i o n o f Km v a l u e s was t h e L i n e w e a v e r - B u r k , 1/V v e r s u s 1/S p l o t . K i v a l u e s f o r v a r i o u s i n h i b i t o r s were d e t e r m i n e d f r o m b o t h t h e L i n e w e a v e r - B u r k p l o t s and D i x o n p l o t s . S l o p e and i n t e r c e p t r e p l o t s d e r i v e d f r o m t h e 42 l i n e w e a v e r - B u r k p l o t s were u s e d t o d e t e r m i n e t h e p a t t e r n o f i n h i b i t i o n . The m e t h o d s d e s c r i b e d t y Webb (1963) were u s e d t o e v a l u a t e c o m p l e x i n h i b i t i o n p a t t e r n s . S i n c e t h e d e t e r m i n a t i o n o f r e a c t i o n r a t e s f r o m t h e l i n e s g i v e n by t h e r e c o r d i n g s p e c t r o p h o t o m e t e r i n v o l v e s u t i l i z i n g a v i s u a l " b e s t . f i t " m e t h od, i f was f e l t t h a t c o m p u t e r drawn l i n e s w o u l d s t i l l be b a s e d upon t h i s p o s s i b l e e x p e r i m e n t a l b i a s . T h u s , l i n e s were f i t t e d t o t h e p o i n t s , v i a a v i s u a l " b e s t f i t " m ethod. I n most c a s e s , t h e b e s t f i t was o b v i o u s . K i n e t i c d e t e r m i n a t i o n s - were p e r f o r m e d on a minimum o f two d i f f e r e n t enzyme p r e p a r a t i o n s , a nd i n many c a s e s , w e r e p e r f o r m e d b e t w e e n 5 and 10 t i m e s . R e p e a t d e t e r m i n a t i o n s i n d i c a t e t h a t Km d e t e r m i n a t i o n s a r e s u b j e c t t o a p p r o x i m a t e l y a 5% e r r o r . M e a s u r e m e n t s o f k i n e t i c e f f e c t s due t o v a r i o u s c a t i o n s u t i l i z e d c a t i o n s w h i c h were n o t c o r r e c t e d f o r p o s s i b l e c h a n g e s i n t h e - d e g r e e o f h y d r a t i o n between d i f f e r e n t e x p e r i m e n t s . P r o t e i n - ' c o n c e n t r a t i o n s were d e t e r m i n e d by m e a s u r i n g t h e a b s o r p t i o n o f t h e s o l u t i o n a t 260 and 280 n a n o m e t e r s . T h e s e v a l u e s were t h e n u s e d t o a r r i v e a t t h e p r o t e i n c o n c e n t r a t i o n i n t h e s o l u t i o n by u s i n g t h e method o f Lane ( 1 9 5 7 ) . 43 P a r t I . C h a n g i n g Enzyme A c t i v i t i e s i n t h e H y p o d e r m i s , G i l l , a n d M u s c l e o f C a n c e r m a g i s t e r d u r i n g t h e M o l t C y c l e . INTRODUCTION S i n c e c r u s t a c e a n t i s s u e s u n d e r g o p r o n o u n c e d s t r u c t u r a l a n d p h y s i o l o g i c a l c h a n g e s d u r i n g t h e m o l t c y c l e , t h e o r g a n i z a t i o n o f m e t a b o l i s m i n t h e t i s s u e s may be s u b j e c t e d t o c h a n g e s i n o v e r a l l o r i e n t a t i o n a s w e l l . To e l u c i d a t e w h e t h e r any marked c h a n g e s i n t h e o r g a n i z a t i o n o f c a r b o h y d r a t e m e t a b o l i s m o c c u r d u r i n g t h e m o l t c y c l e , I m e a s u r e d t h e l e v e l s o f a v a r i e t y o f g l y c o l y t i c a n d g l u c o n e o g e n i c enzymes i n b o t h i n t e r m o l t and f r e s h m o l t m u s c l e , h y p o d e r m i s and g i l l . P h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e , g l y c e r -a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , p h o s p h o g l y c e r a t e k i n a s e , p y r u v a t e k i n a s e , and l a c t a t e d e h y d r o g e n a s e were c h o s e n a s r e p r e s e n t a t i v e g l y c o l y t i c enzymes* The g l u c o n e o g e n i c e n z y m es were f r u c t o s e d i p h o s p h a t a s e , p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e , and p y r u v a t e c a r b o x y l a s e . The l e v e l s o f g l u t a m i n e : f r u c t o s e - 6 -p h o s p h a t e a m i d o t r a n s f e r a s e , t h e f i r s t enzyme i n t h e p a t h w a y t o c h i t i n , were a l s o d e t e r m i n e d i n t h e s e t i s s u e s . MATERIALS The g l y c o l y t i c enzymes measured i n t h e h i g h s p e e d c e n t r i f u g a t i o n a t 27,000 g AND METHODS and f r u c t o s e d i p h o s p h a t a s e were s u p e r n a t a n t r e m a i n i n g a f t e r f o r t w e n t y m i n u t e s . P h o s p h o e n o l -44 p y r u v a t e c a r b o x y k i n a s e , p y r u v a t e c a r b o x y l a s e , p h o s p h o f r u c t o -k i n a s e , a n d g i u t a m i n e : f r u c t o s e - 6 - p h o s p h a t e a m i d o t r a n s f e r a s e were measured i n b o t h t h e h i g h s p e e d s u p e r n a t a n t a n d t h e w h o l e homogenate. P r e p a r a t i o n o f t h e Homogenate f o r Measurement o f Enzyme A c t i v i t i e s A n a e s t h e t i z e d c r a b s were c u t o p e n , t h a g i l l s , s e v e r a l grams o f h y p o d e r m i s and ;- s e v e r a l grams o f l e g m u s c l e were g u i c k l y d i s s e c t e d o u t , b l o t t e d , and w e i g h e d . M u s c l e was h o m o g e n i z e d i n t h r e e v o l u m e s o f c o l d 50 mM i m i d a z o l e — C l , pH 7.0, w h i l e g i l l a n d h y p o d e r m i s were h o m o g e n i z e d i n two v o l u m e s . F o r p r e p a r a t i o n o f t h e h i g h s p e e d s u p e r n a t a n t , a S o r v a l O m n i m i x e r was s e t a t t o p s p e e d a n d r u n f o r 2 m i n u t e s . F o r p r e p a r a t i o n o f t h e w h o l e h o m o g e n a t e , a t e f l o n h o m o g e n i z e r was u s e d and t h e t i s s u e was h o m o g e n i z e d w i t h 20—30 s t r o k e s . I n t h e s e e x p e r i m e n t s * 4 v o l u m e s o f b u f f e r were u s e d w i t h m u s c l e and t h r e e v o l u m e s w i t h g i l l a n d h y p o d e r m i s . The f i n a l volume o f t h e h omogenate was n o t e d f o r b o t h methods o f p r e p a r a t i o n . The s u p e r n a t a n t s o r h o m o g e n a t e s were u s e d d i r e c t l y f o r measurement o f t h e enzyme a c t i v i t y . Enzyme A s s a y s Enzyme l e v e l s i n t h e v a r i o u s t i s s u e s were d e t e r m i n e d by s t a n d a r d s p e c t r o p h o t o m e t r c c o u p l e d enzyme a s s a y s . O p t i m a l c o n d i t i o n s were d e t e r m i n e d f o r e a c h enzyme f r o m e a c h t i s s u e , a nd t h e m a x i m a l v e l o c i t y was u s e d i n c a l c u l a t i n g enzyme l e v e l s . E a c h v a l u e r e p r e s e n t s t h e mean o f t h e v a l u e s f o u n d f o r a t l e a s t 45 t h r e e d i f f e r e n t a n i m a l s , e x c e p t i n t h e c a s e o f t h e g i l l v a l u e s . The a c t i v i t y m e a s u r e m e n t s do n o t i n c l u d e t h e e f f e c t o f m e t a b o l i t e a c t i v a t o r s . The a s s a y t e m p e r a t u r e i n a l l c a s e s was 20 °C. The c o n c e n t r a t i o n s o f r e a g e n t s f o r e a c h enzyme a s s a y a r e g i v e n b e l o w . • P h o s p h o f r u c t o k i n a s e : 0.1 M T r i s - H C l b u f f e r , pH 8. 0, 0.5 mM ATP, 1 mM f r u c t o s e - 6 - p h o s p h a t e , 5 i f MgC12, 100'mM KC1, 0.2 mM NADH, e x c e s s a l d o l a s e , t r i o s e p h o s p h a t e i s o m e r a s e , and c v - g l y c e r o l p h o s p h a t e d e h y d r o g e n a s e . • G ^ y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e : 0.1 M T r i s b u f f e r , pH 7.5, 75 mM K C l , ' 1 mM ATP, 4 mM M g C l 2 , 2 mM 3 - p h o s p h o g l y c e r a t e , 0;3 mM NADH, e x c e s s p h o s p h o g l y c e r a t e k i n a s e . The r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f 3 - p h o s p h o g l y c e r a t e . T h i s a s s a y method was c h o s e n a s t h e p u r i t y and s t a b i l i t y o f t h e d - l ' g l y c e r a l d e h y d e - 3 — p h o s p h a t e was l o w . F u r t h e r m o r e , t h e c o m p a r i s o n o f t h e m a x i m a l g l u c o n e o g e n i c r e a c t i o n r a t e s o f g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e and p h o s p h o g l y c e r a t e k i n a s e was o f i n t e r e s t . P h o s p h o g l y c e r a t e k i n a s e : 50 mM i m i d a z o l e - C l b u f f e r , pH 7,0, 4 mM MgC12, 0.2 mM NADH, 1 mM ATP, 2 mM 3 - p h o s p h o g l y c e r a t e a n d e x c e s s g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e . P y r u v a t e k i n a s e i 50 mM i m i d a z o l e - C l b u f f e r , pH 7.0, 4 mM MgC12, 0.2 mM NADH, '75 mM K C l , 2 mM ADP, 2 mM p h o s p h o e n o l p y r u v a t e , e x c e s s l a c t a t e d e h y d r o g e n a s e . HiSiate d e h y d r o g e n a s e : 0.1 M T r i s - H C l b u f f e r , pH 7.5, 0.3 mM NADH, 3 mM p y r u v a t e . -££JJ£*£se dj.£hgs£hat ase_: 50 mM T r i s - H C l b u f f e r , pH 7.5, 5 mM M g C l 2 , 0.1 mM NADP, 0.01 mM t o 0.03 mM f r u c t o s e 1,6 p h o s p h a t e , 46 e x c e s s p h o s p h o h e x o s e i s o m e r a s e and g l u c o s e - 6 - p h o s p h a t a d e h y d r o -g e n a s e . G l u t a m i n e : _ f r u c t o s e - 6 - g h q s p h a t e a m i d o t r a n s f e r a s e ^  0.1 mM T r i s — H C l b u f f e r , pH 7.6, 5 mM g l u t a m i n e , 3 mM f r u c t o s e - 6 -p h o s p h a t e , 0 . 3 mM NAD, e x c e s s g l u t a m a t e d e h y d r o g e n a s e . I n t h i s a s s a y ; w h i c h m e a s u r e d t h e e v o l u t i o n o f g l u t a m a t e , i t was e s s e n t i a l t o u s e t h e ammonium i o n f r e e g l u t a m a t e d e h y d r o g e n a s e and t o c h e c k t h a t t h e g l u t a m i n e u s e d i n t h e a s s a y was f r e e o f g l u t a m a t e ; P y r u v a t e c a r b o x y l a s e : Two methods were u s e d t o measure t h e a c t i v i t y o f t h i s enzyme. 1) 0.1 M T r i s - H C l b u f f e r , pH 8.4, 10 mM p y r u v a t e , 3 mM ATP, 20.mM KHC03, 0 .1 mM a c e t y l CoA, 8 mM M g C l 2 , 0.2 mM NADH, e x c e s s m a l a t e d e h y d r o g e n a s e . 2) 0.05 M T r i s - H C l b u f f e r , pH 7.5, 0.2 mM DTNB (5,5»-dithiobis-2-nitrobenzoic a c i d ) , 10 m.M MgS04, 2.5 mM ATP, 0.2 mM a c e t y l CoA, 100 mM KHC03, 10 mM p y r u v a t e , e x c e s s c i t r a t e s y n t h a s e . The h i g h e r e x t i n c t i o n c o e f f i c i e n t o f DTNB v e r s u s NADH makes t h i s a s s a y mora s e n s i t i v e t o t h e p r o d u c t i o n o f o x a l o a c e t a t e . T h i s r e a c t i o n i s m o n i t o r e d a t 412 a n g s t r o m s . Iij2S£hoenol£2ruvate c a r b o x y k i n a s e : 0.1 mM T r i s — a a l e a t e b u f f e r , pH 6.0, 100 mM NaHC03, 2 mfl p h o s p h o e n o l p y r u v a t e , 2 mM GDP, I D P , o r CDP, 'V'mM' M g C l 2 , 0.2 mM NADH, e x c e s s m a l a t e d e h y d r o g e n a s e . P h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y i s d e t e r m i n e d f r o m t h e i n c r e a s e i n E340 due t o t h e a d d i t i o n o f HC03 t o t h e c o m p l e t e a s s a y m i x t u r e . 47 RESULTS AND DISCUSSION The r e s u l t s o f t h e enzyme a c t i v i t y m e a s u r e m e n t s a r e shown i n T a b l e 1 . The d e t e r m i n a t i o n o f s e v e r a l o f t h e s e enzyme a c t i v i t i e s r e g u i r e d a d d i t i o n a l c o n t r o l s and e x p e r i m e n t s t o e n s u r e p r o p e r measurement o f enzyme a c t i v i t y . The measurement o f p y r u v a t e c a r b o x y l a s e and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e r e g u i r e s p a r t i c u l a r l y c a r e f u l c o n t r o l s f o r i n t e r f e r i n g enzyme a c t i v i t i e s . The f i r s t a s s a y method f o r p y r u v a t e c a r b o x y l a s e r e g u i r e s a c o n t r o l f o r e n d o g e n o u s l a c t a t e d e h y d r o g e n a s e , and NADH o x i d a s e . The s e c o n d method r e g u i r e s a c o n t r o l f o r e n d o g e n o u s h y d r o l y s i s o f a c e t y l CoA, as w e l l a s c a r e f u l r e g u l a t i o n o f t h e l e v e l s o f s u l f h y d r y l g r o u p s p r e s e n t i n t h e a s s a y . The a s s a y f o r p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a l s o r e g u i r e s p r e c a u t i o n s : s o d i u m b i c a r b o n a t e must ba u s e d t o m i n i m i z e a c t i v a t i o n o f p y r u v a t e k i n a s e and t h e c o u p l i n g enzyme must be d i a l y z e d t o remove NH4+, an a c t i v a t i n g c a t i o n f o r p y r u v a t e k i n a s e . • O b v i o u s l y t h e i m p o r t a n t c o n t r o l f o r t h i s a s s a y i s p y r u v a t e k i n a s e a c t i v i t y . A c o n t r o l f o r s o d i u m a c t i v a t i o n o f p y r u v a t e k i n a s e must be i n c l u d e d . The t w o • m e t h o d s o f m e a s u r i n g p y r u v a t e c a r b o x y l a s e a c t i v i t y y i e l d e d e q u i v a l e n t r e s u l t s . B o t h p y r u v a t e c a r b o x y l a s e a n d p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t i e s a r e a s s o c i a t e d w i t h t h e p a r t i c u l a t e f r a c t i o n . T h u s , measurement o f t h e i r l e v e l s i n t h e w h ole h o m o g e n a t e s y i e l d e d c o n s i d e r a b l y h i g h e r v a l u e s t h a n m e a s u r e m e n t s i n t h e s u p e r n a t a n t . T h i s c o r r e s p o n d s w i t h t h e m i t o c h o n d r i a l l o c a t i o n o f p y r u v a t e c a r b o x y l a s e and t h e p a r t i a l l y 48 Table 1 Specific Activities of Various Enzymes in the Muscle, G i l l and Hypodermis of Cancer magister Specific activities are the mean of the values found for three or four different animals, except in the case of the g i l l values where usually only one animal was measured Comparisons of freshmolt and intermolt enzyme activities were made using Student's T test. The results of the comparison are indicated as follows: (*) significant at P= .0 .05, (**) significant at P= 0.01, and (N.S.) not significant. Enzyme activities are expressed in umoles/min gm wet weight at 20° C, — indicates that the enzyme was not measured in the tissue, 0 indicates that the enzyme was not detectable in the tissue • Intermolt Freshmolt Enzyme Hypodermis G i l l Muscle Hypodermis G i l l Muscle FDPase 0.66 0.44 0.33 1.2 ** 0.56 N.S. 0.484** PFK 0.182 0.241 5.81 0.375** 0.156 2.122** F6PGAT 0.12 0.12 0 0.15* 0.135 0.0025 LDH 4.06 1.205 18.87 7.5* 2.04 19.64 N.S. G3PDH 5.0 43.0 14.5** 12.5** PGK 3.615 3.278 13.02 9.585** 4.34 8.36** PK 6.3 7.7 61.7 15.4** 16.2 51.5TJ.S. PEPCK 0.2 0.08 0 0.5** 0.12 0.105 pyruvate carboxylase 0.48 0.05 1.1** 0.1065** 49 m i t o c h o n d r i a l l o c a t i o n o f p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i n mammalian t i s s u e s ( S a g g e r s o n S ; E v a n s , 1 9 7 5 ) . M e a s u r e m e n t s o f p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i n v a r i o u s r a t t i s s u e s i n d i c a t e t h a t t h e g l u c o n e o g e n i c r e a c t i o n shows a h i g h e r s p e c i f i c a c t i v i t y t h a n t h e - " d o w n h i l l 1 ' r e a c t i o n . However, t h e r a t i o o f a c t i v i t i e s v a r i e s b e t ween t i s s u e s ( P o g s o n S S m i t h , 1975) and v a r i e s c o n s i d e r a b l y f r o m r a t i o s o b t a i n e d w i t h t h e p u r i f i e d m i t o c h o n d r i a l enzyme (Chang e t a l . , 1 9 6 6 ) . T h u s , no c o n c l u s i o n a b o u t t h e a c t u a l r e l a t i o n s h i p o f t h e m e a s u r e d " d o w n h i l l " r e a c t i o n and t h e g l u c o n e o g e n i c r e a c t i o n c a n be made. S i n c e p h o s p h o f r u c t o k i n a s e h a s been r e p o r t e d t o be a s s o c i a t e d w i t h t h e p r e c i p i t a t e a f t e r h i g h s p e e d c e n t r i f u g a t i o n ( O p i e S Newsholme, 1 9 6 7 ) , i t s l e v e l s i n t h e w h o l e h o m o g e n a t e were c o m p a r e d w i t h t h e l e v e l s f o u n d i n t h e s u p e r n a t a n t . No d i f f e r e n c e was f o u n d . I n m e a s u r e m e n t s of 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 , e n d o g e n o u s AMP c o u l d have i n h i b i t e d t h e enzyme. To c h e c k t h i s p o s s i b i l i t y , an "AMP t r a p " c o n s i s t i n g o f 0.2 mM ATP and e x c e s s m y o k i n a s e was added t o t h e a s s a y s y s t e m . T h i s w o u l d c o n v e r t t h e AMP t o ADP and t h u s d e i n h i b i t t h e enzyme. No c h a n g e i n a c t i v i t y was f o u n d upon t h e a d d i t i o n o f t h i s t r a p . P r e s u m a b l y e n d o g e n o u s AMP l e v e l s were low d u r i n g e x t r a c t i o n . - A c o n s i d e r a t i o n o f t h e enzyme a c t i v i t i e s shown i n T a b l e 1 makes i t r e a d i l y a p p a r e n t - t h a t c o n s i d e r a b l e c h a n g e s o c c u r i n l e v e l s o f k e y enzymes b e t w e e n f r e s h m o l t and . i n t e r m o l t i n b o t h m u s c l e a n d h y p o d e r m i s . A c o n s i d e r a t i o n o f enzyme l e v e l s i n t h e i n t e r m o l t and f r e s h m o l t g i l l shows t h a t t h e y d o • n o t u n d e r g o a s marked v a r i a t i o n s a s enzyme l e v e l s i n t h e m u s c l e o r h y p o d e r m i s . 50 G e n e r a l l y , t h e g i l l shows a h i g h g l u c o n e o g e n i c c a p a c i t y . I n t e r m o l t m u s c l e g e n e r a l l y showed h i g h l e v e l s o f a l l enzymes e x c e p t t h e g l u c o n e o g e n i c e n z y m e s , f r u c t o s e d i p h o s p h a t a s e a n d p y r u v a t e c a r b o x y l a s e . P h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e was n o t p r e s e n t i n i n t e r m o l t m u s c l e . F r e s h m o l t m u s c l e showed c o n s i d e r a b l y r e d u c e d l e v e l s o f p h o s p h o f r u c t o k i n a s e , p h o s p h o -g l y c e r a t e k i n a s e , g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e , and p y r u v a t e k i n a s e . By c o n t r a s t , t h e l e v e l s o f f r u c t o s e d i p h o s p h a -t a s e and p y r u v a t e c a r b o x y l a s e were h i g h e r t h a n i n i n t e r m o l t m u s c l e . P h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e was p r e s e n t i n e g u a l l e v e l s w i t h p y r u v a t e c a r b o x y l a s e i n f r e s h m o l t m u s c l e . T h u s , t h e g l y c o l y t i c p o t e n t i a l o f f r e s h m o l t m u s c l e i s c o n s i d e r a b l y r e d u c e d and t h e p r e s e n c e o f t h e v a r i o u s g l u c o n e o g e n i c enzymes makes t h e r e v e r s a l o f g l y c o l y s i s f o r t h e p r o d u c t i o n o f s u g a r o r c h i t i n p r e c u r s o r s a t l e a s t f e a s i b l e . A l l h y p o d e r m a l enzymes a r e p r e s e n t i n h i g h e r c o n c e n t r a t i o n s i n f r e s h m o l t t h a n i n i n t e r m o l t h y p o d e r m i s . T h i s i s i n d i c a t i v e o f t h e g e n e r a l i n c r e a s e i n p r o t e i n s y n t h e s i s d u r i n g p r e m o l t ( A d e l u n g & K e l l e r , 1970; S t e v e n s o n , 1972) a s w e l l a s t h e i n c r e a s e i n t h e amount o f p r o t e i n / D N A d u r i n g p r e m o l t ( S t e v e n s o n , 1 9 7 2 ) . B o t h h y p o d e r m a l and g i l l enzyme l e v e l s a r e c o n s i d e r a b l y l o w e r t h a n t h o s e i n t h e m u s c l e . The l a r g e c o n t r i b u t i o n o f e x t r a c e l l u l a r s p a c e t o t h e wet w e i g h t o f t h e t i s s u e s ( s e e P a r t V) w i l l p a r t i a l l y a c c o u n t f o r t h i s d i f f e r e n c e . T h e low r a t i o o f p h o s p h o f r u c t o k i n a s e / f r u c t o s e d i p h o s p h a t a s e i n d i c a t e s t h a t t h i s r e a c t i o n l o c u s i s s t r o n g l y p o i s e d t o w a r d s g l u c o n e o g e n e s i s . P y r u v a t e k i n a s e , p y r u v a t e c a r b o x y l a s e , and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a l l show t h e same p e r c e n t a g e i n c r e a s e b e t w e e n 51 i n t e r m o l t and f r e s h m o l t t i s s u e s . T h u s , p y r u v a t e k i n a s e r e m a i n s a p p r o x i m a t e l y 20 f o l d t h e l e v e l o f p h o s p h o e n o l p y r u v a t e c a r b o x y -k i n a s e t h r o u g h o u t t h e m o l t c y c l e , g i v i n g t h i s c o n t r o l s i t e a s t r o n g l y g l y c o l y t i c p o i s e . A l t h o u g h t h e a c t i v i t y o f p h o s p h o -e n o l p y r u v a t e c a r b o x y k i n a s e may be somewhat h i g h e r when m e a s u r e d i n t h e g l u c o n e o g e n i c d i r e c t i o n , t h e two f o l d i n c r e a s e f o r t h e enzyme i n most t i s s u e s (Pogson & S m i t h , 1975) w o u l d n o t a l t e r t h e o v e r a l l p o i s e a p p r e c i a b l y * T h u s , p y r u v a t e k i n a s e must be s u b j e c t e d t o c o n s i d e r a b l e r e g u l a t i o n f o r g l u c o n e o g e n i c f l u x t o o c c u r , a n d i t s a c t i v i t y i s a p r i m e d e t e r m i n a n t o f f l u x t h r o u g h t h e r e a c t i o n l o c u s . As was f o u n d by B o u l t o n a n d H u g g i n s ( 1 9 7 0 ) , g l y c e r a l d e h y d e -3 - p h o s p h a t e d e h y d r o g e n a s e l e v e l s were c o n s i d e r a b l y h i g h e r t h a n l a c t a t e d e h y d r o g e n a s e l e v e l s i n m u s c l e . However, t h e l e v e l s o f g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e f o u n d by B o u l t o n a n d H u g g i n s (1970) were c o n s i d e r a b l y h i g h e r t h a n t h e l e v e l s I f o u n d i n M2ister. I n t h i s s t u d y , p h o s p h o g l y c e r a t e k i n a s e l e v e l s a r e l o w e r t h a n g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e l e v e l s i n a l l t i s s u e s . The l e v e l s o f f r u c t o s e d i p h o s p h a t a s e and p y r u v a t e c a r b o x y l a s e f o u n d i n t h e m u s c l e o f G a n c e r - m a a i s t e r a r e s i m i l a r t o t h o s e f o u n d by O p i e and Newsholme (1967) f o r C a r e i n u s maenas and by C r a b t r e e e t a l . (1972) i n Cancer, g a g u r u s . P h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e was n o t d e t e c t e d by C r a b t r e e §t~ a l . (1972) i n ' C a n c e r g a g u r u s m u s c l e , b u t most p r o b a b l y t h e a n i m a l s were i n i n t e r m o l t . I n t h e p r e s e n t s t u d y , p h o s p h o e n o l -p y r u v a t e c a r b o x y k i n a s e was d e t e c t e d i n o n l y f r e s h m o l t m u s c l e . The v a l u e s o f p y r u v a t e c a r b o x y l a s e , p h o s p h o e n o l p y r u v a t e c a r b o x y -k i n a s e , and f r u c t o s e d i p h o s p h a t a s e f o u n d by B e h r i s c h (1976) i n 52 t h e m u s c l e o f P a r a l i t h o d e s c amt c h a t i c a a r e c o n s i d e r a b l y h i g h e r t h a n t h e l e v e l s f o u n d i n C a n c e r p a g u r u s ( C r a b t r e e e t a l . , 1 9 7 2 ) i n - C a r c i n u s maenas ( O p i e & Newsholme, 1967) o r i n C a n c e r m a g i s t e r i n t h e p r e s e n t s t u d y . G e n e r a l l y , t h e enzyme l e v e l s m e a s u r e d i n C a n c e r - m a g i s t e r i n d i c a t e t h a t i n t e r m o l t m u s c l e m e t a b o l i s m p r e s e n t s a h i g h l y g l y c o l y t i c o r i e n t a t i o n , w h i l e f r e s h m o l t m u s c l e shows a c o n s i d e r a b l y r e d u c e d g l y c o l y t i c p o t e n t i a l a n d an i n c r e a s e d g l u c o n e o g e n i c c a p a c i t y . I n t e r m o l t and f r e s h m o l t h y p o d e r m i s b o t h show a r e l a t i v e l y h i g h g l u c o n e o g e n i c c a p a c i t y . F r e s h m o l t h y p o d e r m i s shows h i g h e r s p e c i f i c a c t i v i t i e s , d i a g n o s t i c o f t h e h i g h r a t e o f c h i t i n s y n t h e s i s d u r i n g t h i s p e r i o d . W h i l e t h e enzyme l e v e l s a r e g e n e r a l l y l o w e r t h a n t h o s e i n mammalian t i s s u e s , t h e r e l a t i o n s h i p s b e t w e e n enzyme l e v e l s a r e s i m i l a r . T h u s , i n b o t h t h e mammalian l i v e r a n d t h e c r u s t a c e a n h y p o d e r m i s , f r u c t o s e d i p h o s p h a t a s e l e v e l s a r e 3-4 f o l d t h o s e o f phospho-f r u c t o k i n a s e . P y r u v a t e k i n a s e l e v e l s i n b o t h t i s s u e s a r e a p p r o x i m a t e l y 15 f o l d h i g h e r t h a n t h o s e o f t h e e n z y m a t i c b y p a s s . I n c o n t r a s t t o mammalian s y s t e m s , t h e l e v e l s o f p h o s p h o g l y c e r a t e k i n a s e a r e c o n s i d e r a b l y l o w e r t h a n ' t h o s e o f g l y c e r a l d e h y d e - 3 -p h o s p h a t e d e h y d r o g e n a s e . F u r t h e r m o r e , p y r u v a t e k i n a s e l e v e l s a r e h i g h e r t h a n t h o s e o f p h o s p h o g l y c e r a t e k i n a s e i n b o t h h y p o d e r m i s and m u s c l e . As i n mammalian m u s c l e , t h e r e i s a t e n f o l d • d i f f e r e n c e b e t w e e n p h o s p h o f r u c t o k i n a s e a n d p y r u v a t e k i n a s e l e v e l s i n i n t e r m o l t m u s c l e . The r a t i o o f p h o s p h o f r u c t o k i n a s e t o f r u c t o s e d i p h o s p h a t a s e i n i n t e r m o l t m u s c l e i s 18. T h i s l o w v a l u e i s c h a r a c t e r i s t i c o f m u s c l e s w h i c h e x p e r i e n c e l a r g e c h a n g e s i n g l y c o l y t i c r a t e (Newsholme & S t a r t , 1 9 7 3 ) . F r e s h m o l t m u s c l e shows an e v e n l o w e r v a l u e f o r t h i s r a t i o . T h i s c o u p l e d w i t h t h e i n c r e a s e d l e v e l s o f p y r u v a t e c a r b o x y l a s e and p h o s p h o -e n o l p y r u v a t e c a r b o x y k i n a s e l e n d s a h i g h e r g l u c o n e o g e n i c c a p a c i t y t o t h i s t i s s u e . 54 P a r t I I . C o n t r o l o f H y p o d e r m a l a n d M u s c l e P h o s p h o g l y c e r a t e K i n a s e i n I n t e r m o l t and F r e s h m o l t C a n c e r m a g i s t e r INTBODUCTIGN The r o l e o f p h o s p h o g l y c e r a t e k i n a s e ( E . C . 2 . 7 . 2 . 3 . j i n t h e r e g u l a t i o n o f g l y c o l y s i s a nd g l u c o n e o g e n e s i s i s o p e n t o some • q u e s t i o n . I n some s i t u a t i o n s , i t i s c o n s i d e r e d a p o s s i b l e g l y c o l y t i c c o n t r o l s i t e * w h i l e i n most i t i s c o n s i d e r e d t o c a t a l y z e a n e a r - e q u i l i b r i u m r e a c t i o n . A r e v e r s a l o f t h e r e a c t i o n i s meant t o o c c u r d u r i n g g l u c o n e o g e n e s i s , b u t t h e k i n e t i c c h a r a c t e r i s t i c s o f p r e v i o u s l y s t u d i e d p h o s p h o g l y c e r a t e k i n a s e s do n o t f a v o r t h e r e v e r s a l . A c o m p a r i s o n o f t h e r e g u l a t o r y p r o p e r t i e s o f t h e enzyme f r o m t h e m u s c l e and h y p o d e r m i s c o u l d e l u c i d a t e w h e t h e r d i f f e r e n t c a t a l y t i c m o d i f i c a t i o n s a r e u t i l i z e d by t h e enzyme f r o m a g l u c o n e o g e n i c and - a g l y c o l y t i c t i s s u e . S i n c e t h e h y p o d e r m i s and m u s c l e o f C a n c e r g a g i s t e r u n d e r g o c o n s i d e r a b l e v a r i a t i o n i n t h e d e g r e e o f g l u c o n e o g e n i c p o i s e w i t h t h e m o l t c y c l e , t h e p r o p e r t i e s o f p h o s -p h o g l y c e r a t e k i n a s e f r o m t h e m u s c l e and h y p o d e r m i s o f f r e s h m o l t and i n t e r m o l t a n i m a l s ware a l s o c o m p a r e d . I n most mammalian t i s s u e s , p h o s p h o g l y c e r a t e k i n a s e i s p r e s e n t i n h i g h l e v e l s a l o n g w i t h g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e -h y d r o g e n a s e (Newsholme S S t a r t , 1 9 7 3 ) . I n c r u s t a c e a n t i s s u e s by 55 c o n t r a s t , p h o s p h o g l y c e r a t e k i n a s e l e v e l s a r e c o n s i d e r a b l y l o w e r t h a n t h o s e o f b o t h g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e a nd p y r u v a t e k i n a s e . T h u s , i n t e r m s o f a b s o l u t e enzyme a c t i v i t i e s , p h o s p h o g l y c e r a t e k i n a s e may be a r a t e l i m i t i n g enzyme. The d i f f i c u l t y i n - e l u c i d a t i n g t h e e x a c t r o l e o f p h o s p h o g l y c e r a t e k i n a s e a n d g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e i n t h e c e l l l i e s i n t h e measurement o f t h e i r s h a r e d i n t e r m e d i a t e , 1,3 d i -p h o s p h o g l y c e r a t e . T h u s , t h e r e a c t i o n s a r e g e n e r a l l y e v a l u a t e d on t h e b a s i s o f a c o m b i n e d e q u i l i b r i u m c o n s t a n t . I n g l y c o l y t i c s y s t e m s , an e a r l y a n d t r a n s i e n t r a t e l i m i t a t i o n i s o f t e n f o u n d b e t w e e n g l y c e r a l d e h y d e - 3 - p h o s p h a t e a n d 3 - p h o s p h o g l y c e r a t e . I n mammalian h e a r t , b r a i n , and e r y t h r o c y t e s t h i s was i n t e r p r e t e d a s an e f f e c t due t o g l y c e r a l d e h y d e — 3 - p h o s p h a t e d e h y d r o g e n a s e ( W i l l i a m s o n , 1965; R o l l e s t o n & Newsholme, 1967; Rose & Warms, 1970; R o l l e s t o n , 1 9 7 2 ) . However, o t h e r s t u d i e s w i t h t h e e l e c t r i c o r g a n f r o m t h e e l e c t r i c e e l , and w i t h mammalian e r y t h r o c y t e s , i m p l i c a t e p h o s p h o g l y c e r a t e k i n a s e as a r a t e l i m i t i n g s t e p ( P a r k e r & H o f f m a n , 1967; M a i t r a e t a l . , 1964) S e g a l e t a l . , 1 9 7 5 ) . a l t h o u g h t h e r e i s some q u e s t i o n a b o u t t h e e x a c t - r o u t e s o f c a r b o n f l u x d u r i n g g l u c o n e o g e n e s i s . ( V e n e z i a l e e t a l . , 1 9 7 0 ) , t h e r e i s good e v i d e n c e f o r t h e r e v e r s a l o f b o t h t h e p h o s p h o -g l y c e r a t e k i n a s e a n d g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e r e a c t i o n s . I n a d e g u a t e s u p p l i e s o f NADH c a n c a u s e g l y c e r -a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e t o be a r a t e l i m i t i n g s t e p d u r i n g g l u c o n e o g e n e s i s f r o m a l a n i n e ( S i l l i a m s o n e t a l . , 1 9 6 9 ) . However, -the mammalian enzyme shows c o o p e r a t i v e i n t e r a c t i o n s b e t w een t h e b i n d i n g o f NAD, 1,3 d i p h o s p h o g l y c e r a t e , a n d NADH, 56 w h i c h f a c i l i t a t e t h e r e v e r s a l o f t h e r e a c t i o n f o r g l u c o n e o g e n -e s i s ( S m i t h & V e l i c k , 1 9 7 2 ) . T h u s , u n d e r e n e r g y s a t u r a t i n g c o n d i t i o n s when t h e NAD/NADH r a t i o i s h i g h , t h e r e v e r s e r e a c t i o n i s n o t s u b j e c t t o p r o d u c t i n h i b i t i o n by NAD. I n t h e l i v e r , t h e p h o s p h o g l y c e r a t e k i n a s e and g l y c e r a l d e h y d e - 3 - p n o s p h a t e d e h y -d r o g e n a s e r e a c t i o n s y s t e m was f o u n d t o be n e a r e q u i l i b r i u m u n d e r a v a r i e t y o f m e t a b o l i c c o n d i t i o n s , p r o v i d i n g a means w h e r e b y t h e c y t o p l a s m i c r e d o x b a l a n c e c a n be c o o r d i n a t e d w i t h t h e p h o s p h o r y l a t i o n p o t e n t i a l (Veech e t a l . , 1970; S t u b b s e t a l . , 1 9 7 2 ) . W h i l e t h e means by w h i c h g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y -d r o g e n a s e i s r e v e r s e d d u r i n g g l u c o n e o g e n e s i s h a v e been e l u c i d a t e d , t h e 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 o f p h o s p h o -g l y c e r a t e k i n a s e f r o m g l u c o n e o g e n i c t i s s u e s h a v e r e c e i v e d l i t t l e a t t e n t i o n . P r e v i o u s k i n e t i c s t u d i e s o f p h o s p h o g l y c e r a t e k i n a s e h a v e u t i l i z e d t h e y e a s t and r a b b i t m u s c l e enzyme ( S c o p e s , 1 9 7 3 ) . B o t h enzymes show k i n e t i c s w h i c h s t r o n g l y f a v o r t h e g l y c o l y t i c r e a c t i o n . . I n t h e s e e n z y m e s , a h i g h s e n s i t i v i t y t o MgADP a s an i n h i b i t o r o f t h e g l u c o n e o g e n i c r e a c t i o n i s c o u p l e d w i t h a 1000 f o l d g r e a t e r a f f i n i t y f o r 1,3 d i p h o s p h o g l y c e r a t e o v e r 3 — p h o s p h o g l y c e r a t e . I n t h e c e l l , 3 - p h o s p h o g l y c e r a t e c o n c e n t r a t -i o n s a r e c o n s i d e r e d t o be a p p r o x i m a t e l y t e n f o l d h i g h e r t h a n 1,3 d i p h o s p h o g l y c e r a t e 1 c o n c e n t r a t i o n s . The Km f o r MgADP as a s u b s t r a t e i s h i g h e r t h a n t h e K i f o r MgADP a s an i n h i b i t o r . T h u s , t h e g l u c o n e o g e n i c r e a c t i o n w o u l d be k i n e t i c a l l y l i m i t e d a t v i r t u a l l y a l l MgADP l e v e l s . T h i s k i n e t i c r e g u l a t i o n i s a p p r o p r i a t e f o r a g l y c o l y t i c t i s s u e , b u t p r o b l e m a t i c a l f o r a g l u c o n e o g e n i c one. T h u s , i t seems r e a s o n a b l e t o assume t h a t t h e 57 r e g u l a t o r y p r o p e r t i e s o f t h e enzyme f r o m t h e g l u c o n e o g e n i c a l l y p o i s e d h y p o d e r m i s s h o u l d d i f f e r f r o m t h o s e i n t h e g l y c o l y t i c a l l y p o i s e d m u s c l e . METHODS £lS£aration o f P h o s p h o g l y c e r a t e K i n a s e H y p o d e r m i s and m u s c l e were g u i c k l y d i s s e c t e d o u t o f a n a e s t h e t i z e d c r a b s , r i n s e d i n c o l d e x t r a c t i o n b u f f e r , b l o t t e d d r y and w e i g h e d . T i s s u e s were h o m o g e n i z e d - i n 3 v o l u m e s o f 50 mM i m i d a z o l e - C l b u f f e r , pH 7.0, f o r one m i n u t e . The homogenate was c e n t r i f u g e d a t 27,000 g f o r t w e n t y m i n u t e s . S o l i d ammonium s u l p h a t e was added w i t h c o n s t a n t s t i r r i n g o v e r 30 m i n u t e s , b r i n g i n g t h e s u p e r n a t a n t t o 65% s a t u r a t i o n . The m i x t u r e was c e n t r i f u g e d (12,000 g f o r 10 m i n u t e s ) and s o l i d ammonium s u l p h a t e s l o w l y a d d e d t o b r i n g t h e s u p e r n a t a n t t o 7535. The r e s u l t i n g p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , and t h e n r e d i s s o l v e d i n a s m a l l q u a n t i t y o f e x t r a c t i o n b u f f e r , The enzyme e x t r a c t , u s u a l l y a r o u n d 5 m l , was d i a l y z e d o v e r a H h o u r p e r i o d a g a i n s t two c h a n g e s o f 3 l i t e r s e a c h o f 50 mM i m i d a z o l e - C l b u f f e r , pH 7.0, w i t h 3 mM E D T A . : I n s t u d i e s o f f r e s h m o l t and i n t e r m o