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Temperature adaptation in enzymes from poikilotherms : acetylocholinesterases in the nervous system of… Baldwin, John T 1970

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TEMPERATURE ADAPTATION IN ENZYMES FROM POIKILOTHERMS: ACETYLCHOLINESTERASES IN THE NERVOUS SYSTEM OF FISHES by JOHN BALDWIN B.Sc. (Hons.), Monash University, 1965 M.Sc, Monash University, 196,9 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Zoology We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l 1970 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 d e g r e e 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 t h a t 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 tha 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 . I t i s u n d e r s t o o d t h a t 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 t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^-o-e»l i 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 V a n c o u v e r 8 , Canada i ABSTRACT The e f f e c t s of temperature upon a c e t y l c h o l i n e s t e r a s e (AChE) from the nervous system of f i s h were s t u d i e d to determine i f such compensatory phenomena as thermal accommodation, thermal a c c l i m a t i o n and e v o l u t i o n a r y a d a p t a t i o n to temperature as d i s p l a y e d by t h i s p h y s i o l o g i c a l system c o u l d be observed and i n t e r p r e t e d a t the l e v e l o f enzyme f u n c t i o n . At probable p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s the r a t e of a c e t y l c h o l i n e (ACh) h y d r o l y s i s by AChE from rainbow t r o u t (Salmo g a i r d n e r i i ) and e l e c t r i c e e l remains r e l a t i v e l y u n a f f e c t e d by assay temperature over the temperature ranges normally e x p e r i e n c e d by these animals. P l o t s of Km versus temperature f o r these enzymes y i e l d U shaped curves w i t h minimum Km v a l u e s o c c u r r i n g a t temperatures c l o s e to the minimum h a b i t a t temperature. I t i s proposed t h a t thermal accommodation of r e a c t i o n r a t e i s a c h i e v e d throughout the h a b i t a t temperature range by temperature d i r e c t e d changes i n enzyme-substrate a f f i n i t y . Thermal a c c l i m a t i o n i n rainbow t r o u t , and probably i n s p e c k l e d t r o u t ( S a l v e l i n u s f o n t i n a l i s ) and lake t r o u t ( S a l v e l i n u s  namaychus) i s accompanied by a l t e r a t i o n s i n the r e l a t i v e p r o p o r t i o n s of two 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 AChE v a r i a n t s d i s p l a y i n g d i f f e r e n t and a d a p t i v e Km-temperature r e l a t i o n s h i p s . Since the minimum Km v a l u e s and e n e r g i e s of a c t i v a t i o n of the two rainbow t r o u t enzymes are s i m i l a r , and the s p e c i f i c a c t i v i t i e s of the enzymes are e s s e n t i a l l y i d e n t i c a l f o l l o w i n g a c c l i m a t i o n o f f i s h to 2° and 17°C, i t i s suggested t h a t r a t e compensation o f AChE a c t i v i t y may not occur a t d i f f e r e n t a c c l i m a t i o n temperatures. However, the p o s s i b i l i t y remains t h a t changes i n such f a c t o r s as pH, i o n i c environment and membrane l i p i d s which accompany the a c c l i m a t i o n process may a c t to s t a b i l i z e r e a c t i o n r a t e s . Comparisons o f AChE enzymes from rainbow t r o u t , e l e c t r i c e e l and the A n t a r c t i c f i s h Trematomus b o r c h g r e v i n k i i n d i c a t e t h a t the e v o l u t i o n a r y a d a p t a t i o n of AChE f u n c t i o n i n s p e c i e s i n h a b i t i n g d i f f e r e n t thermal environments i s based upon s e l e c t i o n f o r a Km-temperature r e l a t i o n s h i p t h a t w i l l a l l o w thermal accommodation of r e a c t i o n r a t e over the temperature range normally encountered. S h i f t s i n the Km-temperature r e l a t i o n s h i p d u r i n g s p e c i a t i o n are i n t e r p r e t e d i n terms of changes i n enzyme conformation f o l l o w i n g the accumulation o f amino a c i d s u b s t i t u t i o n s . P o s s i b l e mechanisms by which two AChE enzymes c o u l d be i n c o r p o r a t e d i n t o the t r o u t c e n t r a l nervous system were c o n s i d e r e d and a h y p o t h e s i s i n v o l v i n g h y b r i d i z a t i o n between f i s h p o p u l a t i o n s was t e s t e d w i t h t r o u t i n t e r - s p e c i e s c r o s s e s . I t was observed t h a t h y b r i d s formed between s p e c k l e d and l a k e t r o u t c o n t a i n e d a g r e a t e r number of 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 AChE v a r i a n t s than d i d e i t h e r parent and f u r t h e r , the presence of s i m i l a r t h e r m a l l y c o n t r o l l e d AChE complexes i n rainbow, s p e c k l e d and la k e t r o u t i n d i c a t e d t h a t the o r i g i n a l i n c o r p o r a t i o n of m u l t i p l e AChE enzymes i n t o the rainbow t r o u t p robably o c c u r r e d p r i o r to the e v o l u t i o n a r y divergence o f these three s p e c i e s . I t i s concluded from t h i s study t h a t changes i n enzyme-s u b s t r a t e a f f i n i t y w i t h temperature, and the temperature d i r e c t e d p r o d u c t i o n o f enzyme v a r i a n t s d i s p l a y i n g adaptive Km-temperature r e l a t i o n s h i p s , are both important mechanisms f o r c o n t r o l l i n g c a t a l y t i c a c t i v i t y i n an enzyme system which f u n c t i o n s over a wide range of temperatures. i v TABLE OF CONTENTS Page A b s t r a c t i L i s t of Tables v i i L i s t of F i g u r e s v i i i Acknowledgements x I n t r o d u c t i o n 1 1. Statement of the Problem 1 2. Thermal Accommodation and Thermal A c c l i m a t i o n i n the C e n t r a l Nervous System o f P o i k i l o t h e r m s 3 3. Role of A c e t y l c h o l i n e s t e r a s e i n Nerve Trans m i s s i o n 5 4. Importance of C h o l i n e r g i c Mechanisms i n the C e n t r a l Nervous System 6 Methods 12 1. Experimental Animals 12 2. Enzyme P r e p a r a t i o n s 13 a. P r e p a r a t i o n of Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e 13 b. P r e p a r a t i o n of A c e t y l c h o l i n e s t e r a s e from Trout B r a i n and S p i n a l Cord f o r E l e c t r o -p h o r e s i s 14 c. E l e c t r i c E e l A c e t y l c h o l i n e s t e r a s e 14 d. P r e p a r a t i o n of Trematomus b o r c h g r e v i n k i B r a i n A c e t y l c h o l i n e s t e r a s e 15 3. Assay of A c e t y l c h o l i n e s t e r a s e A c t i v i t y 15 4. Gel E l e c t r o p h o r e s i s 15 5. P r o t e i n Determinations 16 Page 6. Sucrose Gradient C e n t r i f u g a t i o n 16 7. U l t r a v i o l e t D i f f e r e n c e Spectra 18 a. I n t r o d u c t i o n 18 b. Method 18 R e s u l t s and D i s c u s s i o n 20 1. P a r t i a l P u r i f i c a t i o n of Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e 20 2. C h a r a c t e r i z a t i o n of A c e t y l c h o l i n e s t e r a s e from the Rainbow Trout C e n t r a l Nervous System 20 a. I n t r o d u c t i o n 20 b. M u l t i p l e Forms of A c e t y l c h o l i n e s t e r a s e i n the Rainbow Trout C e n t r a l Nervous System 20 c. Substrate S p e c i f i c i t y and I n h i b i t i o n Studies of Rainbow Trout B r a i n A c e t y l -c h o l i n e s t e r a s e 22 d. E f f e c t of pH on AChE A c t i v i t y 2 7 e. Sucrose Gradient C e n t r i f u g a t i o n of Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e 2 7 3. C h a r a c t e r i z a t i o n o f A c e t y l c h o l i n e s t e r a s e from Trematomus b o r c h g r e v i n k i B r a i n 30 4. E f f e c t of Assay Temperature upon the K i n e t i c s of A c e t y l c h o l i n e H y d r o l y s i s by A c e t y l c h o l i n e s t e r a s e 30 a. E f f e c t of Temperature on the Maximum V e l o c i t y of A c e t y l c h o l i n e s t e r a s e H y d r o l y s i s 30 b. E f f e c t o f Assay Temperature on Enzyme-Substrate A f f i n i t y 35 v i Page c. R e l a t i o n s h i p between Thermally Induced Changes i n Km and S t r u c t u r a l Conformation of E l e c t r i c E e l A c e t y l c h o l i n e s t e r a s e 45 5. Thermal Accommodation, Thermal A c c l i m a t i o n and E v o l u t i o n a r y A d a p t a t i o n to Temperature f o r A c e t y l c h o l i n e s t e r a s e from the Nervous System of F i s h . 