l t h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s , t h i s p r e p a r a t i o n was u s e d d i r e c t l y f o r k i n e t i c s t u d i e s , a s no m a j o r c o n t a m i n a n t s w h i c h w o u l d i n t e r f e r e w i t h t h e v a r i o u s e x p e r i m e n t s c o u l d be shown. A l s o t h e h y p o d e r m a l f o r m s were l a r g e l y i n a c t i v a t e d b y t h e s t e p s i n v o l v e d i n t h e f u r t h e r p u r i f i c a t i o n . B o t h m u s c l e p r e p a r a t i o n s , p a r t i c u l a r l y t h e f r e s h m o l t m u s c l e , c o n t a i n e d l a r g e amounts o f m y o k i n a s e . T h u s , 58 f u r t h e r p u r i f i c a t i o n was n e e d e d . Ten grams o f DEAE c e l l u l o s e were p r e t r e a t e d a c c o r d i n g t o m a n u f a c t u r e r ' s d i r e c t i o n s , and e q u i l i b r a t e d w i t h • 10 mM i m i d a z o l e - H C l , 3 mM EDTA, 1 mM m e r c a p t o e t h a n o i , pH 7.5. The enzyme e x t r a c t was d i a l y z e d a g a i n s t t h e same b u f f e r , d i l u t e d u n t i l a p p r o x i m a t e l y t h e same i o n i c s t r e n g t h a s t h e b u f f e r , and t h e n a p p l i e d o n t o t h e c o l u m n . • The a p p l i c a t i o n o f a K C l g r a d i e n t (0—300 mM) l e d t o t h e d i s t i n c t s e p a r a t i o n o f p h o s p h o g l y c e r a t e k i n a s e f r o m m y o k i n a s e . B o t h t h i s p h o s p h o g l y c e r a t e k i n a s e p r e p a r a t i o n a n d t h e c o u p l i n g enzyme were d i a l y z e d a g a i n s t 50 mM i m i d a z o l e - C l , 3 mM EDTA, pH 7.0, and u s e d f o r k i n e t i c s t u d i e s . M e t a b o l i t e M e a s u r e m e n t s One l e g was a m p u t a t e d f r o m t h e e x p e r i m e n t a l a n i m a l , 4 a l i g u o t s , o f a p p r o x i m a t e l y 300 mg e a c h , o f b o t h m u s c l e a nd h y p o d e r m i s were t h e n q u i c k l y d i s s e c t e d o u t and d r o p p e d i n t o l i g u i d n i t r o g e n . S i n c e t h e t i s s u e s a m p l e s were s m a l l , I d i d n o t use t h e f r e e z e c l a m p i n g method. S i n c e t h e e x o s k e l e t o n needed t o be o p e n e d , t h e p r o c e d u r e t o o k b e t w e e n 15 and 30 s e c o n d s . A m p u t a t i o n c o u l d n o t be a c h i e v e d w i t h o u t some r e s i s t a n c e , t h u s , t h e m e a s u r e m e n t s do n o t r e f l e c t r e s t i n g v a l u e s . N e u t r a l i z e d p e r c h l o r a t e e x t r a c t s o f t h e s e f r o z e n t i s s u e s a m p l e s were • made a c c o r d i n g t o t h e method o f W i l l i a m s o n a n d C d r k e y ( 1 9 6 9 ) . The n e u t r a l i z e d e x t r a c t s were s t o r e d a t -20 °C. M e t a b o l i t e a s s a y s were p e r f o r m e d s h o r t l y a f t e r t h e e x t r a c t i o n , w i t h t h e a s s a y f o r t h e u n s t a b l e 1,3 d i p h o s p h o g l y c e r a t e p e r f o r m e d d i r e c t l y a f t e r t i s s u e e x t r a c t i o n . E v e r y e f f o r t was made t o 59 p e r f o r m a l l m a n i p u l a t i o n s g u i c k l y and t o m a i n t a i n t h e e x t r a c t s n e a r 0 °C b e f o r e t h e enzyme a s s a y . A l l a s s a y s were p e r f o r m e d i n d u p l i c a t e f o l l o w i n g s p e c t r o p h o t o m e t r y methods g i v e n by B e r g m e y e r ( 1 9 6 5 ) . Enzyme A s s a y s The enzyme was a s s a y e d a c c o r d i n g t o B u c h e r ( 1 9 4 7 ) : 1,3 d i -p h o s p h o g l y c e r a t e f o r m a t i o n i s c o u p l e d t o g l y c e r a l d e h y d e - 3 - p h o s -p h a t e d e h y d r o g e n a s e , a n d t h e p h o s p h o g l y c e r a t e k i n a s e a c t i v i t y i s m e a s u r e d by t h e d e c r e a s e i n E340 due t o NADH o x i d a t i o n . A l l s t a n d a r d a s s a y s i n c l u d e d 3 - p h o s p h o g l y c e r a t e , ATP, NADH, MgC12, and e x c e s s c o u p l i n g enzyme i n t h e c o n c e n t r a t i o n s shown i n t h e f i g u r e l e g e n d s . I m i d a z o l e — C l b u f f e r s were u s e d t h r o u g h o u t t h e k i n e t i c s t u d i e s . S a t u r a t i n g c o n d i t i o n s f o r t h e enzymes were: 1 mM ATP, 2 mM 3 - p h o s p h o g l y c e r a t e , 4 mM MgC12 f o r t h e i n t e r m o l t f o r m s , 0.6 mM ATP, a n d 2 mM 3 - p h o s p h o g l y c e r a t e f o r t h e f r e s h m o l t f o r m s . RESULTS S p e c i f i c A c t i v i t i e s I n b o t h m u s c l e and h y p o d e r m i s , p h o s p h o g l y c e r a t e k i n a s e l e v e l s show c o n s i d e r a b l e v a r i a t i o n w i t h t h e m o l t c y c l e ( F i g u r e 1 ) . I n t h e h y p o d e r m i s , l e v e l s a r e h i g h a t m o l t (7-8 u n i t s / g m f r e s h w e i g h t ) , f a l l g r a d u a l l y t o a r o u n d 3 u n i t s / g m f r e s h w e i g h t d u r i n g i n t e r m o l t , and: r i s e a g a i n s o m e t i m e i n p r e m o l t . M u s c l e shows t h e o p p o s i t e t r e n d , b e i n g l o w a t m o l t and h i g h d u r i n g i n t e r m o l t . T h e r e i s a p a u c i t y o f p r e m o l t d a t a , a s C a n c e r Ecdysis A B-| C-|. D 1 Ecdysis Molt Cyc l e Stage Figure 1. 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 l e v e l s of phosphoglycerate kinase throughout the molt c y c l e i n muscle and hypodermis. Assay conditions are described i n Materials and Methods, (A) muscle and (O) hypodermis, u n i t s are expressed in umoles product produced per minute. 61 m a g i s t e r d o e s n o t r e a d i l y go i n t o p r e m o l t i n c a p t i v i t y . No a t t e m p t was made t o c o n t r o l f o r e f f e c t s o f s i z e , s e x , o r s e a s o n o n enzyme l e v e l s , a l t h o u g h l i t t l e v a r i a t i o n due t o t h e s e f a c t o r s was o b s e r v e d . T h e s e s p e c i f i c a c t i v i t i e s r e f l e c t t h e c h a n g i n g t i s s u e r o l e s between t h e h i g h l y m o b i l e f e e d i n g i n t e r m o l t a n i m a l and t h e more s e d e n t a r y , b u t a c t i v e l y s y n t h e s i z i n g m o l t i n g a n i m a l , and i n d i c a t e t h a t a t l e a s t i n p a r t , p h o s p h o g l y c e r a t e k i n a s e f u n c t i o n i s r e g u l a t e d by c o n t r o l o f enzyme l e v e l s . D i f f e r e n t Forms o f P h o s p h o g l y c e r a t e K i n a s e The H i c h a e l i s c o n s t a n t s f o r t h e two s u b s t r a t e s o f t h e enzymes f r o m m o l t i n g and i n t e r m o l t m u s c l e a n d h y p o d e r m i s a r e shown i n T a b l e 2. Two s e t s o f c o m p a r i s o n s were made f o r b o t h t h e 3 - p h o s p h o g l y c e r a t e and t h e ATP a f f i n i t i e s : t h e f i r s t l o o k e d f o r t i s s u e d i f f e r e n c e s and t h e s e c o n d f o r m o l t c y c l e r e l a t e d d i f f e r e n c e s . I n i n t e r m o l t a n i m a l s , t h e r e i s no s i g n i f i c a n t d i f f e r e n c e (P>0.05) between m u s c l e and h y p o d e r m a l p h o s p h o -g l y c e r a t e k i n a s e s i n t e r m s o f e i t h e r t h e i r 3 - p h o s p h o g l y c e r a t e o r ATP a f f i n i t y . I n m o l t i n g a n i m a l s , t h e r e i s no s i g n i f i c a n t d i f f e r e n c e (P>0.05) b e t w e e n t h e ATP a f f i n i t i e s o f t h e m u s c l e and h y p o d e r m a l f o r m s , b u t t h e r e i s a 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 b e t w e e n t h e i r 3 - p h o s p h o g l y c e r a t e a f f i n i t i e s ( P < 0 . 0 0 1 ) , e v e n t h o u g h t h e m a g n i t u d e o f t h e d i f f e r e n c e i s s m a l l and p r o b a b l y n o t o f p h y s i o l o g i c a l i m p o r t a n c e . T h u s , t h e 3 - p h o s p h o g l y c e r a t e s i t e s O f t h e m u s c l e and h y p o d e r m a l enzymes b e h a v e d i f f e r e n t l y i n r e s p o n s e t o t h e m o l t c y c l e , i n d i c a t i n g some d i f f e r e n c e b e t w e e n t h e s e t i s s u e f o r m s . Table 2 62 ATP and 3-P-glycerate affinities Michelis constants, here expressed in millimoles/litre were determined at saturating concentrations of the cosubstrate and activating magnesium cation (see Experimental procedures). Values shown are the means with their standard deviations, the number in parentheses indicates the number of different animals used for the determinations. - Intermolt muscle Intermolt Hypodermis Molting Muscle Molting Hypodermis PGA K m ATP K m 0.273+0.057 (8) 0.203+0.045 (7) 0.271+0.054 (9) 0.174+0.063(12) 0.218+0,042 (6) .0.071+0.009 (8) 0.320+0.036 (9) 0.068+0.014(13) 63 C o m p a r i s o n s b e t w e e n i n t e r m o l t and m o l t i n g p h o s p h o g l y c e r a t e k i n a s e s r e v e a l s e v e r a l marked d i f f e r e n c e s . I n t e r m o l t and m o l t i n g h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s d i f f e r s i g n i f i c a n t l y i n b o t h t h e i r ATP and 3 - p h o s p h o g l y c e r a t e a f f i n i t i e s ( P < 0 . 0 1 ) . The ATP a f f i n i t i e s o f m o l t i n g and i n t e r m o l t m u s c l e p h o s p h o -g l y c e r a t e k i n a s e s a r e a l s o s i g n i f i c a n t l y d i f f e r e n t ( P < 0 . 0 1 ) , b u t t h e i r 3 - p h o s p h o g l y c e r a t e a f f i n i t i e s do n o t d i f f e r s i g n i f i c a n t l y (P>0.05) . T h u s , on t h e b a s i s o f s u b s t r a t e a f f i n i t i e s , t h e r e i s good e v i d e n c e f o r c h a n g e s o f t h e p h o s p h o g l y c e r a t e k i n a s e - f o r m s w i t h t h e m o l t c y c l e a s w e l l a s f o r c h a n g e s between t i s s u e f o r m s o f t h e enzyme. W h i l e t h e c h a n g e s i n 3 - p h o s p h o g l y c e r a t e a f f i n i t i e s a r e p r o b a b l y n o t o f 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 , t h e c h a n g e s i n ATP a f f i n i t i e s a r e l a r g e and may be o f r e g u l a t o r y i m p o r t a n c e . S t a r c h g e l e l e c t r o p h o r e s i s and i s o e l e c t r o f o c u s i n g were c a r r i e d o u t t o s e e i f t h e r e was an i s o e n z y m i c b a s i s f o r t h e s e d i f f e r e n t s u b s t r a t e a f f i n i t i e s . I n g e l e l e c t r o p h o r e s i s , b o t h m u s c l e and h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s show a m o l t i n g v a r i a n t w i t h f a s t e r a n o d a l m i g r a t i o n t h a n t h e i n t e r m o l t f o r m ( F i g u r e 2 ) . H y p o d e r m a l and m u s c l e f o r m s o f t h e enzyme show d i f f e r e n t v e l o c i t i e s . The p i v a l u e s a s d e t e r m i n e d by i s o e l e c t r o f o c u s i n g »ere 5.4 f o r t h e i n t e r m o l t m u s c l e , 5.1 f o r t h e m o l t i n g m u s c l e , 5.3 f o r t h e i n t e r m o l t h y p o d e r m i s , and 5.0 f o r t h e m o l t i n g h y p o d e r m i s . The r e l a t i v e m a g n i t u d e s o f t h e p i v a l u e s c o n f i r m t h e r e s u l t s f r o m t h e s t a r c h g e l s , s i n c e t h e l o w e r t h e p i , t h e g r e a t e r " t h e a n o d a l m o b i l i t y a t pH 7.5, g i v e n no g r e a t s i z e d i f f e r e n c e . However, t h e p i v a l u e s f o r t h e two m o l t i n g enzymes a r e g u i t e c l o s e a s a r e t h o s e o f t h e two 64 ORIGIN 1 MOLTING MUSCLE MOLTING HYPODERMIS INTERMOLT MUSCLE INTERMOLT HYPODERMIS + Figure 2. A diagrammatic sketch of a starch gel electrophoretogram showing zones of phosphoglycerate kinase activity extracted from muscle and hypodermis of Cancer magister. The gel was run at 250 mvolts, 25 mamps using a Tris-citrate, pH 7.0, buffer system. Duration of run was 30 hours. 65 i n t e r m o l t f o r m s . The i n s t a b i l i t y o f t h e h y p o d e r m a l f o r m s p r e v e n t e d me' f r o m d e a l i n g w i t h more p u r i f i e d p r o t e i n s , o r f r o m d o i n g e x h a u s t i v e i s o e l e c t r o f o c u s i n g (48 h o u r s o r l o n g e r ) . T h u s , t h e s m a l l d i f f e r e n c e s b e t w e e n 5.0 a n d 5 . 1 , a s ' w e l l a s t h o s e b e t ween 5.3 and 5.4, c a n n o t be t a k e n a s c l e a r i n d i c a t i o n t h a t m u s c l e a n d h y p o d e r m i s h a v e d i s t i n c t p h o s p h o g l y c e r a t e k i n a s e s a t m o l t and d u r i n g i n t e r m o l t . T h e s e d i f f e r e n t p i v a l u e s c o u l d be a r e f l e c t i o n o f d i f f e r i n g r a t i o s o f two gene p r o d u c t s , f o u r d i f f e r e n t gene p r o d u c t s , o r one p r o t e i n u n d e r g o i n g v a r y i n g d e g r e e s o f m o d i f i c a t i o n i n t h e s e t i s s u e s d u r i n g t h e m o l t c y c l e . I n none o f t h e t i s s u e s was t h e r e any e v i d e n c e , d u r i n g e l e c t r o f o c u s i n g o r c o l u m n c h r o m a t o g r a p h y , o f more t h a n one p h o s -p h o g l y c e r a t e k i n a s e f o r m . The m o b i l i t y d i f f e r e n c e s i n d i c a t e t h a t a t l e a s t two enzyme s p e c i e s a r e p r e s e n t i n t h e f o u r c r a b t i s s u e s , one p r e s e n t i n f r e s h m o l t and t h e o t h e r i n i n t e r m o l t t i s s u e s . K i n e t i c P r o p e r t i e s To s e e how p h o s p h o g l y c e r a t e k i n a s e i s r e g u l a t e d u n d e r v a r y i n g m e t a b o l i c ' r e q u i r e m e n t s , a s w e l l as w h e t h e r any d i f f e r e n c e s i n c a t a l y t i c c h a r a c t e r i s t i c s a c c o m p a n i e d t h e s u b s t r a t e and m o b i l i t y d i f f e r e n c e s , an a n a l y s i s o f t h e k i n e t i c s o f t h e p h o s p h o g l y c e r a t e k i n a s e s f r o m t h e f o u r t i s s u e s was u n d e r t a k e n . B o t h h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s show a b r o a d pH optimum w i t h m a x i m a l a c t i v i t y b e t w een pH 6.0 and 8.0. The two m u s c l e f o r m s show a s i m i l a r r e s p o n s e , b u t w i t h m a x i m a l a c t i v i t y b e t w e e n pH 6.5 a n d 7.5. W h i l e d i v a l e n t c a t i o n s ( p a r t i c u l a r l y M g + + and Mn++) p r o v i d e a p r o n o u n c e d and 66 s t o i c h i o m e t r i c a c t i v a t i o n o f t h e r e a c t i o n , i t i s d i f f i c u l t t o d e m o n s t r a t e an a b s o l u t e c a t i o n r e q u i r e m e n t w i t h t h e s e e n z y m e s . T h e s e a l l show a r e s i d u a l enzyme a c t i v i t y i n t h e a b s e n c e o f a d d e d magnesium. 1 W h e t h e r t h i s i s due t o bound m a g n e s i u m , t o a l a c k o f an a b s o l u t e c a t i o n r e q u i r e m e n t , o r t o i n c o m p l e t e d i a l y s i s , c o u l d n o t be c l e a r l y d e m o n s t r a t e d , a s p r o l o n g e d d i a l y s i s (8 h o u r s ) t e n d e d t o i n a c t i v a t e c r a b p h o s p h o g l y c e r a t e k i n a s e s , p a r t i c u l a r l y t h e h y p o d e r m a l f o r m s . G e n e r a l l y , t h e v a r i a t i o n s i n t h e M i c h a e l i s c o n s t a n t s f o r MgCl2 and MnC12 ( T a b l e 3) p a r a l l e l t h o s e o f t h e ATP Km. I n i t i a l k i n e t i c s t u d i e s showed t h a t t h e 3 - p h o s p h o g l y c e r a t e and ATP a f f i n i t i e s a r e i n d e p e n d e n t o f e a c h o t h e r , a n d o f t h e MgCl2 c o n c e n t r a t i o n . W i t h r e s p e c t t o magnesium, t h i s was t r u e i f t h e t o t a l magnesium was h e l d c o n s t a n t a s t h e ATP o r 3-phos-p h o g l y c e r a t e were v a r i e d . Magnesium e f f e c t s up t o 6 mM were c h e c k e d . R e p r e s e n t a t i v e c u r v e s a r e shown i n F i g u r e 3, w i t h t h e same b a s i c r e s u l t s h o l d i n g f o r a l l f o u r enzymes. i n s t u d i e s where magnesium a n d ATP c o n c e n t r a t i o n s were v a r i e d s i m u l t a n e o u s l y , t h e M i c h a e l i s c o n s t a n t f o r MgATP was h i g h e r t h a n t h e c o r r e s p o n d i n g Km i n s t u d i e s where t h e t o t a l magnesium was h e l d c o n s t a n t ( t h i s e f f e c t was l a r g e r i n t h e m u s c l e t h a n i n t h e h y p o d e r m a l f o r m s ) ( T a b l e 4 ) . - - B o t h N a C l and KC1 s t r o n g l y a c t i v a t e t h e s e c r u s t a c e a n p h o s -p h o g l y c e r a t e k i n a s e s l e a d i n g t o a s much a s 150% i n c r e a s e i n t h e i r a c t i v i t y o v e r t h a t i n t h e p r e s e n c e o f s a t u r a t i n g magnesium. The • M i c h a e l i s c o n s t a n t s ( T a b l e 3) a r e f a i r l y h i g h i n d i c a t i n g t h a t t h e s t i m u l a t i o n i s p r o b a b l y due t o a c h a n g e i n Table 3 M i c h e l i s constants f o r a c t i v a t i n g c a t i o n s K m values were determined at s a t u r a t i n g l e v e l s o f the su b s t r a t e s and MgCl2 (except i n the determination of the MgCl2 1^) and i n d i c a t e the c o n c e n t r a t i o n of c a t i o n r e q u i r e d f o r 50% of the maximal a c t i v i t y i n the presence o f th a t c a t i o n . The values are expressed i n m i l l i m o l a r u n i t s . V values are expressed as % of the maximal a c t i v i t y i n the presence of max „ 1 s a t u r a t i n g magnesium l e v e l s . I ntermolt M o l t i n g Muscle Hypodermis Muscle Hypodermis MgCl 2 K m 0.252 0.193 0.09 0.077 MgCl 2 V max 100% 100% 100% 100% MnCl 2 K m 0.164 0.165 0.152 0.058 MnCl 2 V max 88% 95% 75% 95% NaCl K m 100.0 90.0 30.0 19.0 NaCl V max 216% 250% 215% 242% j KCl K 85.0 80.0 65.0 80.0 KCl V max 188.5% 230% 220% 184% 68 T 1 — 1 1 i — I 1 —i T r * 1 1 i j | 0 1 2 3 4 [PGA] in m M F i g u r e 3 ( a ) . M i c h e l i s constants f o r ATP and 3-P-glycerate a t v a r y i n g l e v e l s  o f c o s u b s t r a t e and a c t i v a t o r , (a) 3-P-glycerate (PSA) a f f i n i t y o f the m o l t i n g hypodermal phosphoglycerate k i n a s e : i n the presence o f 1 mM ATP ( • ) , i n the presence o f 3.5 x l<r 4M ATP ( A ) , (b) ATP a f f i n i t y o f the m o l t i n g muscle phosphoglycerate k i n a s e : i n the presence o f 4 mM 3-P-glycerate ( o , £ ) , i n the presence o f 4 x 10"^ M 3-P-glycerate ( © , £ ) . (c) ATP a f f i n i t y o f i n t e r m o l t muscle enzyme i n the presence o f 2 mM MgCl2 t o t a l ( A ) , i n the presence o f 6 x 10"^M MgCl 7, t o t a l ( o ) , and i n the presence o f 6 mM Mg C l 2 ( 3 ) . I 69 O 2.5. 5 7 9 10 [ATP ]X10" 4M Figure 3b. [ A T P ] x 1 0 4 M Figure 3c. 70 Table 4 Michelis Constants for ATP. and MgATP The. Michelis constants for ATP were determined in studies where the total MgC^ was maintained at a fixed total greater than the total ATP. The constants for MgATP were determined by simultaneously varying MgC^ and ATP in a 1:1 ratio. All values in millimolar units. Intermolt Muscle Intermolt Hypodermis Molting Muscle Molting Hypodermis ATP K m 0.16 0.118 0.071 0.067 MgATP Km 0.226 0.142 0.178 0.087 71 t h e s o l v e n t s t r u c t u r e r a t h e r t h a n t o a s p e c i f i c b i n d i n g t o t h e enzyme. N a C l and K C l do n o t a f f e c t t h e s u b s t r a t e a f f i n i t i e s e x c e p t i n t h e c a s e o f t h e m o l t i n g m u s c l e enzyme where t h e y b o t h r a i s e t h e s u b s t r a t e Km v a l u e s . The 3 - p h o s p h o g l y c e r a t e Km c h a n g e s f r o m 0.245 mM t o 0.29 mM and t h e ATP Km r i s e s f r o m 0.085 t o 0 .11 mM. A wide r a n g e o f compounds was t e s t e d f o r t h e i r p o s s i b l e e f f e c t s on t h e s e p h o s p h o g l y c e r a t e k i n a s e s . T hey i n c l u d e d g l y c o l y t i c and K r e b s c y c l e i n t e r m e d i a t e s , i m p o r t a n t a m i n o a c i d s , n u c l e o t i d e s , and c o e n z y m e s . O n l y v e r y c l o s e s u b s t r a t e a n d p r o d u c t a n a l o g u e s a s w e l l as t h e a d e n y l a t e p r o d u c t were i n h i b i t o r s . The d i f f i c u l t y o f 1,3 d i p h o s p h o g l y c e r a t e s y n t h e s i s , and i t s i n s t a b i l i t y made i t s u s e i m p r a c t i c a l . The p r o d u c t , A D P , i s t h e most p o t e n t i n h i b i t o r , b u t AMP, 2,3 d i p h o s p h o g l y c e r a t e , and C a C l 2 a l s o e x e r c i s e i n h i b i t o r y e f f e c t s on t h e r e a c t i o n . The i n h i b i t i o n by ADP was s t r o n g l y i n f l u e n c e d by magnesium l e v e l s ( F i g u r e 4 ) . A t s a t u r a t i n g l e v e l s o f ATP a n d 3 - p h o s p h o -g l y c e r a t e , i n c r e a s i n g magnesium l e v e l s i n c r e a s e t h e e f f e c t i v e n e s s o f t h e ADP i n h i b i t i o n . T h i s i s t r u e f o r t h e enzyme i n a l l f o u r t i s s u e s . B o t h MgADP and ADP a c t e d a s i n h i b i t o r s , b u t t h e K i o f MgADP was c o n s i d e r a b l y l o w e r t h a n t h a t f o r ADP. The p a t t e r n s a r e s u m m a r i z e d i n T a b l e 5; and some r e p r e s e n t a t i v e c u r v e s f r o m t h e i n t e r m o l t m u s c l e a n d m o l t i n g h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s a r e shown i n F i g u r e s 5 a n d 6. ADP and MgADP a f f e c t t h e b i n d i n g o f b o t h 3 - p h o s p h o g l y c e r a t e and ATP. ADP i n h i b i t i o n f o l l o w s t h e same b a s i c p a t t e r n i n a l l f o u r 72 V 0.15 0.10 H 0 . 0 5 [ADP] in mM F i g u r e 4. ADP i n h i b i t i o n o f i n t e r m o l t muscle phosphoglycerate k i n a s e , e f f e c t of i n c r e a s i n g magnesium i o n c o n c e n t r a t i o n . Assay c o n d i t i o n s are des c r i b e d i n M a t e r i a l s .,6 x 1(T 4M MgCl 2 ( • ) , 2 mM MgCl 2 ( o ) , and 6 mM MgCl 2 (A ) . Table 5 values for ADP and MgADP inhibition. values were estimated from Lineweaver-Burk (L-B) and Dixon plots. Assay conditions are as described in Materials and-Methods, with the varying total magnesium concentrations as shown. All K- values are in millimolar units. Total MgCl2 Intermolt Muscle Molting Muscle Intermolt Hypodermis Molting Hypodermis L-B Dixon L-B Dixon L-B Dixon L-B Dixon IC, ADP ys PGA Mg=ATP .13 .23 .27 ,39 .42 .230 .33 IC, MgADP vs PGA 2 mM .012 .12 .13 .19 .18, .20 IC, ADP vs MgATP Mg=ATP .06 .08 .40 .50 .60 .40 .50 Ki, MgADP vs MgATP 2 mM .013 .2009 .20 .19 .20 .20 .23 " 11 " 11 ... 4 mM .05 .10 .16 .145, .15 11 11 " " 6 mM .045 .11 • -4 0 4 8 VPGAX-IO3 F i g u r e 5a. Intermolt muscle phosphoglycerate k i n a s e , ADP and MgADP i n h i b i t i o n  p a t t e r n s . (a) 3-P-glycerate a f f i n i t y i n presence of 2 mM MgCl2 and no ADP ( • ) , 2 mM MgCl 2 and 2 x 10" 5M ADP, 70% of which i s MgADP ( A ) , and 2 mM MgCl 2 and 1 x 10" 4M ADP, 60% of which i s MgADP ( © ) . (b) MgATP a f f i n i t y i n presence of no ADP ( Q ), o f 2 x 10" 5M ADP ( 0 ) , of 1 x 10" 4M ADP ( A ) . (c) ATP a f f i n i t y i n presence o f 2 mM MgCl 2 and no ADP ( • ) , of 2 mM MgCl 2 and 2 x 10" 5M ADP ' (75% o f the ADP present as MgADP) ( A ) , of 2 mM MgCl 2 and 1 x 10" 4M ADP (75% ADP as MgADP) ( o ) . (d) Dixon p l o t o f ADP i n h i b i t i o n i n the presence of 4 mM MgCl 2, 4 mM ATP, 5 mM 3-P-glycerate ( O ) , 4 mM MgCl 2> 4 mM ATP, 5 x 10" 4 M 3-P-glycerate ( A ) , 5 x 10" 4M MgCl 2, 5 x 10" 4M ATP, and . 4 mM 3-P-glycerate ( A ) , 4 mM MgCl 2, 4 x 10" 4M ATP, 5 mM 3-P-g l y c e r a t e ( • ) , and 6 mM MgCl2, 5 x 10" 4M ATP, 5 mM 3-P-glycerate 75 1 / M g A T P x i o 4 Figure 5b. MgATP a f f i n i t y and the e f f e c t of ADP i n h i b i t i o n , intermolt muscle phosphoglycerate kinase. Symbols explained i n the legend to Figure 5a. 8 0 Figure 5c. ATP a f f i n i t y and the e f f e c t of MgADP upon the intermolt muscle phosphoglycerate kinase. Symbols explained i n the legend to f i g u r e 5a. 77 [ADP] in m M Figure 5 d . ADP i n h i b i t i o n of intermolt muscle phosphoglycerate kinase. Symbols given i n the legend to Figure 5 a . T r [ADP] in mM Figure 6 Ca) . Molting hypodermal phosphoglycerate kinase, ADP and MgADP inhibition  patterns. (a) Dixon plot of ADP inhibition in presence of. 4 mM MgCl2, 4 mM 3-P-glycerate, 1 mM ATP (0), of 4 mM MgCl2, 4 x 10"4M 3-P-glycerate, 1 mM ATP (fl ), of 4 mM MgC^, 4 mM 3-P-glycerate, and 1 x 10"4M ATP (A), and 4 mM 3-P-glycerate, 1 mM MgCl2, 1 x 10"4M ATP ( A ) . (b) Dixon plot of ADP inhibition in presence of 1 mM MgCl2, 1 mM ATP, 4 mM 3-P-glycerate ( O ) , of 1 mM MgCl2, 1 mM ATP, 4 x 10"4M 3-P-glycerate ( i ) and of 3.4 x 10"4M MgATP and 4 mM 3-P-glycerate ( A ) . 30 -0.5 0 0.5 1.0 1.5 2.0 [ADP] in mM Figure 6b. 80 enzymes and a d e s c r i p t i o n o f t h e i n t e r m o l t m u s c l e ADP/MgADP r e s p o n s e w i l l i l l u s t r a t e t h e f o r m o f t h e i n h i b i t i o n f o r t h e o t h e r s . At l o w . magnesium, whan e s s e n t i a l l y a l l t h e ADP i s u n c o m p l e x e d , ADP i s a m i x e d c o m p e t i t i v e i n h i b i t o r w i t h b o t h ATP and 3 - p h o s p h o g l y c e r a t e , w i t h K i v a l u e s o f 8 x 1 0 _ s M and 1;3 x 1 0 — * M r e s p e c t i v e l y . As t h e l e v e l s o f magnesium a r e i n c r e a s e d , t h e r e b y i n c r e a s i n g t h e p r o p o r t i o n o f ADP p r e s e n t a s MgADP, t h e K i v a l u e s d r o p s h a r p l y a s t h e i n h i b i t i o n w i t h r e s p e c t t o 3 - p h o s p h o g l y c e r a t e ( K i = 1.2 x 1 0 - s M) becomes more c o m p e t i t i v e , and t h a t v e r s u s ATP ( K i = 1.3 x 1 0 _ s M) becomes c o m p l e t e l y c o m p e t i t i v e . W h i l e t h e ADP i n h i b i t i o n f o l l o w s t h e same b a s i c p a t t e r n i n t h e t h r e e o t h e r p h o s p h o g l y c e r a t e k i n a s e s , t h e o r d e r o f m a g n i t u d e o f t h e i n h i b i t i o n c o n s t a n t s d i f f e r s m a r k e d l y . The m o l t i n g m u s c l e a n d h y p o d e r m a l enzymes a s w e l l a s t h e i n t e r m o l t h y p o d e r m a l enzyme show a K i v a l u e o f 2 x 1Q- 4 M f o r t h e c o m p l e t e l y c o m p e t i t i v e MgADP v e r s u s ATP i n h i b i t i o n . T h i s v a l u e i s t e n t i m e s t h a t f o r t h e i n t e r m c l t m u s c l e enzyme. The MgADP K i v e r s u s 3 - p h o s p h o g l y c e r a t e i s r a i s e d t o 1.8 x 1 0 _ * M f r o m 1;2 x 1 0 — 5 M, a n o t h e r t e n f o l d c h a n g e . The i n h i b i t i o n c o n s t a n t s f o r ADP a r e a l s o i n c r e a s e d , t h o u g h n o t a s m a r k e d l y ( T a b l e 5 ) . W h i l e t h e i n t e r a c t i o n s b etween MgADP, ATP, and 3 - p h o s p h o g l y -c e r a t e a r e much t h e same f o r t h e s e t h r e e e n z y m e s , t h e i n t e r m o l t h y p o d e r m a l enzyme i s l e a s t s e n s i t i v e t o ADP i n h i b i t i o n . A l l t h r e e enzymes a r e an o r d e r o f m a g n i t u d e l e s s s e n s i t i v e t o i n h i b i t i o n by ADP o r MgADP t h a n t h e i n t e r m o l t m u s c l e e n z y m e . The o t h e r c o m p e t i t i v e i n t e r a c t i o n s a r e s u m m a r i z e d i n T a b l e 81 6. I n a l l f o u r f o r m s , AMP i s i n h i b i t o r y o n l y w i t h ATP. F o r m o l t i n g m u s c l e , i n t e r m o l t and m o l t i n g h y p o d e r m i s , t h e K i v a l u e s a r e much t h e same, 2.15-2.5 mM. The i n t e r m o l t m u s c l e enzyme d i f f e r s , h a v i n g a K i f o r AMP o f 7.6 mM. I n a l l f o u r e n z y m e s , 2,3—diphosphoglycerate i s c o m p e t i t i v e w i t h b o t h ATP and 3 - phos-p h o g l y c e r a t e . The K i v e r s u s 3 - p h o s p h o g l y c e r a t e i s much t h e same f o r a l l f o r m s ; a n d r a n g e s f r o m 1.93 t o 2.3 mM. I n t e r m o l t m u s c l e and h y p o d e r m i s a s w e l l a s m o l t i n g m u s c l e p h o s p h o g l y c e r a t e k i n a s e s showed a 2,3 d i p h o s p h o g l y c e r a t e v e r s u s ATP K i o f 4.3 t o 5.0 mM, w h i l e t h e m o l t i n g h y p o d e r m a l enzyme showed a K i v e r s u s ATP o f 16 mM. C a C l 2 i n h i b i t i o n i s c o m p e t i t i v e w i t h 3 - p h o s p h o -g l y c e r a t e , p r o b a b l y due t o t h e f o r m a t i o n o f an u n r e a c t i v e C a * + - 3 - p h o s p h o g l y c e r a t e c o m p l e x . The K i f o r t h e i n t e r a c t i o n o f c a l c i u m w i t h 3 - p h o s p h o g l y c e r a t e i s a n o r d e r o f m a g n i t u d e l o w e r t h a n t h e K i f o r i t s i n t e r a c t i o n w i t h magnesium o r ATP* I n c o n t r a s t t o t h e o t h e r e n z y m e s , t h e i n t e r m o l t m u s c l e enzyme shows a s t r o n g , n o n - l i n e a r r e s p o n s e t o c a l c i u m i n h i b i t i o n v e r s u s magnesium, b u t d o e s n o t show much i n t e r a c t i o n b e t w e e n c a l c i u m and 3 - p h o s p h o g l y c e r a t e . Mass A c t i o n R a t i o s The d i f f e r i n g ATP a f f i n i t i e s and ADP/MgADP s e n s i t i v i t i e s o f t h e s e v a r i o u s p h o s p h o g l y c e r a t e k i n a s e s may a f f e c t t h e p o i s e o f t h e r e a c t i o n s y s t e m i n v i v o . I n o r d e r t o c h e c k - t h i s p o s s i b i l i t y , I m e a s u r e d t h e l e v e l s o f ATP, 3 - p h o s p h o g l y c e r a t e , ADP, and- 1,3 d i p h o s p h o g l y c e r a t e i n m u s c l e a n d - h y p o d e r m i s o f f r e s h m o l t and i n t e r m o l t a n i m a l s ( T a b l e 7 ) . As m e n t i o n e d i n t h e m e t h o d s , t h e s e v a l u e s do n o t r e p r e s e n t r e s t i n g v a l u e s , s i n c e Table 6 K± values for AMP, 2,3 DPG and CaCl 2 K± values were determined from Dixon p l o t s . Assay conditions are described i n Materials and Methods. K. values are expressed i n m i l l i m o l a r u n i t s . Intermolt Muscle Molting Muscle Intermolt Hypodermis Molting Hypodermis V AMP vs ATP 7.6 2.15 2.5 2.3 K.. 2,3 DPG vs P6A 2.0 2.0 2.5 1.93 K., 2,3 DPS vs ATP 4.3 5.0 4.5 16.0 CaCl 2 vs P6A 35.0 2.7 2.3 3.0 V CaCl 2 vs ATP 28.0 24.5 42.0 13.6 K i ' C a C l 2 vs MgCl 2 Parabolic 24.0 43.0 13.6 OO ho 83 Table 7 Mass Action Ratios from Intermolt and Freshmolt Tissues. Each value represents the mean of at least three tissue samples from each animal. Metabolite measurements i n each sample were made i n duplicate. Metabolite concentrations expressed as uMoles/gm fresh weight, _ ATP x 3-phosphoglycerate . "7"^ ^ a s ^ e e n calculated using ADP x 1,3 diphosphoglycerate resting ATP/ADP r a t i o s from Beis and Newsholme (1975). Tissue Animal ATP ADP 3-phospho- 1,3 diphospho-number glycerate glycerate Freshmolt muscle 1 1.69 .449 .233 .0697 12.62 25.2 1.68 .204 .103 .045 18.8 18.8 Freshmolt hypodermis 1 3.14 .383 .158 2.33 .835 .2827 1.794 .381 .2369 3.279 .428 .278 .0726 .0304 .0467 .033 17.8 17.8 26.03 52.06 23.87 47.7 64.64 64.64 Intermolt muscle Intermolt hypodermis 1.986 .837 .1364 3.196 .831 .1571 1.939 .531 .1542 3.04 .535 .3848 2.298 .4933 .219 1.36 .322 .174 *T0134 .0277 .0603 .0427 .033 .0365 24.17 74 21.81 43.62 9.34 18.7 51.27 75.42 31.05 61.1 20.22 40.44 8a a m p u t a t i o n o f l e g s c o u l d n o t be a c h i e v e d w i t h o u t some r e s i s t a n c e by t h e s e l a r g e and m o b i l e a n i m a l s . The mass a c t i o n r a t i o s ( 7 = A T P 3PGA/1,3 DPG ADP) d e r i v e d f r o m t h e c o n c e n t r a t i o n s were s m a l l e r t h a n t h e e q u i l i b r i u m v a l u e o f 1200-2000 (Rose 6 S a r i s , 1970; K r i e t s c h & B u c h e r , 1 9 7 0 ) . I f t h e v a l u e s a r e r e c a l c u l a t e d u s i n g t h e ATP:ADP r a t i o s c h a r a c t e r i s t i c o f r e s t i n g m u s c l e ( B e i s & Newsholme, 1 9 7 5 ) , t h e y become somewhat h i g h e r and c l o s e r t o t h e e q u i l i b r i u m v a l u e ( T a b l e 7 ) . H o w e v e r , i n any one a n i m a l , t h e m u s c l e r a t i o s a r e a l w a y s s m a l l e r t h a n t h e h y p o d e r m a l r a t i o s . G e n e r a l l y , t h e s e • mass a c t i o n r a t i o s a r e n e a r t h e b o r d e r l i n e b e t w e e n e q u i l i b r i u m a n d n o n - e q u i l i b r i u m r e a c t i o n s ("VKeg = 0.05) d e f i n e d by H o l l e s t o n ( 1 9 7 2 ) , w i t h t h e m u s c l e v a l u e s l y i n g f u r t h e r f r o m e q u i l i b r i u m . T h u s , none o f t h e s e e n z y m e s f a l l d e f i n i t e l y w i t h i n t h e r a t e l i m i t i n g c a t e g o r y o c c u p i e d by p y r u v a t e k i n a s e and p h o s p h o f r u c t o k i n a s e . A l l t h e t i s s u e s a m p l e s c o n t a i n e d m e a s u r a b l e , b u t l o w v a l u e s o f 1,3 d i p h o s p h o g l y c e r a t e . The l e v e l s ware n e a r t h e l o w e r l i m i t o f s p e c t r o p h o t o m e t r y s e n s i t i v i t y , s o t h e p e r c e n t a g e a c c u r a c y i s l o w . The l e v e l s o f 3 - p h o s p h o g l y c e r a t e were a l s o l o w . F o r t h e mass a c t i o n r a t i o s t o a p p r o a c h e q u i l i b r i u m , t h e l e v e l s o f 1,3 d i p h o s p h o g l y c e r a t e w o u l d need t o be 50-100 f o l d l o w e r , o r t h e l e v e l s o f 3 - p h o s p h o g l y c e r a t e c o n s i d e r a b l y h i g h e r . The r a t i o s f r o m t h e m u s c l e o f i n t e r m o l t c r a b s show c o n s i d e r a b l e v a r i a t i o n , m a k i n g p r e c i s e c o n c l u s i o n s o f t h e i n v i v o r o l e o f t h e enzyme d i f f i c u l t . W h i l e n e i t h e r t h e m u s c l e n o r t h e h y p o d e r m a l enzymes show a mass a c t i o n r a t i o f a r rem o v e d f r o m e q u i l i b r i u m , g e n e r a l l y , t h e h y p o d e r m a l enzymes show mass a c t i o n r a t i o s w h i c h l i e n e a r e r t o t h e e q u i l i b r i u m c o n s t a n t . 