53 a. Thermal Accommodation 53 b. Thermal A c c l i m a t i o n 59 (i ) Adjustment of the thermal accommodation range 59 ( i i ) Rate compensation of AChE a c t i v i t y 60 c. E v o l u t i o n a r y A d a p t a t i o n t o Temperature 68 (i ) Adjustment of the thermal accommodation range 68 ( i i ) E v o l u t i o n of the rainbow t r o u t b r a i n AChE complex 69 ( i i i ) R e g u l a t i o n o f the composition of the t r o u t b r a i n AChE complex d u r i n g thermal a c c l i m a t i o n 73 Summary 76 A b b r e v i a t i o n s 80 L i t e r a t u r e C i t e d 81 LIST OF TABLES P a r t i a l P u r i f i c a t i o n of AChE from Rainbow Trout B r a i n Summation Experiments w i t h Choline E s t e r s and the E f f e c t of I n h i b i t o r s on the H y d r o l y s i s of ACh by Rainbow Trout and E l e c t r i c E e l AChEs Apparent E n e r g i e s of A c t i v a t i o n (Ea) f o r the Rainbow Trout and E l e c t r i c E e l AChEs a t S e v e r a l Temperatures Rates of ACh H y d r o l y s i s a t Minimum Km L e v e l s o f ACh f o r AChEs from Rainbow Trout, E l e c t r i c E e l and Trematomus E f f e c t o f Temperature upon the Km and Rate of ACh H y d r o l y s i s f o r B r a i n AChE from Trematomus b o r c h g r e v i n k i R e l a t i o n s h i p between Km Change and Q 1 Q of the Rate of ACh H y d r o l y s i s a t Concentra-t i o n s o f ACh Approaching the Minimum Km f o r AChEs from Rainbow Trout, E l e c t r i c E e l and Trematomus b o r c h g r e v i n k i E f f e c t o f S a l t s on the Km and Rate of H y d r o l y s i s of ACh by AChE from 2°C A c c l i m a t e d Rainbow Trout S p e c i f i c A c t i v i t i e s o f B r a i n AChE from n o Rainbow Trout A c c l i m a t e d to 2 and 17 C f o r 35 Days V l l l LIST OF FIGURES F i g u r e F a c i n g Page 1 R e s o l u t i o n of Rainbow Trout B r a i n AChEs by Acrylamide Gel D i s c E l e c t r o p h o r e s i s 23 2 Substrate S p e c i f i c i t y o f AChE from 2°C A c c l i m a t e d Rainbow Trout 24 3 Substrate S p e c i f i c i t y of AChE from 17°C A c c l i m a t e d Rainbow Trout 2 5 4 I n f l u e n c e of pH on the A c t i v i t y of Rainbow Trout B r a i n AChEs 28 5 Sucrose G r a d i e n t C e n t r i f u g a t i o n of Rainbow Trout and E l e c t r i c E e l AChEs 29 6 A r r h e n i u s P l o t s of AChE A c t i v i t y f o r the Rainbow Trout and E l e c t r i c E e l Enzymes 33 7 E f f e c t of Assay Temperature on the Km of ° o AChE f o r AChEs from 17 and 2 C A c c l i m a t e d Rainbow T r o u t 38 8 E f f e c t o f Assay Temperature on the Km of ACh f o r E l e c t r i c E e l AChE 39 9 E f f e c t of Assay Temperature on the Km of ACh f o r Trematomus b o r c h g r e v i n k i AChE. Lineweaver-Burk P l o t s a t 2° and 10°C. 43 10 Sucrose G r a d i e n t Sedimentation P r o f i l e s o f E l e c t r i c E e l AChE a t 15°, 25° and 33°C 48 11 E f f e c t o f Temperature on the Km of ACh f o r E l e c t r i c E e l AChE Assayed i n the C e n t r i f u g a -t i o n Medium. Lineweaver-Burk P l o t s a t 15°, 25° and 33°C. 49 E f f e c t of Temperature upon the Sedimentation Behaviour of E l e c t r i c Eel AChE U l t r a v i o l e t Difference Spectra of E l e c t r i c E e l AChE as a Function of Temperature E f f e c t of Assay Temperature on the Km of ACh for AChEs from Rainbow Trout, E l e c t r i c Eel and Trematomus ACh Saturation Curves of E l e c t r i c Eel AChE o o o at 15 , 25 and 40 C ACh Saturation Curves of Trematomus borchgrevinki AChE at 2° and 10°C ACh Saturation Curves of 2°C Acclimated o o o o Rainbow Trout AChE at 0 , 2 , 12 and 18 C Resolution of Brain AChEs from Speckled Trout, Lake Trout and Splake by Acrylamide Gel Disc Electrophoresis X ACKNOWLEDGEMENTS I am e s p e c i a l l y g r a t e f u l to Drs. Peter Hochachka and George Somero f o r advic e and encouragement throughout the course of t h i s study. I would l i k e to thank Dr. B e v e r l y Green f o r use of the u l t r a c e n t r i f u g e and Dr. S. EL Zbarsky who made a v a i l a b l e the Cary spectrophotometer. I. am? g r a t e f u l to Dr. F. E. J . F r y and T e r r y McFadden a t the U n i v e r s i t y o f Toronto L a b o r a t o r y f o r Experimental Limnology f o r s e t t i n g up the speckled, lake and splake t r o u t experiments, and to T e r r y Gjernes f o r the Pinask Lake rainbow t r o u t . To Drs. G. I. Drujrimond, J . E. P h i l l i p s and D. J . Rand a l l go s p e c i a l thanks f o r t h e i r c r i t i c a l r e a d i n g o f t h i s t h e s i s . I would a l s o l i k e to thank my wif e Wendy, and Mrs. LouAnne Moon f o r t y p i n g innumerable d r a f t s . 1 INTRODUCTION 1. Statement of the Problem The a b i l i t y o f p o i k i l o t h e r m s to u t i l i z e a wide range o f thermal environments and i n many cases to remain a c t i v e through-out r e l a t i v e l y l a r g e and o f t e n r a p i d changes i n body temperature, r a i s e s many i n t e r e s t i n g problems a t the b i o c h e m i c a l l e v e l , p a r t i c u l a r l y w i t h r e s p e c t t o enzyme f u n c t i o n . Before d i s c u s s i n g a number of these q u e s t i o n s i n d e t a i l , i t i s necessary to d e f i n e s e v e r a l terms which w i l l be used i n t h i s t h e s i s to d e s c r i b e the r e a c t i o n s o f b i o l o g i c a l systems to t h e i r environment. The reason f o r t h i s i s not so much to make cla i m s f o r the c o r r e c t n e s s o f c e r t a i n terms, but r a t h e r to give s p e c i f i c meanings to terms which are o f t e n used l o o s e l y i n the l i t e r a t u r e to group processes a t the p h y s i o l o g i c a l l e v e l , so t h a t these concepts may be extended to the m o l e c u l a r l e v e l . The term "thermal accommodation" w i l l r e f e r to the a b i l i t y o f a f u n c t i o n to proceed independently o f temperature throughout a p a r t i c u l a r temperature range, the thermal accommodation range. Thermal accommodation occurs i n s t a n t a n e o u s l y and i s an i n t r i n s i c p r o p e r t y of the f u n c t i o n . "Thermal a c c l i m a t i o n " d e s c r i b e s the a b i l i t y o f a f u n c t i o n to t h e r m a l l y accommodate over a d i f f e r e n t temperature range a f t e r a p e r i o d of exposure of an i n d i v i d u a l organism to a new thermal regime. The time course o f thermal a c c l i m a t i o n f o r v a r i o u s p h y s i o l o g i c a l f u n c t i o n s i n p o i k i l o t h e r m s i s g e n e r a l l y i n the o r d e r of days or weeks. " E v o l u t i o n a r y a d a p t a t i o n " w i l l be used to d e s c r i b e changes which occur over time i n t e r v a l s l o n g e r than the l i f e span o f 2 the i n d i v i d u a l organism. Thermal e v o l u t i o n a r y a d a p t a t i o n would cover such changes as those u n d e r l y i n g the a b i l i t y o f a f u n c t i o n to proceed i n r e l a t e d stenothermic p o i k i l o t h e r m s i n h a b i t i n g d i f f e r e n t thermal environments i n cases where the temperature d i f f e r e n c e i s g r e a t e r than can be compensated f o r by thermal accommodation or thermal a c c l i m a t i o n . The s t u d i e s r e p o r t e d i n t h i s t h e s i s were undertaken w i t h the hope o f p r o v i d i n g a t l e a s t p a r t i a l answers to the f o l l o w i n g g e n e r a l q u e s t i o n s . How are enzyme systems i n many p o i k i l o t h e r m s a b l e to f u n c t i o n over wide temperature ranges when the e f f e c t s o f temperature on the c a t a l y t i c and r e g u l a t o r y p r o p e r t i e s o f many mammalian and b a c t e r i a l enzymes appear to be incompatible w i t h the maintenance o f f u n c t i o n through such thermal extremes? In p a r t i c u l a r , can such phenomena as thermal accommodation, thermal a c c l i m a t i o n and e v o l u t i o n a r y a d a p t a t i o n s to temperature as d i s p l a y e d by p o i k i l o t h e r m i c systems be observed and i n t e r p r e t e d a t the l e v e l o f enzyme f u n c t i o n ? The experimental approach adopted to i n v e s t i g a t e these problems can be o u t l i n e d as f o l l o w s : ( i ) S e l e c t a c r i t i c a l p h y s i o l o g i c a l f u n c t i o n which i s known to d i s p l a y both thermal accommodation and thermal a c c l i m a t i o n , ( i i ) I s o l a t e a key enzyme from t h i s system and i n v e s t i g a t e the e f f e c t s of immediate temperature changes and thermal a c c l i m a t i o n upon the enzyme, ( i i i ) Compare temperature c h a r a c t e r i s t i c s of t h i s enzyme w i t h s i m i l a r c h a r a c t e r i s t i c s of homologous enzymes 3 o b t a i n e d from s p e c i e s i n h a b i t i n g d i f f e r e n t thermal environments. A c e t y l c h o l i n e s t e r a s e from the nervous sytem of f i s h was s e l e c t e d as a s u i t a b l e enzyme f o r such a study. The r a t i o n a l e b e h i n d t h i s c h o i ce i s d i s c u s s e d i n the f o l l o w i n g s e c t i o n s of the i n t r o d u c t i o n . 