85 DISCUSSION One c o n c e p t o f c o n s i d e r a b l e m e t a b o l i c s i g n i f i c a n c e e m e r g e s f r o m t h e s e d a t a . H y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e i s k i n e t i c a l l y m o d i f i e d f o r g l u c o n e o g e n i c f u n c t i o n i n d i s t i n c t c o n t r a s t t o t h e i n t e r m o l t m u s c l e enzyme. The p r e v i o u s l y s t u d i e d p h o s p h o g l y c e r a t e k i n a s e s were f r o m y e a s t and m u s c l e , and t h e i r k i n e t i c s , w h i c h a r e s i m i l a r t o t h o s e o f t h e i n t e r m o l t m u s c l e enzyme f r o m C a n c e r m a g i s t e r , seem t o c u r t a i l r e v e r s a l o f t h e r e a c t i o n . T h e s e enzymes h a v e a h i g h e r a f f i n i t y f o r MgADP a s an i n h i b i t o r t h a n a s a s u b s t r a t e ( K r i e t s c h £ B u c h e r , 1 9 7 0 ; S c o p e s , 1 9 7 3 ) . S i m u l t a n e o u s l y , t h e i r a f f i n i t i e s f o r ATP a n d - 3 - p h o s p h o -g l y c e r a t e a r e an o r d e r o f m a g n i t u d e l o w e r t h a n t h e i r a f f i n i t y f o r MgADP a s an i n h i b i t o r . P h y s i o l o g i c a l l e v e l s o f ATP, ADP, and M g C l 2 a r e w e l l a b o v e t h e i r r e s p e c t i v e Km and K i v a l u e s , w h i l e 3 - p h o s p h o g l y c e r a t e l e v e l s a r e somewhat l o w e r t h a n i t s Km v a l u e . By i n t e r a c t i n g c o m p e t i t i v e l y w i t h t h e b i n d i n g o f b o t h MgATP a n d 3 - p h o s p h o g l y c e r a t e , p h y s i o l o g i c a l l e v e l s o f MgADP w o u l d s t r o n g l y l i m i t t h e g l u c o n e o g e n i c r e a c t i o n . The k i n e t i c s o f p h o s p h o g l y c e r a t e k i n a s e s f r o m g l u c o n e o g e n i c t i s s u e s p r o v i d e a s t r o n g c o n t r a s t t o t h o s e o f t h e i n t e r m o l t m u s c l e enzyme. I n t e r m o l t h y p o d e r m i s , w h i c h s u s t a i n s a c o n t i n u a l b a s a l r a t e o f c h i t i n s y n t h e s i s , a n d t h e m o l t i n g h y p o d e r m i s w h i c h must c o p e w i t h a l a r g e g l u c o n e o g e n i c l o a d , b o t h show p h o s p h o -g l y c e r a t e k i n a s e s c o n s i d e r a b l y m o d i f i e d f o r g l u c o n e o g e n i c f u n c t i o n . The i n t e r m o l t h y p o d e r m a l f o r m s h a r e s t h e s u b s t r a t e a f f i n i t i e s o f t h e " g l y c o l y t i c " enzyme, b u t has c o n s i d e r a b l y i n c r e a s e d K i v a l u e s f o r MgADP i n h i b i t i o n v e r s u s b o t h s u b s t r a t e s . 86 T h u s , t h e e f f e c t i v e n e s s o f p r o d u c t i n h i b i t i o n i s g r e a t l y r e d u c e d . The m o l t i n g h y p o d e r m a l enzyme has b o t h a l o w s e n s i t i v i t y t o p r o d u c t i n h i b i t i o n , a s w e l l a s a s i g n i f i c a n t l y i n c r e a s e d a f f i n i t y f o r ATP. T h u s , i n t h i s t i s s u e where m e t a b o l i c f l e x i b i l i t y , i ; e . e a s y r e v e r s a l o f t h i s r e a c t i o n , i s m a n d a t o r y , t h e g l y c o l y t i c o r i e n t a t i o n o f t h e k i n e t i c s o f t h e i n t e r m o l t m u s c l e p h o s p h o g l y c e r a t e k i n a s e has been o v e r c o m e i n two ways. F i r s t t h e r e i s a s t r o n g i n c r e a s e i n ATP a f f i n i t y , a nd s e c o n d a marked d e c r e a s e i n • s e n s i t i v i t y t o ADP and MgADP i n h i b i t i o n . I n most s t u d i e s o f g l y c o l y t i c c o n t r o l m e c h a n i s m s , p h o s p h o -g l y c e r a t e k i n a s e h a s been c o n s i d e r e d t o c a t a l y z e a n e a r e q u i l i b r i u m , n o n - r a t e l i m i t i n g r e a c t i o n . The mass a c t i o n r a t i o s show t h a t i n C a n c e r m a g i s t e r m u s c l e , t h e g l u c o n e o g e n i c r e a c t i o n a c c o u n t s f o r b e t ween 3 and 8% o f t h e g l y c o l y t i c r e a c t i o n , i f one a c c e p t s an e q u i l i b r i u m c o n s t a n t b e t w e e n 1000 and 1500 (Rose & Harms, 1 9 7 0 ) . T h u s , t h e r e a c t i o n l i e s on t h e b o r d e r l i n e b e t w e e n e q u i l i b r i u m and n o n — e q u i l i b r i u m r e a c t i o n s d e f i n e d by R o l l e s t o n (1972) a s ""1/Keq < 0.05. G i v e n no o t h e r c h a n g e s , i n c r e a s e d l e v e l s o f ADP and 1,3 d i p h o s p h o g l y c e r a t e s h i f t t h e r e a c t i o n away f r o m e q u i l i b r i u m and f a v o r t h e g l y c o l y t i c r e a c t i o n by a mass a c t i o n e f f e c t . I n c r e a s e d l e v e l s o f ADP, as w o u l d o c c u r d u r i n g t h e o n s e t o f m u s c l e ' c o n t r a c t i o n , w o u l d f u r t h e r c u r t a i l r e v e r s a l by a k i n e t i c e f f e c t . The l e v e l s o f 1,3 d i p h o s p h o g l y c e r a t e f o u n d i n c r u s t a c e a n t i s s u e s a r e i n c o n t r a s t t o t h e low l e v e l s i n r a t h e a r t ( W i l l i a m s o n , 1965) and b r a i n (Lowry e t a l , , 1964; R o l l e s t o n & Newsholme, 1 9 6 7 ) . However, Rose and War ms (1 970) , i n a s t u d y o f e r y t h r o c y t e g l y c o l y s i s , h ave shown t h a t u n d e r 87 c e r t a i n c o n d i t i o n s 1,3 d i p h o s p h o g l y c e r a t e l e v e l s r e a c h 110 ^M, l e a d i n g t o a s u b s t a n t i a l d r o p i n t h e mass a c t i o n r a t i o . Y e a s t and a s c i t e s t u m o r c e l l s h a v e been shown t o ha v e 5-20 >J1 1,3 d i -p h o s p h o g l y c e r a t e ( H e s s , 1 9 6 8 ) . R e c e n t l y , E d i n g t o n and c o w o r k e r s (1972) h a v e r e p o r t e d 1,3 d i p h o s p h o g l y c e r a t e l e v e l s up t o 1 mM i n r a t m u s c l e . I n t h e p r e s e n t s t u d y ; a l l s a m p l e s h a d m e a s u r a b l e 1,3 d i p h o s p h o g l y c e r a t e ' c o n c e n t r a t i o n s . A l l i n d i v i d u a l s show h i g h e r mass a c t i o n r a t i o s i n t h e h y p o d e r m i s t h a n i n t h e m u s c l e , i n d i c a t i n g t h a t t h e h y p o d e r m a l s y s t e m l i e s c l o s e r t o e q u i l i b r i u m . However; s i n c e t h e mass a c t i o n r a t i o s were s u b j e c t t o c o n s i d e r a b l e v a r i a t i o n b e t ween a n i m a l s , t h e s e d i f f e r e n c e s may n o t be c o m p l e t e l y m e a n i n g f u l . F u r t h e r m o r e , t h e p r e c i s e p o s i t i o n o f t h e e g u i l i b r i u m u n d e r p h y s i o l o g i c a l c o n d i t i o n s i s u n c l e a r , s i n c e R o s e and Warms (1970) a nd L e a d b e t t e r a n d c o w o r k e r s (1976) h a v e shown t h a t t h e Keg v a r i e s w i t h t h e M g + + l e v e l - p r e s e n t i n t h e s y s t e m . I n any c a s e , i n b o t h m u s c l e and h y p o d e r m i s ; p h o s -p h o g l y c e r a t e k i n a s e i s r e l a t i v e l y n e a r e q u i l i b r i u m c o m p a r e d t o t h e m a j o r r a t e l i m i t i n g e n z y m e s , p h o s p h o f r u c t o k i n a s e a n d p y r u v a t e k i n a s e (Newsholme & S t a r t , 1 9 7 3 ) . S i n c e t h e enzymes i n v o l v e d i n a p o s s i b l e b y p a s s o f t h e r e a c t i o n c o u l d n o t be f o u n d i n ' h y p o d e r m i s ( S t o r e y , 1972) and s i n c e t h e h y p o d e r m a l r e a c t i o n s y s t e m l i e s r e l a t i v e l y c l o s e t o e q u i l i b r i u m , t h e l o g i c a l c o n c l u s i o n i s t h a t p h o s p h o g l y c e r a t e k i n a s e i s r e v e r s e d - d u r i n g g l u c o n e o g e n e s i s . W h i l e some p h o s p h o g l y c e r a t e k i n a s e r e v e r s a l may o c c u r i n t h e i n t e r m o l t m u s c l e , t h e k i n e t i c m o d i f i c a t i o n s f o u n d i n t h e enzyme p r e s e n t i n t h e i n t e r m o l t a n d f r e s h m o l t h y p o d e r m i s a s w e l l a s t h o s e f o u n d f o r t h e f r e s h m o l t m u s c l e enzyme w o u l d c o n s i d e r a b l y f a c i l i t a t e r e v e r s a l o f t h e r e a c t i o n . 88 The k i n e t i c s o f t h e m o l t i n g m u s c l e enzyme a r e r a t h e r i n t r i g u i n g i n v i e w o f t h e p o t e n t i a l f o r g l u c o n e o g e n e s i s i n f r e s h m o l t m u s c l e . ' F r u c t o s e d i p h o s p h a t a s e i s p r e s e n t i n m u s c l e o f f r e s h m o l t - C a n c e r m a g i s t e r i n 2 5 % o f t h e l e v e l s o f p h o s p h o f r u c t o k i n a s e ( T a b l e 1 ) . H i g h l e v e l s o f f r u c t o s e d i p h o s -p h a t a s e h a v e been f o u n d i n t h e l e g m u s c l e o f t h e a r c t i c k i n g c r a b and t h e a r c t i c t a n n e r c r a b ( B e h r i s c h , 1971, 1972; B e h r i s c h & J o h n s o n , 1 9 7 4 ) . B e h r i s c h f o u n d t h e s e m u s c l e enzymes t o s h a r e t h e c o m p l e x r e g u l a t o r y p r o p e r t i e s o f t h e g i l l a n d h y p o d e r m a l enzymes. F u r t h e r m o r e , i s o t o p i c e v i d e n c e f o r g l u c o n e o g e n e s i s f r o m l a c t a t e , a l a n i n e * and p y r u v a t e h a s been f o u n d i n t h e m u s c l e °f £ag§iit]i2<Ies S a m t c h a t i c a ( B e h r i s c h , 1 9 7 6 ) . The low l e v e l s o f p y r u v a t e c a r b o x y l a s e and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i n t h e m u s c l e o f m o l t i n g C a n c e r m a g i s t e r make g l u c o n e o g e n e s i s a t l e a s t f e a s i b l e . The i n c r e a s e d k i n e t i c r e v e r s i b i l i t y o f t h e m o l t i n g m u s c l e ' s p h o s p h o g l y c e r a t e k i n a s e i s r e a s o n a b l e i n t h i s c o n t e x t . However, t h e mass a c t i o n r a t i o d a t a i n d i c a t e no d i f f e r e n c e b e t w e e n t h e p o i s e o f f r e s h m o l t a n d i n t e r m o l t m u s c l e p h o s p h o g l y c e r a t e k i n a s e s . The i n h i b i t i o n p a t t e r n s o f t h e s e c r u s t a c e a n p h o s p h o -g l y c e r a t e k i n a s e s a r e q u a l i t a t i v e l y s i m i l a r t o t h o s e r e p o r t e d f o r t h e y e a s t enzyme ( L a r s s o n - R a z n i k i e w i c z S A r v i d s s o n , 1 9 7 1 ) . I n c r e a s i n g magnesium i n c r e a s e s t h e e f f e c t i v e n e s s o f ADP i n h i b i t i o n , by l o w e r i n g t h e K i v a l u e s f o r t h i s n u c l e o t i d e , a n d i n c r e a s i n g t h e c o m p e t i t i v e n e s s o f i t s i n t e r a c t i o n w i t h ATP. T h e s e c r u s t a c e a n enzymes d i f f e r f r o m t h e y e a s t enzyme i n t h a t i n c r e a s i n g magnesium a l s o i n c r e a s e s t h e c o m p e t i t i v e n e s s o f t h e 89 i n t e r a c t i o n b e t w e e n ADP and 3 - p h o s p h o g l y c e r a t e . I n a l l t i s s u e f o r m s , AMP i s c o m p e t i t i v e o n l y w i t h ATP, a g a i n a s i n t h e y e a s t enzyme. The i n h i b i t i o n i s p r o b a b l y o f l i t t l e 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 , a s t h e K i v a l u e s a r e i n t h e m i l l i m o l a r r a n g e . A c o m p a r i s o n o f t h e s e c r u s t a c e a n p h o s p h o g l y c e r a t e k i n a s e s shows t h a t t h e f o r m s w h i c h h a v e s i m i l a r AMP a f f i n i t i e s a l s o s h a r e t h e K i v a l u e s f o r MgADP i n h i b i t i o n v e r s u s ATP. The two h y p o d e r m a l a nd t h e m o l t i n g m u s c l e enzymes show much t h e same MgADP v e r s u s ATP K i (see T a b l e 5 ) , and t h e i r AMP a f f i n i t i e s a l l l i e n e a r 2.3 mM. The i n t e r m o l t m u s c l e p h o s p h o g l y c e r a t e k i n a s e shows m a r k e d l y d i f f e r e n t v a l u e s f o r b o t h o f t h e s e c o n s t a n t s : t h e AMP K i i s 7.6 mM, a t h r e e f o l d i n c r e a s e o v e r t h e o t h e r f o r m s , and t h e MgADP K i i s 5 x 1 0 _ s M, a f o u r f o l d d e c r e a s e . T hese v a r i a t i o n s i n i n h i b i t o r a f f i n i t i e s a r e i n d e p e n d e n t o f t h e ATP a f f i n i t i e s , s i n c e t h e two h y p o d e r m a l f o r m s show s i g n i f i c a n t l y d i f f e r e n t ATP a f f i n i t i e s , b u t t h e same MgADP v e r s u s ATP K i v a l u e s . ADP must b i n d t o a s i t e w h i c h o v e r l a p s w i t h t h e 3-phos-p h o g l y c e r a t e a n d t h e ATP s i t e s . The a f f i n i t y f o r a d e n y l a t e i n h i b i t o r s d e p e n d s upon v a r i a t i o n s i n t h i s s i t e . I n c r e a s i n g magnesium c o n c e n t r a t i o n s i n c r e a s e t h e i n t e r a c t i o n o f ADP • w i t h b o t h t h e 3 - p h o s p h o g l y c e r a t e and t h e ATP s i t e s . T h e s e d a t a s u p p o r t t h e n o t i o n o f two n u c l e o t i d e b i n d i n g s i t e s f o r t h e s e p h o s p h o g l y c e r a t e ' k i n a s e s . The b u l k o f t h e l i t e r a t u r e o f phos-p h o g l y c e r a t e k i n a s e - s u p p o r t s t h i s b i n d i n g p a t t e r n , on t h e b a s i s o f i n i t i a l v e l o c i t y s t u d i e s ( L a r s s o n - R a z n i k i e w i c z , 1 964, 1 9 6 7 ) , o f - e s s e n t i a l s u l f h y d r y l g r o u p s ( K r i e t s c h S B u c h e r , 1 9 7 0 ) , and on t h e b a s i s o f p r o d u c t i n h i b i t i o n s t u d i e s ( L a r s s o n - R a z n i k i e w i c z & A r v i d s s o n , 1 9 7 1 ) . However, r e c e n t s p e c t r o s c o p i c e v i d e n c e f r o m 90 t h e y e a s t enzyme ( R o u s t a n e t a l . , 1973) i n d i c a t e s o n l y one n u c l e o t i d e b i n d i n g s i t e on t h e p h o s p h o g l y c e r a t e k i n a s e a c t i v e s i t e . I n c o n t r a s t w i t h t h e f i n d i n g s - o f K r i e t s c h and B i i c h e r (1970) i n t h e i r s t u d y of t h e r a b b i t m u s c l e enzyme,* b u t l a - a g r e e m e n t w i t h B o j a n o v s k i e t a l . (1974) and Ponce e t a , l . ( 1 9 7 1 ) , 2 , 3 - d i p h o s p h o g l y c e r a t e i s a c o m p e t i t i v e i n h i b i t o r o f t h e s e p h o s -p h o g l y c e r a t e k i n a s e s . The i n h i b i t i o n i s more p o t e n t v e r s u s 3-p h o s p h o g l y c e r a t e t h a n ATP. I t s i n t e r a c t i o n w i t h b o t h s i t e s i n d i c a t e s t h a t i t may b e h a v e l i k e 1,3 d i p h o s p h o g l y c e r a t e , t h e a c t u a l g l y c o l y t i c s u b s t r a t e . I n t h i s s e n s e , t h e c r u s t a c e a n enzymes must be more f l e x i b l e o r have a r a t h e r d i f f e r e n t s h a p e a t t h e a c t i v e s i t e t h a n t h e mammalian m u s c l e enzyme. T h e s e c r u s t a c e a n enzymes show a marked s t i m u l a t i o n by N a C l and K C l , and some i n h i b i t i o n b y - c a l c i u m . - C a l c i u m i n h i b i t i o n was a l s o f o u n d f o r t h e e r y t h r o c y t e enzyme (Okonkwo e t a l . , 1 9 7 3 ) . The N a C l and K C l e f f e c t s a r e p r o b a b l y due t o c h a n g e s i n t h e s o l v e n t s t r u c t u r e s u r r o u n d i n g t h e enzyme, r a t h e r t h a n t o t h e i r d i r e c t p a r t i c i p a t i o n i n t h e c a t a l y s i s . The Km v a l u e s f o r t h e K C l a c t i v a t i o n l i e w i t h i n t h e i n t r a c e l l u l a r i o n c o n c e n t r a t i o n s . Even t h o u g h c r u s t a c e a n t i s s u e s have h i g h e r i n t r a c e l l u l a r s o d i u m t h a n do mammalian t i s s u e s ( P r o s s e r , 1 9 7 3 ) , t h e i n t e r m o l t e n z y m e s 1 Km - v a l u e s f o r N a C l a c t i v a t i o n a r e w e l l a b o v e t h i s r a n g e . The c o n s i d e r a b l y l o w e r N a C l Km shown by t h e m o l t i n g enzymes may s i m p l y r e s u l t i n a d d i t i o n a l s t i m u l a t i o n o f t h e r e a c t i o n , o r i t may s i g n i f y a d i f f e r e n t c a t a l y t i c m e c h a n i s m . L a r s s o n - R a z n i k i e w i c z a n d A r v i d s s o n (1971) i n c l u d e 0.25 M N a C l i n 91 a l l t h e i r a s s a y s , p e r h a p s f o r i t s a c t i v a t i n g e f f e c t . U n f o r t u n a t e l y , due- t o p r o b l e m s i n s t a b i l i z i n g a n d f u r t h e r p u r i f y i n g t h e s e c r u s t a c e a n p h o s p h o g l y c e r a t e k i n a s e s , I was n o t a b l e t o work o u t t h e e x a c t m o l e c u l a r c h a n g e s w h i c h accompany t h e k i n e t i c d i f f e r e n c e s b e t w e e n t h e t i s s u e s . The r e s u l t s do n o t f a c i l i t a t e t h a c h o i c e between t h e v a r i o u s p o s s i b l e a l t e r n a t i v e s : f o u r gene p r o d u c t s , two p r o t e i n s i n v a r y i n g r a t i o s , o r one gene p r o d u c t s u b j e c t e d t o v a r y i n g d e g r e e s o f m o d i f i c a t i o n i n t h e c e l l . H owever, t h e y do e s t a b l i s h t h a t t h e r e a r e a t l e a s t two k i n e t i c a l l y a nd e l e c t r o p h o r e t i c a l l y d i s t i n c t f o r m s i n t h e s e c r u s t a c e a n s . T h i s s t a n d s i n a g r e e m e n t w i t h work done on t h e b o v i n e l i v e r enzyme s h o w i n g i t t o be d i s t i n c t f r o m t h e b o v i n e m u s c l e enzyme, on e l e c t r o p h o r e t i c and m o l e c u l a r g r o u n d s a s w e l l a s i n o v e r a l l o r i e n t a t i o n o f k i n e t i c s ( B o j a n o v s k i e t a l . , 1 9 7 4 ) . C o m p a r i s o n s o f mammalian m u s c l e , e r y t h r o c y t e , and y e a s t enzymes show d i s t i n c t k i n e t i c a n d i m m u n o l o g i c a l d i f f e r e n c e s (Okonkwo e t a l . , 1 9 7 3 ) . These r e s u l t s a l l c o n t r a s t w i t h t h e o b s e r v a t i o n o f a l a c k o f k i n e t i c , e l e c t r o p h o r e t i c , o r i m m u n o l o g i c a l d i f f e r e n c e s b e t w e e n p h o s p h o g l y c e r a t e k i n a s e s o f r a t l i v e r , muscle-, a n d - h e a r t ( F r i t z & W h i t e ; -1974)* I n C a n c e r m a g _ i s t e r , t h e r e a r e t i s s u e and m o l t c y c l e r e l a t e d d i f f e r e n c e s i n t h e amounts and t h e k i n e t i c p r o p e r t i e s o f t h e p h o s p h o g l y c e r a t e k i n a s e s p r e s e n t . - W i t h i n t h i s enzyme s y s t e m , two b a s i c a l t e r a t i o n s o f t h e r e a c t i o n k i n e t i c s s u f f i c e t o t r a n s f o r m t h e g l y c o l y t i c a l l y p o i s e d enzyme i n f o a n o t h e r g l u c o n e o g e n i c a l l y a d a p t e d f o r m . An i n c r e a s e i n t h e ATP a f f i n i t y w i t h a c o n c o m i t a n t d e c r e a s e i n t h e s e n s i t i v i t y - t o ADP and MgADP p r o d u c t i n h i b i t i o n make t h e r e v e r s a l o f t h e r e a c t i o n more f e a s i b l e . The o t h e r a f f i n i t y d i f f e r e n c e s between t h e f o r m s 92 p r o b a b l y a r i s e a s a c o n s e q u e n c e o f - t h e s e m a j o r c h a n g e s . S i m i l a r a l t e r a t i o n s o f ADP"and ATP a f f i n i t i e s w i l l p r o b a b l y be f o u n d i n p h o s p h o g l y c e r a t e k i n a s e s i n o t h e r g l u c o n e o g e n i c s y s t e m s . 93 P a r t I I I M u s c l e P y r u v a t e K i n a s e INTRODUCTION C r u s t a c e a n m u s c l e . u n d e r g o e s . c o n s i d e r a b l e c h a n g e s w i t h t h e m o l t c y c l e . I t i s a h i g h l y g l y c o l y t i c t i s s u e d u r i n g t h e i n t e r m o l t p o r t i o n o f t h e c y c l e ( H o c h a c h k a e t a l . , 1 9 7 1 ) , and u n d o u b t e d l y m a i n t a i n s a h i g h c a p a c i t y f o r g l y c o l y t i c g e n e r a t i o n o f ATP t h r o u g h o u t t h e p r e m o l t and p o s t m o l t p e r i o d s . W i t h t h e o n s e t o f p r e m o l t , t h e m u s c l e mass d e c r e a s e s m a r k e d l y , d r o p p i n g t o 6 0 1 o f t h e i n t e r m o l t mass ( S k i n n e r , 1 9 6 6 ) . . I n C a n c e r m a g i s t e r , t h e l e v e l s o f g l y c o l y t i c enzymes d r o p , a n d t h e l e v e l s o f g l u c o n e o g e n i c enzymes r i s e ( T a b l e 1 ) . T h u s , t h e g l y c o l y t i c p o t e n t i a l o f t h e t i s s u e i s c o n s i d e r a b l y r e d u c e d , l e a d i n g t o t h e p o s s i b i l i t y o f g l u c o n e o g e n e s i s i n t h e m u s c l e o f m o l t i n g C a n c e r m a g i s t e r . P h o s p h o g l y c e r a t e k i n a s e from f r e s h m o l t m u s c l e shows k i n e t i c m o d i f i c a t i o n s w h i c h f a v o r g l u c o n e o g e n e s i s , i n c o n t r a s t to * t h e enzyme f r o m i n t e r m o l t m u s c l e . T h i s l e n d s f u r t h e r c r e d e n c e t o t h e p o s s i b i l i t y o f g l u c o n e o g e n e s i s i n - f r e s h m o l t m u s c l e . The f r u c t o s e d i p h o s p h a t a s e p r e s e n t i n P a r a l i t h o d e s c a m t c h a t i c a m u s c l e h a s c o m p l e x c o n t r o l c h a r a c t e r i s t i c s w h i c h a r e s u i t e d f o r f u n c t i o n i n a g l u c o n e o g e n i c s y s t e m a s w e l l a s i n a h i g h l y g l y c o l y t i c s y s t e m ( B e h r i s c h > 1 9 7 2 ; B e h r i s c h J o h n s o n , 1 9 7 4 ) . P h o s p h o f r u c t o k i n a s e f r o m P a r a l i t h o d e s - c a m t c h a t i c a m u s c l e i s i n h i b i t e d by h i g h l e v e l s o f ATP and a p t i v a t e d by l o w l e v e l s o f AMP ( F r e e d , 1 9 7 1 ) . These c h a r a c t e r i s t i c s w o u l d a l l o w t h e 94 i n h i b i t i o n o f t h e enzyme d u r i n g p e r i o d s o f g l u c o n e o g e n e s i s , a n d t h e n s t r o n g a c t i v a t i o n o f t h e enzyme f o r g l y c o l y s i s . T h u s , t h e e n z y m a t i c m a c h i n e r y w h i c h - h a s b e e n e x a m i n e d i n c r u s t a c e a n m u s c l e has c o n t r o l c h a r a c t e r i s t i c s w h i c h a r e c o m p a t i b l e w i t h some g l u c o n e o g e n e s i s i n f r e s h m o l t m u s c l e . The c o n t r o l o f p y r u v a t e k i n a s e (E.C. 2.7.1.40) i s i m p o r t a n t u n d e r v a r i o u s m e t a b o l i c c o n d i t i o n s . D u r i n g g l y c o l y s i s , i t i s a m a j o r r a t e l i m i t i n g a n d - t h u s r e g u l a t o r y s i t e ( B e i s £ Newsholme, 1 9 7 5 ; W i l l i a m s o n , 1 9 6 5 ) . D u r i n g g l u c o n e o g e n -e s i s , when t h e p y r u v a t e k i n a s e r e a c t i o n i s r e v e r s e d by an e n z y m a t i c b y p a s s ( S c r u t t o n £ U t t e r , 1 9 6 8 ) , i t s d o w n h i l l f u n c t i o n must be e f f i c i e n t l y c u r t a i l e d . I n mammals, t h e s e r a t h e r d i f f e r e n t c o n t r o l r e q u i r e m e n t s a r e met by t h r e e d i s t i n c t p y r u v a t e k i n a s e f o r m s . The m u s c l e enzyme (M t y p e ) i s u n a f f e c t e d by FDP, shows a h i g h a f f i n i t y f o r i t s s u b s t r a t e s , and a l o w s e n s i t i v i t y t o ATP i n h i b i t i o n ( K a y n e , 1973; Tanaka e t a l . , 1 9 6 7 ) . The m u s c l e enzyme i s a l s o s e n s i t i v e t o c r e a t i n e p h o s p h a t e i n h i b i t i o n (Kemp, 1 9 7 3 ) . The l i v e r enzyme (L t y p e ) shows a s t r o n g s e n s i t i v i t y t o g l u c o s e 1,6 d i p h o s p h a t e a n d FDP a c t i v a t i o n , t o i n h i b i t i o n by ATP, a l a n i n e , c i t r a t e , a n d o t h e r m e t a b o l i t e s , and l o w a f f i n i t y 1 f o r i t s s u b s t r a t e p h o s p h o e n o l -p y r u v a t e ( P E P ) . I n c o n t r a s t t o t h e m u s c l e enzyme, t h e - l i v e r enzyme shows s i g m o i d a l k i n e t i c s (Van B e r k e l gt • a,l.> 1974; C a r d e n a s £ D y s o n , 1 9 7 3 ; T a n a k a e t a l . , 1 9 6 7 ) . The t h i r d f o r m o f mammalian p y r u v a t e k i n a s e , p r e s e n t i n b l o o d c e l l s , - k i d n e y , a d i p o s e t i s s u e , a n d f e t a l t i s s u e i s d i s t i n g u i s h e d a s t h e K o r M2 t y p e . T h i s f o r m d i s p l a y s i n t e r m e d i a t e k i n e t i c s , c o m b i n i n g FDP a c t i v a t i o n a n d s e n s i t i v i t y t o i n h i b i t o r s w i t h h y p e r b o l i c 95 k i n e t i c s ( C a r d e n a s e t a l . , 1975; Van B e r k e l e t al.. , 1 9 7 3 ) . • T h e p y r u v a t e k i n a s e s i n t i s s u e s o f l o w e r v e r t e b r a t e s a n d i n v e r t e b r a t e s f a c e much t h e same g l y c o l y t i c and g l u c o n e o g e n i c c o n t r o l r e q u i r e m e n t s a s t h o s e i n mammalian - t i s s u e s , b u t t h e s y s t e m o f enzymes e v o l v e d t o d e a l w i t h t h e s e n e e d s has n o t been e l u c i d a t e d . - M o s t o f t h e a v a i l a b l e i n f o r m a t i o n c o n c e r n s m u s c l e e n z y m e s , many o f w h i c h show a l l o s t e r i c r e g u l a t o r y p r o p e r t i e s . G e n e r a l l y , t h e s e enzymes a r e r e g u l a t e d by an i n t e r p l a y b e t w e e n i n h i b i t i o n by ATP/ c i t r a t e , a l a n i n e , and v a r i o u s o t h e r m e t a b o l i t e s , and a c t i v a t i o n by-FDP. T h i s p a t t e r n h o l d s t r u e f o r t h e enzyme f r o m Rana £i£i§ns h e a r t , Cori£henoides - m u s c l e , s t u r g e o n m u s c l e , t u r t l e h e a r t , o y s t e r a d d u c t o r , M y _ t i l u s e d u l i s a d d u c t o r , r a i n b o w t r o u t ; T rematomus, a n d c a r p m u s c l e . ( F l a n d e r s , 1971; M u s t a f a e t a l . , 1971; R a n d a l l & A n d e r s o n , 1975; S t o r e y & H o c h a c h k a , 1974; M u s t a f a & H o c h a c h k a , 1971; de Zwaan & H o l w e r d a , 1972; Somero S H o c h a c h k a , 1968; J o h n s t o n , 1 9 7 5 ) . E x c e p t i o n s t o t h e a b o v e a r e t h e m u s c l e enzymes f r o m Rana p i p i e n s , d e s e r t l o c u s t , S c h i c t o s c m a m a n s o n i , o c t o p u s , s h r i m p , and s q u i d w h i c h do n o t show FDP 1 a c t i v a t i o n ( F l a n d e r s e t a l . , 1971; B a i l e y & W a l k e r , 1969; McManus, 1975; G u d e r l e y a t a l . , 1 9 7 6 a ; G u d e r l e y e t a l . , 1976b; S t o r e y & H o c h a c h k a , 1 9 7 5 ) . T h i s s t u d y o f C a n c e r m a g i s t e r m u s c l e p y r u v a t e k i n a s e had two m a j o r g o a l s , • 1 ) t o work o u t t h e r e g u l a t i o n o f - t h e enzyme i n i n t e r m o l t m u s c l e , a n d 2) t o d e t e r m i n e w h e t h e r t h e enzyme a n d / o r t h e p a t t e r n o f r e g u l a t i o n c h a n g e s as t h e m u s c l e c h a n g e s i n p r e p a r a t i o n f o r m o l t . T h i s i n f o r m a t i o n w o u l d e l u c i d a t e b o t h t h e means by w h i c h p y r u v a t e k i n a s e c a n be r e g u l a t e d d u r i n g 96 g l y c o l y s i s a s w e l l a s t h e f e a s i b i l i t y o f g l u c o n e o g e n i c f l u x p a s t t h i s r e a c t i o n l o c u s . METHODS ££§£il££tion o f P y r u v a t e K i n a s e M u s c l e - was q u i c k l y d i s s e c t l y o u t o f a n a e s t h e t i z e d c r a b s , f r e e d o f any a d h e r i n g t i s s u e , b l o t t e d d r y , a n d w e i g h e d . The t i s s u e was t h e n h o m o g e n i z e d i n f o u r v o l u m e s o f 50 mM i m i d a z o l e - H C l , pH 7.0. The homogenate was c e n t r i f u g e d a t 27,000 g f o r t w e n t y m i n u t e s . . S o l i d ammonium s u l p h a t e was a d d e d w i t h c o n s t a n t s t i r r i n g t o b r i n g t h e s u p e r n a t a n t t o 4 5$ s a t u r a t i o n . T h i s m i x t u r e was c e n t r i f u g e d a t 12,000 g f o r 10 m i n u t e s , t h e n s o l i d ammonium s u l p h a t e was g r a d u a l l y a d d e d , w i t h c o n s t a n t s t i r r i n g , t o b r i n g t h e s u p e r n a t a n t t o 6 0 % s a t u r a t i o n . The m i x t u r e was c e n t r i f u g e d as a b o v e , t h e s u p e r n a t a n t d i s c a r d e d , a n d t h e p e l l e t r e d i s s o l v e d i n a s m a l l v o l u m e o f 50 mM i m i d a z o l e - H C l , 3 mM, pH 7.0. I n - s t u d i e s where t h e p r o p e r t i e s o f t h e u n p u r i f i e d enzyme were e x a m i n e d , a p p r o x i m a t e l y 4 ml o f t h i s p r e p a r a t i o n were d i a l y z e d a g a i n s t two c h a n g e s o f 2 l i t e r s - each o f 50 mM i m i d a z o l e - H C l , 3 mM EDTA, pH 7.0, f o r a t o t a l p e r i o d o f 3 h o u r s . Hhen f u r t h e r p u r i f i c a t i o n was d e s i r e d , i t was a c h i e v e d v i a c o l u m n c h r o m a t o g r a p h y on c e l l u l o s e p h o s p h a t e o r on DE AE S e p h a d e x . C e l l u l o s e p h o s p h a t e was p r e t r e a t e d a c c o r d i n g t o m a n u f a c t u r e r ' s i n s t r u c t i o n s , and t h e n ' e q u i l i b r a t e d i n 5 mM p h o s p h a t e , 1 mM • m e r c a p t o e f h a n o l , pH 7.5. The DEkE S e p h a d e x was a l l o w e d t o s w e l l i n 50 mM i m i d a z o l e - H C l , 3 mM EDTA, 4 mM MgC12, 97 pH- 7.5, a n d t h e n e q u i l i b r a t e d i n t h e same b u f f e r . D e p e n d i n g on t h e amount o f t i s s u e u t i l i z e d , e i t h e r 2.5 x 15 cm o r 2.5 x 30 cm c o l u m n s were-used.' The enzyme e x t r a c t was p r e p a r e d f o r - c o l u m n c h r o m a t o g r a p h y by d i a l y s i s a g a i n s t 2 c h a n g e s o f 2 l i t e r s e a c h o f t h e a p p r o p r i a t e c o l u m n b u f f e r . A f t e r a p p r o x i m a t e l y t h r e e h o u r s o f d i a l y s i s , t h e enzyme p r e p a r a t i o n was a p p l i e d t o t h e c o l u m n , f o l l o w e d w i t h 50 ml o f t h e c o l u m n b u f f e r . Then a g r a d i e n t (0-300 mH KC1 i n t h e c o l u m n b u f f e r ) was a p p l i e d t o t h e c o l u m n . F o r t h e s m a l l c o l u m n , 300 ml o f g r a d i e n t were u s e d , f o r t h e l a r g e r c o l u m n , 500 ml were u s e d . The c e l l u l o s e p h o s p h a t e c o l u m n s were e l u t e d a t 35 m l / h o u r , w h i l e t h e DEAE Sephadex c o l u m n s were e l u t e d a t 20 m l / h o u r . The 1.5 ml f r a c t i o n s were c h e c k e d f o r enzyme a c t i v i t y , p r o t e i n , a nd c o n d u c t i v i t y . When e i t h e r o f t h e s e c o l u m n s was u s e d , a p u r i f i c a t i o n o f 800 t o 1000 f o l d was a t t a i n e d , l e a d i n g t o a s p e c i f i c a c t i v i t y o f -100 u n i t s / mg p r o t e i n . The peak f r a c t i o n s were c o n c e n t r a t e d by u l t r a f i l t r a t i o n i n an Amicon c e l l . Enzyme A s s a y s P y r u v a t e k i n a s e a c t i v i t y was m e a s u r e d a c c o r d i n g t o t h e method o f B i i c h e r a n d P f l e i d e r e r (1955) w h i c h c o u p l e s p y r u v a t e f o r m a t i o n t o t h e d e c r e a s e i n o p t i c a l d e n s i t y a t 340 a n g s t r o m s t h r o u g h t h e l a c t a t e d e h y d r o g e n a s e r e a c t i o n . A l l s t a n d a r d a s s a y s i n c l u d e d PEP, ADP, KC1> MgC12, NADH, and e x c e s s l a c t a t e d e h y d r o -g e n a s e i n t h e c o n c e n t r a t i o n s ' shown i n t h e f i g u r e l e g e n d s . K i n e t i c s t u d i e s were done a t 20 °C, u s i n g i m i d a z o l e b u f f e r s . P y r u v a t e k i n a s e a c t i v i t y i n t h e d i r e c t i o n o f p h o s p h o e n o l -98 p y r u v a t e f o r m a t i o n was m e a s u r e d a c c o r d i n g t o Dyson e t a l , ( 1 9 7 5 ) . S t a n d a r d a s s a y s i n c l u d e d ATP, MgC12, K C l , p y r u v a t e , KHC03, NADH, e x c e s s m a l a t e d e h y d r o g e n a s e , and e x c e s s p e a n u t p h o s p h o e n c l p y r u v a t e c a r b o x y l a s e . The a c t i v i t y d u e t o t h e r e v e r s a l o f p y r u v a t e k i n a s e was d e t e r m i n e d by s u b t r a c t i n g t h e two c o n t r o l s , one w i t h o u t p y r u v a t e k i n a s e and one w i t h o u t PEP c a r b o x y l a s e . T h e s e s t u d i e s - were done a t 20 °C u s i n g i m i d a z o l e - H C l b u f f e r e d t o pH 7.5. C o n s i d e r a b l e c a r e was t a k e n d u r i n g m e a s u r e m e n t s o f t h e r e v e r s a l o f p y r u v a t e k i n a s e t o remove a l l t r a c e s o f l a c t a t e d e h y d r o g e n a s e f r o m t h e c u v e t t e s . RESULTS J i j r i ^ f £°Bt*2£g§4§ and I s o e l e c t r o f o c u s i n g E x t e n s i v e e l e c t r o p h o r e t i c c o m p a r i s o n o f t h e i n t e r m o l t m u s c l e p y r u v a t e k i n a s e w i t h t h e enzyme f r o m t h e f r e s h m o l t m u s c l e r e v e a l e d no c o n s i s t e n t d i f f e r e n c e s . Of f o r t y g e l s , f i v e showed a s l i g h t l y f a s t e r m i g r a t i o n f o r t h e f r e s h m o l t m u s c l e enzyme a n d two showed a s l i g h t l y f a s t e r m i g r a t i o n f o r t h e i n t e r m o l t m u s c l e enzyme. T h u s , i n t h e m a j o r i t y o f c a s e s , t h e m o l t i n g a n d i n t e r m o l t m u s c l e p y r u v a t e k i n a s e showed i d e n t i c a l m i g r a t i o n . S e v e r a l d i f f e r e n t m i g r a t i o n s y s t e m s were u s e d t o t e s t o u t t h e v a r i o u s p o s s i b i l i t i e s c o m p l e t e l y . S i n c e no k i n e t i c d i f f e r e n c e s ( s e e b e l o w ) were f o u n d e v e n w i t h t h o s e enzymes w h i c h showed s l i g h t l y d i f f e r e n t m i g r a t i o n p a t t e r n s , i t i s p r o b a b l e t h a t t h e s e s l i g h t d i f f e r e n c e s r e p r e s e n t a l l e l i c d i f f e r e n c e s w h i c h do n o t a f f e c t t h e c a t a l y t i c c h a r a c t e r i s t i c s o f t h e m u s c l e enzyme. The e x p e r i m e n t a l a n i m a l s were a random s a m p l e o f t h e n a t u r a l 99 p o p u l a t i o n and some p h e n o t y p i c v a r i a t i o n b e t ween i n d i v i d u a l s i s n o t s u r p r i s i n g . I s o e l e c t r o f o c u s i n g a l s o a l s o f a i l e d t o r e v e a l d i f f e r e n c e s b e t w e e n t h e : i n t e r m o l t and t h e f r e s h m o l t m u s c l e e n z y m e s . A f t e r t h e 45 t o 6 0 % ammonium s u l p h a t e f r a c t i o n a t i o n , e a c h enzyme was f o c u s e d on a 3—10'pH g r a d i e n t a s w e l l a s a 5-8 pH g r a d i e n t . I n a l l c a s e s , a p i o f 6.4 ± 0.05 was f o u n d . T h u s , I c o n c l u d e d t h a t t h e p y r u v a t e k i n a s e s i n i n t e r m o l t and f r e s h m o l t m u s c l e had much t h e same o v e r a l l c h a r g e and were p r o b a b l y t h e same p r o t e i n . 