2. Thermal Accommodation and Thermal A c c l i m a t i o n i n the  C e n t r a l Nervous System o f P o i k i l o t h e r m s The importance o f the c e n t r a l nervous sytem of p o i k i l o t h e r m s i n s e t t i n g l i m i t s o f thermal t o l e r a n c e , and the probable l i m i t i n g r o l e of changes i n the c e n t r a l nervous system i n the o v e r a l l a c c l i m a t i o n process have been covered i n numerous reviews (e.g. Fry, 1947; B r e t t , 1956; F i s h e r , 1958; Baslow, 1967; Prosser and Nagai, 1968). The f o l l o w i n g o b s e r v a t i o n s are probably the most r e l e v a n t i n e s t a b l i s h i n g a r e l a t i o n s h i p between c e n t r a l nervous f u n c t i o n and the temperature t o l e r a n c e (thermal accommodation range) o f the whole organism. In 1908, Brecht found t h a t heat p a r a l y s i s i n f r o g s was c o n f i n e d to the c e n t r a l nervous system, and o c c u r r e d a t temperatures below those a t which p e r i p h e r a l nerve c o n d u c t i o n and muscle c o n t r a c t i o n were i n h i b i t e d . B a t t l e (1926) determined the upper thermal l i m i t s a t which v a r i o u s t i s s u e s i n skates and flounder were able to e l i c i t p h y s i o l o g i c a l responses and e s t a b l i s h e d t h a t f u n c t i o n s i n v o l v i n g synapses and ephapses such as the h e a r t pacemaker mechanism, cond u c t i o n a c r o s s the nerve muscle j u n c t i o n , and p e r i s t a l s i s i n i n t e n s t i n a l 4 smooth muscle, f a i l e d at, or s l i g h t l y below, the l e t h a l temperature of the organism. In a l a t e r r e p o r t ( B a t t l e , 192 9) i t was noted t h a t a t temperatures approaching the upper thermal l i m i t r e f l e x e s disappeared i n d e f i n i t e sequence, and i t was proposed t h a t death r e s u l t e d p r i m a r i l y from the f a i l u r e of some c e n t r a l c o - o r d i n a t i n g mechanism. Orr (1955) a r r i v e d a t a s i m i l a r c o n c l u s i o n f o r heat death i n Rana p i p i e n s . Prosser and coworkers have demonstrated by both b e h a v i o u r a l and neuro-p h y s i o l o g i c a l techniques a h i e r a r c h y of temperature s e n s i t i v i t y i n the nervous system of f i s h . Mid b r a i n f u n c t i o n s appear to be most temperature s e n s i t i v e , f o l l o w e d by s p i n a l cord, w i t h p e r i p h e r a l nervous f u n c t i o n b e i n g l e a s t s e n s i t i v e (Roots and Prosser, 1962; Prosser and F a r h i , 1965; Prosser and Nagai, 1968). Thus the thermal accommodation range of the c e n t r a l nervous system i n f i s h appears to be a key f a c t o r i n s e t t i n g l i m i t s of thermal t o l e r a n c e f o r the whole organism. The a b i l i t y of the c e n t r a l nervous system i n f i s h t o a c c l i m a t e to thermal s t r e s s has been most c l e a r l y demonstrated by K o n i s h i and Hickman (1964). By m o n i t o r i n g the mid b r a i n response to e l e c t r i c a l s t i m u l a t i o n of the r e t i n a i n rainbow t r o u t h e l d a t both h i g h (16°C) and low (4°C) temperatures they were a b l e to show compensatory changes i n both nerve c o n d u c t i o n v e l o c i t y and c e n t r a l response time over an a c c l i m a t i o n p e r i o d o f s e v e r a l weeks. The e f f e c t o f thermal a c c l i m a t i o n on c e n t r a l nervous f u n c t i o n has a l s o been shown i n q u i t e a d i f f e r e n t way by Roots and Prosser (1962) and Prosser and F a r h i (1965). In a s e r i e s of experiments u t i l i z i n g the e s t a b l i s h m e n t of c o n d i t i o n e d r e f l e x e s i n g o l d f i s h they found t h a t both the 5 lowest temperature a t which a c o n d i t i o n e d r e f l e x c o u l d be e s t a b l i s h e d , and the c o l d b l o c k i n g temperature f o r the c o n d i t i o n e d response v a r i e d d i r e c t l y w i t h the temperature to which the f i s h were a c c l i m a t e d . B i o c h e m i c a l responses of the c e n t r a l nervous system i n f i s h d u r i n g thermal a c c l i m a t i o n have been r e c e n t l y reviewed by Baslow (1967) and i n c l u d e changes i n such f a c t o r s as enzyme l e v e l s , s t r u c t u r a l l i p i d s , e l e c t r o l y t e d i s t r i b u t i o n , and v a r i o u s m e t a b o l i t e s . However, l i t t l e i s known of the r o l e of these changes i n thermal a c c l i m a t i o n of nervous f u n c t i o n . 3. Role of A c e t y l c h o l i n e s t e r a s e i n Nerve Tr a n s m i s s i o n The e a r l y s t u d i e s which e s t a b l i s h e d the r o l e of a c e t y l c h o l i n e (ACh) as a chemical t r a n s m i t t e r a t the neuro-muscular j u n c t i o n have been summarized i n a monograph by Nachmansohn (1959), and l a t e r developments r e l a t i n g to the s t r u c t u r e and f u n c t i o n of c h o l i n e r g i c systems have been d e s c r i b e d i n s e v e r a l r e c e n t reviews (Nachmansohn 1967, 1968, 1969; De R o b e r t i s , 1964). The f o l l o w i n g o u t l i n e o f c h o l i n e r g i c t r a n s m i s s i o n of nerve impulses i s given by Nachmansohn i n h i s 1969 review. ACh i s r e l e a s e d from the nerve membrane f o l l o w i n g e x c i t a t i o n and a c t s as s i g n a l which i s r e c o g n i z e d by a s t e r e o -s p e c i f i c r e c e p t o r p r o t e i n l o c a t e d w i t h i n the membrane. The r e a c t i o n between ACh and the r e c e p t o r induces a conformation change i n the r e c e p t o r molecule, r e l e a s i n g C a + + ions bound to c a r b o x y l groups i n the p r o t e i n . The f r e e C a + + ions induce f u r t h e r c o n f o r m a t i o n a l changes i n membrane p h o s p h o l i p i d s 6 and o t h e r p o l y e l e c t r o l y t e s , l e a d i n g to a change i n nerve membrane p e r m e a b i l i t y and the movement of from 20,000 to 40,000 ions a c r o s s the membrane f o r each molecule o f ACh i n i t i a l l y r e l e a s e d . A c e t y l c h o l i n e s t e r a s e (AChE) r a p i d l y h y d r o l y s e s ACh', p e r m i t t i n g the r e c e p t o r p r o t e i n to r e t u r n to i t s o r i g i n a l conformation thereby r e - e s t a b l i s h i n g the membrane p e r m i a b i l i t y b a r r i e r . Nachmansohn proposes t h a t the ACh-receptor p r o t e i n and AChE are s t r u c t u r a l l y l i n k e d and i t has o f t e n been suggested t h a t AChE may f u n c t i o n as the ACh-receptor molecule. However, there i s no g e n e r a l agreement on t h i s l a t t e r p o i n t (Nachmansohn, 1959; 1969; Changeux, 1966; E h r e n p r e i s , 1967; K a r l i n , 1967; P o d l e s k i , 1969; Hasson-Voloch, 1968; Changeux e t a l , 1968, 1969). While t h i s t h e o r y of c h o l i n e r g i c t r a n s m i s s i o n has gained wide acceptance, a l t e r n a t i v e mechanisms of ACh a c t i o n have been proposed. For example, D u r r e l l , e t a l (1969) suggest t h a t ACh a l t e r s membrane p e r m e a b i l i t y a t the synapse by enhancing enzymatic h y d r o l y s i s of membrane p h o s p h o l i p i d s . I t appears t h a t h y d r o l y s i s o f ACh by AChE would remain an e s s e n t i a l component of t h i s system. 4. Importance of C h o l i n e r g i c Mechanisms i n the C e n t r a l  Nervous System In. s p i t e of a l a r g e volume of data r e l a t i n g to the widespread d i s t r i b u t i o n of ACh, c h o l i n e a c e t y l t r a n s f e r a s e (ChAc, the enzyme i n v o l v e d i n s y n t h e s i s o f ACh) and AChE throughout the v e r t e b r a t e c e n t r a l nervous system and to the c e n t r a l a c t i o n of ACh (Feldberg, 1945; F e l d b e r g and Vogt, 1948; 7 Burgen and Chipman, 1951; Hebb, 1963; A p r i s o n e_t a_l, 1964; E c c l e s , 1964; De R o b e r t i s , 1964; Whittaker, 1965; K o e l l e , 1969; K r n j e v i c , 1969),, there i s s t i l l no c l e a r evidence as to the importance of c h o l i n e r g i c mechanisms i n the c e n t r a l nervous system. Although nerve ending and s y n a p t i c membrane f r a c t i o n s c o n t a i n i n g bound ACh, AChE and ChAC have been i s o l a t e d from mammalian b r a i n c o r t e x p r e p a r a t i o n s by sucrose g r a d i e n t c e n t r i f u g a t i o n (De R o b e r t i s , 1964; Rodriguez DeLores A r n a i z e t a_l, 1967) the motor neurone-Renshaw c e l l synapse remains the f i r s t and o n l y c l e a r demonstration of a c e n t r a l c h o l i n e r g i c j u n c t i o n ( E c c l e s e t a_l, 1954) . I t has been w e l l documented t h a t s i g n i f i c a n t amounts of AChE occur w i t h i n the axonal plasma membrane a t r e g i o n s f a r removed from s y n a p t i c j u n c t i o n s (Schlaepper and Torack, 1966; B r z i n , 1966 ), and Nachmansohn (1959) has proposed t h a t axonal c o n d u c t i o n i s mediated through a c h o l i n e r g i c mechanism e s s e n t i a l l y s i m i l a r to t h a t o p e r a t i n g a t the neuromuscular j u n c t i o n . I f t h i s i s t r u e , then c h o l i n e r g i c mechanisms would be e s s e n t i a l f o r c e n t r a l nervous t r a n s m i s s i o n . C o n s i d e r a t i o n of t h i s p r o p o s a l g e n e r a l l y has been d i s c o u n t e d f o r the f o l l o w i n g reasons: 1. i t i s not p o s s i b l e i n many cases to b l o c k axonal c o n d u c t i o n by the a p p l i c a t i o n of potent AChE i n h i b i t o r s , or i f c o n d u c t i o n i s a f f e c t e d , the c o n c e n t r a t i o n o f i n h i b i t o r f a r exceeds t h a t r e q u i r e d to b l o c k a t the neuromuscular j u n c t i o n . Under these c o n d i t i o n s b l o c k i n g i s assumed to r e s u l t from a non-s p e c i f i c t o x i c e f f e c t of the compounds used; 2. d i r e c t a p p l i c a t i o n of ACh f a i l s to e l i c i t an a c t i o n p o t e n t i a l , although a g e n e r a l d e p o l a r i z a t i o n p o s s i b l y r e l a t i n g to such f a c t o r s as 8 a l t e r e d pH or i o n i c environment may occur. Conclusions based on such r e s u l t s have been r e a d i l y d i s m i s s e d by Nachmansohn on the grounds t h a t not enough i s known about the a v a i l a b i l i t y of AChE and the ACh-receptor to e x t e r n a l l y a p p l i e d pharmacological reagents (Nachmansohn, 1969). Despite c o n s i d e r a b l e experimental evidence i n d i c a t i n g t h a t the outer membranes do i n f a c t mask the plasma membrane from the a c t