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 K i n e t i c a n a l y s e s were c a r r i e d o u t w i t h ammonium s u l p h a t e f r a c t i o n s and p u r i f i e d p y r u v a t e k i n a s e s f r o m b o t h f r e s h m o l t a n d i n t e r m o l t m u s c l e . S i n c e t h e r e s u l t s showed c l o s e a g r e e m e n t between t h e v a r i o u s p r e p a r a t i o n s , o n l y one s e t o f i n f o r m a t i o n w i l l be p r e s e n t e d . The c l o s e a g r e e m e n t o f t h e k i n e t i c r e s u l t s s u p p o r t e d t h e c o n c l u s i o n t h a t o n l y one f o r m o f p y r u v a t e k i n a s e i s p r e s e n t i n t h e m u s c l e t h r o u g h o u t t h e m o l t c y c l e . I n common w i t h o t h e r p y r u v a t e k i n a s e s , t h e c r u s t a c e a n m u s c l e enzyme r e g u i r e d b o t h a m o n o v a l e n t and a d i v a l e n t c a t i o n f o r a c t i v i t y ( K a y n e , 1 9 7 3 ) . B o t h K+ and NH4+ were good a c t i v a t o r s . The enzyme had a h i g h e r a f f i n i t y f o r NH4+ (Ka = 5 mM) t h a n f o r K+ (Ka = 15 mM), b u t showed a t w o - f o l d h i g h e r Vmax w i t h K+ t h a n w i t h NH4+. ' S i m i l a r l y , t h e enzyme showed a h i g h e r a f f i n i t y f o r Mn++ (Ka = 0.15 mM) t h a n f o r M g + + (Ka = 0.6 mM), b u t showed a l o w e r m a x i m a l v e l o c i t y w i t h M n + + ( 5 0 % o f t h a t w i t h M g + + ) . Na* was i n h i b i t o r y i n t h e p r e s e n c e o f 100 o p t i m a l K + , b u t p r o d u c e d a s l i g h t a c t i v a t i o n i n t h e a b s e n c e o f K+ (Ka = 60 mM). C a l c i u m i n h i b i t e d t h e r e a c t i o n and showed a c o m p e t i t i v e i n t e r a c t i o n w i t h b o t h Mg ++ ( K i = 0.1 mM) and ADP ( K i = 0.7 mM). C a l c i u m was l e s s e f f e c t i v e a s an i n h i b i t o r v e r s u s Mn*+ ( K i = 0.66 mM) t h a n M g + + . T h i s d e c r e a s e i n s e n s i t i v i t y was n o t a s p r o n o u n c e d a s t h a t shown by p y r u v a t e k i n a s e f r o m JiY.ti3.us e d u l i s a d d u c t o r (de Zwaan e t a l . , 1 9 7 5 ) . The pH optimum f o r C a n c e r m a g i s t e r m u s c l e p y r u v a t e k i n a s e was pH 7.0. S u b s t r a t e A f f i n i t i e s S u b s t r a t e s a t u r a t i o n c u r v e s f o r b o t h PEP and ADP f o l l o w e d M i c h a e l i s - M e n t e n b e h a v i o r . The a f f i n i t y f o r PEP was h i g h , w i t h Km v a l u e s r a n g i n g f r o m 7 x 1 0 - 5 M t o 1.1 x 1Q-* M. Tha o n l y d i f f e r e n c e b e t w e e n m o l t i n g and i n t e r m o l t p y r u v a t e k i n a s e s l a y i n t h i s p a r a m t e r , t h e m o l t i n g m u s c l e enzyme u s u a l l y showed a h i g h e r a f f i n i t y f o r PEP (Km v a l u e s b e t w e e n 8 x 1 0 - 5 M and 1.0 x 10-* M) t h a n t h e i n t e r m o l t m u s c l e enzyme (Km v a l u e s b e t w e e n 9.0 x 1Q— 5 M and 1.1 x 10-* M). However, t h e r e was c o n s i d e r a b l e o v e r l a y i n t h e s e Km v a l u e s . The ADP a f f i n i t i e s showed much l e s s v a r i a t i o n ; t h e y r a n g e d f r o m 0.23 mM t o 0.29 mM, f o r b o t h t h e i n t e r m o l t and t h e f r e s h m o l t enzyme. S u b s t r a t e a f f i n i t i e s were m e a s u r e d i n t h e p r e s e n c e o f - 4 mM MgCl2 a n d 75 mM K C l i n 50 mM i m i d a z o l e - c l , pH 7;0. The a f f i n i t y f o r e a c h s u b s t r a t e was i n d e p e n d e n t o f v a r i a t i o n s i n t h e l e v e l s o f c o s u b s t r a t e , i n d i c a t i n g a random b i n d i n g m e c h a n i s m . UDP, I D P , and GDP a l l s e r v e d a s n u c l e o t i d e s u b s t r a t e s , b u t ' w i t h s u b s t a n t i a l l y r e d u c e d e f f e c t i v e n e s s c o m p a r e d t o ADP ( T a b l e 8) . 101 TABLE Nucleotide diphosphate a c c e p t a b i l i t y . Nucleotide diphosphate max ADP GDP IDP UDP 0.23 mM 0.8 mM 1.0 mM 1.5 mM 100% 63% 50% 55% 102 A c t i v a t o r s - I n common w i t h many o t h e r i n v e r t e b r a t e - m u s c l e p y r u v a t e k i n a s e s , t h e enzyme was a c t i v a t e d by low l e v e l s o f f r u c t o s e 1,6 d i p h o s p h a t e (Ka - 2 x 1Q-s M ) . ; The m a j o r e f f e c t o f FDP was t o f a c i l i t a t e t h e b i n d i n g o f P E P , 0.05 mM FDP r e d u c e d t h e PEP Km f r o m 8.5 x 10~ 5 M t o 4.5 x 1 0 ~ 5 M. FDP a l s o r e v e r s e d t h e i n h i b i t i o n c a u s e d by MgATP, p r o b a b l y v i a i t s e f f e c t on t h e enzyme's a f f i n i t y f o r PEP ( F i g u r e 7 ) . I n f r e s h l y p r e p a r e d h o m o g e n a t e s , a nd i n ammonium s u l p h a t e f r a c t i o n s , g l u c o s a m i n e - 6 -p h o s p h a t e , U D P — a c e t y l g l u c o s a m i n e and FDP were e q u a l l y g ood a c t i v a t o r s . H o wever, w i t h t h e p u r i f i e d enzyme, o n l y FDP r e m a i n e d a s t r o n g a c t i v a t o r , w h i l e g l u c o s a m i n e - 6 - p h o s p h a t e a nd UDPAG showed a d e c r e a s e d c a p a c i t y t o r e v e r s e ATP i n h i b i t i o n ( s e e T a b l e 9, p. 114) . I n h i b i t o r s As m e n t i o n e d a b o v e , MgATP i s an i n h i b i t o r o f t h i s c r u s t a c e a n m u s c l e enzyme. MgATP showed t h e same e f f e c t i v e n e s s a s an i n h i b i t o r o f t h e p u r i f i e d and u n p u r i f i e d e n z y m e s . I n c o m p a r i s o n w i t h p r e v i o u s l y s t u d i e d m u s c l e p y r u v a t e k i n a s e s , t h i s m u s c l e enzyme i s g u i t e s e n s i t i v e t o MgATP i n h i b i t i o n . The i n h i b i t i o n v e r s u s PEP was c o m p e t i t i v e w i t h a K i o f 1.8 mM ( F i g u r e 8) w h i l e t h a t v e r s u s ADP was n o n - c o m p e t i t i v e a n d showed a K i o f 5 mM ( F i g u r e 9 ) . The enzyme showed much l o w e r s e n s i t i v i t y t o - t h e o t h e r n u c l e o t i d e t r i p h o s p h a t e s (MgUTP, M g l T P , and MgGTP) t h a n t o MgATP. A r g i n i n e p h o s p h a t e p l a y s an i m p o r t a n t r o l e a s t h e s t o r a g e p h o sphagen i n c r u s t a c e a n m u s c l e s ( B e i s 6 103 0 1 2 3 4 5 1 / P E P X 1 0 4 F i g . 7. E f f e c t o f MgATP and FDP on PEP a f f i n i t y o f C_. magister muscle pyruvate kinase. A l l experiments were performed i n 50 mM imidazo l e - C l , pH 7.0 with 75 mM KC1, 5 mM MgCl 2, 1.2 mM ADP, 0.1 mM NADH, and excess l a c t a t e dehydrogenase. (0) i n the presence of 2 mM MgATP; (•) i n the presence o f 2 mM MgATP and 0.5 mM FDP; (A) c o n t r o l ; (A) i n the presence of 0.5 mM FDP. 8. MgATP i n h i b i t i o n , e f f e c t on PEP a f f i n i t y . Experimental c o n d i t i o were as given i n the legend f o r Figure 7. Treatments are as f o l l o w s : (0) 5 mM MgATP; (•) 2.5 mM MgATP; (A) c o n t r o l . 105 0 1 2 1/ADP x 10 4 F i g . 9. MgATP i n h i b i t i o n , e f f e c t on ADP a f f i n i t y . Experimental conditions a r e as i n Figure 7 except that PEP was held constant a t 2 mM and A D P was va r i e d . (A) 5 mM MgATP; (•) 2.5 mM MgATP; (0) c o n t r o l . 106 Newsholme, 1 9 7 5 ) , s o I c h e c k e d f o r i t s e f f e c t on t h e p y r u v a t e k i n a s e r e a c t i o n . P h y s i o l o g i c a l l e v e l s o f a r g i n i n e p h o s p h a t e s u b s t a n t i a l l y r e d u c e d enzyme a c t i v i t y . A r g i n i n e p h o s p h a t e was c o m p e t i t i v e w i t h t h e enzyme f o r ADP { K i = 2.8 mM) ( F i g u r e 1 0 ) , a s w e l l a s p a r t i a l l y h i n d e r i n g t h a b i n d i n g o f PEP ( K i = 11 mM) ( F i g u r e 1 1 ) . W h i l e FDP r e v e r s e d t h e i n h i b i t i o n due t o ATP, i t d i d n o t r e v e r s e t h a t due t o a r g i n i n e p h o s p h a t e . A r g i n i n e p h o s p h a t e i n h i b i t i o n was n o t due t o ATP f o r m a t i o n by a r g i n i n e p h o s p h o k i n a s e . A v a r i e t y o f m e t a b o l i t e s , i n c l u d i n g amino a c i d s , g l y c o l y t i c and K r e b s c y c l e i n t e r m e d i a t e s h a v e b e e n i m p l i c a t e d i n t h e c o n t r o l o f p y r u v a t e k i n a s e f r o m v a r i o u s t i s s u e s . I n o r d e r t o s e e w h i c h , i f a n y , i n t r a c e l l u l a r compounds c o u l d f u r t h e r m o d u l a t e t h e a c t i v i t y o f t h i s m u s c l e enzyme, t h e f o l l o w i n g compounds were t e s t e d a t t h e l e v e l s shown i n b r a c k e t s : t a u r i n e (1 -5 mM), a r g i n i n e (1-5 mM) , a l a n i n e ( 2 , 5, 10, and 20 mM) , p h e n y l a l a n i n e ( 1 ; 3, and 20 mM) , g l u t a m a t e (5 mM) -, p r o l i n e (5 mM) , v a l i n e (5 mM) , a s p a r a g i n e (5 mM) , a s p a r t a t e (5 mM) , l e u c i n e (10 mM), h i s t i d i n e (10 mM), l y s i n e (10 mM), c y s t e i n e (10 mM) t g l y c i n e (10 mM) , g l u t a m i n e (10 mM) , m e t h i o n i n e (10 mM) , i s o l e u c i n e (10 mM), t h r e o n i n e (10 mM), s e r i n e (10 raM a n d 20 mM), 3—PGA (1 mM) , AMP (1-4 mM) , t r y p t o p h a n (1-5 mM) , a c e t y l CoA (1 x 1 0 - * M) , f r u c t o s e - 6 - p h o s p h a t e (0.5 mM) , g l u c o s e - 6 - p h o s p h a t e (1 mM), NADH (0 . 0 5 - 0 . 2 mM) , c y c l i c AMP (0.05 mM), M g 2 c i t r a t e (1-5 mM) , M g^malate (1-5 mM) , < * - k e t o g l u t a r a t e (1-5 mM) , o C - g l y c e r o l p h o s p h a t e (1-5 mM), o x a l o a c e t a t e (0.01-0.6 mM) , 2,3 d i p h o s p h o g l y c e r a t e (1 mM), and CoA (0.1 -0.8 mM). T h e s e t e s t s were r u n b o t h a t s a t u r a t i n g and Km l e v e l s o f s u b s t r a t e s , and 107 0 . 3 0 h F i g . 1 0 . Arginine phosphate i n h i b i t i o n , e f f e c t on ADP a f f i n i t y . Experimental conditions are as i n Figure 9. (•) 2 mM a r g i n i n e phosphate; j ( 0 ) 1 . 2 mM arginine phosphate; and (A) c o n t r o l . -108 0.15 h 1 / P E P x l O 4 F i g . 1 1 . Arginine phosphate i n h i b i t i o n , e f f e c t on PEP a f f i n i t y . The e x p e r i -mental conditions are those given i n Figure 7 . ( • ) 4 mM a r g i n i n e phosphate; (0) 2 mM arginine phosphate; and (A) c o n t r o l . 109 were c a r r i e d o u t w i t h p u r i f i e d e n z y m e s . A l a n i n e , v a l i n e , s e r i n e , t r y p t o p h a n , M g 2 c i t r a t e , M g - m a l a t e , o ( — k e t o g l u t a r a t e , a nd ©<—glycerophosphate a f f e c t e d enzyme a c t i v i t y , a l t h o u g h none o f t h e s e was as' p o t e n t a s MgATP o r a r g i n i n e p h o s p h a t e . A l a n i n e a n d s e r i n e b o t h showed m i x e d c o m p e t i t i v e i n h i b i t i o n v e r s u s PEP, w i t h K i v a l u e s a r o u n d 30-mM* V a l i n e d e m o n s t r a t e d o n l y a weak, n o n — c o m p e t i t i v e i n h i b i t i o n . T r y p t o p h a n was c o m p e t i t i v e w i t h ADP ( F i g u r e 12) a n d showed a K i o f 1*6 mM. o < — g l y c e r o l p h o s p h a t e showed c o m p e t i t i v e i n h i b i t i o n w i t h b o t h ADP and PEP, h o w e v e r , t h e K i v a l u e s were w e l l o u t s i d e t h e p h y s i o l o g i c a l r a n g e f o r t h i s compound ( K i v e r s u s PEP = 20 mM; K i v e r s u s ADP = 40 mM). The i n h i b i t i o n due t o t h e t r i c a r b o x y l i c a c i d c y c l e i n t e r m e d i a t e s was more c o m p l e x t h a n t h a t due t o any o f t h e a b o v e i n h i b i t o r s . M a l a t e , t e s t e d a s M g — m a l a t e t o a v o i d c h e l a t i o n e f f e c t s , showed c o m p e t i t i v e i n h i b i t i o n v e r s u s b o t h s u b s t r a t e s ( K i v e r s u s PEP = 5 mM; K i v e r s u s ADP = 17 mM). K i n e t i c p l o t s f o r m a l a t e i n h i b i t i o n were l i n e a r , e x c e p t i n t h e r e g i o n o f h i g h m a l a t e a n d l o w s u b s t r a t e c o n c e n t r a t i o n s ( F i g u r e • 1 3 ) . I n h i b i t i o n by »<.— k e t o g l u t a r a t e showed s i m i l a r k i n e t i c s b u t i t s e f f e c t i v e n e s s was l o w e r ( K i v e r s u s PEP o f 8 mM; K i v e r s u s ADP o f 15 mM) . F o r n e i t h e r m a l a t e n o r < * — k e t o g l u t a r a t e was t h e r e any i n d i c a t i o n i n t h e k i n e t i c s t h a t t h e i n h i b i t o r was c o m p l e x i n g w i t h t h e s u b s t r a t e s . H i t h c i t r a t e a s an i n h i b i t o r , t h e d e v i a t i o n s f r o m l i n e a r i t y became more p r o n o u n c e d . B o t h M g - c i t r a t e a n d M g 2 c i t r a t e were t e s t e d a s i n h i b i t o r s . The k i n e t i c s o f M g - c i t r a t e i n h i b i t i o n i n d i c a t e d t h a t t h e i n h i b i t o r was c o m p l e x i n g w i t h b o t h ADP and PEP. I n t h i s c a s e , t h e i n h i b i t i o n showed b o t h c u r v e d L i n e w e a v e r - B u r k p l o t s a n d c u r v e d no 10 [Tryptophan] m M 12. Dixon p l o t o f tryptophan i n h i b i t i o n . A l l experiments were performed at 20°C i n 50 mM imid a z o l e - C l , pH 7.0, 75 mM KCl, 0.12 mM NADH, 4 mM MgCl2» and excess l a c t a t e dehydrogenase. (A) 0.12 mM ADP, 1.0 mM PEP; (•) 0.2 mM ADP, 1.0 mM PEP; (0) 1.2 mM ADP, 0.1 mM PEP; and (A) 1.2 mM ADP and 1.0 mM PEP. I l l 0 . 1 4 h _j i i i 0 1 0 2 0 3 0 Mg-Ma!ate mM F i q . 13. Dixon p l o t o f Mg-malate i n h i b i t i o n . Experimental c o n d i t i o n s are given i n the legend to Figure 12, (0) 2 mM PEP and 0.12 mM ADP; (0) 2 mM PEP and 0.2 mM ADP; (A) 0.12 mM PEP and 2 mM ADP; (±) 0.2 mM PEP and 2 mM ADP; and (a ) 2 mM PEP and 2 mM ADP. 112 D i x o n p l o t s . M g 2 c i t r a t e k i n e t i c s d i d n o t p r o d u c e c u r v e d L i n e w e a v e r — B u r k p l o t s , b u t D i x o n p l o t s o f M g 2 c i t r a t e i n h i b i t i o n v e r s u s PEP s t i l l showed c u r v e d l i n e s ( F i g u r e 1 4 ) . A c c o r d i n g t o Webb ( 1 9 6 3 ) , t h i s i s an i n d i c a t i o n t h a t more t h a n one m o l e c u l e o f c i t r a t e i s a f f e c t i n g PEP b i n d i n g . I n h i b i t i o n v e r s u s ADP i n d i c a t e d s i m p l e l i n e a r k i n e t i c s w i t h a K i o f 27 mM. A gauge o f M g 2 c i t r a t e * s e f f e c t i v e n e s s a s an i n h i b i t o r l i e s i n t h e s e f i g u r e s : 5 mM M g 2 c i t r a t e c a u s e s 2 3 % i n h i b i t i o n , 20 mM M g 2 c i t r a t e c a u s e s 5 0 % i n h i b i t i o n . T a b l e 9 a l l o w s a c o m p a r i s o n o f t h e e f f e c t i v e n e s s o f t h e s e v a r i o u s i n h i b i t o r s , a d d e d s i n g l y o r i n c o n c e r t , o n t h e a c t i v i t y o f t h e p u r i f i e d enzyme a t Km l e v e l s o f b o t h s u b s t r a t e s . S i n c e , i n t h i s c a s e , t h e Km v a l u e s a r e r e a s o n a b l e a p p r o x i m a t i o n s o f p h y s i o l o g i c a l l e v e l s o f b o t h PEP and ADP, t h i s a l l o w s e v a l u a t i o n o f t h e a c t i v i t y o f t h a enzyme i n v i v o . From t h i s t a b l e i t c a n be s e e n t h a t t h e i n h i b i t i o n due t o ATP was a d d i t i v e w i t h t h a t c a u s e d by a r g i n i n e p h o s p h a t e . F u r t h e r m o r e , t h e i n h i b i t i o n due t o t h e K r e b s c y c l e i n t e r m e d i a t e s was a d d i t i v e w i t h t h a t due t o MgATP and a r g i n i n e p h o s p h a t e . However, t h e e f f e c t i v e l e v e l s o f c i t r a t e , m a l a t e ; a n d c < — k e t o g l u t a r a t e were w e l l a b o v e t h o s e t o be e x p e c t e d i n t h e c y t o p l a s m . R e c e n t work w i t h r a b b i t m u s c l e p y r u v a t e k i n a s e i n d i c a t e s t h a t t h e r e v e r s a l o f t h e r e a c t i o n i s f e a s i b l e i n t h e t i s s u e (Dyson e t a l . , 1975; G i l e s e t a l . , 1 9 7 5 ) , and t h a t t h i s r e v e r s a l may - a c c o u n t f o r p r e v i o u s l y m e a s u r e d r a t e s o f g l y c o g e n s y n t h e s i s f r o m l a c t a t e i n f r o g a n d r a b b i t m u s c l e ( B e n d a l l & T a y l o r * 1 9 7 0 ) . T h e r e i s some e v i d e n c e t h a t c r u s t a c e a n m u s c l e h a s an i n c r e a s e d J i 1—: 1— O 10 20 30 Mg 2 -C i t ra te m M F i g . 14. Dixon p l o t o f Mg 2<ritrate i n h i b i t i o n . Experimental c o n d i t i o n s are given in the legend to Figure 12. {•) 2 mM PEP nad 0.12 mM ADP; (A) 2 mM PEP and 0.2 mM ADP; (0) 0.12 mM PEP and 2 mM ADP; (A) 0.2 mM PEP and 2 mM ADP; (•) 2 mM PEP and 2 mM ADP. TABLE 9 E f f e c t i v e n e s s o f a c t i v a t o r s and i n h i b i t o r s . -4 A l l experiments except the l a s t two were done with 2 x 10 M PEP, -4 -4 2 x 10 M ADP, 75 mM KCI, 1 x 10 M NADH, 4 mM MgCl 2, with excess l a c t a t e dehydrogenase (Sigma) i n 50 mM imidaz o l e - C l , pH 7.0. The l a s t two were the same except f o r a PEP concentration o f 4 x 10 5M and an ADP concentration o f 2 mM. A c t i v i t y was measured as AOD 3 4 Q/min. A l l values are the mean o f three determinations. Treatment % Control Control 100 5 mM MgATP 47 5 mM MgATP + 0.05 mM FDP 58 5 mM MgUTP 94 5 mM arginine phosphate 72 5 mM arginine phosphate + 0.05 mM FDP 72 10 mM arginine phosphate 57 .5 mM M g 2 c i t r a t e 78 5 mM M g 2 c i t r a t e + 0.05 mM FDP 78 5 mM a-ketoglutarate 80 5 mM Mg-malate 80 1 mM tryptophan 73 5 mM a-glycerol-phosphate 88 5 mM MgATP + 5 mM arginine phosphate 27 5 mM MgATP + 5 mM M g 2 c i t r a t e 35 5 mM MgATP + 5 mM ketoglutarate 33 5 mM MgATP + 5 mM ketoglutarate + 5 mM M g 2 c i t r a t e 27 5 mM MgATP .+ 5 mM ketoglutarate + 5 mM M g 2 c i t r a t e + 5 mM arginine phosphate 18 5 x 10~5M acetylCoA 102 -4 1 x 10 M UDP-acetyl glucosamine 116 5 X 10 _ 5M FDP 130 115 g l u c o n e o g e n i c p o t e n t i a l c o m p a r e d t o o t h e r i n v e r t e b r a t e and v e r t e b r a t e m u s c l e . However, the k i n e t i c c h a r a c t e r i s t i c s o f t h i s m u s c l e p y r u v a t e k i n a s e w o u l d n o t p e r m i t a p p r e c i a b l e f l u x t h r o u g h t h e l o w l e v e l s o f t h e b y p a s s enzymes. An e f f e c t i v e r e v e r s a l o f t h e r e a c t i o n m i g h t s i g n i f i c a n t l y augment g l u c o n e o g e n i c f l u x . M e a s u r e m e n t s o f t h e r e v e r s e r e a c t i o n ( r e s u l t s shown i n T a b l e 10) i n d i c a t e t h a t - a t s a t u r a t i n g p y r u v a t e (10 mM) and a t s a t u r a t i n g MgATP (2 mM), t h e m a x i m a l v e l o c i t y o f t h e r e a c t i o n d i d n o t r i s e a bove 0.5% o f t h e m a x i m a l v e l o c i t y o f t h e f o r w a r d r e a c t i o n . R e c e n t work w i t h r a t l i v e r p y r u v a t e k i n a s e showed t h a t a l a n i n e and ATP, w h i c h a r e b o t h a l l o s t e r i c i n h i b i t o r s , s t a b i l i z e t h e enzyme a g a i n s t t h e r m a l d e n a t u r a t i o n (Van B e r k e l e t a l . , 1 9 7 5 ) . S i n c e t h e i s o e l e c t r i c p o i n t a n d t h e e l e c t r o p h o r e t i c m o b i l i t y o f t h i s c r u s t a c e a n m u s c l e enzyme a r e more l i k e t h o s e o f t h e mammalian l i v e r enzyme t h a n t h e o t h e r mammalian f o r m s , and s i n c e a v a r i e t y o f m e t a b o l i t e s a f f e c t t h e a c t i v i t y o f t h e enzyme, I c h a r a c t e r i z e d t h e t h e r m a l d e n a t u r a t i o n b e h a v i o r o f t h e p u r i f i e d enzyme ( F i g u r e 1 5 ) . I n c o n t r a s t t o t h e b e h a v i o r o f t h e mammalian l i v e r e nzyme, ATP a c c e l e r a t e d t h e d e n a t u r a t i o n o f t h e enzyme. T h i s o c c u r r e d when t h e enzyme was i n c u b a t e d w i t h ATP a l o n e , a s w e l l a s w i t h ATP and p o s s i b l e p r o t e c t a n t s l i k e ADP, PEP, and FDP. C l o s e e x a m i n a t i o n o f t h e d e n a t u r a t i o n c u r v e s i n d i c a t e s t h a t i n t h e f i r s t 15 m i n u t e s , t h e p r e s e n c e o f PEP, ADP, a n d FDP • i n • c o m b i n a t i o n , p r e v e n t e d t h e a c c e l e r a t e d d e n a t u r a t i o n c a u s e d by ATP. However, a f t e r 20 m i n u t e s , t h i s c o m b i n a t i o n f o l l o w e d t h e same p a t t e r n as a l l t h e o t h e r ATP c o n t a i n i n g i n c u b a t i o n s . S i n c e a l a n i n e i s o n l y a weak i n h i b i t o r , I d i d n o t t e s t i t s e f f e c t on t h e d e n a t u r a t i o n . TABLE 10 Pyruvate kinase r e v e r s a l . The v e l o c i t y o f the reverse r e a c t i o n i s expressed i n AOD^ 4Q / 2 0 minutes.. The V , , ,. f o r the i d e n t i c a l quantity of enzyme p r o t e i n used i n the max(forward) r e v e r s a l experiments was 8.0 AOD^Q/minute. A l l values are the mean of at l e a s t three determinations, and were the d i f f e r e n c e between the r e a c t i o n r a t e i n the presence of a l l substrates, pyruvate kinase, PEP carboxylase and malate dehydrogenase and the sum of the rate shown by the two c o n t r o l s . The controls were (1) substrates and pyruvate kinase alone as w e l l as (2) substrates and coupling enzymes (PEP carboxylase and malate dehydrogenase) A l l r e a c t i o n mixtures contained'75 mM KCl except where i n d i c a t e d . V / V b max(forward) 2 mM pyruvate 2 mM MgATP 0.6 0.00375 4 mM MgATP 2 mM pyruvate 0.5 0.0031 2 mM MgATP 10 mM pyruvate 0.8 0.005 4 mM MgATP 10 mM pyruvate 0.6 0.00375 2 mM MgATP 10 mM pyruvate - KCl 0.02 0.000125 2 mM ATP 10 mM pyruvate - Mg 0.03 0.0001875 1117 20 40 60 80 Denaturation Time in Minutes Figure 15 Thermal denaturation of C_. magister muscle pyruvate kinase. o Experiments were run at 40 C under the i n d i c a t e d i n c u b a t i o n con^-d i t i o n s . Enzyme a c t i v i t y was measured under s a t u r a t i n g c o n d i t i o n s of substrates and cof a c t o r s . (©) c o n t r o l ; ( ° ) 4 mM MqCl^ and 4 mM ATP; (A) 0.1 mM FDP; ( & ) 0.1 mM FDP, 4 mM ATP, and 4 mM MgCl (B) 2 mM PEP; (0) 2 mM ADP and 2 mM PEP; U) 2 mM ADP, 2 mM PEP, and 0.1 mM FDP; (• ) 2 mM ADP, 2 mM PEP, 0.1 mM FDP, 4 mM MgCl 2, and 4 mM ATP; (X) 75 mM KCl. 118 DISCUSSION A l t h o u g h c r u s t a c e a n - m u s c l e g o e s t h r o u g h r a t h e r marked m e t a b o l i c c h a n g e s a s t h e a n i m a l g o e s t h r o u g h t h e m o l t c y c l e , t h e s e c h a n g e s a r e n o t r e f l e c t e d by c h a n g e s i n t h e i s o z y m e o f p y r u v a t e k i n a s e p r e s e n t i n t h e t i s s u e . ' The p y r u v a t e k i n a s e w h i c h i s f o u n d i n t h e m u s c l e o f - C a n c e r m a g i s t e g h a s c o m p l e x r e g u l a t o r y p r o p e r t i e s w h i c h w o u l d a l l o w c a r e f u l m o d u l a t i o n o f i t s a c t i v i t y u n d e r a v a r i e t y o f m e t a b o l i c c o n d i t i o n s . A l t h o u g h i t i s s u b j e c t t o a l l o s t e r i c i n h i b i t i o n , i t d o e s n o t show c o o p e r a t i v i t y o f s u b s t r a t e b i n d i n g . I t s h i g h a f f i n i t y f o r s u b s t r a t e s , h i g h s e n s i t i v i t y t o i n h i b i t i o n , a s w e l l a s i t s a c t i v a t i o n by FDP, make t h e k i n e t i c c h a r a c t e r i s t i c s o f t h e enzyme s i m i l a r t o t h o s e o f t h e mammalian M2 o r K t y p e p y r u v a t e k i n a s e (Van B e r k e l e t a l . , 1 9 7 3 a ) . However, t h i s m u s c l e enzyme showed no i n d i c a t i o n o f t h e EDTA, M g + + , o r FDP i n d u c e d c h a n g e s i n c o n f o r m a t i o n w h i c h h a v e been f o u n d f o r mammalian M2 enzyme ( P o g s o n , 1968; Van B e r k e l , 1974) and i s more s e n s i t i v e t o MgATP i n h i b i t i o n t h a n t h e mammalian M2 enzyme ( V a n B e r k e l , 1 9 7 4 ) . I n v i v o t h i s m u s c l e enzyme i s p r o b a b l y r e g u l a t e d p r i m a r i l y by ATP, a r g i n i n e p h o s p h a t e , and FDP l e v e l s . I n c r a b a n d l o b s t e r m u s c l e , r e s t i n g ATP l e v e l s a r e b e t w e e n 4 and 6 mM ( F r e e d * 1971; B e i s S Newsholme, 1 9 7 5 ) . A r g i n i n e p h o s p h a t e l e v e l s a s h i g h a s 33 mM h a v e b e e n m e a s u r e d i n l o b s t e r m u s c l e ( B e i s & Newsholme, 1 9 7 5 ) . T h e s e v a l u e s a r e c o n s i d e r a b l y a b o v e t h e r e s p e c t i v e K i v a l u e s f o r t h e two i n h i b i t o r s . C o n s i d e r i n g t h a t PEP l e v e l s a r e p r o b a b l y b e l o w t h e Km v a l u e , and t h a t ADP l e v e l s a r e - n e a r t h e Km v a l u e ( B e i s S Newsholme, 1 9 7 5 ) , t h e s e h i g h l e v e l s o f ATP and 119 a r g i n i n e p h o s p h a t e w o u l d l e a d t o a s t r o n g i n h i b i t i o n o f t h e enzyme i n r e s t i n g m u s c l e . I f any g l u c o n e o g e n e s i s were t o o c c u r i n m u s c l e , i t w o u l d l o g i c a l l y o c c u r when t h e m u s c l e i s a t r e s t . As t h e m u s c l e i s a c t i v a t e d and c o n t r a c t i o n b e g i n s , ATP i s h y d r o l y z e d t o ADP and i n o r g a n i c p h o s p h a t e . The ADP i s t h e n r e c o n v e r t e d t o ATP by t h e a c t i o n o f a r g i n i n e p h o s p h o k i n a s e a n d a r g i n i n e p h o s p h a t e l e v e l s d r o p . S u c h a d r o p i n a r g i n i n e p h o s p h a t e w o u l d s e r v e t o d a i n h i b i t t h e p y r u v a t e k i n a s e r e a c t i o n . F u r t h e r d e i n h i b i t i o n w o u l d come t h r o u g h FDP a c t i v a t i o n . T h i s w o u l d s e r v e a s a f e e d — f o r w a r d mechanism whereby i n c r e a s e d l e v e l s o f an e a r l y g l y c o l y t i c i n t e r m e d i a t e s e r v e t o i n c r e a s e f l u x t h r o u g h a l a t e r r a t e l i m i t i n g g l y c o l y t i c r e a c t i o n . S i n c e FDP i s one o f t h e m a j o r a c t i v a t o r s o f p h o s p h o f r u c t o k i n a s e ( T o r n h e i m & L o w e n s t e i n , 1 9 7 5 ) , t h i s mechanism s e r v e s t o i n t e g r a t e f l u x t h r o u g h t h e t w o p o r t i o n s o f g l y c o l y s i s . F u r t h e r i n t e g r a t i o n i s a c h i e v e d t h r o u g h a d e n y l a t e c o u p l i n g , a n d i n h i b i t i o n by h i g h l e v e l s o f t h e s t o r a g e p h o s p h a g e n and ATP. C i t r a t e , m a l a t e , and < * - k e t o g l u t a r a t e may p l a y some r e g u l a t o r y r o l e i n v i v o . However, t h e e f f e c t i v e l e v e l s o f t h e s e compounds a r e u n l i k e l y t o o c c u r i n t h i s m a i n l y g l y c o l y t i c t i s s u e ( H o c h a c h k a e t ' a l . , • 1 9 7 1 ; F r e e d , 1971; W i l l i a m s o n , 1965; W i l l i a m s o n e t a l . , 1 9 6 9 ) . Any e f f e c t s w h i c h w o u l d o c c u r w o u l d have t h e g e n e r a l e f f e c t o f m o d e r a t i n g p y r u v a t e k i n a s e a c t i v i t y a t t i m e s o f h i g h K r e b s c y c l e f l u x . T h i s w o u l d p r e v e n t an o v e r s h o o t o f p y r u v a t e a n d e v e n t u a l l y a c e t y l CoA f o r m a t i o n . S i m i l a r l y , an e f f e c t by < x - g l y c e r c l p h o s p h a t e w o u l d o c c u r v e r y r a r e l y , a n d t h e n o n l y i n s i t u a t i o n s o f e x t r e m e a n a e r o b i o s i s when t h e t i s s u e has l o s t t h e c a p a c i t y t o b a l a n c e r e d o x t h r o u g h t h e 120 l a c t a t e d e h y d r o g e n a s e r e a c t i o n . I n t h i s c o n t e x t , t h e o x i d a t i o n o f o C - g l y c e r o l p h o s p h a t e by t i s s u e h o mogenates f r o m v a r i o u s o r g a n s o f Cambarus a f f i n i s i s o f i n t e r e s t ( K e l l e r , 1 9 6 5 ) . However, t h e r e was- no i n d i c a t i o n o f u t i l i z a t i o n o f t h i s s u b s t r a t e by m u s c l e , s i n c e m u s c l e homogenates showed no m e a s u r a b l e o x y g e n u p t a k e ( K e l l e r , 1 9 6 5 ) . T r y p t o p h a n l e v e l s i n c r u s t a c e a n m u s c l e a r e l o w ( S c h o f f e n i e l s S G i l l e s , 1 9 7 0 ) , and t h e r e - i s no e v i d e n c e o f f l u c t u a t i o n s w h i c h a r e c o r r e l a t e d w i t h e n e r g y m e t a b o l i s m . I t s e f f i c i e n c y a s a n i n h i b i t o r i s p r o b a b l y b a s e d on a s t r u c t u r a l s i m i l a r i t y w i t h ADP. The m i x e d c o m p e t i t i v e i n h i b i t i o n w h i c h b o t h a l a n i n e and s e r i n e e v i d e n c e t o w a r d PEP i s p r o b a b l y o f s m a l l 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 s i n c e l a r g e f l u c t u a t i o n s w o u l d be n e e d e d f o r an e f f e c t . The r e v e r s a l o f t h e m u s c l e p y r u v a t e k i n a s e f r o m C a n c e r mag.ist.er amounted t o l e s s t h a n 0.5% o f t h e f o r w a r d r e a c t i o n a t s a t u r a t i n g l e v e l s o f p y r u v a t e (10 mM) and ATP (2 mM) . ATP l e v e l s l i e b e t w e e n U and 6 mM i n r e s t i n g m u s c l e ( B e i s S Newsholme, 1 9 7 5 ) , and a t t h e s e c o n c e n t r a t i o n s ATP s l i g h t l y i n h i b i t e d t h e r e v e r s a l o f t h e r e a c t i o n . P y r u v a t e l e v e l s r a r e l y r i s e ' a b o v e 0.1 mM i n ' e i t h e r c r u s t a c e a n ' o r mammalian m u s c l e ( H o c h a c h k a e t a l . , 1 9 7 1 ; F r e e d , 1S71; W i l l i a m s o n , 1 9 6 5 ) , and t h u s a p p r o a c h o n l y 1% o f t h e l e v e l s n e eded f o r s a t u r a t i o n . T h e r e f o r e i t i s u n l i k e l y t h a t t h e r e v e r s a l a p p r o a c h e s more t h a n 0.005% o f t h e f o r w a r d r e a c t i o n r a t e . The r a b b i t m u s c l e enzyme showed a r e v e r s e - r e a c t i o n w i t h s i m i l a r o p t i m a l c o n d i t i o n s w h i c h amounted t o 5% o f t h e f o r w a r d r a t e (Dyson e t a l . , 1975; G i l e s e t a l . , 1 9 7 5 ) . S i n c e t h e l e v e l s o f p y r u v a t e k i n a s e i n mammalian m u s c l e a r e h i g h , p y r u v a t e k i n a s e r e v e r s a l may a c c o u n t f o r t h e 121 g l u c o n e o g e n e s i s w h i c h h a s been o b s e r v e d i n r a b b i t m u s c l e ( B e n d a l l & T a y l o r , 1 9 7 0 ) . However, i t seems u n l i k e l y t h a t g l u c o n e o g e n e s i s o c c u r s v i a t h i s r o u t e i n c r u s t a c e a n m u s c l e . I n t e r m o l t m u s c l e i n C a n c e r m a c j i s t e r l a c k s a f u n c t i o n a l e n z y m a t i c b y p a s s f o r t h e p y r u v a t e k i n a s e r e a c t i o n . W h i l e f r e s h m o l t m u s c l e h a s b o t h p y r u v a t e c a r b o x y l a s e and p h o s p h o e n o l p y r u v a t e c a r b o x y -k i n a s e ( T a b l e 1 ) , t h e l e v e l s a r e l o w c o m p a r e d t o p y r u v a t e k i n a s e l e v e l s . P y r u v a t e c a r b o x y l a s e e x h i b i t s o p t i m a l a c t i v i t y a t 2 mM p y r u v a t e (Ashman e t a l . , 1 9 7 2 ) ; h e n c e i t s f u n c t i o n i s f e a s i b l e u n d e r p h y s i o l o g i c a l c o n d i t i o n s . However, f o r any n e t g l u c o n e o g e n i c c a r b o n f l u x t o o c c u r , t h e p y r u v a t e k i n a s e r e a c t i o n must be i n h i b i t e d t o l e s s t h a n 0.2% o f i t s m a x i m a l a c t i v i t y . S i n c e 5 mM ATP and 5 mM a r g i n i n e p h o s p h a t e o n l y i n h i b i t t h e enzyme by 7 3 % , g l u c o n e o g e n i c f l u x t h r o u g h t h i s r e a c t i o n l o c u s i s u n l i k e l y . 122 P a r t I V . H y p o d e r m a l P y r u v a t e K i n a s e INTRODUCTION As o u t l i n e d b e f o r e , t h e h y p o d e r m i s i s most c l o s e l y a s s o c i a t e d w i t h t h e p r o d u c t i o n o f t h e new e x o s k e l e t o n . D u r i n g t h e e a r l y p o s t m o l t p e r i o d , t h e h y p o d e r m i s c a r r i e s o u t c o n s i d e r a b l e g l u c o n e o g e n e s i s f o r c h i t i n s y n t h e s i s . H i g h r a t e s o f c a r b o h y d r a t e o x i d a t i o n s u p p o r t t h i s b i o s y n t h e t i c a c t i v i t y ( S p e c k & U r i c h , 1 9 7 1; Speck e t a l . , 1 9 7 2 ) . F o r any g l u c o n e o g e n e s i s t o o c c u r , p y r u v a t e k i n a s e must be e f f e c t i v e l y i n h i b i t e d ; h o w e v e r , d u r i n g c a r b o h y d r a t e u t i l i z a t i o n , i t f o r m s a g l y c o l y t i c c o n t r o l s i t e . T h u s , t h e c o n t r o l o f p y r u v a t e k i n a s e i s c e n t r a l t o h y p o d e r m a l m e t a b o l i s m . The f r e s h m o l t h y p o d e r m i s shows b o t h a c o n s i d e r a b l e i n c r e a s e i n p y r u v a t e k i n a s e a c t i v i t y ( T a b l e 1) and a marked i n c r e a s e i n o x y g e n c o n s u m p t i o n ( S k i n n e r , 1962; M c W h i nnie e t a l . , 1972; B u l n h e i m , 1 9 7 4 ) . I n - c r a y f i s h , a l l t h e ' c a r b o n u t i l i z e d f o r c h i t i n s y n t h e s i s a n d e n e r g y m e t a b o l i s m d u r i n g t h e l a t e p r e m o l t and e a r l y p o s t m o l t p e r i o d i s d e r i v e d f r o m h e x o s e u n i t s r e s o r b e d f r o m t h e o l d e x o s k e l e t o n ( S p e c k & U r i c h ; 1 9 7 1 ) . I n l a r g e r m a r i n e c r u s t a c e a n s , s u c h a s C a n c e r m a g i s t e r , t h e ~ i n c r e a s e d o x y g e n c o n s u m p t i o n d u r i n g l a t e p r e m o l t and e a r l y p o s t m o l t i s n o t s o l e l y due t o t h e u t i l i z a t i o n o f c a r b o h y d r a t e s , s i n c e l i p i d s , and p o s s i b l y p r o t e i n s may be m o b i l i z e d a t t h i s t i m e a s w e l l (Renaud, 1949; T r a v i s , 1 9 5 5 a ; H e a t h S B a r n e s , 1 9 7 0 ) . H o w e v e r , c a r b o h y d r a t e s f o r m a m a j o r 123 s u b s t r a t e d u r i n g t h i s p e r i o d , and t h u s d o w n h i l l f l u x t h r o u g h t h e p y r u v a t e k i n a s e r e a c t i o n i s m a n d a t o r y . D u r i n g t h i s p e r i o d c h i t i n s y n t h e s i s f r o m a mino a c i d s and i n t e r m e d i a r y c o m p o u n d s , s u c h a s l a c t a t e , - i s m a x i m a l a s w e l l ( H e r z - H u b n e r & U r i c h , 1973; H e r z - H u b n e r e t a l . , 1 9 7 3 ) . T h u s , t h e m e t a b o l i c c o n t r o l r e q u i r e m e n t s f a c e d by t h e p y r u v a t e k i n a s e r e a c t i o n a r e s t r i n g e n t s i n c e i t must be a b l e t o s w i t c h f r o m v i r t u a l l y t o t a l l y i n h i b i t e d d u r i n g p e r i o d s o f g l u c o n e o g e n e s i s t o h i g h r a t e s o f c a t a l y s i s w i t h i n s h o r t p e r i o d s o f t i m e . S i n c e u n d e r s t a n d i n g o f t h i s c o n t r o l s i t e i s m a n d a t o r y f o r a d e s c r i p t i o n o f b o t h g l u c o n e o -g e n i c and g l y c o l y t i c c o n t r o l m e c h a n i s m s i n t h e h y p o d e r m i s , I s t u d i e d t h e r e g u l a t o r y p r o p e r t i e s o f t h e p y r u v a t e k i n a s e . S i n c e t h e h y p o d e r m i s u n d e r g o e s marked p h y s i o l o g i c a l c h a n g e s a s a f u n c t i o n o f t h e m o l t c y c l e ( S k i n n e r , 1 9 6 2 ) ; a c o m p a r i s o n o f t h e p r o p e r t i e s o f t h e f r e s h m o l t and i n t e r m o l t h y p o d e r m a l e n z y m e s was c a r r i e d o u t a s w e l l . METHODS P r e p a r a t i o n o f h y p o d e r m a l p y r u v a t e k i n a s e f o l l o w e d t h e m ethods d e s c r i b e d i n P a r t I I I f o r t h e m u s c l e enzyme. I n i t i a l h o m o g e n i z a t i o n p r o c e e d e d i n two r a t h e r t h a n f o u r v o l u m e s . Column c h r o m a t o g r a p h y on DEAE S e p h a d e x and c e l l u l o s e p h o s p h a t e l e d t o a p p r o x i m a t e l y 100 f o l d p u r i f i c a t i o n , w i t h s p e c i f i c a c t i v i t y o f 50 u n i t s / m g p r o t e i n f r o m t h e c e l l u l o s e p h o s p h a t e c o l u m n . 