i o n of AChE i n h i b i t o r s and ACh i n the e x t e r n a l medium (Walsh and Deal, 1957; Dettbarn, 1960a;b; Armett and R i c h i e , 1960; Rosenberg, 1965; B r z i n , 1966; M a r t i n and Rosenberg, 1968), the concept of c h o l i n e r g i c a l l y mediated axonal conductions has gained l i t t l e support from workers i n t h i s f i e l d and the f u n c t i o n of axonal AChE remains an open q u e s t i o n . The r e l e a s e of s u b s t a n t i a l amounts o f ACh i n the mammalian c e r e b r a l c o r t e x f o l l o w i n g s t i m u l a t i o n o f a f f e r e n t pathways and the mid b r a i n r e t i c u l a r f o r m a t i o n has a l s o been c i t e d as evidence f o r the importance o f c h o l i n e r g i c mechanisms i n the c e n t r a l nervous system (Kanai and Szerb, 1965; P h i l l i s and Chong, 1965; C e l e s i a and Jasper, 1966). K r n j e v i c (1969) has suggested t h a t t h i s type o f slow and d i f f u s e r e l e a s e i s not s u i t a b l e f o r r a p i d t r a n s m i s s i o n but may p l a y a more g e n e r a l f u n c t i o n i n c o r t i c a l a r o u s a l , or i n m a i n t a i n i n g d i f f e r e n t a c t i v i t y l e v e l s i n the c e n t r a l nervous system. In r e c e n t years a number of i n v e s t i g a t o r s have searched f o r p o s s i b l e r e l a t i o n s h i p s between c e n t r a l c h o l i n e r g i c mechanisms and animal behaviour. These s t u d i e s , and the problems i n v o l v e d i n t h i s type of approach have been reviewed by R u s s e l l (1969) and Weiss and H e l l e r (1969). In the 1950's 9 Rosenzweig and coworkers i n v e s t i g a t e d the e f f e c t s o f "enric h e d " and "impoverished" b e h a v i o u r a l environments upon the l e v e l s o f AChE a c t i v i t y i n the r a t c e n t r a l nervous system, and evidence was presented f o r i n c r e a s e d AChE a c t i v i t i e s i n animals s u b j e c t e d to such b e h a v i o u r a l s i t u a t i o n s as maze t r a i n i n g (Rosenzweig, 1957; Bennett e t a_l, 1964) . R u s s e l l e s t a b l i s h e d a dose-response r e l a t i o n s h i p between i n h i b i t i o n o f b r a i n AChE wi t h organo-phosphates, and the e x t i n c t i o n of l e a r n e d b e h a v i o u r a l responses i n r a t s . The r e l a t i o n s h i p was not l i n e a r , but below a c r i t i c a l l e v e l o f 40 to 50 percent i n h i b i t i o n o f normal AChE a c t i v i t y , the speed o f e x t i n c t i o n was d i r e c t l y r e l a t e d to AChE i n h i b i t i o n (see R u s s e l l , 1969). S i m i l a r experiments by Glow and coworkers (Glow and Rose, 1966; Glow e t a_l, 1966) demonstrated t h a t r e d u c t i o n o f AChE a c t i v i t y below 40 percent o f the normal value leads to a sudden i n c r e a s e i n b r a i n ACh l e v e l s . While i t i s d i f f i c u l t a t present to e v a l u a t e the importance of such experiments i n terms o f u n d e r l y i n g c h o l i n e r g i c mechanisms, t h i s b e h a v i o u r a l approach, together w i t h the b i o c h e m i c a l and n e u r o p h y s i o l o g i c a l evidence f o r the presence and a c t i o n o f ACh and AChE i n the c e n t r a l nervous system, seems to i n d i c a t e a d e f i n i t e r o l e f o r c h o l i n e r g i c mechanisms i n c e n t r a l nervous i n t e g r a t i o n . Thus AChE from the f i s h c e n t r a l nervous system appears to meet the c r i t e r i a o u t l i n e d a t the b e g i n n i n g o f t h i s d i s c u s s i o n f o r a s u i t a b l e enzyme system w i t h which to study thermal acclimmation and e v o l u t i o n a r y a d a p t a t i o n to temperature a t the l e v e l of enzyme f u n c t i o n . i t i s an enzyme i n c o r p o r a t e d i n t o a c r i t i c a l p h y s i o l o g i c a l process t h a t i s known to d i s p l a y both thermal 10 accommodation and thermal a c c l i m a t i o n i n response to changing environmental temperature. An i n v e s t i g a t i o n i n t o the e f f e c t s of immediate temperature changes upon AChE a c t i v i t y r e v e a l e d t h a t thermal accommodation of r e a c t i o n r a t e does occur a t probable p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s . The b a s i s f o r t h i s phenomenon l i e s i n the i n f l u e n c e of temperature upon enzyme s u b s t r a t e a f f i n i t y . The e f f e c t s o f thermal a c c l i m a t i o n upon AChE were s t u d i e d i n s e v e r a l s p e c i e s of t r o u t . F o l l o w i n g a c c l i m a t i o n of these f i s h to d i f f e r e n t temperatures, a l t e r a t i o n s i n the r e l a t i v e p r o p o r t i o n s of two AChE v a r i a n t s were observed. K i n e t i c a n a l y s i s of these enzymes showed t h a t when the environmental temperature was maintained a t a l e v e l where one form of the enzyme c o u l d no longer t h e r m a l l y accommodate f o r r e a c t i o n r a t e , o r where r e g u l a t i o n of c a t a l y t i c a c t i v i t y might be l o s t , a second form i s produced f o r which the enzyme s u b s t r a t e a f f i n i t y -temperature r e l a t i o n s h i p i s b e t t e r s u i t e d f o r c o n t r o l of these f u n c t i o n s . Comparisons of the p r o p e r t i e s o f probably homologous AChE enzymes from d i f f e r e n t s p e c i e s o f f i s h i n h a b i t i n g markedly d i f f e r e n t thermal environments l e a d to the c o n c l u s i o n t h a t e v o l u t i o n a r y a d a p t i o n o f AChE f u n c t i o n to temperature i s based upon s e l e c t i o n f o r an enzyme-substrate a f f i n i t y - t e m p e r a t u r e r e l a t i o n s h i p p e r m i t t i n g thermal accommodation of r e a c t i o n r a t e over the temperature range normally e x p e r i e n c e d by the s p e c i e s . In answer to the q u e s t i o n i n i t i a l l y posed i n d e s i g n i n g these experiments, i t can be s t a t e d t h a t thermal accommodation, thermal a c c l i m a t i o n and e v o l u t i o n a r y a d a p t a t i o n to temperature as d i s p l a y e d by many p o i k i l o t h e r m systems can be observed and i n t e r p r e t e d a t the l e v e l of enzyme f u n c t i o n . 12 METHODS 1. Experimental Animals A d u l t rainbow t r o u t (Salmo g a i r d n e r i i ) a veraging about 250 g were o b t a i n e d from the Sun V a l l e y Trout Farm, Port Moody, B.C. The f i s h were h e l d i n a l a r g e outdoor tank w i t h c i r c u l a t i n g water and f e d ad l i b on C l a r k ' s New Age 'F i s h Feed' ( J . R. C l a r k Co., S a l t Lake C i t y , Utah). Rainbow t r o u t g i l l n e t t e d i n Pinask Lake, B.C. d u r i n g both summer and w i n t e r were used i n a number of experiments. S_. g a i r d n e r i i can t o l e r a t e temperatures i n the range of 0° to 2 5°C. In a c c l i m a t i o n experiments, groups o f t r o u t ( g e n e r a l l y 18 f i s h ) were taken from the outdoor h o l d i n g pool and p l a c e d i n 60 g a l l o n s t a i n l e s s s t e e l tanks i n which the temperature c o u l d be c o n t r o l l e d a c c u r a t e l y w i t h h e a t i n g and r e f r i g e r a t i o n u n i t s . As i t was not p o s s i b l e to c i r c u l a t e water through these tanks, one q u a r t e r of the volume was changed d a i l y . The n a t u r a l p h o t o period of the outdoor tank was maintained throughout the a c c l i m a t i o n p e r i o d o f from 30 to 36 days, and the f i s h were f e d d a i l y w i t h C l a r k ' s ' F i s h Feed'. Speckled t r o u t ( S a l v e l i n u s f o n t i n a l i s ) , lake t r o u t ( S a l v e l i n u s namaychus) and the s p e c k l e d - l a k e h y b r i d , splake, were made a v a i l a b l e by the U n i v e r s i t y o f Toronto L a b o r a t o r y f o r Experimental Limnology, Southern Research S t a t i o n , Maple, O n t a r i o . Immature f i s h (6-15 cm i n length) were t h e r m a l l y a c c l i m a t e d i n c i r c u l a t i n g water tanks a t the r e s e a r c h s t a t i o n . 13 Trematonus b o r c h g r e v i n k i were cap t u r e d i n McMurdo Sound, A n t a r c t i c a d u r i n g the summer of 1965 by Dr. G. N. Somero. Brains from 6 f i s h were f r e e z e d r i e d and s t o r e d a t -20°C u n t i l assayed i n November 1969. The temperature of the waters i n h a b i t e d by t h i s f i s h average -1.9°C, w i t h annual v a r i a t i o n s i n the o r d e r of 0.1°C. T h i s s p e c i e s has not been reco r d e d i n waters w i t h temperatures h i g h e r than 2°C (see Somero and DeVries, 1967). 