1 2 4 Measurement o f Bound F r u c t o s e J. x6 D i p h o s p h a t e The method u s e d was e s s e n t i a l l y t h a t o f B e r g m e y e r ( 1 9 6 5 ) . Two ml o f enzyme f r o m t h e peak t u b e s o f t h e DEAE S e p h a d e x a nd t h e c e l l u l o s e p h o s p h a t e c o l u m n s were c a l i b r a t e d f o r enzyme a c t i v i t y , p l a c e d i n b o i l i n g w a t e r f o r 2 m i n u t e s , c o o l e d i n i c e , and c e n t r i f u g e d f o r 10 m i n u t e s a t 27,000 g. The s u p e r n a t a n t was us e d d i r e c t l y f o r t h e a s s a y o f f r u c t o s e d i p h o s p h a t e ; v i a t h e a l d o l a s e , t r i o s e p h o s p h a t e i s o m e r a s e and < * - g l y c e r o l p h o s p h a t e d e h y d r o g e n a s e r e a c t i o n s . The c o n c e n t r a t i o n o f NADH i n t h e c u v e t t e was 0.08 mn. T h i s method o f m e a s u r i n g f r u c t o s e d i p h o s p h a t e y i e l d s two m o l e c u l e s o f o W g l y c e r o l p h o s p h a t e and NAD p e r f r u c t o s e d i p h o s p h a t e , t h u s a l l AOD340 v a l u e s were d i v i d e d by 2 t o c a l c u l a t e t h e o r i g i n a l c o n c e n t r a t i o n o f f r u c t o s e d i p h o s p h a t e . Enzyme A s s a y s P y r u v a t e k i n a s e a c t i v i t y was measured f o l l o w i n g p r o c e d u r e s g i v e n i n P a r t I I I . RESULTS JiS£tro£horesis and E l e c t r o f o c u s i n g E x t e n s i v e e l e c t r o p h o r e t i c c o m p a r i s o n o f i n t e r m o l t a n d f r e s h l y m o l t e d h y p o d e r m a l p y r u v a t e k i n a s e s r e v e a l e d no c o n s i s t e n t d i f f e r e n c e s . H o s t o f t e n t h e i r m i g r a t i o n v e l o c i t i e s were' t h e same, b u t i n .5 o u t o f 25 e x p e r i m e n t s , t h e i n t e r m o l t h y p o d e r m a l enzyme showed s l i g h t l y f a s t e r m i g r a t i o n . I n one 125 c a s e , ' t h e f r e s h m o l t h y p o d e r m a l enzyme was f a s t e r . E l e c t r o p h o r e t i c c o m p a r i s o n o f t h a i n t e r m o l t and f r e s h m o l t m u s c l e and h y p o d e r m a l p y r u v a t e k i n a s e s showed o n l y s l i g h t m i g r a t i o n a l d i f f e r e n c e s w i t h t h e m u s c l e enzyme s h o w i n g somewhat f a s t e r a n o d a l m i g r a t i o n . S e v e r a l d i f f e r e n t b u f f e r s y s t e m s were u s e d t o t e s t o u t t h e p o s s i b i l i t i e s c o m p l e t e l y , b u t u n d e r no c i r c u m s t a n c e s c o u l d p r o u n o u n c e d d i f f e r e n c e s b e t w e e n m o l t s t a g e s b e • d e m o n s t r a t e d . I n a l l c a s e s p y r u v a t e k i n a s e a c t i v i t y was c o n f i n e d t o one band. E l e c t r o f o c u s i n g a l s o f a i l e d t o r e v e a l any d i f f e r e n c e s b e t w e e n t h e f r e s h m o l t and t h e i n t e r m o l t h y p o d e r m a l p y r u v a t e k i n a s e s . F r e s h m o l t h y p o d e r m a l p y r u v a t e k i n a s e showed an i s o e l e c t r i c p o i n t o f 6.1, w h e r e a s i n t e r m o l t h y p o d e r m a l p y r u v a t e k i n a s e showed an i s o e l e c t r i c p o i n t o f 6.005. B o t h i n t e r m o l t a n d f r e s h m o l t p y r u v a t e k i n a s e s f o c u s e d i n s h a r p , s y m m e t r i c a l p e a k s . T h u s , I c o n c l u d e d t h a t t h e p y r u v a t e k i n a s e s p r e s e n t i n i n t e r m o l t a nd f r e s h m o l t h y p o d e r m i s have t h e same n e t c h a r g e a n d a r e p r o b a b l y t h e same p r o t e i n . The m u s c l e e n z y m e s , i n c o n t r a s t , show i s o e l e c t r i c p o i n t s o f 6.4, i n d i c a t i n g some c h a r g e d i f f e r e n c e , C a t a l y t i c and R e g u l a t o r y C h a r a c t e r i s t i c s I n i n i t i a l e x p e r i m e n t s , i t was f o u n d t h a t t h e p r e s e n c e o f EDTA a n d magnesium d u r i n g t h e p r e p a r a t i o n and s t o r a g e o f t h e enzyme had p r o f o u n d e f f e c t s upon t h e c a t a l y t i c c h a r a c t e r i s t i c s o f t h e enzyme.' I n t h e a b s e n c e o f EDTA, t h e enzyme showed a l o w e r e d a f f i n i t y f o r i t s s u b s t r a t e , p h o s p h o e n o l p y r u v a t e , and was 126 more s e n s i t i v e t o a c t i v a t i o n by f r u c t o s e d i p h o s p h a t e . The p r e s e n c e o f EDTA p r e v e n t e d t h e s e c h a n g e s i n PEP and FDP a f f i n i t i e s . W i t h C a r c i n u s maenas h e p a t o p a n c r e a s p y r u v a t e k i n a s e , EDTA was f o u n d t o m i m i c t h e k i n e t i c e f f e c t s o f FDP ( G i l e s e t a l . , 1 9 7 6 ) . I n c o n t r a s t , EDTA p r o m o t e s t h e t r a n s i t i o n o f b o t h t h e mammalian B2 and L t y p e p y r u v a t e k i n a s e s i n t o a l o w a f f i n i t y s t a t e ( B a i l e y e t a l . , 1968; Badwey & W e s t h e a d , 1975; P o g s o n , 1968; Van B e r k e l , 1 9 7 4 ) . The k i n e t i c s o f t h e m u s c l e enzyme f r o m C a n c e r m a g i s t e r were n o t a f f e c t e d by t h e p r e s e n c e o r a b s e n c e o f EDTA. I n - f u r t h e r e x p e r i m e n t s , I s o u g h t t o d e t e r m i n e w h e t h e r t h e s e EDTA p r o m o t e d e f f e c t s were a r t i f a c t s o r c o n f o r m a t i o n a l c h a n g e s o f p o t e n t i a l r e g u l a t o r y s i g n i f i c a n c e . F u r t h e r p u r i f i c a t i o n i n d i c a t e d t h a t p y r u v a t e k i n a s e f r o m i n t e r m o l t and f r e s h m o l t h y p o d e r m i s c o u l d e x i s t i n two f u n c t i o n a l f o r m s . P u r i f i c a t i o n on DEAE S e p h a d e x s t a b i l i z e d t h e enzyme i n a h i g h a f f i n i t y f o r m w h i c h I w i l l c a l l PK I . I n a l l work w i t h t h i s f o r m o f t h e enzyme I i n c l u d e d b o t h 3 mM EDTA a n d 4 mM MgC12, s i n c e M g + + h a s i m p o r t a n t e f f e c t s upon some p y r u v a t e k i n a s e s (Van B e r k e l , 1 9 7 4 ) . P u r i f i c a t i o n o f t h e enzyme upon c e l l u l o s e - p h o s p h a t e p r o m o t e d t h e t r a n s i t i o n o f the enzyme i n t o a n o t h e r f u n c t i o n a l f o r m w i t h a l o w e r e d PEP a f f i n i t y (PK I I ) . T h e - s t a b i l i t y and c l o s e s i m i l a r i t y o f t h e two f o r m s i n a l l b u t a few k i n e t i c c r i t e r i a ; a s w e l l a s t h e l a c k o f i n f o r m a t i o n p o i n t i n g t o s e p a r a t e enzymes w i t h i n t h e h y p o d e r m i s , l e d me t o assume t h a t t h e s e a r e i n t e r c o n v e r t i b l e f o r m s o f - t h e same p r o t e i n . F u r t h e r m o r e , i n c u b a t i o n o f PK I I w i t h f r u c t o s e d i p h o s p h a t e ( s e e below) p r o d u c e d an enzyme w i t h c h a r a c t e r i s t i c s o f PK I , w h i l e e x t e n s i v e d i a l y s i s o f PK I i n t h e a b s e n c e o f EDTA 127 a n d ng** l e d t o an enzyme w i t h a l o w e r e d a f f i n i t y f o r PEP and an i n c r e a s e d s e n s i t i v i t y t o f r u c t o s e d i p h o s p h a t e . M u s c l e p y r u v a t e k i n a s e d i d n o t show t h e s e e f f e c t s . T h e s e r e s u l t s s u p p o r t t h e c o n c l u s i o n t h a t t h e s e e f f e c t s a r e n o t a r t i f a c t s , b u t r e p r e s e n t i n t e r c o n v e r t i b l e f o r m s o f t h e enzyme w h i c h may be o f r e g u l a t o r y s i g n i f i c a n c e i n v i v o . T h e r e f o r e , t h e k i n e t i c b e h a v i o r o f b o t h PK I and PK I I was c o m p l e t e l y c h a r a c t e r i z e d i n f u r t h e r s t u d i e s , A l l t h e k i n e t i c a n d p u r i f i c a t i o n e x p e r i m e n t s were p e r f o r m e d on b o t h i n t e r m o l t and- f r e s h m o l t h y p o d e r m a l p y r u v a t e k i n a s e s . S i n c e t h e r e s u l t s were t h e same f o r b o t h t i s s u e s o n l y o n e s e t o f r e s u l t s w i l l be p r e s e n t e d . The c l o s e a g r e e m e n t o f t h e k i n e t i c r e s u l t s s u p p o r t s t h e c o n c l u s i o n t h a t d i f f e r e n t f o r m s o f p y r u v a t e k i n a s e a r e n o t s y n t h e s i z e d as t h a h y p o d e r m i s c h a n g e s d u r i n g t h e m o l t c y c l e . The l e v e l s o f p y r u v a t e k i n a s e i n t h e h y p o d e r m i s r i s e a s t h e a n i m a l g o e s f r o m i n t e r m o l t i n t o p r e m o l t . ( T a b l e 1 ) . T h u s , c o n t r o l a t t h i s r e a c t i o n l o c u s i s p a r t l y v e s t e d i n c h a n g i n g enzyme l e v e l s . I n common w i t h o t h e r p y r u v a t e k i n a s e s , t h e h y p o d e r m a l enzyme r e q u i r e d b o t h a m o n o v a l e n t and a d i v a l e n t c a t i o n f o r a c t i v i t y . B o t h f o r m s showed a h i g h e r a f f i n i t y f o r NH4 + (Km = 6;0-mM) t h a n f o r K+ (Km = 10 mfl) b u t t h e Vmax w i t h HH4.+ a s t h e a c t i v a t i n g c a t i o n was o n l y 38% o f t h a t w i t h K+ a s t h e a c t i v a t i n g c a t i o n . A t o p t i m a l K + c o n c e n t r a t i o n s , Na+ i n h i b i t e d c a t a l y s i s , s h o w i n g a K i v e r s u s PEP o f 40 mM and a K i v e r s u s K+ o f 85 mM. A t a p p r o x i m a t e l y Km l e v e l s o f K + , Na+ p r o v i d e d a s l i g h t a c t i v a t i o n - B o t h M n + + a n d Mg++ c o u l d a c t a s t h e a c t i v a t i n g d i v a l e n t c a t i o n f o r b o t h PK I and PK I I . M n + + showed a l o w e r Km 128 v a l u e (0.15 mM) t h a n d i d Mg** (0.6 mM). However, t h e Vraax w i t h Mh+* was o n l y 3 9 % o f t h a t w i t h Mg + + . C a * + was a c o m p e t i t i v e i n h i b i t o r v e r s u s b o t h o f t h e s e d i v a l e n t c a t i o n s , and showed a t e n f o l d h i g h e r e f f e c t i v e n e s s v e r s u s M g + + ( K i = 0.075 mM) t h a n v e r s u s Mn++ ( K i = 0 . 6 mM) . B o t h t h e h y p o d e r m a l PK I a n d PK I I show a b r o a d pH optimum f r o m pH 6.5 t o pH 8.0. S u b s t r a t e A f f i n i t i e s F o r b o t h PK I and PK I I , s u b s t r a t e s a t u r a t i o n c u r v e s f o r p h o s p h o p h o e n o l p y r u v a t e a n d ADP f o l l o w e d M i c h a e l i s - M e n t e n b e h a v i o u r . PK I showed a h i g h a f f i n i t y f o r p h o s p h o e n o l p y r u v a t e w i t h Km v a l u e s r a n g i n g between 0.09 mM and 0.18 mM ( F i g u r e 1 6 ) . I n t h i s c h a r a c t e r i s t i c t h e r e was some v a r i a t i o n b e t w e e n i n t e r m o l t and - f r e s h m o l t p y r u v a t e k i n a s e s ; t h e i n t e r m o l t enzyme g e n e r a l l y had Km v a l u e s b e t w e e n 0.1 mM and 0.18 mM, w h i l e t h e f r e s h m o l t enzyme had Km v a l u e s b e t w e e n 0.09 mM and 0.11 mM. S i m i l a r v a r i a t i o n s i n t h e s e p h o s p h o e n o l p y r u v a t e Km v a l u e s were f o u n d f o r t h e m u s c l e enzyme f r o m t h e s e a n i m a l s w F o r PK I , PEP a f f i n i t y d e c r e a s e d a s t h e pH was c h a n g e d f r o m pH 7*0. A f f i n i t y c u r v e s were h y p e r b o l i c a t pH v a l u e s o f 6.5,- 7.0, and 7.5, i n c o n t r a s t t o t h e mammalian M2 and L t y p e enzymes (Van B e r k e l e t a l . , 1 9 73a; S e u b e r t £•Schoner, 1971).- PK I I showed a much l o w e r a f f i n i t y f o r p h o s p h o e n o l p y r u v a t e ( F i g u r e 1 7 ) . Km v a l u e s r a n g e d f r o m 0.4-0.8 mM w i t h no p a r t i c u l a r v a r i a t i o n b etween i n t e r m o l t and f r e s h m o l t p r e p a r a t i o n s . By c o n t r a s t t o t h e PEP Km v a l u e s , t h e ADP Km v a l u e s were s i m i l a r f o r a l l p r e p a r a t i o n s . B o t h PK I and PK I I showed Km v a l u e s f o r ADP t h a t r a n g e d f r o m 0.2-0.3 mM. I n c o n t r a s t w i t h r e c e n t f i n d i n g s w i t h c r a b h e p a t o p a n c r e a s .129 1/PEP x 10 4 F i g . 16. Phosphoenolpyruvate a f f i n i t y of PK I. Assay conditions were as . follows: 50 mM imidazole-Cl, pH 7.0, 75 mM KC1, 4 mM MgCl 2, 0.1 mM -4 i NADH, PEP and ADP as shown. (A) 2 mM ADP, (A) 4 x 10 M ADP, -4 (•) 2 x 10 M ADP. 130 0.30 r 1 / P E P x 10 F i g . 1 7 . Phosphoenolpyruvate a f f i n i t y of PK II. Assay conditions as in the figure legend to Figure (A) 2 mM ADP, (0) 0.8 mM ADP, (•) 0.2 mM ADP. 131 p y r u v a t e k i n a s e ( G i l e s e t a l . , 1 9 7 6 ) , t h e a f f i n i t y f o r e a c h s u b s t r a t e was i n d e p e n d e n t o f v a r i a t i o n i n t h e c o n c e n t r a t i o n s o f c o s u b s t r a t e , i n d i c a t i n g a random b i n d i n g m echanism. UDP, GDP, a n d I D P a l l s e r v e d a s n u c l e o t i d e s u b s t r a t e s , b u t w i t h s u b s t a n t i a l l y l o w e r a f f i n i t i e s t h a n ADP; Km v a l u e s were 1.5 mM f o r UDP, 1.2 mM f o r I D P , and 0.9 mM f o r GDP. A c t i v a t o r s B o t h p y r u v a t e k i n a s e I a n d p y r u v a t e k i n a s e I I were s t r o n g l y a c t i v a t e d by l o w l e v e l s o f f r u c t o s e d i p h o s p h a t e ( F D P ) . The p r i m e e f f e c t o f f r u c t o s e d i p h o s p h a t e was t o f a c i l i t a t e t h e b i n d i n g o f PEP. F o r PK I I , t h e a d d i t i o n o f 0.05 mM FDP r e d u c e d t h e PEP Km f r o m 0.7 t o 0.15 mM ( F i g u r e 1 8 ) . These l e v e l s o f FDP a l s o r e v e r s e d MgATP i n h i b i t i o n f o r b o t h f o r m s . P y r u v a t e k i n a s e I showed a Ka f o r FDP o f 1.3 x 10-SM, w h i l e p y r u v a t e k i n a s e I I showed a much i n c r e a s e d a f f i n i t y f o r FDP w i t h Ka v a l u e o f 9 x 10~8M. PK I I t h u s b i n d s FDP 100 t i m e s a s s t r o n g l y a s PK I . I n c r u d e h o m o g e n a t e s o r ammonium s u l p h a t e f r a c t i o n s , U D P - a c e t y l -g l u c o s a m i n e (UDPAG), FDP a n d g l u c o s a m i n e - 6 - p h o s p h a t e - • were e q u a l l y good a c t i v a t o r s . T h e s e a c t i v a t o r s a l l r e v e r s e d t h e i n h i b i t i o n due t o MgATP. W i t h t h e p u r i f i e d e n z y m e s , FDP r e m a i n e d a p o t e n t a c t i v a t o r , w h i l e UDPAG and g l u c o s a m i n e - 6 -p h o s p h a t e p r o d u c e d o n l y 25% o f t h e a c t i v a t i o n c a u s e d by FDP. 132 Figure 18. Influence of FDP on the phosphoenolpyruvate affinity of PK I and PK II. Assay conditions are as given in the legend to Figure 15. ADP concentration was 2 mM in a l l experiments. 133 I n h i b i t o r s B o t h PK I and PK I I were a f f e c t e d by a v a r i e t y o f i n h i b i t o r s , w i t h PK I I g e n e r a l l y s h o w i n g a g r e a t e r s e n s i t i v i t y t o i n h i b i t i o n , c e r t a i n l y a t ' p h y s i o l o g i c a l l e v e l s o f s u b s t r a t e s and i n h i b i t o r s . One o f t h e p r i m e i n h i b i t o r s f o r b o t h PK I and PK I I was MgATP. ; MgATP K i v a l u e s v e r s u s PEP r a n g e d f r o m 0.95-1.8 mM, w i t h t h e i n t e r m o l t enzymes s h o w i n g v a l u e s a t t h e l o w e r p o r t i o n o f t h e r a n g e and t h e f r e s h m o l t enzymes l y i n g a t t h e u p p e r e n d . B o t h p u r i f i e d and u n p u r i f i e d enzymes showed t h e same r e s p o n s e t o MgATP. MgATP i n h i b i t i o n was m i x e d c o m p e t i t i v e v e r s u s PEP ( F i g u r e 19) as w e l l a s ADP ( K i v a l u e s o f 2 mM). F o r p y r u v a t e k i n a s e I I > MgATP was a c o m p e t i t i v e i n h i b i t o r v e r s u s PEP ( K i = 1 . 2 mM) ( F i g u r e 20) and a n o n c o m p e t i t i v e i n h i b i t o r v e r s u s ADP. F o r b o t h f o r m s FDP r e v e r s e d t h e e f f e c t o f MgATP on PEP b i n d i n g ( F i g u r e s 19 8 20) b u t d i d n o t a f f e c t t h e i n h i b i t i o n v e r s u s ADP. The o t h e r n u c l e o t i d e t r i p h o s p h a t e s (MgUTP, M g l T P , and MgGTP) p r o d u c e d o n l y a s l i g h t i n h i b i t i o n o f t i i e s e h y p o d e r m a l p y r u v a t e k i n a s e f o r m s . A r g i n i n e p h o s p h a t e was an i n h i b i t o r o f b o t h PK I and PK I I . F o r PK I t h e i n h i b i t i o n was c o m p e t i t i v e w i t h b o t h s u b s t r a t e s , and showed K i o f 7.0 mM v e r s u s ADP and a K i " o f 7.7 mM v e r s u s PEP. A r g i n i n e p h o s p h a t e i n h i b i t i o n was c o m p e t i t i v e v e r s u s PEP ( K i = 6 mM) and u n c o m p e t i t i v e v e r s u s ADP f o r PK I I . F o r b o t h f o r m s t h e i n h i b i t i o n c o u l d ba p a r t i a l l y r e v e r s e d by t h e a d d i t i o n o f 0.05 mM FDP. The a r g i n i n e p h o s p h a t e i n h i b i t i o n was n o t due t o ATP f o r m a t i o n by a r g i n i n e p h o s p h o k i n a s e . A v a r i e t y o f m e t a b o l i t e s , i n c l u d i n g amino a c i d s , K r e b s 134 1 2 1 /PEP x 1 0 4 19. MgATP i n h i b i t i o n of PK I, influence of MgATP on PEP a f f i n i t y and r e v e r s a l of MgATP i n h i b i t i o n by FDP. Assay conditions are as i n the legend to Figure 16. (A) 2 mM ADP, no MgATP, (A) 2 mM ADP, 2 . 5 mM MgATP, (0) 2 mM ADP, 5 mM MgATP, ( D ) 2 mM ADP, no MgATP, 0 . 0 1 mM FDP, (•) 2 mM ADP, 5 mM MgATP, 0 . 0 1 mM FDP. 135 1 /PEPx 1 0 3 F i g . 20. MgATP i n h i b i t i o n of phosphoenolpyruvate bin d i n g by PK II and the r e v e r s a l of t h i s i n h i b i t i o n by FDP. Assay conditions as i n the legend to Figure 16, symbols as fo l l o w s : (A) 2 mM ADP, no MgATP, (•) 2 mM ADP, 2 mM MgATP, (0) 2 mM ADP, 4 mM MgATP, ( • ) 2 mM ADP, no MgATP, 0.05 mM FDP, (A) 2 mM ADP, 4 mM MgATP, 0.05 mM FDP. 136 c y c l e • a n d g l y c o l y t i c i n t e r m e d i a t e s h a v e been i m p l i c a t e d i n t h e c o n t r o l o f p y r u v a t e k i n a s e f r o m v a r i o u s t i s s u e s . To s e e w h i c h , i f a n y , i n t r a c e l l u l a r compounds c o u l d f u r t h e r m o d u l a t e t h e a c t i v i t y o f t h e - h y p o d e r m a l f o r m s o f p y r u v a t e k i n a s e , t h e f o l l o w i n g compounds' were t e s t e d a t l e v e l s shown i n b r a c k e t s : t a u r i n e (1-5 mM), a r g i n i n e (1-5 mM), a l a n i n e ( 2 , 5, 10, and 20 mM), p h e n y l a l a n i n e ( 1 , 3, and 20 mM), g l u t a m a t e (5 mM), p r o l i n e (5 mM) , v a l i n e (5 mM), a s p a r a g i n e (5 mM), a s p a r t a t e (5 mM) , l e u c i n e (10 mM), h i s t i d i n e (10 mM) , l y s i n e (10 mM) , c y s t e i n e (10 mM), g l y c i n e (10 mM) , g l u t a m i n e (10 mM) , m e t h i o n i n e (10 mM) , i s o l e u c i n e (10 mM) , t h r e o n i n e (10 mM) , s e r i n e (10 and 20 mM) , 3 - P - g l y c e r a t e (1 mM) t AMP (1-4 mM) , t r y p t o p h a n (1-5 mM) , a c e t y l CoA (1 x 10 -*H) , f r u c t o s e d i p h o s p h a t e (0.5 mM) , g l u c o s e -6 ^ p h o s p h a t e (1 mM) , NADH (0.05-0.2 mM) , c y c l i c AMP (0.05 mM) , M g 2 c i t r a t e (1-5 mM) , M g - m a l a t e (1-5 mM) , ©^-ketoglutarate ( 1 -5 mM) , < * - g l y c e r o l p h o s p h a t e (1-5 mM) , o x a l o a c e t a t e ( 0 . 0 1 -0.6 mM) , 2,3 d i p h o s p h o g l y c e r a t e (1 mM) , and CoA ( 0 . 1 - 0 . 8 mM) . T h e s e t e s t s were r u n b o t h a t s a t u r a t i n g and Km l e v e l s o f s u b s t r a t e s f o r b o t h t h e f r e s h m o l t a n d i n t e r m o l t PK I a n d PK I I , and were c a r r i e d o u t w i t h p u r i f i e d enzymes. I f o u n d t h a t a l a n i n e , s e r i n e , t r y p t o p h a n * M g 2 c i t r a t e , M g - m a l a t e , b t - k e t o g l u t a r a t e , and ©C-glycerol p h o s p h a t e a f f e c t e d b o t h PK I and PK I I , a l t h o u g h none o f t h e s e was a s p o t e n t an i n h i b i t o r a s MgATP. PK I I was more a f f e c t e d by t h e i n h i b i t o r s t h a n PK I . A l a n i n e , s e r i n e , and v a l i n e were weak i n h i b i t o r s f o r PK I . A l l t h r e e were n o t c o m p l e t e l y c o m p e t i t i v e w i t h e i t h e r PEP o r ADP, and t h e a d d i t i o n o f more t h a n a p a r t i c u l a r l e v e l o f t h e a mino a c i d d i d n o t l e a d t o a f u r t h e r i n h i b i t i o n . The K i f o r 137 a l a n i n e i n h i b i t i o n v e r s u s PEP was 30 mM ( F i g u r e 2 1 ) , t h e K i f o r s e r i n e i n h i b i t i o n 35 mM> and t h a t f o r v a l i n e i n h i b i t i o n 45 mM. FDP d i d n o t a f f e c t t h e i n h i b i t i o n c a u s e d by t h e s e a m i n o a c i d s . P y r u v a t e k i n a s e I I showed no r e s p o n s e t o v a l i n e , b u t showed a more marked r e s p o n s e t o s e r i n e and a l a n i n e t h a n PK I . S e r i n e was a m i x e d c o m p e t i t i v e i n h i b i t o r v e r s u s b o t h PEP and ADP, w i t h K i v a l u e s o f 10;5 mM and 36 mM, r e s p e c t i v e l y . The a d d i t i o n o f 0.01 mM FDP p a r t i a l l y r e v e r s e d t h e i n h i b i t i o n , r a i s i n g t h e K i v a l u e f o r t h e s e r i n e v e r s u s PEP i n h i b i t i o n t o 19 mM and c h a n g i n g t h e i n h i b i t i o n p a t t e r n v e r s u s ADP t o u n c o m p e t i t i v e ( F i g u r e 2 2 ) . The K i f o r a l a n i n e i n h i b i t i o n v e r s u s PEP was 7 mM and t h e a d d i t i o n o f 0.01 mM FDP r a i s e d t h i s K i v a l u e t o 26 mM ( F i g u r e 2 1 ) . The K i f o r a l a n i n e i n h i b i t i o n v e r s u s ADP was 26 mM. F o r PK I I , i n h i b i t i o n due t o s e r i n e and a l a n i n e was a d d i t i v e ( T a b l e 12, p. 1 4 4 ) . T r y p t o p h a n was a c o m p e t i t i v e i n h i b i t o r v e r s u s ADP f o r b o t h PK I a n d PK I I ( K i v a l u e s o f 1.8 a n d 2.5 mM, r e s p e c t i v e l y ) . T r y p t o p h a n i n h i b i t i o n was n o t r e v e r s e d by FDP. M g 2 c i t r a t e , o t - k e t o g l u t a r a t e , ©t-glycerol p h o s p h a t e , a n d Mg - m a l a t e a f f e c t b o t h PK I and PK I I , b u t p a r t i c u l a r l y f o r t h e l a s t t h r e e , t h e e f f e c t i v e l e v e l s were u n p h y s i o l o g i c a l ( W i l l i a m s o n e t a l . , 1 9 6 9 ) . S i n c e t h e l e v e l s o f m a l a t e , 6 < - k e t o g l u t a r a t e , and o < - g l y c e r o l p h o s p h a t e w h i c h c a u s e 5 0 % i n h i b i t i o n a r e t h e same f o r b o t h PK I and PK I I , I o n l y i n v e s t i g a t e d t h e mode o f i n h i b i t i o n f o r PK I . M g 2 c i t r a t e a f f e c t s t h e two f o r m s d i f f e r e n t l y , so I e x a m i n e d t h e mode o f i n h i b i t i o n f o r b o t h . & c - g l y c e r o l p h o s p h a t e showed l i n e a r i n h i b i t i o n k i n e t i c s , and was m i x e d c o m p e t i t i v e v e r s u s b o t h s u b s t r a t e s w i t h a K i o f 24 mM v e r s u s a PEP and a K i o f 29 mM 0.15 r I I I ; ; I I 20 40 [Alanine] mM 21. Alanine i n h i b i t i o n of PK I and PK I I and the influence of FDP or the i n h i b i t i o n pattern. Assay conditions as i n the legend to Figure 16, ADP concentration was held constant at 2 mM. PK I, PK I I , ( • ) 2 mM PEP, (A) 1.2 mM PEP, (0) 0.4 mM PEP, ( 0 ) 2 mM PEP, 0.01 mM FDP, (Q ) 0.4 mM PEP, 0.01 mM FDP, ( 0 ) 2 mM PEP, (A) 0.2 mM PEP, (©) 0.12 mM PEP. 139 i i i i i 20 40 [Serine] m M F i g . 22. Serine i n h i b i t i o n of PK II and the influence of FDP upon the i n h i b i t i o n pattern. Assay conditions as i n the legend to Figure 16, (0) 0.4 mM PEP, 2 mM ADP, ( 0 ) 0.8 mM PEP, 2 mM ADP, ( 0 ) 2 mM PEP, 2 mM ADP, (A) 2 mM PEP, 0.4 mM ADP, ( $ ) 2 mM PEP, 2 mM ADP, 0.01 mM FDP, ( • ) 2 mM ADP, 0.4 mM PEP, 0.01 mM FDP, ( • ) 2 mM PEP, 0.2 mM ADP, 0.01 mM FDP. 140 v e r s u s ADP. o < — K e t o g l u t a r a t e showed a 8 mM K i i n t h e i n t e r a c t i o n w i t h PEP a n d a 20 mM K i v e r s u s ADP. M g - m a l a t e i n h i b i t i o n showed l i n e a r i n h i b i t i o n v e r s u s ADP w i t h a K i o f 20 !mM, b u t t h e i n h i b i t i o n v e r s u s PEP showed c u r v e d D i x o n p l o t s . L i n e w e a v e r - B u r k r e p l o t s o f t h e d a t a were l i n e a r , i n d i c a t i n g t h a t i n t h e i n h i b i t i o n v e r s u s PEP* M g - m a l a t e i s i n t e r a c t i n g a t more t h a n one s i t e ( Sebb, 1 9 6 3 ) . F o r PK I , M g 2 c i t r a t e showed c u r v e d D i x o n p l o t s v e r s u s b o t h PEP and ADP. I n b o t h c a s e s , L i n e w e a v e r -B u r k r e p l o t s o f t h e d a t a were l i n e a r * a g a i n i n d i c a t i n g i n t e r a c t i o n a t more t h a n one s i t e . By c o n t r a s t , PK I I showed l i n e a r k i n e t i c s f o r t h e i n t e r a c t i o n o f M g 2 c i t r a t e w i t h b o t h PEP and ADP ( K i v a l u e s o f 7.5 and 8.0 mM). I n t h e p r e s e n c e o f 0.01 mM FDP t h e K i v e r s u s PEP r o s e t o 10 mM and t h e i n h i b i t i o n v e r s u s ADP became u n c o m p e t i t i v e ( F i g u r e 23a & b ) . A t 5 mM l e v e l s , M g 2 c i t r a t e i n h i b i t e d PK I by 4 3 % and PK I I by 5 2 % . T a b l e s 11 amd 12 a l l o w an a n a l y s i s o f t h e e f f e c t i v e n e s s o f t h e v a r i o u s m o d u l a t o r s a d d e d s i n g l y o r i n c o n c e r t , i n c o n t r o l l i n g t h e a c t i v i t y o f t h e s e two p y r u v a t e k i n a s e f o r m s . T h e t a b l e s show t h a t f o r v b o t h PK I and PK I I , MgATP, a r g i n i n e p h o s p h a t e , a n d M g 2 c i t r a t e i n h i b i t i o n was a d d i t i v e . Amino a c i d i n h i b i t i o n was a d d i t i v e w i t h t h a t due t o MgATP and a r g i n i n e p h o s p h a t e , b u t t h i s i n h i b i t i o n was m e a n i n g f u l o n l y f o r PK I I . The c o m b i n a t i o n o f 5 mM MgATP and 5 mM M g 2 c i t r a t e i n h i b i t e d PK II" by 9 9 . 5 % , w h i l e t h e same c o m b i n a t i o n o n l y i n h i b i t e d PK I by 6 3 % . FDP was a l s o f a r more e f f e c t i v e a s an a c t i v a t o r f o r PK I I t h a n f o r PK I. I n c o m p a r i s o n w i t h PK I , PK I I showed a d e c r e a s e d a f f i n i t y 1 0 2 0 [Mg 2 citrate] m M F i g . 23a. I - ^ c l t r a t e i n h i b i t i o n of PK I I , i n h i b i t i o n versus ADP and inf l u e n c e of FDP upon the i n h i b i t i o n p a t t e r n versus ADP and PEP. Assay con-d i t i o n s are as i n the legend to Figure 16 (0) 2 mM ADP, 2 mM PEP, (O ) 2 mM PEP, 0.2 mM ADP, (•) 2 mM PEP, 0.8 mM ADP, ( H ) 2 mM PEP, 2 mM ADP, 0.01 mM FDP, (A) 0.8 mM ADP, 2 mM PEP, 0.01 mM FDP, (A) 2 mM ADP, 0.4 mM PEP, 0.01 mM FDP. 142 5 10 15 [Mg 2 c i t ra te ] m M Figure M g 2 c i t r a t e i n h i b i t i o n of PK I I , i n h i b i t i o n versus PEP. Assay con-23b. d i t i o n s as i n the legend to Figure 16 ADP concentration constant at 2 mM. (0) 0.4 mM PEP, (0)-0.8 mM PEP, (A) 4 mM PEP. 143 TABLE 11 E f f e c t i v e n e s s of various i n h i b i t o r s of hypodermal pyruvate kinase I . Enzyme a c t i v i t y was measured at 0.2 mM PEP, 0.2 mM ADP, 4 mM MgCl 2, 75 mM KCl, 0.1 mM NADH, and excess LDH i n 50 mM imidazole-HCl, pH 7.0. Enzyme a c t i v i t y i s expressed as percent o f c o n t r o l a c t i v i t y . I f FDP was added to the treatment, the concentration was 0.05 mM. Treatment % Control- A c t i v i t y P l a i n + FDP 50% 68% 85% 67% 82% , -5 mM MgATP 5 mM arginine phosphate 5 mM Mg2citrate 5 mM a - k e t o g l u t a r a t e 5 mM MgATP + 5 mM arginine phosphate J 3 j 5 mM a-ketoglutarate + 5 mM arginine phosphate 57% 5 mM arginine phosphate + 5 mM MgATP + 5 mM Mg2citrate 3 Q % 4 2 % + 5 mM a-ketoglutarate 5 mM MgATP + 5 mM Mg2citrate + 5 mM a-ketoglutarate 41% 49% 5 mM Mg2citrate + 5 mM a - k e t o g l u t a r a t e 54% 61% 5 mM MgATP + 5 mM "-ke t o g l u t a r a t e 43% - . 5 mM MgATP + 5 mM Mg2citrate 35% 48% 35% 46% 68% TABLE 12 Ef f e c t i v e n e s s of various i n h i b i t o r s of hypodermal pyruvate kinase I I . Conditions of the experiments were the same as those given i n the legend f o r Table 11, with the exception that when FDP was added, the f i n a l concentration was 0.01 mM. % Control A c t i v i t y Treatment P l a i n + FDP 5 mM MgATP 18% 35% 5 mM arginine phosphate 64% -20 mM serine 69% -20 mM alanine 78% -5 mM Mg2citrate 50% 120% 10 mM a-ketoglutarate 75% -5 mM arginine phosphate + 5 mM MgATP 14% -5 mM MgATP + 5 mM M g 2 c i t r a t e 0.5% 34% 5 mM MgATP + 20 mM alanine 12% 20% 20 mM alanine + 20 mM serine 57% 85% 20 mM alanine + 20 mM serine + 5 mM MgATP 0.9% -5 mM arginine phosphate + 5 mM MgATP + 20 mM alanine 14% 25% 2.5 mM tryptophan + 5 mM MgATP 13% -5 mM arginine phosphate + 5 mM Mg2citrate 45% 116% 0.01 mM FDP 180% -0.01 mM UDP-acetylglucosamine 116% -145 f o r PEP and a c o n s i d e r a b l y i n c r e a s e d a f f i n i t y f o r FDP a s an a c t i v a t o r . T h e r e was no e l e c t r o p h o r e t i c o r i s o e l e c t r o f o c u s i n g e v i d e n c e f o r d i f f e r e n t p r o t e i n s c o r r e s p o n d i n g t o PK I a n d PK I I i n t h e h y p o d e r m i s . S i n c e t h e two f o r m s were s i m i l a r , b u t c o u l d be r e p r o d u c i b l y s e p a r a t e d i n t o two s t a b l e f o r m s w h i c h d i s p l a y e d t h i s ' r e c i p r o c a l r e l a t i o n s h i p b e t ween s u b s t r a t e and a c t i v a t o r a f f i n i t i e s , I f e l t t h e y c o u l d r e p r e s e n t two i n t e r c o n v e r t i b l e f o r m s o f t h e same p r o t e i n ^ F o r t h i s i n t e r c o n v e r s i o n t o be o f 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 , my p u r i f i c a t i o n - t e c h n i q u e s must i n some way* h a v e m i m i c k e d t h e method by w h i c h t h e s e f o r m s a r e i n t e r c o n v e r t e d w i t h i n t h e c e l l . P h o s p h o r y l a t i o n , b i n d i n g o f Mg*+ o r o f C a + + , o x i d a t i o n o f s u l p h y d r y l g r o u p s , a n d b i n d i n g a n d r e l e a s e o f FDP h a v e a l l b een i m p l i c a t e d i n i n t e r c o n v e r s i o n s o f e i t h e r t h e L t y p e o r t h e M2 t y p e o f mammalian p y r u v a t e k i n a s e (Hess & K u t z b a c h * 1971; Van B e r k e l e t a l . , 1973b; Van B e r k e l , 1974; L j u n g s t r o m e t a l . , 1974; F l i k w e e r t e t a l . , 1 9 7 5 ) . S i n c e t h e two enzymes a l r e a d y p o s s e s s a b i n d i n g s i r e f o r FDP, and s i n c e t h e a f f i n i t y o f t h i s s i t e v a r i e s s t r o n g l y b e t ween t h e two f o r m s , m e d i a t i o n o f t h e t r a n s i t i o n by c h a n g e s i n t h e amount o f FDP a s s o c i a t e d w i t h t h e enzyme seemed t h e s i m p l e s t m e c h a n i s m . S i n c e FDP i s n o t d i r e c t l y i n v o l v e d w i t h t h e c a t a l y s i s , i t seemed f e a s i b l e t h a t FDP m o l e c u l e s r e m a i n bound t o t h e enzyme f o r r e l a t i v e l y l o n g d u r a t i o n s . ' S u c h l o n g t e r m b i n d i n g o f t h e a c t i v a t o r c o u l d s t a b i l i z e t h e p r o t e i n i n a more a c t i v e c o n f o r m a t i o n . To e v a l u a t e t h i s h y p o t h e s i s , I t e s t e d t h e e f f e c t - o f i n c u b a t i o n w i t h FDP upon PK I I . A t h i r t y m i n u t e i n c u b a t i o n w i t h 10 yUM FDP was s u f f i c i e n t t o c o n v e r t t h e l o w a f f i n i t y f o r m i n t o 146 t h e h i g h a f f i n i t y PK I ( F i g u r e 2 4 ) . 0.5 uM FDP d i d n o t a f f e c t a c o m p l e t e c o n v e r s i o n , w h i l e MgATP i n c o m b i n a t i o n w i t h 10 uM FDP p r e v e n t e d t h e f u l l c o n v e r s i o n o f PK I I i n t o PK I ( T a b l e 1 3 ) . A f t e r t h e v a r i o u s i n c u b a t i o n s , t h e enzyme r e m a i n e d s e n s i t i v e t o FDP a c t i v a t i o n o f PEP b i n d i n g ( F i g u r e 2 4 ) , t h u s s u p p o r t i n g t h e c o n c e p t o f i n t e r c o n v e r t i b l e enzyme f o r m s . The r e s u l t s o f t h i s e x p e r i m e n t i n d i c a t e t h a t k i n e t i c e x p e r i m e n t s i n v o l v i n g t h e a d d i t i o n o f FDP t o PK I I a r e a c t u a l l y m o n i t o r i n g a m i x t u r e o f PK I I and PK I , o r some i n t e r m e d i a t e f o r m . The " t w o s t a g e a c t i v a t i o n " , - i e . t h e i n i t i a l s h i f t f r o m a l o w t o a h i g h a f f i n i t y f o r m , a n d t h e f u r t h e r a c t i v a t i o n by FDP, s u p p o r t s t h e e x i s t e n c e o f two c o n f o r m a t i o n a l s t a t e s . A f u r t h e r e v a l u a t i o n o f t h e h y p o t h e s i s o f FDP m e d i a t e d c o n v e r s i o n came f r o m m e a s u r e m e n t s o f t h e amount o f FDP w h i c h o c c u r r e d w i t h PK I and PK I I . I f o u n d t h a t b o t h PK I a n d PK I I had m e a s u r a b l e amounts o f FDP bound t o o r c l o s e l y a s s o c i a t e d w i t h them. S i n c e I was n o t w o r k i n g w i t h homogeneous p r o t e i n s , I c o u l d n o t e x p r e s s t h e v a l u e s a s m o l e s FDP/mole enzyme, b u t I was a b l e t o f i n d t h a t p e r u n i t o f enzyme a c t i v i t y , PK I was a s s o c i a t e d w i t h 0.0014 m o l e s FDP a n d PK I I w i t h 0.0003 m o l e s FDP.• W h i l e t h e r e was some v a r i a t i o n i n t h e v a i u e s b e t w e e n d i f f e r e n t e x p e r i m e n t s , f o r any one a n i m a l , t h e FDP c o n t e n t o f PK I a n d PK I I were a l w a y s i n a p p r o x i m a t e l y a 4:1 r a t i o . I t was a l s o i n t e r e s t i n g t o - f i n d t h a t t h e m u s c l e enzyme f r o m C a n c e r m a g i s t e r c o n t a i n e d bound FDP, b u t i n more v a r i a b l e a m o u n t s . C o n t r o l e x p e r i m e n t s u s i n g c o l u m n e f f l u e n t o f e q u a l p r o t e i n c o n c e n t r a t i o n , b u t no p y r u v a t e k i n a s e a c t i v i t y , showed no FDP. 147 1 1/PEP x 10 4 F i g . 24. Conversion of PK II in t o PK I by incubation with 0.05 mM FDP. F i l l e d symbols show the PEP a f f i n i t y curves measured i n the presence of . 0.05 mM FDP, while the open symbols show the a f f i n i t y curves i n the absence of FDP. C i r c l e s represent the enzyme before the t h i r t y -minute incubation with 0.05 mM FDP, while t r i a n g l e s show the enzyme a f t e r incubation. 