2. Enzyme P r e p a r a t i o n s (a) P r e p a r a t i o n of Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e Pooled brains were homogenized i n a s m a l l volume of c o l d d i s t i l l e d water and f r e e z e d r i e d . The procedure f o r s o l u b i l i z a t i o n of the membrane bound enzyme was based on the bu t a n o l e x t r a c t i o n technique d e s c r i b e d by Morton (1955). A l l steps were c a r r i e d out i n a 4°C c o l d room. Freeze d r i e d b r a i n s were d i s p e r s e d i n c o l d n-butanol (1, g d r i e d t i s s u e to 50 ml s o l v e n t ) by g r i n d i n g w i t h a mortar and p e s t l e . The suspension was s t i r r e d f o r 2 hours, then c e n t r i f u g e d a t 10,000 x g r a v i t y f o r 15 minutes. The supernatant was d i s c a r d e d and the p e l l e t r e - e x t r a c t e d w i t h n-butanol as b e f o r e . A f t e r c e n t r i f u g i n g the p e l l e t was d i s p e r s e d i n dry acetone a t -20°C, s t i r r e d f o r 5 minutes and c e n t r i f u g e d a t 10,000 x g r a v i t y f o r 20 minutes. The p e l l e t was d r i e d in. vacuo over c a l c i u m c h l o r i d e a t -20°C. A f t e r complete removal of the o r g a n i c s o l v e n t s the acetone d r i e d powder was taken up i n c o l d 10 M t r i s - H C l b u f f e r , pH 7.2, s t i r r e d f o r 2 hours and c e n t r i f u g e d a t 30,000x g r a v i t y f o r one hour to remove i n s o l u b l e m a t e r i a l . The supernatant was brought to 20 percent s a t u r a t i o n w i t h s o l i d ammonium sulphate, l e f t to s e t t l e f o r one hour, then c e n t r i f u g e d a t 10, 000 x gravity f o r 10 minutes. The p e l l e t was d i s c a r d e d and the supernatant a d j u s t e d to 50 percent s a t u r a t i o n w i t h s o l i d ammonium s u l p h a t e . A f t e r p r e c i p i t a t i o n o v e r n i g h t the sediment 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 a t 10,000 x g r a v i t y f o r 20 minutes and taken up i n d i s t i l l e d water. T h i s p r e p a r a t i o n c o u l d be s t o r e d f r o z e n i n 5 percent s a t u r a t e d ammonium sulphate f o r a t l e a s t s i x months without l o s s o f a c t i v i t y . The enzyme s o l u t i o n was d i a l y s e d a g a i n s t 10 -^ M t r i s - H C l b u f f e r , pH 7.2 b e f o r e use. (b) P r e p a r a t i o n of A c e t y l c h o l i n e s t e r a s e from Trout B r a i n and S p i n a l Cord f o r E l e c t r o p h o r e s i s B r a i n and s p i n a l c o r d were d i s s e c t e d out, washed and homogenized i n a s m a l l volume o f c o l d d i s t i l l e d water. The p r e p a r a t i o n was f r o z e n and thawed s i x times then spun a t 2,000 rpm f o r 20 minutes on a bench c e n t r i f u g e . The supernatant was drawn o f f and used as a source o f AChE f o r e l e c t r o p h o r e s i s . (c) E l e c t r i c E e l A c e t y l c h o l i n e s t e r a s e A p a r t i a l l y p u r i f i e d p r e p a r a t i o n of AChE e x t r a c t e d from the e l e c t r i c organ o f e l e c t r i c e e l was purchased from the Sigma Chemical Company ( E l e c t r i c e e l A c e t y l c h o l i n e s t e r a s e type V) . T h i s m a t e r i a l had an a c t i v i t y o f l,00Cyu Molar units/mg (one^jL molar u n i t w i l l h y d r o l y z e 1/AMole of a c e t y l c h o l i n e per minute a t pH 8.0 a t 37°C) and gave a s i n g l e band of AChE a c t i v i t y on acrylamide d i s c e l e c t r o p h o r e s i s . (d) P r e p a r a t i o n of Trematomus b o r c h g r e v i n k i b r a i n A c e t y l c h o l i n e s t e r a s e Freeze d r i e d b r a i n s from 6 f i s h were homogenized i n 4 ml o f 1 0 - 2 M t r i s - H C l b u f f e r , pH 7.2. T h i s p r e p a r a t i o n was used as a source of b r a i n AChE. 3. Assay o f A c e t y l c h o l i n e s t e r a s e A c t i v i t y AChE c a t a l y s e s the r e a c t i o n a c e t y l c h o l i n e + H^O ~ • ^ Choline + a c e t i c a c i d AChE a c t i v i t y can be c o n v e n i e n t l y determined by f o l l o w i n g the r a t e of hydrogen i o n p r o d u c t i o n . In t h i s study, AChE a c t i v i t y was assayed i n an automatic t i t r a t o r (Radiometer, Copenhagen, type TTA 31) operated as a pH s t a t w i t h sodium hydroxide as t i t r a n t . The volume o f sodium hydroxide added per u n i t time g i v e s a measure of the r a t e of a c e t y l c h o l i n e (ACh) h y d r o l y s i s . The b a s i c r e a c t i o n mixture c o n t a i n e d b u f f e r , enzyme and s u b s t r a t e i n a t o t a l volume o f 2 ml. Temperature was c o n t r o l l e d a c c u r a t e l y w i t h a c i r c u l a t i n g water bath (Lauda Brinkman, K-2/R) coupl e d to a water j a c k e t s u r r ounding the r e a c t i o n v e s s e l . Sodium hydroxide ( g e n e r a l l y 1 0 - 2 M) was s t a n d a r d i z e d by t i t r a t i o n a g a i n s t potassium b i p h t h a l a t e . In a l l experiments a p p r o p r i a t e blanks were run to compensate f o r any uptake o f atmospheric carbon d i o x i d e or non-enzymic h y d r o l y s i s o f s u b s t r a t e . 4. Gel E l e c t r o p h o r e s i s E l e c t r o p h o r e t i c s e p a r a t i o n of e s t e r a s e s was c a r r i e d out by s t a n d a r d acrylamide d i s c e l e c t r o p h o r e s i s (Davis, 1964), u s i n g 16 a 4 percent s t a c k i n g g e l , a 7 percent s e p a r a t i n g g e l , and 5 x 1 0 - 3 M t r i s - g l y c i n e tank b u f f e r , pH 8.7. Samples were a p p l i e d to the top o f the s t a c k i n g g e l and run f o r 90 minutes a t 3 mA per tube and a t 4°C. Es t e r a s e a c t i v i t y was l o c a l i z e d w i t h i n the g e l by the «C naphthyl acetate-diazonium s a l t technique (Market and Hunter, 1959) . A f t e r completion o f the run, ge l s were p l a c e d i n 4 x 1 0 - 2 M t r i s - H C l b u f f e r pH 7.1 f o r 10 minutes to i n h i b i t p r e c i p i t a t i o n o f the dye d u r i n g s t a i n i n g ( A l l e n e t a_l, 1965) and then t r a n s f e r r e d to a s o l u t i o n containing«^naphthylacetate (Sigma Chemical Co., 40 mg/100 ml) and F a s t Blue R.R. s a l t (Sigma Chemical Co., 70 mg/100 ml) i n 4 x 10~ 2 M t r i s - H C l b u f f e r , pH 7.1. Gels were r e a c t e d f o r 20 minutes a t 2 5°C then p l a c e d i n an a c i d - a l c o h o l s o l u t i o n ( e t h a n o l : .10 percent a c e t i c a c i d , 3:2) f o r 30 minutes to s t o p the r e a c t i o n and reduce n o n s p e c i f i c s t a i n i n g . A f t e r r e h y d r a t i o n i n d i s t i l l e d water, the g e l s c o u l d be s t o r e d i n d e f i n i t e l y a t 4°C. 5. P r o t e i n Determinations P r o t e i n c o n c e n t r a t i o n s o f AChE p r e p a r a t i o n s were es t i m a t e d by the method .of Lowry e t a__ (1951) . Samples were d i l u t e d to a c o n c e n t r a t i o n o f approximately 20/u.g p r o t e i n / m l . A s t a n d a r d curve was p l o t t e d w i t h ovalbumin i n the range 5 to 100/<A.g/ml f o r each s e t o f d e t e r m i n a t i o n s . 6. Sucrose G r a d i e n t C e n t r i f u g a t i o n Sucrose g r a d i e n t s were prepared i n 5 ml tubes w i t h a dual chamber g r a d i e n t maker. Samples were run on the Spinco model L p r e p a r a t i v e u l t r a c e n t r i f u g e equipped w i t h an SW 39 r o t o r . For s t u d i e s o f the e f f e c t o f temperature on the sedimentation o f e l e c t r i c e e l AChE, 5 to 20 percent sucrose g r a d i e n t s were prepared i n 2 x 1 0 - 2 M sodium b a r b i t o n e b u f f e r , pH 7.2, and c o n t a i n e d 2 x 10-"'" M magnesium c h l o r i d e . A 0.2 ml sample of AChE d i s s o l v e d i n the same buffer-magnesium c h l o r i d e s o l u t i o n was l a y e r e d onto the top of each g r a d i e n t and run f o r s i x hours a t 35,000 rpm a t the s p e c i f i e d temperature. Drop count f r a c t i o n s were c o l l e c t e d by g r a v i t y and assayed f o r AChE a c t i v i t y by the standard method. Gr a d i e n t d e n s i t i e s a t each temperature were c a l c u l a t e d from r e f r a c t o m e t e r r e a d i n g s . In the case o f . t r o u t b r a i n AChE p r e p a r a t i o n s , where the a c t i v i t y was too low to measure c o n v e n i e n t l y by the standar d assay, the enzyme was d e t e c t e d i n s i t u by the method of J o l l e y e t a l (1967). In t h i s technique sucrose g r a d i e n t s are prepared i n acrylamide s o l u t i o n and photopolymerized a f t e r completion of the run. Gradients were formed by p l a c i n g 2.3 ml of a 7.5 percent acrylamide g e l s o l u t i o n c o n t a i n i n g 2 x 10 ^ M magnesium c h l o r i d e i n the d i s t a l chamber of the g r a d i e n t maker and 2.5 ml of the same s o l u t i o n c o n t a i n i n g 20 percent sucrose i n the o u t l e t chamber. The sample, d i s s o l v e d i n 0.2 ml of the 7.5 percent acrylamide g e l s o l u t i o n , was a p p l i e d to the top of the prepared g r a d i e n t immediately b e f o r e c e n t r i f u g i n g and run f o r ten hours a t 35,000 rpm and a t 4°C. A f t e r completion of the run, water was l a y e r e d onto the top of each tube to give a f l a t s u r f a c e , and the g e l polymerized under a f l u o r e s c e n t l i g h t . The g e l s were then removed from 18 the c e n t r i f u g e tubes and s t a i n e d f o r e s t e r a s e a c t i v i t y as d e s c r i b e d p r e v i o u s l y f o r d i s c e l e c t r o p h o r e s i s . 