148 TABLE 13 Effect of FDP, ATP and alanine on the interconversions of PK I and PK I I . The percentage of PK I and PK II i n each experiment were determined from the reaction rate at 0.5 mM PEP. For PK I the rate at this concentration of substrate was 95% of V , while for PK II the rate was only 50% of V m„ . max max V for PK I, PK II, and the incubated enzyme were determined from Lineweaver-znax Burk plots. The following equation was used i n the calculations: % PK I = °^erved rate - rate with PK II p R = _ % p K rate with PK I - rate with PK II Treatment no incubation 30 minutes incubation with 50 uM FDP 2 minutes incubation with 50 uM FDP no incubation 30 minutes incubation with 10 uM FDP 30 minutes incubation with 0.5 uM FDP 30 minutes incubation with 5 mM MgATP, 20 mM alanine, and 10 yM FDP % PK II 93% 15.5% 25% 75% 13% 56% 23% 149 R e c e n t work w i t h t h e r a b b i t m u s c l e p y r u v a t e k i n a s e i n d i c a t e s t h a t t h e r e v e r s a l o f t h e r e a c t i o n i s f e a s i b l e i n t h e t i s s u e (Dyson e t a l . , 1975; G i l e s e t a l . , 1 9 7 5 ) . S i n c e t h e l e v e l s o f p y r u v a t e k i n a s e i n t h e f r e s h m o l t h y p o d e r m i s a r e g u i t e h i g h c o m p a r e d t o t h o s e o f p y r u v a t e c a r b o x y l a s e and p h o s p h o e n o l -p y r u v a t e c a r b o x y k i n a s e ( T a b l e 1 ) , a h i g h r a t e o f r e v e r s a l w o u l d be one way o f f a c i l i t a t i n g g l u c o n e o g e n e s i s . I f o u n d , h o w e v e r , t h a t t h e r a t e o f t h e r e v e r s e r e a c t i o n was t h e same f o r PK I and PK I I and a t Vmax c o n s i s t e d o f 0.4% o f t h e Vmax o f t h e f o r w a r d r e a c t i o n . S i n c e t h e t h e r m a l d e n a t u r a t i o n b e h a v i o r o f p r o t e i n s c a n p r o v i d e one means by w h i c h d i f f e r e n t p r o t e i n s c a n be d i s t i n g u i s h e d , I c h a r a c t e r i z e d t h e e f f e c t o f p r o l o n g e d i n c u b a t i o n a t 40 °C on b o t h PK I and PK I I and c o m p a r e d t h i s w i t h t h e r e s p o n s e shown by t h e m u s c l e enzyme ( F i g u r e 1 5 ) . The two h y p o d e r m a l enzymes were c o n s i d e r a b l y more a f f e c t e d by t h e i n c u b a t i o n s t h a n t h e m u s c l e enzyme (compare F i g u r e 15 w i t h F i g u r e s 25 and 2 6 ) * a f t e r 80 m i n u t e s a t 40 °C, 9 5 % o f t h e o r i g i n a l enzyme a c t i v i t y r e m a i n e d i n t h e m u s c l e p r e p a r a t i o n , w h i l e PK I h a d 7 5 % and PK I I o n l y 4 5 % o f t h e o r i g i n a l a c t i v i t y . F o r a l l e n z y m e s , ATP a c c e l e r a t e d d e n a t u r a t i o n . H o w e v e r , t h e m u s c l e enzyme r e t a i n e d 8 0 % o f i t s a c t i v i t y a f t e r 80 m i n u t e s i n c u b a t i o n w i t h 4 mM MgATP, w h i l e b o t h PK I and PK I I r e t a i n e d o n l y 2 5 % o f t h e i n i t i a l a c t i v i t y . A c o m p a r i s o n o f t h e d e n a t u r a t i o n b e h a v i o r o f PK I and PK I I i n d i c a t e s t h a t PK I I i s g e n e r a l l y more p r o n e t o w a r d s d e n a t u r a t i o n . None o f t h e p r o t e c t i v e t r e a t m e n t s s u c c e e d e d i n m a i n t a i n i n g PK I I a c t i v i t y a b o v e 6 0 % a f t e r 80 m i n u t e s d e n a t u r a t i o n t i m e , w h i l e t h e p r e s e n c e 150 Ql—: 1 1 1 I i I i l _ 0 20 4 0 6 0 8 0 Denaturat ion Time in Minutes Figure 25. Thermal denaturation of Cancer magister hypodermal pyruvate kinase I. Experiments were run at 40°C under the indicated incubation conditions. Enzyme activity was measured under saturating conditions of substrates and cofactors. (A) control; (CD 4 mM MgATP; (x ) 0.1 mM FDP; (^ ) 0.1 mM FDP,.4 mM ATP, and 4 mM MgCl2; ( +) 2 mM P E P ; ( o) 2 mM ADP and 2 mM PEP; (Q) 2 mM ADP, 2 mM PEP, and 0.1 mM F D P ; ( © ) 2 mM ADP, 2 mM PEP, 0.1 mM FDP, and 4 mM MgATP; ( A ) 75 mM KC1. 151 conditions. Enzyme activity was measured under saturating conditions of substrates and cofactors. (V)control; (•) 4 mM MgATP; (0) 0.1 mM F D P ; ( +) 0.1 mM FDP, 4 mM MgATP; ( @) 2 mM PEP; ( x) 2 mM ADP and 2 mM P E P ; ( A ) 2 mM ADP, 2 mM PEP, and 0.1 mM FDP; ( o) 2 mM ADP, 2 mM P E P , 0.1 mM FDP and, 4mM MgATP; ( A ) 75 mM KC1. 152 o f 2mM ADP + 2 mM1 PEP + 0.1 mM FDP m a i n t a i n s PK I a t 8 5 % o f t h e o r i g i n a l a c t i v i t y . B o t h PK I and PK I I snow a c c e l e r a t e d d e n a t u r a t i o n i n t h e p r e s e n c e o f 2 mM PEP, ' w h i l e 2 mM PEP a n d 2 mM ADP a f f o r d - some p r o t e c t i o n . N e i t h e r PEP n o r PEP + ADP a f f e c t t h e d e n a t u r a t i o n o f t h e m u s c l e enzyme. • -•• C o n s i d e r a b l e d i f f e r e n c e s seem t o e x i s t b e t w e e n t h e d e n a t u r a t i o n p a t t e r n s o f t h e m u s c l e enzyme and t h e two h y p o d e r m a l f o r m s . H o w e v e r , t h e o v e r a l l d e n a t u r a t i o n p a t t e r n s o f PK I and PK I I a r e g u i t e s i m i l a r . A d i f f e r e n c e b e t w e e n t h e two f o r m s comes i n t h e s l i g h t d e g r e e o f p r o t e c t i o n a f f o r d e d by 0.1 mM FDP t o PK I I . T h i s may r e f l e c t t h e d e c r e a s e d l e v e l s o f FDP bound t o PK I I . The h i g h e r d e g r e e o f d e n a t u r a t i o n shown by PK I I may i n d i c a t e t h a t t h i s f o r m i s i n a l e s s s t a b l e c o n f o r m a t i o n a l s t a t e t h a n PK I . One e f f e c t w h i c h I d i d n o t e x a m i n e e x p e r i m e n t a l l y i n v o l v e d t h e 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 o f t h e d i f f e r e n t enzyme p r e p a r a t i o n s . The p r o t e i n c o n c e n t r a t i o n i n t h e t h r e e e x p e r i m e n t s was t h e same, b u t t h e s p e c i f i c a c t i v i t y -of t h e p y r u v a t e k i n a s e was n o t . T h u s , t h e p o s s i b i l i t y o f i n c r e a s e d s e l f - a s s o c i a t i o n may' p a r t i a l l y e x p l a i n t h e t h e r m a l t o l e r a n c e o f t h e m u s c l e enzyme. H o w e v e r , t h e c e n t r a l c o n c l u s i o n w h i c h emerges f r o m t h e s e d e n a t u r a t i o n e x p e r i m e n t s i s t h a t t h e m u s c l e p y r u v a t e k i n a s e i s d i f f e r e n t f r o m t h e two h y p o d e r m a l e n z y m e s . 153 DISCUSSION I n f r e s h m o l t h y p o d e r m i s , t h e p y r u v a t e k i n a s e b r a n c h p o i n t f a c e s two u r g e n t r e q u i r e m e n t s : (1) f o r g l u c o n e o g e n e s i s f r o m amino a c i d s and l a c t a t e , t h e p y r u v a t e k i n a s e r e a c t i o n must be i n h i b i t e d , and (2) p y r u v a t e k i n a s e must be a b l e t o s h i f t f r o m l o w t o h i g h a c t i v i t y t o s u p p o r t h i g h r a t e s o f c a r b o h y d r a t e o x i d a t i o n d u r i n g t h i s p e r i o d ( H e r z - H u b n e r & U r i c h , - 1 9 7 3 ) . H y p o d e r m a l p y r u v a t e k i n a s e a c t i v i t i e s a r e c o n s i d e r a b l y h i g h e r t h a n t h o s e o f t h e e n z y m a t i c b y p a s s , s o f l u c t u a t i o n s i n p y r u v a t e k i n a s e r a t e s a r e p r o b a b l y t h e p r i m e d e t e r m i n a n t o f f l u x t h r o u g h t h i s b r a n c h p o i n t . H y p o d e r m a l p y r u v a t e k i n a s e a p p e a r s t o be a c o m p l e x a l l o s t e r i c p r o t e i n c a p a b l e o f making t h e r e q u i r e d t r a n s i t i o n s i n a c t i v i t y . H y p o d e r m a l p y r u v a t e k i n a s e was f o u n d t o e x i s t i n t w o f o r m s , one w h i c h c a n be c o n s i d e r e d t o be a h i g h a f f i n i t y f o r m (PK I ) , and t h e o t h e r a l o w a f f i n i t y f o r m (PK I I ) . I n v i t r o i n t e r c o n v e r s i o n by FDP as w e l l a s t h e d i f f e r e n t l e v e l s o f I D P a s s o c i a t e d w i t h t h e two f o r m s i n d i c a t e d t h a t t h e i n v i v o i n t e r c o n v e r s i o n b e t w e e n t h e two i s p r o b a b l y m e d i a t e d by FDP. T h e r e a r e q u i t e a number o f p r e c e d e n t s f o r i n t e r c o n v e r t i b l e p y r u v a t e k i n a s e s , t h e b e s t e s t a b l i s h e d b e i n g t h e FDP m e d i a t e d c o n v e r s i o n o f mammalian L t y p e p y r u v a t e k i n a s e f r o m a s i g m o i d a l l o w a f f i n i t y s t a t e t o a h y p e r b o l i c h i g h a f f i n i t y s t a t e (Hess S K u t z b a c h , 1971; I b s e n e t a l . , 1 S 7 5 ) . P h o s p h o r y l a t i o n o f t h e L t y p e enzyme l e a d s ' t o an enzyme w i t h l o w e r e d PEP a f f i n i t y ( L j u n g s t r o m e t a l . , 1 9 7 4 ) . W h i l e ATP i n d u c e s a d i s s o c i a t i o n o f g r o u n d s g u i r r e l l i v e r p y r u v a t e k i n a s e i n t o i n a c t i v e d i m e r s , FDP m e d i a t e s t h e r e a s s o c i a t i o n o f t h e s e d i m e r s i n t o t e t r a m e r s ( B e h r i s c h , 1 9 7 4 ) . The L t y p e enzyme a l s o u n d e r g p e s 154 i n t e r c o n v e r s i o n s u n d e r t h e i n f l u e n c e o f o x i d i z e d . a n d r e d u c e d g l u t a t h i o n e . The o x i d i z e d enzyme shows a m a r k e d l y r e d u c e d a f f i n i t y f o r b o t h FDP and PEP. T h i s e f f e c t i s r e v e r s e d by t h e a d d i t i o n o f r e d u c e d g l u t a t h i o n e (Van B e r k e l e t a l i , 1 9 7 3 b ) . The M2 o r K t y p e p y r u v a t e k i n a s e h a s a l s o b een f o u n d t o u n d e r g o s e v e r a l t y p e s o f t r a n s i t i o n s . P o g s o n (1968) f o u n d t h a t EDTA t r a n s f o r m e d t h e enzyme f r o m a h i g h t o a l o w a f f i n i t y s t a t e . T h ese i n t e r c o n v e r t i b l e f o r m s h a v e been shown t o e x i s t , i n v i v o ( W a l k e r & P o t t e r , 1973; F e l i u e t a l . , 1 9 7 5 ) . Van B e r k e l (1974) f o u n d t h a t t h i s e f f e c t was due t o t h e r e m o v a l o f Mg+*-from t h e v i c i n i t y o f t h e enzyme. C a + * h a s - a l s o been f o u n d t o p r o d u c e i n t e r c o n v e r s i o n s o f t h e M2 enzyme ( F l i k w e e r t e t a l . , 1 9 7 5 ) , a s h a s FDP (Van B e r k e l , 1 9 7 4 ) . An a l a n i n e m e d i a t e d i n a c t i v e d i m e r t o a c t i v e t e t r a m e r t r a n s i t i o n , a n d a magnesium m e d i a t e d d i m e r -t e t r a m e r t r a n s i t i o n h a v e been f o u n d f o r t h e mammalian M2 enzyme ( S c h u l z e t a l . , 1 975; I b s e n e t a l . , 1 9 7 5 ) . W i t h t h e c r u s t a c e a n h y p o d e r m a l p y r u v a t e k i n a s e , I f o u n d no a c t i v a t i o n due t o t h e r e d u c e d m e r c a p t o e t h a n o l u s e d i n t h e p u r i f i c a t i o n * and i n a g r e e m e n t w i t h G i l e s e t a l . , ( 1 9 7 6 ) , I f o u n d t h a t t h e r e s p o n s e s t o EDTA were o p p o s i t e t o t h o s e shown by t h e mammalian e n z y m e s . The m a j o r f u n c t i o n a l d i s t i n c t i o n b e t w e e n t h e two h y p o d e r m a l p y r u v a t e k i n a s e f o r m s i s t h e i r d i s p a r a t e PEP a f f i n i t y . I n t h e v a r i o u s v e r t e b r a t e a n d i n v e r t e b r a t e t i s s u e s where PEP l e v e l s have' been m e a s u r e d , i t s l e v e l s r a r e l y r e a c h o r e x c e e d 0.2 mM ( E x t o n 6 P a r k , 1969; B e i s & Newsholme, 1 9 7 5 ) . T h e r e f o r e , PK I I , w i t h i t s Km f o r PEP o f 0.55 mM, w o u l d b e f u n c t i o n i n g a t a p p r o x i m a t e l y 15% - o f - Vmax, w h i l e PK I , w i t h i t s PEP Km o f 0.1 mM, w o u l d be f u n c t i o n i n g a t a p p r o x i m a t e l y 8 0 % o f Vmax. T h i s 155 r e c k o n i n g assumes an a b s e n c e o f m o d u l a t o r s . ATP l e v e l s i n t h e h y p o d e r m i s l i e b e tween 2 and 3 mM ( T a b l e 7) . Such l e v e l s w o u l d d r o p p y r u v a t e k i n a s e I I t o a p p r o x i m a t e l y 2% o f m a x i m a l a c t i v i t y , w h i l e r e d u c i n g p y r u v a t e k i n a s e I t o between 50 and 60% o f i t s Vmax. M a r i n e c r u s t a c e a n s have h i g h i n t r a c e l l u l a r l e v e l s o f amino a c i d s w i t h a l a n i n e a t a p p r o x i m a t e l y 20 mM a n d s e r i n e a t 5 ma ( S c h o f f e n i e l s - S G i l e s , 1 S 7 0 ) . W h i l e PK I a c t i v i t y i s r e l a t i v e l y u n a f f e c t e d by t h e s e l e v e l s , t h e p r e s e n c e o f b o t h o f t h e s e compounds w o u l d s e r v e t o f u r t h e r i n h i b i t PK I I t o l e s s t h a n 0.2% o f m a x i m a l a c t i v i t y , a s w o u l d any a c c u m u l a t i o n o f M g 2 c i t r a t e ( F i g u r e 2 7 ) . T h u s , p y r u v a t e k i n a s e a c t i v i t y c a n e s s e n t i a l l y be t u r n e d o f f , a l l o w i n g g l u c o n e o g e n i c f l u x p a s t t h e r e a c t i o n . R e v e r s a l o f p y r u v a t e k i n a s e i n h i b i t i o n c a n be b r o u g h t a b o u t by an i n c r e a s e i n t h e l e v e l s o f FDP. D u r i n g g l u c o n e o g e n e s i s , i n c r e a s i n g F D P - l e v e l s w o u l d i n d i c a t e some i n h i b i t i o n a t t h e FDPase r e a c t i o n . I n k i n g c r a b s , h y p o d e r m a l FDPase e x i s t s i n two f o r m s , one w h i c h i s h i g h l y s e n s i t i v e t o f e e d b a c k i n h i b i t i o n by compounds s u c h as U D P - a c e t y l g l u c o s a m i n e and U D P - g l u c o s e ( H o c h a c h k a 1 9 7 2 ) . Whenever t h e s e compounds a c c u m u l a t e due t o a s l o w d o w n o f c h i t i n o r g l y c o g e n s y n t h e s i s , t h i s FDPase w o u l d be i n h i b i t e d , l e a d i n g t o an a c c u m u l a t i o n o f FDP. The s e c o n d f o r m o f FDPase i s a l s o s u b j e c t t o some f e e d b a c k i n h i b i t i o n by h i g h l e v e l s o f O D P - a c e t y l g l u c o s a m i n e ( H o c h a c h k a , 1 9 7 2 ) . D u r i n g e x t r e m e i n h i b i t i o n o f c h i t i n s y n t h e s i s , t h i s FDPase w o u l d be l i m i t e d a s w e l l . W h i l e FDP i s t h e most p o t e n t a c t i v a t o r o f b o t h PK I a n d PK' I I * U D P - a c e t y i g l u c o s a m i n e a n d g l u c o s a m i n e - 6 -p h o s p h a t e a r e a l s o a c t i v a t o r s . The a c c u m u l a t i o n o f FDP w o u l d do Figure 27 Cont ro l of C a r b o h y d r a t e Metabol ism in the C r u s t a c e a n Hypodermis GLYCOGEN P G A f Alanine Serine M g 2 C i t r a t e OXA / / , acetyl CoA A T P -A r g - P h o s C a C U 157 two t h i n g s , i n i t i a l l y an a c t i v a t i o n o f PK I I and t h e n a s t h e numbers o f FDP m o l e c u l e s bound t o t h e enzyme i n c r e a s e d * PK I I w o u l d c h a n g e c o n f o r m a t i o n , f o r m i n g PK I . The h i g h a f f i n i t y o f PK I I f o r FDP w o u l d f a c i l i t a t e t h e t r a n s i t i o n . PK I w o u l d t h e n f u n n e l c a r b o n i n t o K r e b s c y c l e , l e a d i n g t o b o t h a d r o p i n FDP and an i n c r e a s e i n ATP l e v e l s . An e x t e n s i o n o f t h i s model p r e d i c t s t h a t PK I w o u l d r e m a i n i n t h i s c o n f o r m a t i o n u n t i l a s u f f i c i e n t d r o p i n FDP l e v e l s w o u l d f a v o u r d i s s o c i a t i o n r a t h e r t h a n b i n d i n g . The d e c r e a s e d a f f i n i t y f o r FDP c o u l d be s i g n i f i c a n t i n t h i s c o n t e x t . T h e o r e t i c a l l y , o t h e r m o d u l a t o r s c o u l d a f f e c t t h e r a t e o f t h i s t r a n s i t i o n , s i n c e a l a n i n e and MgATP p a r t i a l l y p r e v e n t t h e FDP i n d u c e d i n t e r c o n v e r s i o n o f t h e two f o r m s ( T a b l e 1 3 ) . However, I was n o t a b l e t o f i n d a n o t h e r l i g a n d c a p a b l e o f c o n v e r t i n g PK I i n t o PK I I . T h u s , t h e b i n d i n g and r e l e a s e o f FDP seems t h e p r i m e v e h i c l e f o r c o n v e r s i o n o f t h e two f o r m s . The d e g r e e t o w h i c h PK I I c a n be i n h i b i t e d w o u l d a l l o w c o n s i d e r a b l e f l u x t h r o u g h t h e p y r u v a t e c a r b o x y l a s e and p h o s p h o -e n o l p y r u v a t e c a r b o x y k i n a s e b y p a s s . The h y p o d e r m a l l e v e l s o f t h e s e enzymes a r e c o n s i d e r a b l y h i g h e r i n f r e s h m o l t t h a n i n i n t e r m o l t a n i m a l s ( T a b l e 1 ) . I n f r e s h l y m o l t e d a n i m a l s , p h o s o h o e n o l p y r u v a t e c a r b o x y k i n a s e , t h e r a t e l i m i t i n g enzyme i n t h e b y p a s s ( S c r u t t o n & U t t e r , 1S68; E x t o n & P a r k , 1 9 6 9 ) , h a s r i s e n t o 4% o f t h e p y r u v a t e k i n a s e a c t i v i t y . T h i s i s t e n f o l d h i g h e r t h a n t h e m a x i m a l r a t e o f p y r u v a t e k i n a s e r e v e r s a l . F u r t h e r m o r e , t h e m a x i m a l v e l o c i t y f o r PEP c a r b o x y k i n a s e m e a s u r e d i n ' t h e g l u c o n e o g e n i c d i r e c t i o n may be h i g h e r ( P o g s o n & S m i t h , 1 9 7 5 ) . S i n c e , a t p h y s i o l o g i c a l l e v e l s o f PEP, ADP, ATP, a l a n i n e 158 a n d s e r i n e , PK I I shows l e s s t h a n 0.5% o f i t s m a x i m a l v e l o c i t y , n e t f l u x t h r o u g h t h e e n z y m a t i c b y p a s s i s f e a s i b l e . Some e x p e r i m e n t s i n mammalian s y s t e m s i n d i c a t e t h a t a l t e r n a t e r o u t e s o f g l u c o n e o g e n i c f l u x p a s t t h e p y r u v a t e k i n a s e r e a c t i o n may e x i s t ( V e n e z i a l e e t a l . , 1 9 7 0 ) . W h i l e I have no i n f o r m a t i o n a s t o t h e i r e x i s t e n c e i n c r u s t a c e a n h y p o d e r m i s , t h e i n h i b i t i o n o f p y r u v a t e k i n a s e w o u l d a l s o be n e c e s s a r y w i t h a n y a l t e r n a t i v e r o u t e w h i c h e n t e r e d b e l o w t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n . The r e g u l a t o r y p r o p e r t i e s o f t h i s p y r u v a t e k i n a s e a r e d i s t i n c t f r o m t h o s e o f p r e v i o u s l y d e s c r i b e d p y r u v a t e k i n a s e s i n t h a t FDP m e d i a t e s a t r a n s i t i o n b e t w e e n two f o r m s , e a c h o f w h i c h i s s e n s i t i v e t o FDP a c t i v a t i o n . The mammalian L t y p e shows a FDP m e d i a t e d t r a n s i t i o n b e t w e e n two f o r m s , b u t t h e a c t i v a t e d f o r m i s n o t s u b j e c t t o f u r t h e r a c t i v a t i o n ( S e u b e r t & S c h o n e r , 1 9 7 1 ) . The M2 t y p e shows a h i g h and a low a f f i n i t y f o r m w i t h M g + + and FDP p r o m o t i n g a s l o w t r a n s i t i o n b e t ween t h e f o r m s . Of t h e two f o r m s , o n l y t h e h i g h a f f i n i t y f o r m i s s e n s i t i v e t o FDP a c t i v a t i o n (Van B e r k e l , 1 9 7 4 ) . A d i m e r - t e t r a m e r t r a n s i t i o n may be i n v o l v e d i n t h i s a c t i v a t i o n ( I b s e n e t a l . , 1 9 7 5 ) . A f u r t h e r d i s t i n c t i o n o f t h e h y p o d e r m a l p y r u v a t e k i n a s e i s t h e l a c k o f c o o p e r a t i v i t y o f t h e s u b s t r a t e o r m o d u l a t o r b i n d i n g . T h i s i s i n c o n t r a s t t o ' t h e "enzyme f r o m t h e h e p a t o p a n c r e a s o f C a r c i n u s maenas ( G i l e s e t a l . , 1976) and o t h e r l i v e r * t y p e e n z y m e s f r o m v e r t e b r a t e s a n d i n v e r t e b r a t e s ( K a y n e , 1 9 7 3 ) . G e n e r a l l y ; t h e p y r u v a t e k i n a s e s w h i c h show c o o p e r a t i v i t y o f s u b s t r a t e b i n d i n g show a d e c r e a s e i n c o o p e r a t i v i t y and an i n c r e a s e i n a f f i n i t y a s t h e pH i s d e c r e a s e d . T h i s i s t a k e n t o i n d i c a t e t h e i n v o l v e m e n t o f an i o n i z a b l e g r o u p i n t h e r e g u l a t i o n o f t h e enzyme ( S e u b e r t & 159 S c h o n e r , 1 9 7 1 ) . I n c o n t r a s t , h y p o d e r m a l p y r u v a t e k i n a s e showed a d e c r e a s e i n a f f i n i t y f o r PEP a s t h e pH w a s ' e i t h e r i n c r e a s e d o r d e c r e a s e d f r o m pH 7.0. I s i n b o t h t h e L and M2 mammalian enzymes, FDP s t r o n g l y a f f e c t e d PEP b i n d i n g by b o t h • P K I - and PK I I . However, FDP a c t i v a t i o n o f PK I I showed a n o t h e r - i n t e r e s t i n g f e a t u r e ; w i t h b o t h s e r i n e and M g 2 c i t r a t e i n h i b i t i o n , a d d i t i o n o f FDP n o t o n l y r e l i e v e d t h e e f f e c t on PEP b i n d i n g , b u t a l s o c h a n g e d t h e p a t t e r n o f i n h i b i t i o n v e r s u s ADP. I n b o t h c a s e s , t h e i n h i b i t i o n v e r s u s ADP became u n c o m p e t i t i v e . T h u s , t h e r e g u l a t o r y e f f e c t s o f FDP e x t e n d t o b o t h s u b s t r a t e b i n d i n g s i t e s . Of a i l - t h e - i n h i b i t o r s , o n l y a r g i n i n e p h o s p h a t e i s o f q u e s t i o n a b l e s i g n i f i c a n c e , s i n c e i t s p r e s e n c e i n t h e t i s s u e h a s n o t b e e n d e m o n s t r a t e d . The p r e s e n c e o f a r g i n i n e p h o s p h o k i n a s e i n h e p a t o p a n c r e a s m i t o c h o n d r i a (Chen & L e h n i n g e r , 1973) i n d i c a t e s t h a t t h e enzyme and t h e p h o s p h a g e n may h a v e a w i d e r d i s t r i b u t i o n t h a n p r e v i o u s l y c o n s i d e r e d . However, t h e e f f e c t e x e r t e d by a r g i n i n e p h o s p h a t e may s i m p l y t e s t i f y t o an e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n t h e h y p o d e r m a l a n d m u s c l e e n z y m e s , s i n c e i n many ways, t h e k i n e t i c s o f PK I a r a s i m i l a r t o t h o s e • o f t h e m u s c l e enzyme ( s e e d i s c u s s i o n ) . H o w e v e r , t h e m u s c l e enzyme e x i s t s - o n l y i n o n e f u n c t i o n a l f o r m and e x h i b i t s r a t h e r d i f f e r e n t t h e r m a l d e n a t u r a t i o n b e h a v i o r t h a n e i t h e r PK I o r PK I I f r o m t h e h y p o d e r m i s . 160 P a r t V C a t i o n i c C o m p o s i t i o n o f I n t e r m o l t and F r e s h m o l t H y p o d e r m i s and M u s c l e INTBODUCTION The enzymes w h i c h have been e x a m i n e d i n t h i s s t u d y a r e a l l a f f e c t e d by t h e f o u r m a j o r i n t r a c e l l u l a r c a t i o n s , s o d i u m , p o t a s s i u m , magnesium, and c a l c i u m . The p h o s p h o g l y c e r a t e k i n a s e s were p a r t i c u l a r l y s e n s i t i v e t o - t h e l e v e l o f magnesium, s i n c e h i g h l e v e l s o f magnesium m a r k e d l y i n c r e a s e t h e e f f i c i e n c y o f ADP i n h i b i t i o n . B o t h t h e p y r u v a t e k i n a s e s a n d - t h e p h o s p h o g l y c e r a t e k i n a s e s d i s p l a y e i t h e r a c o m p l e t e o r a p a r t i a l d i v a l e n t c a t i o n r e q u i r e m e n t . P o t a s s i u m i s r e q u i r e d f o r p y r u v a t e k i n a s e a c t i v i t y and s t r o n g l y ' a c t i v a t e s p h o s p h o g l y c e r a t e k i n a s e a c t i v i t y . C a l c i u m i s a s t r o n g i n h i b i t o r o f a l l o f t h e enzymes- e x a m i n e d . Sodium* i s an a c t i v a t o r o f t h e p h o s p h o g l y c e r a t e k i n a s e s and ah i n h i b i t o r o f t h e p y r u v a t e k i n a s e s . The Km and K i v a l u e s f o r t h e s e i o n i c e f f e c t s a r e w i t h i n o r n e a r t h e r a n g e of p r e v i o u s l y d e t e r m i n e d i o n c o n c e n t r a t i o n s i n c r u s t a c e a n m u s c l e . W h i l e v a r i o u s s t u d i e s h a v e • been made o f m u s c l e i o n c o n c e n t r a t i o n s (Shaw, 1955; Dunham & G a i n e r , 1968; L a n g & G a i n e r , 1 9 6 9 a ) , I was n o t a b l e t o " f i n d 1 any m e a s u r e m e n t s f o r h y p o d e r m a l i o n c o n c e n t r a t i o n s . F u r t h e r m o r e , no v a l u e s e x i s t f o r t h e 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 i n m u s c l e o f C a n c e r m a g i s t e r . T h u s , i n o r d e r t o b e t t e r e v a l u a t e t h e s i g n i f i c a n c e o f t h e i o n i c e f f e c t s upon t h e s e p y r u v a t e k i n a s e s a n d p h o s p h o g l y c e r a t e 161 k i n a s e s , I measured- the' i o n i c c o m p o s i t i o n of hypodermis and muscle from Cancer m a g i s t e r . I n t r a c e l l u l a r i o n c o n c e n t r a t i o n s s e r e determined by measuring t h e t o t a l t i s s u e i o n c o n t e n t and t h e n c o r r e c t i n g t h i s f o r the i o n i c c o n t r i b u t i o n of the e x t r a c e l l u l a r space. F i n a l l y * t h e i o n i c c o m p o s i t i o n s were e x p r e s s e d i n terms o f the c o n c e n t r a t i o n o f the i o n w i t h i n the p o r t i o n of t h e t i s s u e o c c u p i e d by t h e c e l l s . Thus, e x t r a c e l l u l a r space v a l u e s f o r both muscle and hypodermis, hemolymph i o n c o n c e n t r a t i o n s , and t i s s u e i o n c o n t e n t s needed t o be determined. D u r i n g t h e molt c y c l e , the i o n i c c o m p o s i t i o n o f c r u s t a c e a n hemolymph undergoes c o n s i d e r a b l e v a r i a t i o n . G e n e r a l l y * t h e i o n s r i s e i n c o n c e n t r a t i o n b e f o r e e c d y s i s and then g r a d u a l l y drop d u r i n g t h e p o s t m o l t p e r i o d . I n t h e marine c r a b , - C a r c i n u s maenas, c a l c i u m shows the most marked v a r i a t i o n , i n c r e a s i n g by 30% between i n t e r m o l t and p r e m o l t * and then d r o p p i n g 30% below the i n t e r m o l t v a l u e i n p o s t m o l t . Sodium and p o t a s s i u m show i n c r e a s e s o f - a p p r o x i m a t e l y - -10% ( R o b e r t s o n , 1960), The f r e s h w a t e r c r a y f i s h , Orconectes l i m o s u s , shows more marked premolt i n c r e a s e s i n N a + , C l - , and K + and m a i n t a i n s e l e v a t e d Ca++ l e v e l s d u r i n g p o s t m o l t (Andrews, 1967). While c o n s i d e r a b l e i n f o r m a t i o n c o n c e r n i n g c r u s t a c e a n body f l u i d c o m p o s i t i o n and i t s v a r i a t i o n w i t h r e s p e c t to the molt c y c l e ( R o b e r t s o n , 1960; Andrews, 1967; G l y n n , 1968) , s a l i n i t y ( E n g e l h a r d t , 1970; A l s p a c h , 1972; S c h m i d t - N i e l s e n , 1975; Hunter & Rudy* 1975), and season (Dehnel & C a r e f o o t , 1965; C o l v o c o r e s s e s e t a l . , 1974) i s a v a i l a b l e , t i s s u e a n a l y s e s - a r e much l e s s common. Most s t u d i e s o f t i s s u e i o n i c c o m p o s i t i o n have concerned muscle and nerves 162 (Shaw, 1955;- Dunham £ G a i n e r , 1968; La n g & G a i n e r , 1969a; S c h m i d t - N i e l s e n , 1975) and ha v e been c o n c e r n e d w i t h t h e p r o p e r t i e s o f e x c i t a b l e membranes, v o l u m e r e g u l a t i o n , a n d t h e r e s p o n s e t o ' c h a n g i n g e x t e r n a l s a l i n i t i e s . No s t u d i e s h a v e s p e c i f i c a l l y c o n s i d e r e d m o l t c y c l e c o r r e l a t e d c h a n g e s i n i o n i c c o m p o s i t i o n . S i n c e hemolymph i o n c o n c e n t r a t i o n s c h a n g e w i t h t h e m o l t c y c l e , t i s s u e i o n c o n c e n t r a t i o n s may f l u c t u a t e - a s w e l l . Any m a j o r s h i f t s i n i o n i c c o m p o s i t i o n w o u l d a f f e c t a v a r i e t y o f c e l l u l a r c o n t r o l f u n c t i o n s , w o u l d s h i f t t h e e q u i l i b r i a o f m e t a l i o n - m e t a b o l i t e c o m p l e x f o r m a t i o n (Cumme e t a l . , 1 9 7 3 ) , a nd c e r t a i n l y w o u l d a f f e c t t h e f u n c t i o n o f numerous enzymes. As e x p l a i n e d a b o v e , p h o s p h o g l y c e r a t e k i n a s e s and p y r u v a t e k i n a s e s f r o m h y p o d e r m i s a n d m u s c l e o f C a n c e r m a g i s t e r a r e a f f e c t e d by t h e f o u r m a j o r c a t i o n s . F u r t h e r m o r e , p h o s p h o f r u c t o k i n a s e f r o m l o b s t e r m u s c l e and f r u c t o s e d i p h o s p h a t a s e f r o m k i n g c r a b h y p o d e r m i s a r e a l s o s e n s i t i v e t o t h e l e v e l s o f K* and M g + + ( H o c h a c h k a , 1972 ; Sugden 6 Newsholme, 1975b) .• S i n c e t h e s e f o u r enzymes a r e i n v o l v e d i n t h e c o n t r o l o f b o t h g l y c o l y s i s and g l u c o n e o g e n e s i s , c h a n g e s i n i o n l e v e l s c o u l d be o f r e g u l a t o r y s i g n i f i c a n c e . To e v a l u a t e t h e r e g u l a t o r y p o t e n t i a l - o f s u c h c h a n g e s , I m e a s u r e d t h e i o n c o n c e n t r a t i o n s i n - m u s c l e a nd h y p o d e r m i s o f b o t h i n t e r m o l t a n d f r e s h m o l t C a n c e r m a g i s t e r . METHODS C o l l e c t i o n and M a i n t e n a n c e o f A n i m a l s "- • A d u l t C a n c e r lagiSiSI were c o l l e c t e d by SCUBA d i v i n g a t J e r i c h o P i e r , V a n c o u v e r , B. C. B o t h male and f e m a l e a n i m a l s 163 were c o l l e c t e d . The a n i m a l s were m a i n t a i n e d i n a r e f r i g e r a t e d r e c i r c u l a t i n g s a l t w a t e r s y s t e m . W a t e r f o r t h i s s y s t e m comes f r o m an i n t a k e 6 m b e l o w mean l o w w a t e r s p r i n g t i d e s , a t F i r s t N a r r o w s i n t h e V a n c o u v e r H a r b o u r . The s a l i n i t y o f t h e s y s t e m was a b o u t 27.3±1°/oo t h r o u g h o u t t h e p e r i o d o f s t u d y . The i o n i c c o n c e n t r a t i o n s o f l o c a l s e a w a t e r c o r r e s p o n d i n g t o t h i s s a l i n i t y a r e Na++ 3 7 0 m M , K+ 8.2 mM, Ca++ 8.05 mM, a n d Mg** 42 mM ( E n g e l h a r d t , 1 9 7 0 ) . The t e m p e r a t u r e o f t h e s a l t w a t e r s y s t e m was m a i n t a i n e d a t 5-7 °C t h r o u g h o u t t h e s t u d y . A l l a n i m a l s were a c c l i m a t e d t o t h e new c o n d i t i o n s f o r a t l e a s t one week. O n l y f r e s h l y m o l t e d a n i m a l s and i n t e r m o l t a n i m a l s were u s e d f o r t h i s s t u d y , s i n c e t h e s e were t h e s t a g e s f r o m w h i c h enzyme p r e p a r a t i o n s had been made. T i s s u e P r e p a r a t i o n f o r I o n M e a s u r e m e n t s P r e l i m i n a r y e x p e r i m e n t s i n d i c a t e d t h a t t h e f o l l o w i n g method y i e l d e d t h e most c o n s i s t e n t r e s u l t s o f v a r i o u s p o s s i b l e ways o f s a m p l e p r e p a r a t i o n . I n d i v i d u a l c r a b l e g s were a m p u t a t e d , c u t o p e n , and u s e d f o r p r e p a r a t i o n o f 3-5 s a m p l e s , o f a p p r o x i m a t e l y 0.4 g , o f b o t h m u s c l e and h y p o d e r m i s . E a c h s a m p l e was q u i c k l y d i p p e d i n t o 0.4 M s u c r o s e ( l e s s t h a n 3 s e c o n d s s u b m e r s i o n ) , b l o t t e d b e t w e e n two p i e c e s o f No. 1 f i l t e r p a p e r , and w e i g h e d . S a m p l e s were w e i g h e d , a s h e d , and m e a s u r e d i n g l a s s s c i n t i l l a t i o n v i a l s . T h e s e v i a l s were f o u n d n o t t o c o n t r i b u t e - s o d i u m o r p o t a s s i u m t o t h e s a m p l e s . The s a m p l e s were a s h e d a t 450 °C {24 h o u r s ) , c o o l e d > and t h e n t a k e n up i n t h e a p p r o p r i a t e swamp. B l o o d s a m p l e s were t a k e n by i n s e r t i n g a h y p o d e r m i c s y r i n g e i n t o t h e a r t h r o d i a l membrane and w i t h d r a w i n g a p p r o x i m a t e l y a 0.5 ml 164 s a m p l e . T h i s was t h e n e m p t i e d o n t o a c l e a n p e t r i p l a t e . A c a l i b r a t e d L a n g - L e v y p i p e t t e was t h e n u s e d t o d e l i v e r 50 j u l o f b l o o d i n t o t h e s a m p l e v i a l s . T h e s e b l o o d s a m p l e s were t h e n s i m p l y d i l u t e d w i t h t h e a p p r o p r i a t e swamp. D i s t i l l e d w a t e r was t h e swamp u s e d f o r t h e measurement o f s o d i u m , 500 p a r t s p e r m i l l i o n Na + t h e swamp u s e d f o r p o t a s s i u m , -1.5% EDTA t h e swamp u s e d f o r magnesium, and 0.5% l a n t h a n u m c h l o r i d e ( a s L a + ) t h e swamp u s e d f o r c a l c i u m measurement. D e t e r m i n a t i o n o f I o n C o n c e n t r a t i o n s T i s s u e i o n i c c o n t e n t was d e t e r m i n e d by f l a m e p h o t o m e t r y o f t h e s a m p l e s . S odium was me a s u r e d by i t s e m i s s i o n a t 589.5 mn, p o t a s s i u m by i t s e m i s s i o n a t 766.8 mn, magnesium by i t s a b s o r b a n c e a t 285.8 mn, and c a l c i u m by i t s a b s o r b a n c e a t 423.5 mn. A T e c h t r o n AA 120 A t o m i c A b s o r p t i o n S p e c t r o p h o t o m e t e r was u s e d i n t h e s e s t u d i e s w i t h an a i r - a c e t y l e n e f l a m e . Once t i s s u e and b l o o d i o n c o n t e n t s were d e t e r m i n e d , t h e i n t r a c e l l u l a r i o n i c c o n c e n t r a t i o n s were d e t e r m i n e d by c o r r e c t i n g t h e t i s s u e i o n c o n t e n t f o r t h e i o n s due t o t h e e x t r a c e l l u l a r s p a c e . T h i s p r o c e e d e d a c c o r d i n g t o t h e f o l l o w i n g c a l c u l a t i o n : i „i ^ v - ; ^ „ _ Tissue Ion Content - Ions i n ECS I n t r a c e l l u l a r i o n c o n c e n t r a t i o n : Tissue grams - ECS grams D e t e r m i n a t i o n o f E x t r a c e l l u l a r S_pa.ce • T h r e e i n t e r m o l t and t h r e e f r e s h m o l t a n i m a l s were i n j e c t e d w i t h i * c i n u l i n ( d o s e a p p r o x i m a t e l y 1 >ucurie p e r 10 g b o d y w e i g h t ) . A t h o u r l y i n t e r v a l s , b l o o d s a m p l e s were t a k e n and a n a l y z e d f o r r a d i o a c t i v i t y . When t h e **C i n u l i n i n t h e b l o o d 165 had e g u i l i b r a t e d , 1 l e g p e r i n d i v i d u a l was a m p u t a t e d b e l o w t h e main j o i n t t o t h e body, s o t h a t t h e a n i m a l w o u l d n o t n e e d t o be s a c r i f i c e d . S i x h o u r s l a t e r f u r t h e r s a m p l e s were t a k e n . I m m e d i a t e l y a f t e r s a m p l i n g , t h e l e g s were c u t o p e n , and-3 s e m i -u n i f o r m s a m p l e s o f b o t h m u s c l e and h y p o d e r m i s d i s s e c t e d o u t . The s a m p l e s were q u i c k l y d i p p e d i n t o i s o s m o t i c s u c r o s e , t h o r o u g h l y b l o t t e d b e t ween two p i e c e s o f No. 1 f i l t e r p a p e r t o remove a d h e r i n g s u r f a c e b l o o d , and t h e n w e i g h e d . F o u r d r o p s o f 10% KOH were a d d e d t o t h e v i a l s and a l l o w e d t o d i g e s t o v e r n i g h t . The d i g e s t e d s a m p l e s were n e u t r a l i z e d w i t h H2S04, and c o u n t e d i n 10 ml o f A g u a s o l o r - S c i n t i v e r s e . B l o o d s a m p l e s t a k e n j u s t b e f o r e t h e t i s s u e s a m p l e s were c o u n t e d s i m u l t a n e o u s l y and u s e d t o d e t e r m i n e t h e - amount o f e x t r a c e l l u l a r s p a c e w i t h i n t h e r e s p e c t i v e t i s s u e s . S e p a r a t e e x t r a c e l l u l a r s p a c e d e t e r m i n a t i o n s were n o t made f o r a l l t h e t i s s u e s u s e d i n t h i s s t u d y ; r a t h e r , t h e means f o r f r e s h m o l t and i n t e r m o l t m u s c l e a n d h y p o d e r m i s were u s e d t o c a l c u l a t e t h e i n t r a c e l l u l a r i o n i c c o n c e n t r a t i o n s i n t h e r e s p e c t i v e t i s s u e s . RESULTS AND DISCUSSION I n t r a c e l l u l a r S p a c e D e t e r m i n a t i o n T a b l e 14 shows t h e r e s u l t s c f t h e e x t r a c e l l u l a r s p a c e d e t e r m i n a t i o n s f o r m u s c l e and h y p o d e r m i s . The r e s u l t s show c l e a r l y t h a t t h e r e i s a l a r g e d i f f e r e n c e i n e x t r a c e l l u l a r s p a c e between t h e m u s c l e and t h e h y p o d e r m i s , b u t t h a t t h e d i f f e r e n c e b e t w e e n i n t e r m o l t and f r e s h m o l t t i s s u e s i s s m a l l . The m u s c l e e x t r a c e l l u l a r s p a c e v a l u e s a r e s i m i l a r t o t h o s e f o u n d by Table 14 E x t r a c e l l u l a r Space i n Muscle and Hypodermis. Measurements were made as described i n Materials and Methods. Each measure-ment represents the mean of four determinations on one i n d i v i d u a l animal. Mean Intermolt muscle 14.16% 10.94% 12.04% 12.04% Intermolt hypodermis 39.62% 41.40% 43.64% . 49.94% Freshmolt hypodermis 41.90% 48.62% 45.27% 45.30% Freshmolt muscle 11.16% 13.61% 12.44% 12.34% 167 R o b e r t s o n (1961) f o r t h e Norway l o b s t e r , h i g h e r t h a n t h e 9% v a l u e f o u n d by Dunham & - G a i n e r (1968) f o r Homarus a m e r i c a n u s , l o w e r t h a n t h o s e f o r C a l l i n e c t e s s a p i d u s (Lang & G a i n e r , 1 9 6 9 b ) , and somewhat h i g h e r t h a n t h o s e f o u n d by A l s p a c h (1972) f o r C a n c e r m a g i s t e r i n h i g h s a l i n i t i e s . A l s p a c h (1972) i n d i c a t e d t h a t t h e m u s c l e e x t r a c e l l u l a r s p a c e i n c r e a s e d a s t h e a n i m a l s a d a p t e d t o d i l u t e s a l i n i t i e s . The l a c k o f c h a n g e i n e x t r a c e l l u l a r s p a c e w i t h t h e t r a n s i t i o n f r o m i n t e r m o l t t o f r e s h m o l t i n d i c a t e s t h a t t h e u p t a k e o f f l u i d d u r i n g t h e m o l t d o es n o t a f f e c t t h e v o l u m e o f e x t r a c e l l u l a r f l u i d a s s o c i a t e d w i t h a t i s s u e . I o n i c C o m p o s i t i o n o f t h e Hemolymph T a b l e 15 shows t h e mean v a l u e s f o r hemolymph c a t i o n c o n c e n t r a t i o n s o f i n t e r m o l t a n d f r e s h m o l t a n i m a l s . The volume c o r r e c t e d mean r e f l e c t s t h e c a l i b r a t i o n o f t h e L a n g - L e v y p i p e t t e . T h e r e was no s i g n i f i c a n t d i f f e r e n c e b e t ween i n t e r m o l t and f r e s h m o l t K + and Mg*+ c o n c e n t r a t i o n s . T h e r e was a s i g n i f i c a n t d i f f e r e n c e b e t ween c a l c i u m and s o d i u m c o n c e n t r a t i o n s i n i n t e r m o l t and f r e s h m o l t hemolymph, w i t h s o d i u m b e i n g h i g h e r and c a l c i u m l o w e r i n f r e s h m o l t hemolymph. The low c a l c i u m l e v e l may r e f l e c t i n c r e a s e d c a l c i u m m o b i l i z a t i o n d u r i n g t h e i n i t i a l c a l c i f i c a t i o n p e r i o d . R o b e r t s o n (1960) f o u n d i n c r e a s e d s o d i u m v a l u e s d u r i n g l a t e p r e m o l t and j u s t a f t e r m o l t , i n p a r a l l e l w i t h t h e r e s u l t s o f t h i s s t u d y . When t h e s e i o n c o n c e n t r a t i o n s a r e co m p a r e d t o t h o s e o f t h e medium ( s e e M e t h o d s i n t h i s s e c t i o n ) , magnesium s t a n d s ' o u t a s t h e m a j o r i o n whose c o n c e n t r a t i o n i s c o n t r o l l e d . C a n c e r m a g i s t e r s t r o n g l y h y p o r e g u l a t e s magnesium 168 Table 15 Hemolymph Ion Concentrations. Each mean value represents the mean of the ion concentrations i n as many i n -d i v i d u a l animals as i s i n d i c a t e d under "N". The volume corrected mean expresses these ion concentrations i n terms of the a c t u a l volume t r a n s f e r r e d by the Lang-Levy p i p e t t e s used f o r sample measurement. P expresses the r e s u l t s of Student's t t e s t comparison of molting and inte r m o l t i on concentrations. V.C. mean indicates volume corrected mean. P = 0.05 was the l e v e l o f s i g n i f i c a n c e . Intermolt Freshmolt Mean Stan. Dev. N V.C. Mean Mean Stan. Dev. N V.C. Mean P_ K + 8.15 mM ±1.13 9 8.57 mM 8.19 mM ±0.68 8 8.62 mM N.S. Na + 346.5 mM ±23.5 9 364.7 mM 376.85 mM ±16.7 8 396.7 mM =0.01 Mg + + 14.53 mM ±2.36 9 15.29 mM 15.03 mM ±0.67 8 15.82 mM N.S. C a + + 9.35 mM ±0.89 10 9.84 mM 6.72 mM ±0.3 8 7.07 mM =.000001 169 c o n c e n t r a t i o n s (14 mM i n t h e b l o o d v e r s u s 42 mM i n t h e medium). I n t e r m o l t c a l c i u m l e v e l s a r e v e r y s i m i l a r t o t h o s e i n - t h e medium w h i l e f r e s h m o l t l e v e l s were somewhat l o w e r . H y p o r e g u l a t i o n o f magnesium and h y p e r r e g u l a t i o n o f c a l c i u m was shown by E n g a l h a r d t (1970) f o r C a n c e r m a g i s t e r f r o m l o c a l w a t e r s , by H u n t e r and Rudy (1975) f o r C a n c e r m a g i s t e r f r o m O r e g o n , as w e l l a s by C o l v o c o r e s s e s e t a l . , (1974) f o r C a l l i n e c t e s s a o i d u s . I o n i c C o m p o s i t i o n o f M u s c l e and H l £ O d e r m i s • T a b l e 16 shows t h e i n t e r m o l t a nd f r e s h m o l t m u s c l e i o n c o n c e n t r a t i o n s . F o r t h e s a m p l e s i z e e x a m i n e d , o n l y s o d i u m l e v e l s showed a s i g n i f i c a n t d i f f e r e n c e (P=0.05) b e t w e e n f r e s h m o l t and i n t e r m o l t m u s c l e . The h i g h e r s o d i u m l e v e l s i n f r e s h m o l t m u s c l e c o r r e s p o n d t o t h e i n c r e a s e i n hemolymph s o d i u m i n f r e s h m o l t a n i m a l s . The c a l c i u m , magnesium and p o t a s s i u m c o n c e n t r a t i o n s were s i m i l a r f o r i n t e r m o l t a n d f r e s h m o l t m u s c l e . T a b l e 17 shows t h e i n t e r m o l t and f r e s h m o l t h y p o d e r m a l i o n c o n c e n t r a t i o n s . The mean v a l u e s f o r f r e s h m o l t . and i n t e r m o l t h y p o d e r m i s were s i m i l a r f o r e a c h i o n , a l t h o u g h t h e r e was c o n s i d e r a b l e v a r i a t i o n i n t h e s a m p l e s , p a r t i c u l a r l y f o r i n t e r m o l t c a l c i u m and p o t a s s i u m v a l u e s . F o r e a c h i o n t h e n u l l h y p o t h e s i s t h a t t h e c a t i o n c o n c e n t r a t i o n s i n f r e s h m o l t a n d i n t e r m o l t h y p o d e r m i s were t h e same was a c c e p t e d ( P > 0 . 0 5 ) . The v a r i a b i l i t y o f t h e s a m p l e s may be due t o a l a c k o f h o m o g e n e i t y w i t h i n t h e t i s s u e s , o r t o s m a l l v a r i a t i o n s i n t h e amount o f e x t r a c e l l u l a r s p a c e i n t h e t i s s u e s . The l a t t e r w o u l d be a p a r t i c u l a r p r o b l e m i n t h e measurement and c a l c u l a t i o n o f s o d i u m 170 Table 16 Freshmolt and Intermoit Muscle Ion Concentrations. For each experimental animal a mean ion concentration was determined from three to four separate samples. The mean value presented i n t h i s table represents the mean of these means. A l l values have been corrected f o r the i o n i c c o n t r i -bution of the e x t r a c e l l u l a r space i n the t i s s u e according to the formula i n Materials and, Methods . P = 0.05 was the significance l e v e l u t i l i z e d . Intermolt Mean - Stan. Dev. N Freshmolt Mean Stan. Dev. N Na 45.32 mM ±22.7 K + 112.70 mM ±10.67 ++ Ca Mg ++ 2.79 mM ±1.40 -12.23 mM ±2.74 5 4 5 4 89.81 mM ±4.77 114.00 mM ±19.8 2.87 mM ±1.43 13.43 mM ±2.1 4 =.045 4 N.S. 4 N.S. 4 N.S. 171 Table 17 Freshmolt and Intermolt Hypodermal Ion Concentrations. For each experimental animal, a mean ion concentration was determined from three to f o u r - t i s s u e samples. The mean value presented i n t h i s t a b l e represents the mean of these means. A l l values have been corrected f o r the i o n i c c o n t r i -bution of the\ e x t r a c e l l u l a r space. P = 0.05 was chosen as the l e v e l of s i g n i f i c a n c e . Intermolt Mean Stan. Dev. Freshmolt Mean Stan. Dev. N Na + K •i Ca Mg"1 ++ 49.51, mM ±22.7 4 74.7 mM ±19.7 5 16.78 mM ±11.88 5 19.33 mM ±4.23 4 52.4 mM ±19.77 70.81 mM ±7.64 16.07 mM ±4.86 15.90 mM ±3.02 3 N.S, .4 N.S. 4 N.S, 4 N.S. 172 c o n c e n t r a t i o n s , s i n c e s o d i u m l e v e l s i n t h e hemolymph a r e 5-6 f o l d t h o s e i n t h e h y p o d e r m i s . I n c o n t r a s t t o t h e m u s c l e , h y p o d e r m i s shows no i n c r e a s e i n i n t r a c e l l u l a r s o d i u m b e t w e e n i n t e r m o l t and f r e s h m o l t a n i m a l s . ••••* C o m p a r i s o n o f h y p o d e r m a l and m u s c l e i o n c o n c e n t r a t i o n s f r o m b o t h i n t e r m o l t and f r e s h m o l t . a n i m a l s r e v e a l s s e v e r a l marked d i f f e r e n c e s ( T a b l e 1 8 ) . I n b o t h m o l t i n g and i n t e r m o l t t i s s u e s , c a l c i u m l e v e l s a r e s i g n i f i c a n t l y h i g h e r (P=Q.05) i n t h e h y p o d e r m i s t h a n i n m u s c l e . P o t a s s i u m i s s i g n i f i c a n t l y h i g h e r i n i n t e r m o l t m u s c l e t h a n i n t e r m o l t h y p o d e r m i s , and- w h i l e t h e d i f f e r e n c e i s n o t s i g n i f i c a n t f o r - m o l t i n g t i s s u e s , i t i s s t i l l l a r g e . N e i t h e r magnesium n o r s o d i u m show a s i g n i f i c a n t d i f f e r e n c e b e t w e e n m u s c l e and h y p o d e r m i s . T h e r e a p p e a r s t o be a d i f f e r e n c e i n s o d i u m l e v e l s o f m o l t i n g t i s s u e s , b u t a l a r g e r s a m p l e s i z e w o u l d be n e c e s s a r y t o c o n f i r m t h i s ( n o t s i g n i f i c a n t a t P =0.05). The l e v e l s o f p o t a s s i u m and s o d i u m a r e more s i m i l a r i n t h e h y p o d e r m i s t h a n i n m u s c l e . C a l c i u m p r o v i d e s a g r e a t e r i o n i c c o n t r i b u t i o n i n h y p o d e r m i s t h a n i n m u s c l e . Magnesium l e v e l s a r e a p p r o x i m a t e l y - t h e same as c a l c i u m l e v e l s i n h y p o d e r m i s , w h i l e i n m u s c l e , magnesium i s p r e s e n t i n f o u r - f o l d h i g h e r c o n c e n t r a t i o n s t h a n c a l c i u m . T h u s , t h e r e l a t i o n s h i p s b e t w een t h e m a j o r c a t i o n s show n o t a b l e d i f f e r e n c e s i n m u s c l e and h y p o d e r m i s . A c o m p a r i s o n o f t h e s e m u s c l e i o n c o n c e n t r a t i o n s - w i t h i o n c o n c e n t r a t i o n s i n o t h e r c r u s t a c e a n s shows t h a t C a n c e r m a g i s t e r m u s c l e h a s a l o w e r - p o t a s s i u m l e v e l t h a n f o u n d i n Homarus SlS£icanus and Homarus v u l g a r i s (Dunham & G a i n e r , 1 9 6 8 ) . 173 Table 18 Comparison of Muscle and Hypodermal Ion Concentrations. Means are taken from the other t a b l e s . Although P = 0.05 i s the chosen l e v e l of s i g n i f i c a n c e , P values which l i e near t h i s l e v e l are also presented. Na K. Ca Mean Muscle Stan. Dev. Mg ++ 42.32 mM ±22.7 112.7 mM ±10.67 2.79 mM ±1.40 12.23 mM ±2.74 INTERMOLT Hypodermal Mean Stan. Dev. N 5 4 5 4 49.51 mM ±22.7 74.7 mM ±19.7 16.78 mM ±11.88 19.33 mM ±4.23 N 4 5 5 4 P_ N.S. =0.014 =0.05 N.S. Na K Ca Mg ++ Mean Muscle Stan. Dev. 89.82 mM ±4.77 114.0 mM ±19.8 ^ 2 . 8 7 mM ±1.43 .-13.43 mM ±2.1 FRESHMOLT Hypodermal Mean Stan. Dev. N N 4 4 4 4 52.4 mM ±19.77 3 70.8 mM ±7.64 4 16.08 mM ±4.86 4 15.91 mM ±3.02 4 P =0.08 = .06 = ,013 N.S. 174 However K + l e v e l s a r e s i m i l a r t o t h o s e i n C a r c i n u s - m u s c l e (Shaw, 1 9 5 5 ) . The s o d i u m v a l u e s a r e i n t h e same r a n g e o r l o w e r t h a n most p r e v i o u s l y p u b l i s h e d r e s u l t s ( R o b e r t s o n , 1961; Dunham & G a i n e r , 1968; Mackay & P r o s s e r , 1 9 7 0 ) . The c a l c i u m c o n c e n t r a t i o n f o u n d i n C a n c e r m a g i s t e r m u s c l e was c o n s i d e r a b l y l o w e r t h a n t h a t i n k i n g c r a b m u s c l e (Mackay & P r o s s e r , 1970) o r C a r c i n u s m u s c l e (Shaw, 1955) By c o n t r a s t , magnesium l e v e l s were h i g h e r " t h a n - t h o s e o f k i n g c r a b s , and l o w e r t h a n t h o s e o f C a r c i n u s maenas. The c o n c e n t r a t i o n s o f b o t h magnesium and c a l c i u m i n C a r c i n u s maenas hemolymph (Shaw, 1 9 5 5 ) , and magnesium i n k i n g c r a b hemolymph (Mackay S P r o s s e r , 1 9 7 0 ) , were c o n s i d e r a b l y h i g h e r t h a n t h e c o r r e s p o n d i n g c o n c e n t r a t i o n s i n t h e hemolymph o f C a n c e r m a g i s t e r . T h i s may a c c o u n t f o r t h e d i f f e r e n c e i n m u s c l e magnesium and c a l c i u m l e v e l s . No v a l u e s f o r i o n c o n c e n t r a t i o n s i n h y p o d e r m i s o f o t h e r c r u s t a c e a n s a r e a v a i l a b l e f o r c o m p a r i s o n . However, i n o t h e r a n i m a l s , when i n t r a c e l l u l a r s o d i u m i s h i g h , t h e i n t r a c e l l u l a r p o t a s s i u m c o n c e n t r a t i o n i s l o w ( S c h m i d t - N i e l s e n , 1 9 7 5 ) . T h i s p a t t e r n h o l d s t r u e f o r h y p o d e r m i s . The t o t a l sum o f t h e s e f o u r c a t i o n s i n b o t h \ m u s c l e and h y p o d e r m i s i s s i m i l a r t o t h e v a l u e s f o u n d i n o t h e r o r g a n i s m s (Mackay & P r o s s e r , 1 9 7 0 ; S c h m i d t - N i e l s e n , 1 9 7 5 ) . When one c o m p a r e s t h e c a t i o n c o n c e n t r a t i o n s i n t h e two t i s s u e s ( T a b l e s " 16 6 17, o r 18) w i t h t h e c a t i o n c o n c e n t r a t i o n s i n t h e hemolymph, s e v e r a l c o n c l u s i o n s c a n be made. B o t h h y p o d e r m i s and m u s c l e a c c u m u l a t e p o t a s s i u m a n d e x t r u d e s o d i u m . The c o n c e n t r a t i o n s o f magnesium i n t h e two t i s s u e s a r e o n l y s l i g h t l y d i f f e r e n t f r o m t h e c o n c e n t r a t i o n s i n t h e hemolymph. The m u s c l e m a i n t a i n s s l i g h t l y l o w e r magnesium c o n c e n t r a t i o n s , 175 w h i l e t h e h y p o d e r m i s h a s s l i g h t l y e l e v a t e d l e v e l s . The t i s s u e s r e g u l a t e t h e i r c a l c i u m c o n c e n t r a t i o n s i n o p p o s i t e ways; t h e h y p o d e r m i s seems t o a c c u m u l a t e c a l c i u m , w h i l e t h e m u s c l e seems t o e x c l u d e c a l c i u m . I n t h e k i n g c r a b , m u s c l e m a i n t a i n s o n l y s l i g h t l y l o w e r l e v e l s o f c a l c i u m t h a n t h e hemolymph, w h i l e t h e magnesium c o n c e n t r a t i o n i s c o n s i d e r a b l y l o w e r t h a n t h e hemolymph c o n c e n t r a t i o n (Mackay £ P r o s s e r , 1 9 7 0 ) . I n C a r c i n u . s maenas m u s c l e , magnesium and c a l c i u m l e v e l s a r e r e g u l a t e d i n much t h e same way as i n C a n c e r m a g i s t e r (Shaw, 1 9 5 5 ) . The h i g h e r l e v e l s o f c a l c i u m i n t h e h y p o d e r m i s a r e p r o b a b l y r e l a t e d t o c a l c i f i c a t i o n a n d r e p a i r o f t h e e x o s k e l e t o n . A l t h o u g h t h e s e c o n c e n t r a t i o n m e a s u r e m e n t s do n o t r e p r e s e n t t h e a c t u a l a c t i v i t i e s o f t h e i o n s i n t h e c e l l s , t h e y do p r e s e n t g u i d e l i n e s a s t o t h e c o n c e n t r a t i o n s w h i c h t h e enzymes e n c o u n t e r i n t h e c e l l . T h u s , one c a n s a y t h a t t h e c o m p e t i t i v e i n h i b i t i o n b e t w e e n C a + + and M g + + w i l l be o f s m a l l i m p o r t a n c e i n m u s c l e , where Mg** l e v e l s a r e 4 - f o l d h i g h e r t h a n c a l c i u m l e v e l s . I n t h e h y p o d e r m i s , h o w e v e r , • Mg++ and C a + + c o n c e n t r a t i o n s a r e a p p r o x i m a t e l y t h e same. T h u s , u n l e s s t h e r e i s s i g n i f i c a n t c o m p a r t m e n t a l i z a t i o n and b i n d i n g o f t h e s e d i v a l e n t c a t i o n s , c o m p e t i t i v e i n h i b i t i o n - b y C a + + o f Mg+ + a c t i v a t i o n may p l a y a s i g n i f i c a n t r o l e i n r e g u l a t i n g t h e a c t i v i t y o f s u c h e n z y m es a s p y r u v a t e k i n a s e a n d p h o s p h o g l y c e r a t e k i n a s e . P y r u v a t e k i n a s e i s p a r t i c u l a r l y s e n s i t i v e t o c a l c i u m i n h i b i t i o n , w i t h a K i v e r s u s magnesium o f 7. 5 x 10~ SM. F o r p h o s p h o g l y c e r a t e k i n a s e * Ca++ i n h i b i t i o n i s most e f f e c t i v e v e r s u s t h e b i n d i n g o f 3 - p h o s p h o -g l y c e r a t e , Ki = 2mM, w h i l e t h e i n h i b i t i o n v e r s u s M g + + shows a K i v a l u e o f 20 mM. Of t h e two e n z y m e s , p y r u v a t e k i n a s e i s t h u s 1 7 6 more s u s c e p t i b l e t o i n h i b i t i o n by c a l c i u m . P y r u v a t e k i n a s e must be i n h i b i t e d f o r g l u c o n e o g e n e s i s t o o c c u r w h i l e p h o s p h o g l y c e r a t e k i n a s e ' i s one o f t h e f u n c t i o n a l enzymes i n t h e g l u c o n e o g e n i c p a t h w a y . T h u s , ' ' i n t h e c o n t e x t o f g l u c o n e o g e n e s i s , - t h e s e d i f f e r e n t i a l s e n s i t i v i t i e s t o c a l c i u m i n h i b i t i o n make f u n c t i o n a l s e n s e . T h e r e " i s -no - s i g n i f i c a n t d i f f e r e n c e 1 b e t w e e n c a l c i u m l e v e l s i n i n t e r m o l t and f r e s h m o l t h y p o d e r m i s , s o d i f f e r e n t i a l c a l c i u m i n h i b i t i o n d u r i n g ' t h e v a r i o u s p a r t s o f t h e m o l t c y c l e d o e s n o t come i n t o p l a y i n t h e r e g u l a t i o n o f t h e s e e n z y m e s . P o t a s s i u m l e v e l s i n a l l t i s s u e s a r e h i g h e n o u g h t o f u l l y a c t i v a t e p y r u v a t e k i n a s e (Ka = 15 mM). S i n c e t h e K i f o r s o d i u m i n h i b i t i o n v e r s u s p h o s p h o e n o l p y r u v a t e b i n d i n g i s 40 mM, a n d t h e K i v e r s u s K + i s 85 mM, s o d i u m i n h i b i t i o n w i l l p r o b a b l y be o f l i t t l e s i g n i f i c a n c e . P o t a s s i u m and s o d i u m b o t h a c t i v a t e p h o s -p h o g l y c e r a t e k i n a s e - . F o r i n t e r m o l t t i s s u e s , t h e Ka v a l u e s f o r s o d i u m a c t i v a t i o n a r e 100 mM, w e l l above i n t r a c e l l u l a r s o d i u m l e v e l s . I n m o l t i n g t i s s u e s , s o d i u m l e v e l s a r e h i g h e r t h a n i n i n t e r m o l t t i s s u e s . The Ka v a l u e s f o r s o d i u m a c t i v a t i o n i n m o l t i n g t i s s u e s ' d r o p t o 30 mM and 19 mM f o r m u s c l e a n d h y p o d e r m i s , r e s p e c t i v e l y . T h u s , s o d i u m a c t i v a t i o n i n m o l t i n g t i s s u e s i s more f e a s i b l e . P o t a s s i u m Ka v a l u e s do n o t f l u c t u a t e much t h r o u g h o u t ' t h e m o l t c y c l e , r e m a i n i n g a r o u n d 80 mM f o r b o t h h y p o d e r m a l and m u s c l e p h o s p h o g l y c e r a t e k i n a s e . I n m u s c l e , p o t a s s i u m l e v e l s a r e a p p r o x i m a t e l y 110 mM, w h i l e i n h y p o d e r m i s , p o t a s s i u m l e v e l s ' a r e l o w e r , n e a r 70 mM. T h u s , a c t i v a t i o n o f p h o s p h o g l y c e r a t e k i n a s e by p o t a s s i u m w o u l d be a p p r e c i a b l e , b u t w o u l d n o t f l u c t u a t e b e t w e e n i n t e r m o l t and f r e s h m o l t . w h i l e t h e d a t a on t h e e f f e c t s o f t h e s e f o u r c a t i o n s on c r u s t a c e a n p h o s p h o -177 f r u c t o k i n a s e a r e - n o t a v a i l a b l e , magnesium i s r e g u i r e d f o r i t s c a t a l y t i c a c t i v i t y a n d p o t a s s i u m a c t i v a t e s -between 0 a n d 50 mM and i n h i b i t s between 100 and 200 mM (Sugden 6 Newsholme, 1 9 7 5 b ) . D e p e n d i n g upon t h e d e g r e e o f a s s o c i a t i o n o f K* w i t h t i s s u e p r o t e i n s , K+ l i e s i n t h e i n h i b i t o r y c o n c e n t r a t i o n r a n g e w i t h i n t h e m u s c l e c e l l s , a n d on t h e b o r d e r l i n e o f t h i s i n h i b i t o r y r a n g e i n t h e h y p o d e r m i s . K+, Na+, and Mg* + a l l - have p r o f o u n d e f f e c t s upon t h e a c t i v i t y o f h y p o d e r m a l f r u c t o s e d i p h o s p h a t a s e ( H o c k a c h k a , 1 9 7 2 ) . Sodium i s i n h i b i t o r y a t a l l c o n c e n t r a t i o n s t e s t e d , M g + + o r M n + + i s r e g u i r e d f o r c a t a l y s i s , and K+ a c t i v a t e s a t l e v e l s up t o 100 mM. K + a l s o a c c e n t u a t e s t h e AMP i n h i b i t i o n o f t h e enzyme, w h i l e M g + + r e v e r s e s t h i s i n h i b i t i o n . The i o n i c c o n c e n t r a t i o n s - w h i c h a f f e c t enzyme a c t i v i t y a r e w i t h i n t h e 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 r a n g e . T h e r e f o r e , t h e i n t e r a c t i o n s b e t w e e n K*, M g + + , AMP, and FDP p r o v i d e an i m p o r t a n t e l e m e n t i n t h e c o n t r o l o f h y p o d e r m a l F D P a s e s ( H o c h a c h k a , 1 9 7 2 ) . T h u s , e v e n t h o u g h t h e s e c a t i o n s p r o b a b l y do n o t p l a y a r o l e i n g u i c k c h a n g e s o f enzyme a c t i v i t y , t h e i r c o n c e n t r a t i o n s c o u l d p a r t i a l l y d e f i n e t h e l i m i t s o f c a t a l y t i c a c t i v i t y . 178 GENERAL DISCUSSION AND SUMMARY T h e s e s t u d i e s o f p h o s p h o g l y c e r a t e k i n a s e and p y r u v a t e k i n a s e from- C a n c e r - m a g i s t e r h a v e l e d t o s e v e r a l i m p o r t a n t c o n c l u s i o n s c o n c e r n i n g b o t h t h e r e g u l a t o r y p r o p e r t i e s o f t h e enzymes f r o m • m u s c l e and h y p o d e r m i s a s w e l l a s t h e - p o s s i b l e e n z y m a t i c a d a p t a t i o n s w h i c h f a c i l i t a t e e f f i c i e n t g l u c o n e o g e n e s i s and g l y c o l y s i s i n = t h e same t i s s u e . T h i s s t u d y h a s a l s o p r o v i d e d some i n s i g h t i n t o t h e c h a n g e s i n m e t a b o l i c o r g a n i z a t i o n w h i c h o c c u r i n t h e m u s c l e and h y p o d e r m i s d u r i n g t h e m o l t c y c l e , a s w e l l a s a l l o w i n g e v a l u a t i o n o f t h e p o t e n t i a l f o r g l u c o n e o g e n e s i s i n t h e m u s c l e o f C a n c e r m a g i s t e r . To c o n c l u d e t h i s s t u d y , t h e m a j o r i m p l i c a t i o n s o f t h e s e f i n d i n g s w i l l be d i s c u s s e d , a l o n g w i t h q u e s t i o n s l e a d i n g t o f u r t h e r e x p e r i m e n t a t i o n . The r e q u i r e m e n t f o r e f f i c i e n t g l u c o n e o g e n e s i s i n c r u s t a c e a n s a r i s e s f r o m t h e p e r i o d i c s h e d d i n g o f t h e o l d e x o s k e l e t o n and t h e s u b s e q u e n t s y n t h e s i s o f a new l a r g e r c h i t i n o u s e x o s k e l e t o n . I n most c r u s t a c e a n s , t h e s y n t h e s i s o f t h e new e x o s k e l e t o n i s s u p p o r t e d - by r e s o r b e d m a t e r i a l * - a s w e l l a s by e n d o g e n o u s s t o r e s ( R e n a u d , 1949; H e a t h S B a r n e s * 1970: S p e c k e t a l . , 1 9 7 1 ) . E x p e r i m e n t s w i t h O r c o n e c t e s l i m o s u s i n d i c a t e t h a t t h e r e s o r b e d m a t e r i a l u n d e r g o e s i n i t i a l c o n v e r s i o n i n t o c h i t i n , amino a c i d s , and l a c t a t e . The l a t t e r - t w o compounds a r e - s u b s e q u e n t l y u t i l i z e d f o r b o t h c h i t i n s y n t h e s i s a n d e n e r g y p r o v i s i o n ( H e r z - H u b n e r a t a l , 1 9 7 3 ) . S i m i l a r c o n v e r s i o n s i n t o amino a c i d s and l a c t a t e o c c u r w i t h hemolymph g l u c o s e and i n g e s t e d g l u c o s e i n O r c o n e c t e s l i m o s u s (Speck S U r i c h , 1969; U r i c h e t a l . , 1973; H e r z - H u b n e r S U r i c h , 1973) and i n o t h e r 179 c r u s t a c e a n s ( H u g g i n s , 1966; H u g g i n s , 1969; S h e w b a r t e t a l , , 1 9 7 2 ) . T h u s , t h e m e t a b o l i c r o u t e s f o r t h e c o n v e r s i o n o f amino a c i d s and l a c t a t e i n t o p o l y s a c c h a r i d e s a r e e m p h a s i z e d d u r i n g t h e b i o s y n t h e s i s o f t h e c h i t i n o u s e x o s k e l e t o n . U t i l i z a t i o n o f e n d o g e n o u s s t o r e s w o u l d augment t h e s e b a s i c m e t a b o l i c f l u x p a t t e r n s . lil£2^S£ial G l u c o n e o g e n e s i s Measurement o f enzyme a c t i v i t i e s i n d i c a t e s t h a t g l u c o n e o -g e n e s i s i n c r u s t a c e a n s c a n f o l o w t h e same p a t h w a y u t i l i z e d i n v e r t e b r a t e s ( S c r u t t o n S U t t e r , 1 9 6 8 ) . T h u s , p y r u v a t e c a r b o x y l a s e and f r u c t o s e d i p h o s p h a t a s e a r e p r e s e n t i n a l l t i s s u e s , w h i l e p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i s p r e s e n t i n a l l t i s s u e s e x c e p t i n t e r m o l t m u s c l e . The e f f e c t i v e f u n c t i o n i n g o f t h e s e b y p a s s enzymes i s p a r t i a l l y d e p e n d e n t upon t h e a c t i v i t y o f t h e c o r r e s p o n d i n g g l y c o l y t i c r e a c t i o n . I n b o t h t h e f r e s h m o l t and i n t e r m o l t h y p o d e r m i s , f r u c t o s e d i p h o s p h a t a s e l e v e l s a r e t h r e e f o l d h i g h e r t h a n t h o s e o f p h o s p h o f r u c t o k i n a s e . T h u s , n e t g l u c o n e o g e n i c f l u x t h r o u g h t h i s r e a c t i o n l o c u s i s f e a s i b l e . By c o n t r a s t , p y r u v a t e k i n a s e l e v e l s a r e a p p r o x i m a t e l y -20 f o l d h i g h e r t h a n t h o s e ' O f t h e - b y p a s s enzymes. G l u c o n e o g e n i c f l u x t h r o u g h t h i s r e a c t i o n l o c u s n e c e s s i t a t e s e f f e c t i v e i n h i b i t i o n o f p y r u v a t e k i n a s e . A t p h y s i o l o g i c a l l e v e l s o f PEP, ADP, ATP, a l a n i n e , and ' s e r i n e , • t h e l o w a f f i n i t y f o r m o f h y p o d e r m a l p y r u v a t e k i n a s e (PK I I ) - shows l e s s t h a n 0.2% o f i t s m a x i m a l a c t i v i t y . T h u s , n e t f l u x t h r o u g h t h e p y r u v a t e c a r b o x y l a s e , p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e b y p a s s i s p o s s i b l e . The m a x i m a l g l u c o n e o g e n i c f l u x t h r o u g h t h e b y p a s s i s a p p r o x i m a t e l y 180 3 0 % o f t h e m a x i m a l FDPase a c t i v i t y . As m e n t i o n e d b e f o r e , t h e v a l u e s f o r PEPCK may be low> s i n c e t h e enzyme was n o t m e a s u r e d i n t h e g l u c o n e o g e n i c d i r e c t i o n (Chang e t a l . , 1966; P o g s o n & S m i t h , 1 9 7 5 ) . T h e o t h e r p o s s i b l e " b o t t l e n e c k " i n t h e g l u c o n e o -g e n i c p a t h w a y o c c u r s a t t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n , P h o s p h o g l y c e r a t e k i n a s e l e v e l s , m e a s u r e d i n t h e g l u c o n e o g e n i c d i r e c t i o n , a r e c o n s i d e r a b l y h i g h e r t h a n t h o s e o f PEPCK, p y r u v a t e c a r b o x y l a s e , and P D P a s e ; F u r t h e r m o r e , b o t h t h e h y p o d e r m a l a n d t h e f r e s h m o l t m u s c l e enzymes show k i n e t i c m o d i f i c a t i o n s w h i c h f a c i l i t a t e t h e r e v e r s a l o f t h e r e a c t i o n f o r g l u c o n e o g e n e s i s . The p r e s e n c e o f g l u t a m i n e : f r u c t o s e - 6 - p h o s p h a t e a m i d o t r a n s f e r a s e i n t h e h y p o d e r m i s and i n f r e s h m o l t m u s c l e i n d i c a t e s t h a t t h e f r u c t o s e - 6 - p h o s p n a t e g e n e r a t e d by t h e FDPase r e a c t i o n c a n be d i r e c t e d t o w a r d s c h i t i n s y n t h e s i s . M u s e l e G l u c o n e o g e n e s i s 1 The v a r i o u s enzymes i n t h e g l u c o n e o g e n i c s e q u e n c e a r e p r e s e n t i n f r e s h m o l t m u s c l e . However, t h e r a t i o s o f p h o s p h o -f r u c t o k i n a s e t o FDPase and o f p y r u v a t e k i n a s e t o p h o s p h o e n o l -p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e a r e c o n s i d e r a b l y h i g h e r t h a n i n t h e h y p o d e r m i s . The c o n t r o l mechanisms o f C a n c e r m a g i s t e r m u s c l e p h o s p h o f r u c t o k i n a s e a r e n o t known e x a c t l y . H owever, e x t r a p o l a t i o n f r o m t h e c o n t r o l p r o p e r t i e s o f l o b s t e r and k i n g c r a b - p h o s p h o f r u c t o k i n a s e s i n d i c a t e s t h a t a t p h y s i o l o g i c a l f r u c t o s e - 6 - p h o s p h a t e l e v e l s , h i g h l e v e l s o f • ATP and a r g i n i n e p h o s p h a t e would- c a u s e more t h a n 7 5 % i n h i b i t i o n ( F r e e d , 1971; Sugden & Newsholme, 1 9 7 5 b ) . T h i s w o u l d a l l o w n e t g l u c o n e o g e n i c f l u x t h r o u g h t h e r e a c t i o n l o c u s . P y r u v a t e k i n a s e 181 i s p r e s e n t a t n e a r l y 500 f o l d t h e l e v e l s o f i t s b y p a s s enzymes. U n l i k e t h e h y p o d e r m a l p y r u v a t e k i n a s e , t h e m u s c l e enzyme d o e s n o t e x i s t i n i n t e r c o n v e r t i b l e h i g h a n d l o w a f f i n i t y s t a t e s . T h u s , t h e k i n e t i c " c h a r a c t e r i s t i c s o f m u s c l e p y r u v a t e k i n a s e make i t h i g h l y u n l i k e l y t h a t any c o m b i n a t i o n o f i n h i b i t o r s c o u l d r e d u c e enzyme a c t i v i t y t o l e s s t h a n 0.2% o f Vmax. I n c o n t r a s t t o * r a b b i t m u s c l e p y r u v a t e k i n a s e , where t h e r e v e r s e r e a c t i o n a c c o u n t s f o r 5% o f t h e g l y c o l y t i c r e a c t i o n (Dyson e t a l - . , 1975) , t h e r e v e r s e r e a c t i o n o f t h e enzyme f r o m C a n c e r m a q i s t e r m u s c l e a c c o u n t e d f o r - o n l y 0.5% o f t h e f o r w a r d r e a c t i o n . - M a x i m a l r e v e r s a l o c c u r r e d a t 10 mM p y r u v a t e , a h i g h l y u n p h y s i o l o g i c a l c o n c e n t r a t i o n . -• A l t h o u g h t h e k i n e t i c m o d i f i c a t i o n s o f f r e s h m o l t m u s c l e p h o s p h o g l y c e r a t e k i n a s e w o u l d f a c i l i t a t e g l u c o n e o g e n e s i s , t h e c h a r a c t e r i s t i c s o f t h e p y r u v a t e k i n a s e l o c u s a r e s u c h a s t o p r e v e n t n e t g l u c o n e o g e n i c f l u x f r o m l a c t a t e , p y r u v a t e , o r a l a n i n e . T h i s c o n c l u s i o n c o n t r a s t s w i t h t h e f i n d i n g s o f B e h r i s c h (1976) who r e p o r t s b o t h h i g h l e v e l s o f t h e b y p a s s enzymes and h i g h r a t e s o f g l u c o n e o g e n e s i s f r o m l a c t a t e a n d p y r u v a t e i n P a r a l i t h o d e s c a m t c h a t i c a m u s c l e ( B e h r i s c h , - 1976) . The l e v e l s o f FDPase> PEPCK, and p y r u v a t e c a r b o x y l a s e f o u n d by B e h r i s c h (1972, 1976) a r e c o n s i d e r a b l y h i g h e r t h a n t h o s e f o u n d i n t h e p r e s e n t ' s t u d y o r t h o s e f o u n d by O p i e and Newsholme (1967) and - i y C r a b t r e e e t a l . (1972) i n t h e -muscle -of v a r i o u s i n v e r t e b r a t e s i n c l u d i n g C a r c i n u s maenas and M a i a s q u i n a d o . U n l e s s t h e r e i s s i g n i f i c a n t , c o m p a r t m e n t a l i z a t i p n o f enzyme a c t i v i t i e s , w h i c h somehow s u c c e e d s i n s e p a r a t i n g t h e p h o s p h o -e n o l p y r u v a t e u s e d by p y r u v a t e k i n a s e a s a s u b s t r a t e f r o m t h e p h o s p h o e n o l p y r u v a t e p r o d u c e d by p h o s p h o e n o l p y r u v a t e c a r b o x y -182 k i n a s e , n e t g l u c o n e o g e n i c f l u x t h r o u g h t h i s r e a c t i o n l o c u s i s n o t l i k e l y t o o c c u r i n C a n c e r m a g i s t e r m u s c l e . M o l t C y c l e V a r i a t i o n s i n Enzyme L e v e l s B o t h h y p o d e r m a l and m u s c l e enzyme a c t i v i t i e s were f o u n d t o c h a n g e d u r i n g t h e m o l t c y c l e . Enzyme l e v e l s i n t h e h y p o d e r m i s g e n e r a l l y i n c r e a s e d d u r i n g t h e t r a n s i t i o n f r o m i n t e r m o l t t o m o l t . T h i s i s d i a g n o s t i c o f t h e i n c r e a s e i n m e t a b o l i c • a c t i v i t y o f t h e h y p o d e r m i s d u r i n g p r e m o l t and p o s t m o l t , a s w a l l a s t h e i n c r e a s e i n amount o f p r o t e i n p e r DNA d u r i n g p r e m o l t ( S t e v e n s o n , 1 9 7 2 ) . W h i l e m u s c l e enzyme a c t i v i t i e s g e n e r a l l y d e c r e a s e d u r i n g t h e t r a n s i t i o n f r o m i n t e r m o l t t o f r e s h m o l t , some e n z y m e s , s u c h a s F D P a s e , PEPCK and p y r u v a t e c a r b o x y l a s e a r e p r e s e n t i n i n c r e a s e d l e v e l s i n t h e m u s c l e o f f r e s h l y m o l t e d a n i m a l s . As d i s c u s s e d i n t h e p r e v i o u s p a r a g r a p h , t h i s i n c r e a s e d g l u c o n e o -g e n i c p o t e n t i a l i s p r o b a b l y n o t u t i l i z e d f o r s y n t h e s i s o f p o l y s a c c h a r i d e s f r o m amino a c i d s and l a c t a t e . O t h e r g l u c o n e o -g e n i c p r e c u r s o r s s u c h a s g l y c e r o l may be u t i l i z e d . I n t h i s c o n t e x t , some' o f t h e o b s e r v a t i o n s o f S p e c k a n d U r i c h (1972) on O r c o n e c t e s l i m o s u s a r e o f i n t e r e s t . T h e s e w o r k e r s f o u n d t h a t w h i l e c o n s i d e r a b l e c a r b o n f r o m r e s o r b e d a c e t y l g l u c o s a m i n e was i n c o r p o r a t e d i n t o v a r i o u s compounds i n t h e m u s c l e , t h e d i s t r i b u t i o n o f l a b e l l e d c a r b o n i n t h e s e compounds d i d n o t c h a n g e i f one c o m p a r e d t h e m u s c l e o f l a t e p r e m o l t , e a r l y p o s t m o l t and p a r t i a l l y h a r d e n e d a n i m a l s . W h i l e some o f t h e c a r b o n f r o m a c e t y l g l u c o s a m i n e was c o n v e r t e d i n t o p o l y s a c c h a r i d e s i n t h e m u s c l e , t h e b u l k o f t h e l a b e l s t a y e d i n t h e f r a c t i o n w i t h t h e i n t e r m e d i a r y compounds, i n c l u d i n g amino a c i d s , s m a l l o r g a n i c 183 a c i d s , a n d s u g a r s . I n o t h e r s t u d i e s ( H e r z - H u b n e r & O r i c h , 1 9 7 3 ) , i t was f o u n d t h a t o n l y a s m a l l p r o p o r t i o n o f a c e t y l g l u c o s a m i n e i s r e l e a s e d f r o m t h e r e s o r b i n g t i s s u e s u n c h a n g e d . T h u s , l a b e l w h i c h e n t e r e d t h e m u s c l e was i n t h e f o r m o f s m a l l i n t e r m e d i a r y compounds. T h e s e s t u d i e s i n d i c a t e t h a t i n t h e s e c r a y f i s h , m u s c l e p l a y s a r e l a t i v e l y q u i e s c e n t r o l e d u r i n g t h e b i o s y n t h e s i s o f t h e e x o s k e l e t o n d u r i n g p r e m o l t and p o s t m o l f . However, t h i s s i t u a t i o n may be somewhat a l t e r e d i n a n i m a l s w i t h l a r g e , l o n g a p p e n d a g e s , where t h e m u s c l e f o r m s t h e b u l k o f t h e t i s s u e i n t h e v i c i n i t y o f the h y p o d e r m i s and t h e - n a s c e n t e x o s k e l e t o n . W h i l e t h i s i s an i n t e r e s t i n g c o n c e p t , t h e p r e s e n t s t u d y d o e s n o t p r o v i d e s u p p o r t i v e e v i d e n c e . C o n t r o l o f G l y c o l y s i s and G l u c o n e o g e n e s i s i n t h e H y p o d e r m i s W h i l e h y p o d e r m a l enzyme a c t i v i t i e s i n d i c a t e t h a t t h e t i s s u e i s p o i s e d t o w a r d s g l u c o n e o g e n e s i s , t h e y a l s o show t h a t i t m a i n t a i n s an a m ple g l y c o l y t i c c a p a c i t y . The i n f o r m a t i o n g l e a n e d f r o m t h i s s t u d y , c o u p l e d w i t h p r e v i o u s work o n - t h e c o n t r o l p r o p e r t i e s o f t h e h y p o d e r m a l - FDPase f r o m P a r a l i t h o d e s camt-c h a t i c a ( H o c h a c h k a , 1 9 7 2 ) , a l l o w s t h e f o r m u l a t i o n o f a m e t a b o l i c c o n t r o l t h e o r y d e s c r i b i n g t h e means by w h i c h h y p o d e r m a l m e t a b o l i s m s w i t c h e s f r o m g l u c o n e o g e n e s i s t o g l y c o l y s i s . - D u r i n g g l u c o n e o g e n e s i s f r o m l a c t a t e o r amino a c i d s s u c h a s a l a n i n e , g l u t a m a t e and a s p a r t a t e , t h e l e v e l s o f ATP and t h e o v e r a l l e n e r g y c h a r g e a r e h i g h . The h i g h l e v e l s o f ATP and t h e h i g h e n e r g y c h a r g e w o u l d c o m b i n e t o i n h i b i t p y r u v a t e k i n a s e and p r e s u m a b l y p h o s p h o f r u c t o k i n a s e . • S i n c e h y p o d e r m a l l e v e l s o f p h o s p h o f r u c t o k i n a s e l i e b e l o w t h o s e o f f r u c t o s e d i p h o s p h a t a s e . 