7. U l t r a V i o l e t D i f f e r e n c e Spectra a. I n t r o d u c t i o n P r o t e i n s e x h i b i t c h a r a c t e r i s t i c a b s o r p t i o n p a t t e r n s i n the u l t r a v i o l e t r e g i o n of the spectrum. The b a s i s of t h i s phenomenon has been reviewed by W e t l a u f e r (1962), and i t i s g e n e r a l l y c o n s i d e r e d t h a t a b s o r p t i o n i n the 260-300 rnytf. i n t e r v a l i s caused by e l e c t r o n t r a n s i t i o n s i n the amino a c i d s p h e n y l a l a n i n e , tryptophan and t y r o s i n e , w h i l s t a b s o r p t i o n i n the 230 my\A r e g i o n r e s u l t s mainly from e l e c t r o n t r a n s i t i o n s i n the c a r b o x y l moeity of the peptide group. In a number of s t u d i e s , s h i f t s i n a b s o r p t i o n s p e c t r a have been i n t e r p r e t e d i n terms of changes i n the secondary and t e r t i a r y s t r u c t u r e of the p r o t e i n molecule (Yanari and Bovey, 1960; Foss, 1960, 1961; Massey e_t a_l, 1966) . Thus, by p l o t t i n g the UV a b s o r p t i o n s p e c t r a o b t a i n e d a t d i f f e r e n t temperatures r e l a t i v e to the spectrum a t a r e f e r e n c e temperature t h e r m a l l y induced changes i n p r o t e i n conformation can be d e t e c t e d . I d e a l l y , the d i f f e r e n c e s p e c t r a are o b t a i n e d i n a twin beam spectrophotometer i n which the samples can be read r e l a t i v e to a c e l l h e l d a t the r e f e r e n c e temperature. b. Method UV s p e c t r a were determined w i t h a Cary 15 r e c o r d i n g spectrophotometer equipped w i t h a c i r c u l a t i n g water bath. A l l measurements were made i n 1 cm c e l l s . E l e c t r i c e e l AChE was d i s s o l v e d i n 2 x 10"^ M sodium phosphate b u f f e r , pH 7.2, to a c o n c e n t r a t i o n of 0.15 mg/ml, and e x t e n s i v e l y d i a l y z e d a g a i n s t t h i s b u f f e r b e f o r e use. The r e f e r e n c e c u v e t t e c o n t a i n e d sample b u f f e r o n l y and was maintained a t the sample cuvette temperature. S p e c t r a l scans were run i n t r i p l i c a t e and a d i f f e r e n c e spectrum f o r each temperature was o b t a i n e d by r e p l o t t i n g the r e s u l t s r e l a t i v e to the spectrum o b t a i n e d RESULTS AND DISCUSSION 1. P a r t i a l P u r i f i c a t i o n of Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e The r e l a t i v e a c t i v i t i e s of f r a c t i o n s taken throughout the e x t r a c t i o n procedure are given i n Table 1. Although the s p e c i f i c a c t i v i t y of the f i n a l p r e p a r a t i o n r e p r e s e n t s o n l y about a f o u r f o l d p u r i f i c a t i o n over the o r i g i n a l crude homogenate, t i t r a t i o n p l o t s gave s t r a i g h t l i n e s down to an AChE c o n c e n t r a t i o n of 2 x lO -'* M. With crude homogenates non l i n e a r p l o t s were encountered and a c t i v i t y c o u l d not be a c c u r a t e l y measured below about 5 x 1 0 - ^ M ACh. 2. C h a r a c t e r i z a t i o n of A c e t y l c h o l i n e s t e r a s e s from the Rainbow  Trout C e n t r a l Nervous System a. I n t r o d u c t i o n A c e t y l c h o l i n e s t e r a s e s have been d e f i n e d by Augustinsson (1957) as e s e r i n e - s e n s i t i v e e s t e r a s e s which are i n h i b i t e d by h i g h a c e t y l c h o l i n e c o n c e n t r a t i o n s ( g e n e r a l l y 3 to 5 x 10 M) and which s p l i t a c e t y l c h o l i n e a t a much h i g h e r r a t e than b u t y r y l c h o l i n e . The compound 284C51 (Burroughs Wellcome) a t - 6 - 5 c o n c e n t r a t i o n s from 10 to 10 M g i v e s about 100,000 f o l d g r e a t e r i n h i b i t i o n of AChE than of o t h e r c h o l i n e s t e r a s e s ( A u s t i n and Berry, 1953). b. M u l t i p l e Forms of A c e t y l c h o l i n e s t e r a s e i n the Rainbow Trout C e n t r a l Nervous System At l e a s t 7 bands o f e s t e r a s e a c t i v i t y were observed f o l l o w i n g acrylamide g e l d i s c e l e c t r o p h o r e s i s of e x t r a c t s from rainbow t r o u t b r a i n and s p i n a l c o r d . S p e c i f i c AChE bands Table 1. P a r t i a l P u r i f i c a t i o n o f AChE from Rainbow Trout B r a i n . b F r a c t i o n A c t i v i t y P u r i f i c a t i o n ( X ) Recovery(%) B r a i n homogenate 8.5 100 Butanol s o l u b i l i z e d enzyme 11.9 1.4 107 20% ( N H 4 ) 2 SO. f r a c t i o n 23.7 2.8 53 20-50% (NH 4)„ S0 4 f r a c t i o n 32.3 3.8 2 5 o a. 2 C a c c l i m a t e d t r o u t b. S p e c i f i c a c t i v i t y i s expressed a s ^ M ACh h y d r o l y s e d / mg p r o t e i n / h o u r were d e t e c t e d by t h e i r i n h i b i t i o n w i t h 10 ^ M e s e r i n e and w i t h -5 . ~ 10 M 284C51. The AChE bands o b t a i n e d w i t h b r a i n p r e p a r a t i o n s from rainbow t r o u t a c c l i m a t e d to 2°, 12° and 17°C f o r 32 days are shown i n F i g u r e 1. I d e n t i c a l r e s u l t s were o b t a i n e d w i t h both p u r i f i e d e x t r a c t s and crude homogenates of b r a i n , and w i t h the s p i n a l c o r d p r e p a r a t i o n s . AChE from c o l d a c c l i m a t e d t r o u t shows a d i s t i n c t l y slower m i g r a t i o n r a t e than does the enzyme from warm a c c l i m a t e d f i s h . T r o ut a c c l i m a t e d to 12°C possess both enzyme types. On t h e b a s i s of s t a i n i n g i n t e n s i t y i t was e s t i m a t e d t h a t equal amounts of the two rainbow enzymes were present i n the 12°C f i s h . B r a i n e x t r a c t s from w i l d t r o u t p o p u l a t i o n s were a l s o examined. In f i s h c a p t u r e d d u r i n g the wi n t e r , o n l y the 2°C enzyme was present; i n summer f i s h , both enzymes u s u a l l y o c c u r r e d , w i t h the 17°C form i n excess . F i s h h e l d a t 9°C i n the outdoor pool d u r i n g autumn a l s o had both enzymes, w i t h the c o l d form predominating. c. Substrate S p e c i f i c i t y and I n h i b i t i o n Studies o f Rainbow Trout B r a i n A c e t y l c h o l i n e s t e r a s e s Substrate s a t u r a t i o n p l o t s f o r b r a i n e x t r a c t s from 2 ° c and 17°C a c c l i m a t e d rainbow t r o u t are g i v e n i n F i g u r e 2 and F i g u r e 3. Both enzymes show g r e a t e r a c t i v i t y w i t h ACh than w i t h p r o p i o n y l - c h o l i n e or b u t y r y l c h o l i n e , and i n each case s u b s t r a t e i n h i b i t i o n occurs a t c o n c e n t r a t i o n s above about _ o 3 x 10 M ACh. The r e l a t i v e a c t i v i t i e s o f rainbow t r o u t p r e p a r a t i o n s and e l e c t r i c e e l AChE w i t h i n d i v i d u a l and p a i r e d s u b s t r a t e s and i n h i b i t o r s are g i v e n i n Table 2. No summation 23 F i g u r e 1. R e s o l u t i o n o f Rainbow Trout B r a i n AChEs by Acrylamide Gel D i s c E l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s c o n d i t i o n s : 90 minutes a t 3 mA and 400 v o l t s per g e l . T r i s -g l y c i n e tank b u f f e r , pH 8.7 2°C a c c l i m a t e d t r o u t 17°C a c c l i m a t e d t r o u t 12°C a c c l i m a t e d t r o u t 24 Figure 2. Substrate S p e c i f i c i t y of AChE from 2°C Acclimated Rainbow Trout, Standard assay i n 10~ 2 M t r i s - H C l buffer, pH 7.2 with -2 . . 10 M sodium hydroxide as t i t r a n t . Assay temperature 2°C. © Acetylcholine iodide _ Propionylcholine iodide A Butyrylcholine iodide 25 Figure 3. Substrate S p e c i f i c i t y of AChE from 17°C Acclimated Rainbow Trout. Standard assay as i n Figure 2. Assay temperature 15°C. © Acetylcholine -iodide E2 Propionylcholine iodide A Butyrylcholine iodide Table 2. Surnmation Experiments w i t h Choline E s t e r s and the E f f e c t of I n h i b i t o r s on H y d r o l y s i s of ACh by Rainbow Trout and E l e c t r i c E e l AChEs Reac t i o n r a t e (JK M s u b s t r a t e hydrolysed/mg p r o t e i n / h r ) Substrate (2.5 x 10~3M) A c e t y l c h o l i n e P r o p i o n y l c h o l i n e B u t y r y l c h o l i n e A c e t y l c h o l i n e and p r o p i o n y l c h o l i n e A c e t y l c h o l i n e and b u t y r y l c h o l i n e P r o p i o n y l c h o l i n e and b u t y r y l c h o l i n e A c e t y l c h o l i n e and 5 x 10~ 3 M e s e r i n e A c e t y l c h o l i n e and 10" 6 M 284C51 2 C t r o u t AChE 54.0 9.2 0 22.1 11.9 0 0 0 17 C t r o u t AChE 43.2 7.3 0 15.9 5.3 3.5 0 0 e l e c t r i c e e l AChE ( x l 0 ~ 3 ) 71.0 61.0 4.3 64.0 9.9 8.0 Assays were c a r r i e d out a t 10 C f o r the rainbow t r o u t enzymes and a t 2 5°C f o r the e l e c t r i c e e l AChE S u b s t r a t e s : A c e t y l c h o l i n e i o d i d e ; p r o p i o n y l c h o l i n e i o d i d e ; b u t y r y l c h o l i n e i o d i d e . 