184 o n l y p a r t i a l i n h i b i t i o n o f p h o s p h o f r u c t o k i n a s e x s n e c e s s a r y t o a c h i e v e a p p r e c i a b l e g l u c o n e o g e n i c f l u x . The h i g h e n e r g y c h a r g e w o u l d s e r v e t o a c t i v a t e b o t h F D P a s e s p r e s e n t i n t h e h y p o d e r m i s , s i n c e h i g h e n e r g y c h a r g e i n d i c a t e s low l e v e l s o f AMP, a p o t e n t i n h i b i t o r o f t h e s e a n d o t h e r F D P a s e s ( H o c h a c h k a , 1 9 7 2 ) . The l o w l e v e l s o f U D P - a c e t y i g l u c o s a m i n e w h i c h w o u l d o c c u r d u r i n g t h e i n i t i a t i o n o f c h i t i n s y n t h e s i s a c t i v a t e FDPase I I by d e c r e a s i n g t h e Km f o r f r u c t o s e 1,6 d i p h o s p h a t e ( H o c h a c h k a , 1 3 7 2 ) . T h i s a c t i v a t i o n o f FDPase w o u l d l e a d t o a d e c r e a s e i n l e v e l s o f f r u c t o s e 1,6 d i p h o s p h a t e ^ T h i s d e c r e a s e i n FDP w o u l d s h i f t t h e e g u i l i b r i u m b e t w e e n h y p o d e r m a l PK I and PK I I i n f a v o r o f t h e l o w a f f i n i t y f o r m , PK I I , w h i c h i s more s u s c e p t i b l e t o i n h i b i t i o n by amino a c i d s and c i t r a t e ( F i g u r e 27, p. 1 5 6 ) . I n a d d i t i o n , PK I I h a s s u c h a l o w a f f i n i t y f o r p h o s p h o e n o l p y r u v a t e t h a t i t s g l y c o l y t i c f u n c t i o n w o u l d be l i m i t e d i n t h e p r e s e n c e o f p h y s i o l o g i c a l PEP c o n c e n t r a t i o n s . T h u s , t h e s h i f t i n p y r u v a t e k i n a s e enzymes f r o m -the h i g h a f f i n i t y t o t h e l o w a f f i n i t y c o n f o r m a t i o n w o u l d e f f e c t i v e l y i n h i b i t p y r u v a t e k i n a s e and a l l o w f l u x t h r o u g h t h e e n z y m a t i c b y p a s s * The e x a c t means w h e r e b y f l u x t h r o u g h t h i s b y p a s s i s a c t i v a t e d a r e n o t known. I n t h e mammalian l i v e r , h i g h l e v e l s o f ATP f a v o r b o t h the c o n v e r s i o n o f p y r u v a t e t o o x a l o a c e t a t e and t h e c o n v e r s i o n o f o x a l o a c e t a t e t o p h o s p h o e n o l p y r u v a t e (Newsholme & S t a r t , 1973; G a r b e r £ S a l g a n i c o f f * 1 9 7 3 ) . C y c l i n g a r o u n d t h i s r e a c t i o n l o c u s may p r o v i d e c o s t l y , b u t p r e c i s e r e g u l a t i o n o f n e t g l u c o n e o g e n i c f l u x (Newsholme 6 S t a r t , 1 9 7 3 ) , o r i t may be u t i l i z e d t o s u p p o r t t h e t r a n s f e r o f r e d u c i n g u n i t s , f o r p u r p o s e s o t h e r t h a n g l u c o n e o g e n e s i s , f r o m t h e m i t o c h o n d r i a t o t h e c y t o p l a s m ( M e i j e r 185 & W i l l i a m s o n , 1974) . H i g h l e v e l s o f ATP and l o w l e v e l s o f ADP, c h a r a c t e r i s t i c o f a h i g h e n e r g y c h a r g e , a l s o f a v o r t h e g l u c o n e o g e n i c r e v e r s a l o f p h o s p h o g l y c e r a t e k i n a s e . The enzyme p r e s e n t i n t h e h y p o d e r m i s o f t h e f r e s h l y m o l t e d a n i m a l shows a h i g h a f f i n i t y f o r ATP com p a r e d t o t h e i n t e r m o l t m u s c l e enzyme. F u r t h e r m o r e , t h e s e n s i t i v i t y t o ADP a n d MgADP i n h i b i t i o n i s c o n s i d e r a b l y r e d u c e d i n t h e h y p o d e r m a l f o r m s . T h e s e two k i n e t i c m o d i f i c a t i o n s c o m b i n e t o f a c i l i t a t e t h e r e v e r s a l o f t h e r e a c t i o n . R e m o v a l o f 1,3 d i p h o s p h o g l y c e r a t e by t h e g l y c e r a l d e h y d e - 3 - p a o s p h a t e d e h y -d r o g e n a s e , w o u l d a l s o f a v o r r e v e r s a l o f t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n by a mass a c t i o n a f f e c t . Mammalian g l y c e r -a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e shows k i n e t i c m o d i f i c a t i o n s w h i c h f a c i l i t a t e t h e r e d u c t i v e d e p h o s p h o r y l a t i o n - d u r i n g t h e h i g h NAD/NADH r a t i o s c h a r a c t e r i s t i c o f e n e r g y s a t u r a t i n g c o n d i t i o n s { S m i t h & V e l i c k , * 1 9 7 2 ) . S i n c e s i m i l a r k i n e t i c s a r e shown by b o t h t h e mammalian l i v e r and m u s c l e enzymes, a n d s i n c e c r u s t a c e a n m u s c l e and mammalian m u s c l e g l y c e r a l d e h y d e - 3 ~ p h o s -p h a t e d e h y d r o g e n a s e s a r e much t h e same ( T r e n t h a m , 1 9 7 1 ; De V i j l d e r e t a l . , 1 9 6 9 ; H a r r i g a n & T r e n t h a m , 1974) , s i m i l a r k i n e t i c m o d i f i c a t i o n s p r o b a b l y h o l d f o r t h e h y p o d e r m a l enzyme. T h u s , a t low- l e v e l s o f NADH • and-1,3 d i p h o s p h o g l y c e r a t e , NAD s t i m u l a t e s t h e b i n d i n g o f t h e s e s u b s t r a t e s a t one s u b u n i t , w h i l e i t s e l f b i n d i n g a t t h e o t h e r t h r e e . A t h i g h l e v e l s o f NADH a n d 1,3 d i p h o s p h o g l y c e r a t e , NAD c o m p e t e s f o r t h e b i n d i n g s i t e , b u t a t l o w l e v e l s , K i t p r o m o t e s t h e b i n d i n g o f 1,3 d i p h o s p h o -g l y c e r a t e . NAD " b i n d i n g a t t h e a c y l a t e d s i t e i s r e s t r i c t e d , w h i l e NADH b i n d i n g i s f a v o r e d . T h u s , i n t h e f a c e o f t h e h i g h 186 NAD/NADH r a t i o w h i c h a c c o m p a n i e s e n e r g y s a t u r a t i n g c o n d i t i o n s , t h e h i g h a f f i n i t y o f t h e d e h y d r o g e n a s e f o r NAD d o e s n o t i n h i b i t t h e g l u c o n e o g e n i c r e v e r s a l o f t h e r e a c t i o n , b u t i n s t e a d f a c i l i t a t e s i t ( S m i t h & V e l i c k , 1 972) . ATP and c r e a t i n e p h o s p h a t e i n h i b i t " t h e g l y c o l y t i c r e a c t i o n o f m a m m a l i a n - m u s c l e g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e ( O g u c h i e t a l . * 1 9 7 3 ) . T h e s e compounds may p o s s i b l y f a c i l i t a t e t h e • r e v e r s e r e a c t i o n , t h u s s t i m u l a t i n g g l u c o n e o g e n i c f l u x t h r o u g h b o t h p hospho-g l y c e r a t e k i n a s e and g l y c e r a l d e h y d e - 3 - p h o s p h a t e d e h y d r o g e n a s e . I n t e g r a t i o n o f G l u c o n e o g e n e s i s and L i p i d O x i d a t i o n T h i s m o d e l o f g l u c o n e o g e n i c c c n t r o l m e c h a n i s m s i n t h e c r u s t a c e a n h y p o d e r m i s h a s n o t t o u c h e d upon a number o f a d d i t i o n a l r e g u l a t o r y i n t e r a c t i o n s . T h e s e d e a l p r i m a r i l y w i t h t h e e f f e c t s o f l i p i d o x i d a t i o n on g l u c o n e o g e n e s i s . I n s p e c i e s w i t h an a p p r e c i a b l e c y t o s o l i c p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y , l i p i d o x i d a t i o n d u r i n g p e r i o d s o f g l u c o n e o g e n e s i s h a s a s t i m u l a t o r y e f f e c t ( W i l l i a m s o n e t a l . 1969; C o r n e l l e t a l . , 1 9 7 4 ) . T h i s e f f e c t i s t h o u g h t t o a r i s e f r o m t h e h i g h l e v e l s o f c i t r a t e and a c e t y l CoA w h i c h r e s u l t f r o m l i p i d u t i l i z a t i o n . T h e r e i s e v i d e n c e f o r s i m i l a r e f f e c t s d u r i n g g l u c o n e o g e n e s i s i n c r u s t a c e a n s . C i t r a t e i s a p o t e n t i n h i b i t o r o f h y p o d e r m a l PK I I , and p r e s u m a b l y o f p h o s p h o f r u c t o k i n a s e . As i s t r u e f o r - o t h e r p y r u v a t e c a r b o x y l a s e s (Ashman e t a l . , 1 9 7 2 ) , t h e enzyme p r e s e n t i n C a n c e r m a g i s t e r t i s s u e s shows a d e p e n d e n c e upon a c e t y l CoA f o r c a t a l y t i c a c t i v i t y * F u l l s t i m u l a t i o n o f a c t i v i t y came a t 0.05 mn a c e t y l CoA. H i g h l e v e l s o f a c e t y l CoA w o u l d i n h i b i t p y r u v a t e d e h y d r o g e n a s e , l e a d i n g t o an a c c u m u l a t i o n o f p y r u v a t e 187 (Guder S W i e l a n d , 1 9 7 4 ) . I n c r e a s e d l e v e l s o f p y r u v a t e w o u l d t h e n a c t i n c o n j u n c t i o n w i t h t h e h i g h l e v e l s o f a c e t y l CoA t o a c t i v a t e p y r u v a t e c a r b o x y l a s e * I n c r e a s e d f o r m a t i o n o f o x a l o a c e t a t e w o u l d t h e n f a c i l i t a t e PEP f o r m a t i o n f r o m o x a l o a c e t a t e by PEPCK. By c o n t r a s t , i n s p e c i e s w h i c h h a v e h i g h e r l e v e l s o f p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i n t h e m i t o c h o n d r i a t h a n i n t h e c y t o p l a s m , l i p i d o x i d a t i o n i n h i b i t s g l u c o n e o g e n e s i s ( S o l i n g S i Si*» 1973; Jomain-Baum & Ha n s o n , 1 9 7 5 ) . T h i s i n h i b i t o r y e f f e c t i s d i r e c t l y r e l a t e d t o a d e c r e a s e i n t h e m i t o c h o n d r i a l NAD/NADH r a t i o , w h i c h l e a d s t o a marked d e c r e a s e i n t h e r a t e o f p h o s p h o e n o l p y r u v a t e f o r m a t i o n . - I n c r e a s e d l e v e l s o f ammonium c h l o r i d e (1 mM) r e v e r s e t h i s i n h i b i t i o n and l e a d t o an i n c r e a s e i n t h e NAD/NADH r a t i o , v i a t h e g l u t a m a t e d e h y d r o g e n a s e r e a c t i o n . D u r i n g p e r f u s i o n o f i s o l a t e d l i v e r , f u r t h e r i n c r e a s e s i n ammonium i o n (2 mM) d i d n o t r e l i e v e t h e i n h i b i t i o n u n t i l - t h e i r a d d i t i o n was t e r m i n a t e d . T h e s e h i g h l e v e l s o f ammonium i o n l e d t o c o n s i d e r a b l e i n c r e a s e s i n t h e l e v e l s o f b o t h g l u t a m a t e a n d a s p a r t a t e . A s p a r t a t e was d e r i v e d f r o m o x a l o a c e t a t e , t h u s r e d u c i n g t h e a v a i l a b i l i t y o f t h i s compound f o r PEP f o r m a t i o n (Jomain-Baum & H a n s o n , 1 9 7 5 ) . T h u s , t h e NAD/NADH r a t i o p l a y s a c e n t r a l r o l e i n - m o d u l a t i n g - t h e r a t e o f m i t o c h o n d r i a l PEP f o r m a t i o n . H o w e v e r , c o m p e t i t i o n b e t ween a s p a r t a t e a m i n o t r a n s f e r a s e and p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e w i l l a l s o s t r o n g l y a f f e c t t h e r a t e o f PEP f o r m a t i o n . The s e p a r a t e r e g u l a t i o n o f t h e c y t o p l a s m i c and t h e m i t o c h o n d r i a l NAD/NADH r a t i o s may e x p l a i n t h e l a c k o f i n h i b i t i o n o f c y t o p l a s m i c PEP f o r m a t i o n d u r i n g f a t t y a c i d o x i d a t i o n . I n C a n c e r m a g i s t e r , 188 p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y i s p r e s e n t i n b o t h t h e m i t o c h o n d r i a a n d t h e c y t o p l a s m , b u t i t s e x a c t - d i s t r i b u t i o n b e t w e e n t h e two c o m p a r t m e n t s has n o t been d e t e r m i n e d . T h u s , no c o n c l u s i o n s c a n be drawn a b o u t t h e n e t e f f e c t s o f l i p i d o x i d a t i o n on h y p o d e r m a l g l u c o n e o g e n e s i s . However, t h e e f f e c t s o f c i t r a t e u p o n P K I I and a c e t y l CoA upon p y r u v a t e c a r b o x y l a s e shown i n t h e p r e s e n t s t u d y c o u l d w e l l be o f 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 . Inhibition o f G l u c o n e o g e n e s i s and A c t i v a t i o n o f G l y c o l y s i s I n h i b i t i o n o f - g l u c o n e o g e n e s i s c a n o c c u r u n d e r v a r i o u s m e t a b o l i c c o n d i t i o n s i n c l u d i n g t h e d e p l e t i o n o f ATP s u p p l i e s o r t h e p r o d u c t i o n o f a d e q u a t e q u a n t i t i e s o f p o l y s a c c h a r i d e . I n f o r m a t i o n r e g a r d i n g a d e q u a t e s y n t h e t i c a c t i v i t y w o u l d p r o b a b l y be h o r m o n a l and w o u l d have i t s i n i t i a l e f f e c t upon g l y c o g e n s y n t h e t a s e o r c h i t i n s y n t h e t a s e f u n c t i o n . The • • i n h i b i t i o n - d u e t o l a c k o f ATP w o u l d o c c u r p a r t i c u l a r l y f r e q u e n t l y i f c a r b o h y d r a t e f o r m e d t h e m a j o r s u b s t r a t e f o r e n e r g y p r o v i s i o n as w e l l a s t h e s u b s t r a t e f o r b i o s y n t h e t i c a c t i v i t y . S i n c e c a r b o h y d r a t e i s one o f t h e m a j o r s u b s t r a t e s u t i l i z e d f o r e n e r g y p r o v i s i o n d u r i n g p r e m o l t and p o s t m o l t i n t h e h y p o d e r m i s (Speck & U r i c h , 1971; H e r z - H u b n e r & U r i c h , 1 9 7 3 ) , c y c l i c v a r i a t i o n s i n - g l y c o l y t i c a nd g l u c o n e o g e n i c f l u x c a n be e x p e c t e d . When ATP l e v e l s d e c l i n e , ADP and AMP l e v e l s r i s e . As i n d i c a t e d a b o v e , AMP i s a p o t e n t i n h i b i t o r o f most F D P a s e s . F u r t h e r m o r e , i t i s a s t r o n g a c t i v a t o r o f p h o s p h o f r u c t o k i n a s e s . - T h u s , t h e e f f e c t o f i n c r e a s e s i n AMP i s a m p l i f i e d , s i n c e i t a f f e c t s t h e two s i d e s o f t h e p h o s p h o f r u c t o k i n a s e - f r u c t o s e d i p h o s p h a t a s e c y c l e i n o p p o s i t e 189 ways (Newsholme & S t a r t , 1 9 7 3 ) . A p a u c i t y o f ATP w o u l d l e a d t o a n a c c u m u l a t i o n o f c a r b o h y d r a t e p r e c u r s o r s , i n c l u d i n g f r u c t o s e -s - p h o s p h a t e and e i t h e r U D P - a c e t y l g l u c o s a m i n e o r U D P - g l u c o s e ( d e p e n d i n g upon t h e p o l y s a c c h a r i d e c u r r e n t l y b e i n g s y n t h e s i z e d ) . An a c c u m u l a t i o n o f f r u c t o s e - 6 - p h o s p h a t e w o u l d c a u s e an a c t i v a t i o n o f p h o s p h o f r u c t o k i n a s e , w h i l e U D P - a c e t y l g l u c o s a m i n e and U D P - g l u c o s e b o t h i n h i b i t h y p o d e r m a l FDPase I I ( H o c h a c h k a , 1 9 7 2 ) . The i n h i b i t i o n o f g l u c o n e o g e n e s i s due t o d e c r e a s e d l e v e l s o f ATP w o u l d a l s o a f f e c t t h e s e c o n d - c o n t r o l s i t e , by d e i n h i b i t i n g t h e p y r u v a t e k i n a s e r e a c t i o n . The i n c r e a s e d l e v e l s o f FDP, w h i c h w o u l d o c c u r a f t e r t h e i n h i b i t i o n o f f r u c t o s e d i -p h o s p h a t a s e , w o u l d a c t i v a t e p y r u v a t e k i n a s e i n two ways. F i r s t , FDP w o u l d i n c r e a s e t h e c a t a l y t i c r a t e by a c t i v a t i n g t h e b i n d i n g o f PEP and r e v e r s i n g t h e i n h i b i t i o n due t o ATP and a m i n o a c i d s . F u r t h e r m o r e , t h e i n c r e a s e d l e v e l s o f FDP w o u l d p r o m o t e t h e c o n v e r s i o n o f t h e l o w a f f i n i t y PK I I i n t o t h a h i g h a f f i n i t y f o r m , PK I ( F i g u r e 2 7 , p. 1 5 6 ) . T h i s c o n v e r s i o n w o u l d l e a d t o a c o n s i d e r a b l e i n c r e a s e i n g l y c o l y t i c f l u x t h r o u g h t h i s r e a c t i o n l o c u s . I n c r e a s e d l e v e l s o f ADP w o u l d a l s o a c t i v a t e g l y c o l y t i c f l u x , w h i l e i n h i b i t i n g b o t h t h e p y r u v a t e c a r b o x y l a s e a n d t h e PEP c a r b o x y k i n a s e r e a c t i o n s (Newsholme & S t a r t , 1973; G a r b e r S S a l g a n i c o f f , 1 9 7 3 ) . When g l u c o n e o g e n e s i s i s i n h i b i t e d due t o an e f f e c t upon c h i t i n s y n t h e t a s e o r g l y c o g e n s y n t h e t a s e , t h e r e g u l a t o r y e f f e c t s w o u l d be e x p r e s s e d a t s e v e r a l l o c i a s w e l l . The i n i t i a l e f f e c t , i n t h e c a s e o f an i n h i b i t i o n o f c h i t i n s y n t h e t a s e , w o u l d be an 190 a c c u m u l a t i o n o f U D P - a c e t y l g l u c o s a m i n e . T h i s w o u l d l e a d t o an i n h i b i t o n o f b o t h g l u t a m i n e : f r u c t o s e - 6 - p h o s p h a t e a m i d o t r a n s f e r -a s e and f r u c t o s e d i p h o s p h a t a s e ( W i n t e r b u r n & P h e l p s , 1 9 7 1 a ; H o c h a c h k a , 1 9 7 2 ) . W h i l e g l u t a m i n e : f r u c t o s e - 6 - p h o s p h a t e a m i d o -t r a n s f e r a s e h a s n o t b e e n e x a m i n e d i n c r u s t a c e a n s , i t s - p r o p e r t i e s h a v e been c h a r a c t e r i z e d i n r a t l i v e r , l o c u s t h y p o d e r m i s , and P r o s o p h l l a s a l i v a r y g l a n d s ( S i n t e r b u r n & P h e l p s , 1971a; W i n t e r b u r n S P h e l p s , 1971b; K r e s s & E n g h o f e r , 1975; S u r h o l t , 1 9 7 5 ) . The enzyme f r o m a l l s o u r c e s i s s u b j e c t t o f e e d b a c k i n h i b i t i o n by U D P - a c e t y l g l u c o s a m i n e , a m e c h a n i s m w h i c h e f f e c t i v e l y p r e v e n t s f u r t h e r a c c u m u l a t i o n o f t h e compound. B o t h FDPase I and FDPase I I f r o m k i n g c r a b h y p o d e r m i s a r e s e n s i t i v e t o i n h i b i t i o n by t h i s m e t a b o l i t e . FDPase I I i s s u b j e c t t o 8 0 % i n h i b i t i o n by 1 mM U D P - a c e t y l g l u c o s a m i n e w h i l e F D P a s e I i s i n h i b i t e d by 5 0 % by 2.5 mM l e v e l s o f t h i s compound ( H o c h a c h k a , 1 9 7 2 ) . T h u s , p a r t i a l i n h i b i t i o n o f f l u x t h r o u g h t h e FDPase r e a c t i o n c a n be a c h i e v e d by p r e s u m a b l y m o d e r a t e l e v e l s o f UDP-a c e t y l g l u c o s a m i n e . • B e s i d e s i n h i b i t i n g F D P a s e , UDP-a c e t y l g l u c o s a m i n e a c t i v a t e s p y r u v a t e k i n a s e . T h i s a c t i v a t i o n o c c u r s v i a a f a c i l i t a t i o n o f s u b s t r a t e b i n d i n g and a r e v e r s a l o f ATP i n h i b i t i o n . I f g l y c o g e n s y n t h e s i s i s a c t i v a t e d - d u r i n g i n h i b i t i o n o f c h i t i n s y n t h e s i s , F D P a s e I w o u l d c o n t i n u e t o p r o v i d e f r u c t o s e - 6 - p h o s p h a t e and m a i n t a i n low l e v e l s o f f r u c t o s e 1,6 d i p h o s p h a t e . T h e s e w o u l d keep p y r u v a t e k i n a s e i n i t s l o w a f f i n i t y - c o n f o r m a t i o n . T h u s , i n t h e a b s e n c e of c o m p l e t e i n h i b i t i o n o f b o t h FDPase I and FDPase I I , high, l e v e l s o f UDP-a c e t y l g l u c o s a m i n e •• w o u l d - o n l y c r e a t e a s m a l l i n c r e a s e i n p y r u v a t e k i n a s e a c t i v i t y . The s e n s i t i v i t y o f b o t h FDPase I I a n d p y r u v a t e 191 k i n a s e t o t h e e n d p r o d u c t s o f c h i t i n s y n t h e s i s d i s t r i b u t e s c o n t r o l o v e r ' t h e two r a t e l i m i t i n g s t e p s i n t h e p a t h w a y , a n d a l l o w s s e l e c t i v e c h a n n e l i n g o f f r u c t o s e - 6 - p h o s p h a t e i n t o g l y c o g e n , c h i t i n o r e n e r g y m e t a b o l i s m . - D u r i n g p r e m o l t and p o s t m o l t , when c h i t i n i s t h e p r i m a r y p r o d u c t o f t h e h y p o d e r m i s , F D P a s e I I l e v e l s may i n c r e a s e , a l l o w i n g g r e a t e r r e g u l a t o r y s p e c i f i c i t y . T h u s , t h e enzymes i n t h e c r u s t a c e a n h y p o d e r m i s h a v e a number o f s p e c i f i c m o d i f i c a t i o n s w h i c h a l l o w a h i g h d e g r e e o f c o n t r o l o v e r - t h e - two m a j o r " b o t t l e n e c k s " i n g l y c o l y s i s a nd g l u c o n e o g e n e s i s . S p e c i f i c c h a r a c t e r i s t i c s w h i c h a r e u n i q u e t o t h e c r u s t a c e a n h y p o d e r m i s a r e t h e m u l t i p l e - f o r m s o f f r u c t o s e d i -p h o s p h a t a s e and t h e h i g h and l o w a f f i n i t y f o r m s - o f p y r u v a t e k i n a s e . A f u r t h e r d i s t i n c t l y g l u c o n e o g e n i c m o d i f i c a t i o n o c c u r s i n t h e m o l t c y c l e s p e c i f i c v a r i a n t s o f p h o s p h o g l y c e r a t e k i n a s e . A c o m p a r i s o n o f t h e c o n t r o l m e c h a n i s m s p r e s e n t i n t h e h y p o d e r m i s w i t h t h o s e p r e s e n t i n t h e m u s c l e , i n d i c a t e s t h a t t h e s e c o n t r o l c h a r a c t e r i s t i c s must h a v e e v o l v e d i n a n s w e r t o t h e c r i t i c a l n e e d f o r e f f i c i e n t g l u c o n e o g e n e s i s d u r i n g - t h e i n i t i a l p o s t m o l t p e r i o d . A number o f g u e s t i o n s n e e d t o be a n s w e r e d b e f o r e t h e e x a c t p i c t u r e o f c o n t r o l o f g l u c o n e o g e n e s i s a n d g l y c o l y s i s i n t h e h y p o d e r m i s - c a n be d e f i n i t i v e l y e x p l a i n e d . P e r c e n t a g e c h a n g e s i n t h e f o r m s o f FDPase p r e s e n t i n t h e d i f f e r e n t p o r t i o n s o f t h e m o l t c y c l e , a nd t h e e x a c t r e g u l a t i o n o f p h o s p h o f r u c t o -k i n a s e , p y r u v a t e - c a r b o x y l a s e , and p h o s p h o e n o l p y r u v a t e c a r b o x y -k i n a s e r e m a i n t o be e s t a b l i s h e d * F u r t h e r m o r e , g u e s t i o n s o f i n v i v o f l u x p a t t e r n s a s w e l l ; a s t h e i n f l u e n c e o f h o r m o n a l r e g u l a t i o n a l s o n e e d t o be a n s w e r e d . However, t h i s s t u d y h a s 192 p r o v i d e d c o n s i d e r a b l e e l u c i d a t i o n o f a v a i l a b l e g l u c o n e o g e n i c and g l y c o l y t i c c o n t r o l m echanisms i n b o t h t h e h y p o d e r m i s a n d m u s c l e o f C a n c e r m a g i s t e r . P h o s ^ h o g l j c e r a t e K i n a s e i n G l u c o n e o g e n i c T i s s u e s • The r e g u l a t o r y p r o p e r t i e s o f t h e p h o s p h o g l y c e r a t e k i n a s e s i n v e s t i g a t e d i n t h i s s t u d y may e l u c i d a t e t h e means by w h i c h t h e r e v e r s a l o f t h e r e a c t i o n f o r g l u c o n e o g e n e s i s i s f a c i l i t a t e d i n o t h e r o r g a n i s m s . The k i n e t i c m o d i f i c a t i o n s o f f r e s h m o l t m u s c l e and h y p o d e r m a l p h o s p h o g l y c e r a t e k i n a s e s h a v e c o n s i d e r a b l e p o t e n t i a l f o r o v e r c o m i n g t h e MgADP i n h i b i t i o n o f t h e g l u c o n e o -g e n i c r e a c t i o n . The c o m b i n a t i o n o f i n c r e a s e d a f f i n i t y f o r - ATP and d e c r e a s e d s e n s i t i v i t y t o ADP and MgADP i n h i b i t i o n may p r o v i d e t h e means f o r t h e e a s y r e v e r s a l o f p h o s p h o g l y c e r a t e k i n a s e i n o t h e r g l u c o n e o g e n i c t i s s u e s . The mass a c t i o n r a t i o s show t h a t t h e r e a c t i o n c a t a l y z e d by : p h o s p h o g l y c e r a t e k i n a s e i s r e l a t i v e l y n e a r e q u i l i b r i u m i n a l l t h e t i s s u e s e x a m i n e d . E v i d e n t l y t h e a c t i v i t y o f t h e enzyme i s h i q h enough i n b o t h h y p o d e r m i s and m u s c l e t o b r i n g t h e r e a c t i o n c l o s e t o e q u i l i b r i u m . C e r t a i n l y t h e mass a c t i o n r a t i o s f o r p h o s p h o -f r u c t o k i n a s e and p y r u v a t e k i n a s e a r e o r d e r s o f m a g n i t u d e f u r t h e r r emoved f r o m e q u i l i b r i u m t h a n t h o s e o f t h e p h o s p h o g l y c e r a t e k i n a s e r e a c t i o n . < However, q u a l i t a t i v e d i f f e r e n c e s i n d i c a t e t h a t t h e r e a c t i o n i s f u r t h e r - f r o m e q u i l i b r i u m i n m u s c l e t h a n i n h y p o d e r m i s . " The k i n e t i c c h a r a c t e r i s t i c s o f t h e h y p o d e r m a l a n d f r e s h m o l t m u s c l e enzymes w o u l d f a c i l i t a t e t h e r e v e r s a l o f t h e r e a c t i o n , i n c o n t r a s t t o t h e p r o p e r t i e s o f t h e i n t e r m o l t m u s c l e enzyme. 193 Isajfliatory. P r o p e r t i e s o f P y r u v a t e K i n a s e f r o m C a n c e r m a g i s t e r The r e g u l a t o r y p r o p e r t i e s o f p y r u v a t e k i n a s e f r o m m u s c l e and h y p o d e r m i s ( T a b l e 19) a r e i n t e r e s t i n g b o t h i n * r e l a t i o n t o o t h e r p y r u v a t e k i n a s e s and a s e x a m p l e s o f p r o t e i n a d a p t a t i o n f o r s p e c i f i c m e t a b o l i c p u r p o s e s . M u s c l e p y r u v a t e k i n a s e i s d i s t i n c t f r o m t h e enzyme i n o t h e r m u s c l e s i n t h a t i t i s s e n s i t i v e t o a c t i v a t i o n by O D P - a c e t y l g l u c o s a m i n e and g l u c o s a m i n e - 6 - p h o s p h a t e . T h i s e f f e c t may w e l l be s p e c i f i c t o c r u s t a c e a n s and r e f l e c t s t h e i n f l u e n c e o f c h i t i n m e t a b o l i s m . A r g i n i n e p h o s p h a t e i n h i b i t i o n p r o b a b l y o c c u r s i n a l l a n i m a l s w h i c h u t i l i z e a r g i n i n e p h o s p h a t e a s t h e i r s t o r a g e p h o s p h a g e n , a n d h a s b e e n f o u n d f o r p y r u v a t e k i n a s e f r o m o c t o p u s - ( G u d e r l e y e t a l . , 1975) and s h r i m p ( G u d e r l e y §£ S i * # 1 9 7 6 ) . A l t h o u g h t h e m u s c l e enzyme shows a l l o s t e r i c e f f e c t s , i . e . a c t i v a t i o n by FDP, i n h i b i t i o n by c i t r a t e a n d a r g i n i n e - p h o s p h a t e , i t d o e s n o t show s i g m o i d a l k i n e t i c s . V a r i o u s o t h e r p y r u v a t e k i n a s e s show s i g m o i d a l k i n e t i c s a t h i g h pH v a l u e s ( S e u b e r t & S c h o n e r , 1 9 7 1 ) , i n t h e p r e s e n c e o f M g + + r a t h e r t h a n M n + + (de Zwaan e t a l . , 1 9 7 5 ) , o r show s i g m o i d a l k i n e t i c s w h i c h a r e c o n v e r t e d t o h y p e r b o l i c k i n e t i c s by t h e a d d i t i o n o f FDP ( K a y n e , 1 9 7 3 ) . The p r e s e n c e o f m e a s u r a b l e l e v e l s o f FDP a s s o c i a t e d w i t h t h e enzyme i n d i c a t e s t h a t t h i s l a c k o f s i g m o i d a l - k i n e t i c s may be due t o bound FDP w h i c h somehow p r e v e n t s c o o p e r a t i v e i n t e r a c t i o n s b e t w e e n • s u b u n i t s . F u r t h e r e x a m i n a t i o n o f t h i s a s p e c t o f p y r u v a t e k i n a s e r e g u l a t i o n w o u l d be o f c o n s i d e r a b l e i n t e r e s t . The s i m i l a r i t y • o f t h e r e g u l a t o r y p r o p e r t i e s o f m u s c l e p y r u v a t e k i n a s e and h y p o d e r m a l PK I ( T a b l e 19) makes i t t e m p t i n g 194 Table 19 Comparison of Muscle and Hypodermal Pyruvate Kinases Muscle PK Hypodermal PK I Hypodermal PK I I K PEP m K ADP m K FDP a K± MgATP a r g i n i n e phosphate tryptophan a l a n i n e Kj. a l a n i n e (with FDP) 0.1 mM 0.2 mM 2 x 10" 5M 1.8 mM (vs PEP) 2.8 mM (vs ADP) 1.6 mM 30 mM 30 mM 0.1 mM 0.2 mM 1.3 x 10" 5M 1.2 mM (vs PEP) 7.0 mM (vs ADP) 2.0 mM 30 mM 30 mM 0.55mM 0.2 mM 9 x 10" 8M 1.2 mM (vs PEP) 7.0 mM (vs ADP) 2.0 mM 7.0 mM 26 mM ser i n e K s e r i n e (with FDP) M g 2 c i t r a t e K ± C a C l 2 K K a K aNH+ K a Ms" K Mn"" a P i 30 mM 30 mM complex two s i t e i n t e r a c t i o n 0.1 mM (vs Mg""") 0.66 mM (vs Mn44") 15 mM 5 mM 0.6 mM 0.15 mM 6.4 35 mM 35 mM complex two s i t e i n t e r a c t i o n .075 mM (vs Mg""") .6 mM (vs Mn ) 10 mM 6 mM 0.6 mM 0.1 mM 6.1 10.5 mM 26 mM 7.5 mM .075 mM (vs Mg"") .6 mM (vs Mn"") 10 mM 6 mM 0.6 mM 0.1 mM 6.1 195 t o s p e c u l a t e t h a t t h e h y p o d e r m a l p y r u v a t e k i n a s e e v o l v e d f r o m t h e m u s c l e enzyme. However, t h e two enzymes a r e d e f i n i t e l y d i f f e r e n t p r o t e i n s , s i n c e t h e m u s c l e enzyme d o e s n o t show i n t e r c o n v e r s i o n s b e t w e e n a h i g h and a l o w a f f i n i t y s t a t e . The d i f f e r e n c e b e t w e e n t h e h y p o d e r m a l and t h e m u s c l e p y r u v a t e k i n a s e may be due t o a c o n s i d e r a b l y h i g h e r a f f i n i t y o f t h e m u s c l e enzyme f o r FDP o r t o a few amino a c i d s u b s t i t u t i o n s w h i c h f a c i l i t a t e c o n f o r m a t i o n a l c h a n g e s a t t h e PEP b i n d i n g s i t e o f h y p o d e r m a l p y r u v a t e k i n a s e . s i n c e t h e a p p a r e n t FDP a f f i n i t y o f t h e m u s c l e enzyme was n o t h i g h , t h e f o r m e r a l t e r n a t i v e seems u n l i k e l y . H y p o d e r m a l p y r u v a t e k i n a s e i s d i s t i n c t f r o m o t h e r p r e v i o u s l y e x a m i n e d p y r u v a t e k i n a s e s i n t h a t FDP p r o m o t e s a c o n v e r s i o n b e t w e e n two f o r m s , e a c h o f w h i c h i s s e n s i t i v e t o f u r t h e r a c t i v a t i o n o f s u b s t r a t e b i n d i n g . The e x a c t m e c h a n i s m o f t h i s i n t e r c o n v e r s i o n i s n o t c l e a r , ••but t h e i n c r e a s e d l e v e l s o f FDP a s s o c i a t e d w i t h t h e h i g h a f f i n i t y f o r m i n d i c a t e t h a t t h e i n t e r c o n v e r s i o n s may i n v o l v e l o n g t e r m b i n d i n g o f FDP t o t h e p r o t e i n . Long t e r m b i n d i n g h a s b e e n i n d i c a t e d f o r t h e mammalian L and M2 f o r m s (Hess 6 K u t z b a c h , 1 9 7 1 ; S e u b e r t S S c h o n e r , 1971; I b s e n e t a l . , 1 9 7 5 ) . S u b u n i t c o o p e r a t i v i t y d o e s n o t seem t o be i n v o l v e d i n t h e r e g u l a t i o n o f t h e h y p o d e r m a l enzyme. A c o m p a r i s o n o f t h e h y p o d e r m a l enzyme w i t h t h e enzyme f r o m t h e h e p a t o p a n c r e a s m i g h t e l u c i d a t e s t r u c t u r a l d i f f e r e n c e s w h i c h u n d e r l i e t h e l a c k "of s u b u n i t i n t e r a c t i o n s on t h e p a r t o f t h e h y p o d e r m a l enzyme* An e x a m i n a t i o n o f t h e e n z y m e f r o m t h e h e p a t o p a n c r e a s f o r s i m i l a r s t a b l e h i g h and l o w a f f i n i t y s t a t e s w o u l d a s c e r t a i n w h e t h e r s u b u n i t i n t e r a c t i o n s a n d t r a n s i t i o n s b e t w e e n h i g h and l o w a f f i n i t y c o n f o r m a t i o n s r e p r e s e n t a l t e r n a t e 196 f o r m s o f a d a p t a t i o n f o r g l u c o n e o g e n i c f u n c t i o n . 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