27 o f a c t i v i t y o c c u r r e d w i t h any s u b s t r a t e p a i r t e s t e d , i n d i c a t i n g t h a t b u t y r y l or p r o p i o n y l c h o l i n e s t e r a s e s are not c o n t r i b u t i n g to the r a t e o f h y d r o l y s i s . H y d r o l y s i s o f 2.5 x 1 0 - 3 M ACh by e i t h e r the 2°.and 17°C t r o u t AChE or the e e l enzyme, was -5 -6 completely i n h i b i t e d w i t h 5 x 10 M e s e r i n e , and w i t h 10 M 284C51. These r e s u l t s o b t a i n e d w i t h s p e c i f i c s u b s t r a t e s and i n h i b i t o r s i n d i c a t e t h a t e s s e n t i a l l y a l l o f the e s t e r a s e a c t i v i t y o f the t r o u t b r a i n e x t r a c t s d e t e c t e d by the assay method can be a t t r i b u t e d to AChE. d. E f f e c t o f pH on A c e t y l c h o l i n e s t e r a s e A c t i v i t y The b e l l shaped p H - a c t i v i t y curves f o r the rainbow t r o u t AChEs shown i n F i g u r e 4 are s i m i l a r t o those o b t a i n e d w i t h AChEs from a v a r i e t y o f sources (Bernsohn e t a_l, 1963; B u l l and L i n d q u i s t , 1968; Silman and K a r l i n , 1967). e. Sucrose G r a d i e n t C e n t r i f u g a t i o n o f Rainbow Tr o u t B r a i n A c e t y l c h o l i n e s t e r a s e s The r e s u l t s o f the experiments i n which rainbow t r o u t b r a i n p r e p a r a t i o n s were c e n t r i f u g e d on sucrose g r a d i e n t s made up i n acrylamide g e l s o l u t i o n are shown i n F i g u r e 5. I d e n t i c a l r e s u l t s were o b t a i n e d w i t h e x t r a c t s from both 2° and 17°C a c c l i m a t e d f i s h . In each case 3 bands o f e s t e r a s e a c t i v i t y were detected, the c e n t r a l band sedimenting i n the same p o s i t i o n as e l e c t r i c e e l AChE. I n h i b i t i o n w i t h 10~ 5 M 284C51 and -4 10 M e s e r i n e d i d not remove any one band, but g r e a t l y reduced the i n t e n s i t y o f the c e n t r a l band. 28 Figure 4. Influence of pH on the A c t i v i t y of Rainbow Trout Brain AChEs. Sodium phosphate buffer, -2 10 M, was used i n the range pH 6-8, and -2 -2 t r i s - H C l buffer, 10 M, containing 10 M sodium chloride from pH 7.5 to 9.5. © 17°C acclimated rainbow trout. Assay temperature 17°C. o E3 2 C acclimated rainbow trout. Assay temperature 2°C. Rate (uM ACh hydrolyzed /mg protein /hr) 29 F i g u r e 5. Sucrose G r a d i e n t C e n t r i f u g a t i o n o f Rainbow Tr o u t and E l e c t r i c E e l AChEs. A 5 ml 0 to 20 percent sucrose g r a d i e n t was formed i n 7.5 percent acrylamide g e l s o l u t i o n c o n t a i n i n g 2 x 1 0 - 1 M MgC-L_. Samples o f 0.2 ml were l a y e r e d on top. C e n t r i f u g a t i o n c o n d i t i o n s : 10 hours a t 100,000 x g r a v i t y a t 4°C. A f t e r c e n t r i f u g a t i o n the g e l s were photopolymerized and s t a i n e d f o r e s t e r a s e a c t i v i t y . electric eel 2 ° C acclimated 17°C acclimated 2° or 17°C acclim-AChE frout trout ated trout. Stained in the presence of T0 _ 6/M 284C51 30 The f o l l o w i n g c o n c l u s i o n s can be made on the b a s i s o f these r e s u l t s . 1. Rainbow t r o u t b r a i n e s t e r a s e s o c c u r i n 3 mol e c u l a r s i z e c l a s s e s , AChE probably s h a r i n g a c l a s s w i t h a t l e a s t one ot h e r e s t e r a s e . 2. The AChEs from both warm and c o l d a c c l i m a t e d t r o u t and from e l e c t r i c e e l are of s i m i l a r m o l e c u l a r weight, about 260,000 (Leuzinger e t a_l, 1969). 3. C h a r a c t e r i z a t i o n o f A c e t y l c h o l i n e s t e r a s e from Trematomus  b o r c h g r e v i n k i Bra-in Because of the sm a l l amount o f m a t e r i a l a v a i l a b l e i t was not p o s s i b l e to c h a r a c t e r i z e t h i s enzyme system as f u l l y as was done f o r the rainbow t r o u t AChE's. I t was concluded from the f o l l o w i n g r e s u l t s t h a t the t o t a l a c t i v i t y d e t e c t e d by the assay c o u l d be a t t r i b u t e d to a s i n g l e AChE s p e c i e s . — 6 1. The presence o f 2 x 10 M 284C51 i n the assay c o m p l e t e l y i n h i b i t e d ACh h y d r o l y s i s . 2. Acrylamide g e l e l e c t r o p h o r e s i s of the crude b r a i n homogenate gave f o u r bands o f e s t e r a s e a c t i v i t y ; o n l y — ft one o f these bands was i n h i b i t e d by 5 x 10 M 284C51 -4 and 10 M e s e r m e . 4. E f f e c t o f Assay Temperature upon the K i n e t i c s o f A c e t y l c h o l i n e  H y d r o l y s i s by A c e t y l c h o l i n e s t e r a s e a. E f f e c t o f Temperature on the Maximum V e l o c i t y o f A c e t y l c h o l i n e H y d r o l y s i s Numerous attempts have been made to e s t a b l i s h r e l a t i o n s h i p s between the thermal t o l e r a n c e s o f p o i k i l o t h e r m s and temperature dependent c h a r a c t e r i s t i c s o f t h e i r enzymes. The 31 c h a r a c t e r i s t i c s most commonly i n v e s t i g a t e d are the temperatures f o r o p t i m a l enzyme a c t i v i t y a t V max l e v e l s o f s u b s t r a t e , and enzyme t h e r m o s t a b i l i t y . Studies of t h i s type have been reviewed recentlyby L i c h t (1967), Read (1967), and Ushakov (1967) among o t h e r s . In most cases both thermal optima and thermal d e n a t u r a t i o n occur a t temperatures above those encountered i n the environment. Of p a r t i c u l a r i n t e r e s t w i t h r e f e r e n c e to the present study are r e s u l t s o b t a i n e d f o r f i s h c h o l i n e s t e r a s e s . Kusakina (1963) has demonstrated a p o s i t i v e r e l a t i o n s h i p between the temperature a t which muscle e x c i t a b i l i t y i s l o s t , the temperature of muscle c h o l i n e s t e r a s e i h a c t i v a t i o n , and the thermal environment o f c o t t o i d f i s h . The temperature a t which 50 percent o f c h o l i n e s t e r a s e a c t i v i t y was l o s t a f t e r i n c u b a t i o n f o r 30 minutes i n c r e a s e d w i t h i n c r e a s i n g h a b i t a t temperature, and o c c u r r e d a t temperatures 12° to 15°C above those a t which muscle e x c i t a b i l i t y ceased. S i m i l a r l y , Baslow and K f i g r e l l i (1964) found no d i f f e r e n c e i n the l e v e l s o f c h o l i n e s t e r a s e a c t i v i t y o f c o n t r o l and heat k i l l e d k i l l i f i s h . Alexandrov (1969) has proposed t h a t i n cases where a c o r r e l a t i o n e x i s t s between h a b i t a t temperature and the r e s i s t a n c e of p r o t e i n s to thermal d e n a t u r a t i o n , but where t h e r m o s t a b i l i t y i s c l e a r l y not s e t t i n g temperature l i m i t s f o r the organism, the p r o p e r t y s u b j e c t to s e l e c t i o n may not be t h e r m o s t a b i l i t y per se, but r a t h e r , a c e r t a i n c o n f o r m a t i o n a l f l e x i b i l i t y o f the p r o t e i n molecule r e q u i r e d f o r such f u n c t i o n s as c a t a l y t i c a c t i v i t y and a l l o s t e r i c r e g u l a t i o n . Changes i n t h e r m a l l y dependent c o n f o r m a t i o n a l f l e x i b i l i t y may i n t u r n a l t e r the thermal d e n a t u r a t i o n c h a r a c t e r -i s t i c s of the molecule. C e r t a i n aspects o f t h e r m a l l y dependent c o n f o r m a t i o n a l changes i n p r o t e i n s t r u c t u r e are d i s c u s s e d i n d e t a i l i n f o l l o w i n g s e c t i o n s o f t h i s t h e s i s . C r i t i c i s m a g a i n s t the use o f thermal optima f o r maximum enzyme a c t i v i t y and t h e r m o s t a b i l i t y as measures o f thermal a d a p t a t i o n i s g e n e r a l l y l e v e l e d a t the n o n - p h y s i o l o g i c a l nature o f the experimental methods. For example, the a c t u a l temperature a t which enzyme a c t i v i t y i s maximal depends on such f a c t o r s as assay time, and the V max l e v e l s o f s u b s t r a t e employed are g e n e r a l l y f a r i n excess o f probable p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s . S i m i l a r l y , p r o t e i n t h e r m o s t a b i l i t y i s h i g h l y dependent upon such f a c t o r s as presence or.absence o f s u b s t r a t e , pH, i o n i c environment, and b i n d i n g of the enzyme to membranes and o t h e r c e l l u l a r s t r u c t u r e s ( V e s s e l l and Y i e l d i n g , 1966, Bowen and Kerwin, 1956; Cheeseman e_t a_l, 1967) . Thus any c o r r e l a t i o n between i r i v i t r o enzyme thermal optima or t h e r m o s t a b i l i t y w i t h environmental temperature, may be simply f o r t u i t o u s . A r r h e n i u s p l o t s o f l o g V opt. versus 1/T f o r AChEs from rainbow t r o u t a c c l i m a t e d to 2° and 17°C and f o r e l e c t r i c e e l are shown i n F i g u r e 6. i n each case the maximum v e l o c i t y o f the r e a c t i o n i n c r e a s e s w i t h temperature beyond the thermal range o f the f i s h . With b r a i n AChE from Trematomus b o r c h g r e v i n k i the a c t i v i t y o f the enzyme a t h i g h l e v e l s o f s u b s t r a t e (10 M ACh) i s twice as g r e a t a t 10°C as i t i s a t 2°C (see Table 5 ) . Th i s f i s h has not been rec o r d e d i n waters above 2°C. Thus, the op t i m a l temperature f o r enzyme a c t i v i t y , and thermal s t a b i l i t y o f enzyme s t r u c t u r e , are probably not important f a c t o r s i n 33 Figure 6. Arrhenius Plots of AChE A c t i v i t y for the Rainbow Trout and E l e c t r i c Eel Enzymes. -2 Standard assay i n 10 M t r i s - H C l buffer, pH 7.2 with V opt. l e v e l s of ACh. o © 17 C acclimated trout AChE 13 2°C acclimated trout AChE A e l e c t r i c e e l AChE .do^ 6o| 34 s e t t i n g thermal l i m i t s f o r AChE a c t i v i t y i n these s p e c i e s . I t has been argued t h a t r a t e s of enzyme a c t i v i t y i n c o l d adapted organisms where a v a i l a b l e thermal energy i s low may be maintained through a lowering of the energy of a c t i v a t i o n (Ea). Although such a c o r r e l a t i o n between Ea and the environmental temperature has been demonstrated i n a number of cases (Vroman and Brown, 1963; Kwon and O l c o t t , 1965; Somero and Hochachka, 1968) i t i s by no means u n i v e r s a l (Read, 1964; Hochachka and Somero, 19 68). In the case of the rainbow t r o u t and e l e c t r i c e e l AChEs the curved A r r h e n i u s p l o t s y i e l d e n e r g i e s of a c t i v a t i o n which decrease as the temperature i s r a i s e d . S i m i l a r l y curved p l o t s have been 'reported p r e v i o u s l y f o r an e l e c t r i c e e l AChE (Wilson and Cabib, 1956), and f o r c h o l i n e s t e r a s e s from a v a r i e t y of sources (Chadwick, 1957). Wilson and Cabib (1956) i n t e r p r e t e d the decrease i n Ea w i t h temperature f o r e l e c t r i c e e l AChE i n terms of a change i n the r a t e l i m i t i n g s t e p f o r the o v e r a l l r e a c t i o n . They proposed t h a t A r r h e n i u s p l o t s would give a s t r a i g h t l i n e through the temperature range where one of the steps i s r a t e l i m i t i n g , and a d i f f e r e n t s t r a i g h t l i n e i n the range where the o t h e r s t e p i s slower. These two l i n e s would be j o i n e d by a curved p o r t i o n over the thermal range where both r a t e s are comparable. The curve would f l a t t e n a t h i g h e r temperatures as the r a t e approached the s t e p which had the lower energy of a c t i v a t i o n . I t i s a l s o suggested t h a t l o g V max versus 1/T p l o t s need not give s t r a i g h t l i n e s when both V max and Km v a r y w i t h temperature (see below). N o n - l i n e a r A r r h e n i u s p l o t s o b t a i n e d w i t h amino oxidase have been r e l a t e d to a temperature dependent t r a n s i t i o n of the enzyme between two conformations (Koster and Veeger, 1968; Massey e t a_l, 1966). Recently, evidence has been presented f o r s i m i l a r temperature-dependent t r a n s i t i o n s between m u l t i p l e forms of serum c h o l i n -e s t e r a s e s and AChE from e r y t h r o c y t e s (Main, 1969). Energies o f a c t i v a t i o n f o r the t r o u t and e l e c t r i c e e l AChEs were c a l c u l a t e d from the r e l a t i o n s h i p l o g (K - K )= —* E a (-^— - -ji—) where Kn and are r e a c t i o n v e l o c i t i e s a t 4.6 1^ T-/ 1 2 a b s o l u t e temperatures and T_ and Ea i s the energy of a c t i v a t i o n . Ea v a l u e s a t a s p e c i f i c temperature was o b t a i n e d by drawing a tangent to the A r r h e n i u s curve a t t h a t temperature. The r e l a t i o n s h i p s between Ea and temperature are g i v e n i n Table 3. For the rainbow t r o u t enzymes the Ea v a l u e s a t any g i v e n temperature are e s s e n t i a l l y the same. The r e s u l t s are d i f f i c u l t to i n t e r p r e t i n terms of adaptive advantage, as the value f o r the 17°C enzyme i s lower a t 17°C than the value of the 2°C enzyme at 2°C. The value o f 1.9 K cal/mole o b t a i n e d f o r the e l e c t r i c e e l enzyme a t the probable h a b i t a t temperature ( 2 5°C) i s lower than e i t h e r of the t r o u t enzymes a t 2°C. I t must be concluded t h a t no c l e a r r e l a t i o n s h i p e x i s t s between the apparent e n e r g i e s of a c t i v a t i o n of AChE and h a b i t a t temperature i n the s p e c i e s s t u d i e d . b. E f f e c t of Temperature on Enzyme-Substrate A f f i n i t y C urrent models of enzyme r e g u l a t i o n s t r e s s the importance of e f f e c t o r s i n m o d i f y i n g enzyme-substrate a f f i n i t i e s (Atkinson, 3 6 Table 3. Apparent Energies of A c t i v a t i o n (Ea) f o r the Rainbow-Trout and E l e c t r i c E e l AChEs a t s e v e r a l temperatures AChE source (°C) E a (^cal/mole) 2°C a c c l i m a t e d t r o u t 2 3.6 17 1.8 o 17 C a c c l i m a t e d t r o u t 2 4.1 17 2.1 E l e c t r i c e e l 25 1.9 17 3.0 1966; Stadtman, 1966). I t has been suggested r e c e n t l y t h a t i n the case of enzymes from p o i k i l o t h e r m s , temperature may p l a y a r o l e analogous to t h a t of p o s i t i v e and negative e f f e c t o r s by a l t e r i n g enzyme-substrate a f f i n i t i e s i n such a way as to b r i n g about compensatory changes i n the r a t e s of enzyme r e a c t i o n s throughout the b i o l o g i c a l thermal range of the organism (Hochachka and Somero, 1968). The r e l a t i o n s h i p s between the a f f i n i t y of rainbow t r o u t and e l e c t r i c e e l AChE f o r ACh (as measured by the r e c i p r o c a l of the apparent M i c h a e l i s constant, Km) and assay temperature, are shown i n F i g u r e s 7 and 8. In the case of the t r o u t AChEs i t i s apparent t h a t over the upper p a r t of the b i o l o g i c a l thermal range of each enzyme, the a f f i n i t i e s of the enzymes f o r the ACh v a r y w i t h temperature and approach maximal v a l u e s (minimum Km) a t temperatures c o r r e s p o n d i n g to those a t which the f i s h were a c c l i m a t e d . T h i s r e l a t i o n s h i p between h a b i t a t temperature and Km a l s o h o l d s f o r the e l e c t r i c e e l enzyme; the minimum Km i n t h i s case occurs a t about 25°C, a temperature c o r r e s p o n d i n g c l o s e l y to the probable minimum h a b i t a t temperature. A s i m i l a r correspondence between minimum Km and environmental temperature has been observed f o r pyruvate kinases from rainbow t r o u t and the a n t a r c t i c f i s h Trematomus b e r n a c c h i i (Somero and Hochachka, 1968) f o r l a c t a t e dehydrogenases from t r o u t , tuna, l u n g f i s h , Trematomus (Hochachka and Somero, 1968) and k i n g - c r a b (Somero and Hochachka, 1969), f o r glucose-6-phsophate dehydrogenase and 6-phospho-gluconate dehydrogenases from k i n g - c r a b (Somero, 1969) , f o r salmon and l u n g f i s h f r u c t o s e diphosphatases (Behrisch, 38 Figure 7. E f f e c t of Assay Temperature on the Km of ACh for AChEs from 17° and 2°C Acclimated -2 Rainbow Trout. Standard assay xn 10 M t r i s - H C l buffer, pH 7.2. The enzymes were assayed at.ACh concentrations i n the range - 4 - 3 10 to 5 x 10 M and Km values were determined from double-reciprocal plots (1/V versus 1/ ACh ). @ 17°C acclimated trout AChE El 2°C acclimated trout AChE -Temperature (°C) 39 F i g u r e 8. E f f e c t o f Assay Temperature on the Km o f ACh f o r E l e c t r i c E e l AChE. Standard assay i n 10~ 2 M t r i s - H C l b u f f e r , pH 7.2. The enzyme was assayed a t ACh c o n c e n t r a t i o n s -4 -3 i n the range 10 to 5 x 10 M and Km va l u e s were determined from d o u b l e - r e c i p r o c a l p l o t s (1/V versus 1/ ACh ). 5 01 l I i 1 1 1 1 l 0 5 10 15 20 25 30 35 40 Temperature (°C) 1969; B e h r i s c h and Hochachka, 1969), f o r rainbow t r o u t NADP i s o c i t r a t e dehydrogenases (Moon, p e r s o n a l communication) and c i t r a t e synthases (Hochachka and Lewis, 1970). A t temperatures above t h a t a t which Km i s minimum, the 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 o f the Km-temperature r e l a t i o n s h i p i s c l e a r . Although a r i s e i n temperature would be expected to in c r e a s e the v e l o c i t y o f the enzyme r e a c t i o n , t h i s e f f e c t i s co u n t e r a c t e d by a decrease i n enzyme-substrate a f f i n i t y . Hence, the o v e r a l l r e a c t i o n r a t e remains r e l a t i v e l y independent of temperature. T h i s type of temperature-independence i s c h a r a c t e r i s t i c o f both forms o f t r o u t AChE. The 'warm' form shows t h i s r e l a t i o n s h i p a t temperatures above about 17°C; the ' c o l d ' form shows the r e l a t i o n s h i p above about 2°C. Th i s e f f e c t i s r e f l e c t e d i n the r a t e s of a c e t y l c h o l i n e h y d r o l y s i s a t s u b s t r a t e c o n c e n t r a t i o n s approaching the minimum Km (Table 4 ) . -4 In t h i s connection, a r e c e n t estimate o f 2.16 x 10 M f o r the c o n c e n t r a t i o n of ACh r e l e a s e d i n t o the s y n a p t i c space of the v e r t e b r a t e motor endplate corresponds c l o s e l y to the minimum Km value o f both t r o u t AChEs (Namba and Grob, 1969). A c o n s i d e r a b l e overcompensation f o r the a c c e l e r a t i n g e f f e c t s of temperature upon r e a c t i o n r a t e i s observed w i t h the _o o 2 C t r o u t enzyme a t 18 C, and w i t h the e l e c t r i c e e l AChE a t 40°C (Table 4 ) . However, t h i s e f f e c t may be of l i t t l e p h y s i o l o g i c a l importance. In the case of the 2°C t r o u t enzyme the decrease i n r e a c t i o n r a t e i s presumably compensated f o r i n v i v o by the appearance o f the 17°C enzyme, while the e l e c t r i c e e l may not encounter water temperatures as h i g h as 40°C. 41 Table 4. Rates of ACh H y d r o l y s i s a t Minimum Km Leve l s of ACh f o r AChEs from Rainbow Trout, E l e c t r i c E e l and Trematomus AChE source Temperature Assay. V/V a ACh (M) a t minimum temperature (minimum Km (°C) (°C) Km) 2°C 2.5 x 10~ 4 a c c l i m a t e d t r o u t 17°C 2.5 x 10 4 17 a c c l i m a t e d t r o u t E l e c t r i c 10 4 25 e e l 0 0.79 5 1.10 8 1.10 12 0.90 18 0.61 11 0.61 13 0.80 22 1.00 27 1.20 15 0.42 20 0.92 30 1.10 35 1.10 40 0.65 10 0.51 -4 Trematomus 1.5 x 10 b o r c h g r e v i n k i a. Rate of h y d r o l y s i s a t assay temperature/rate a t temperature of minimum Km b. The r a t e o f ACh h y d r o l y s i s a t 10°C i s expressed r e l a t i v e to the r a t e a t 2°C A t lower thermal extremes the Km-temperature r e l a t i o n s h i p i s r e v e r s e d . For the 'warm' form of the t r o u t enzyme the Km r i s e s s h a r p l y as the temperature f a l l s below 15°C, r e a c h i n g a value a t 10°C t h a t i s about 4 times the minimum v a l u e . Thus, a t 2°C the Km of the enzyme i s probably so h i g h as to make the enzyme e s s e n t i a l l y i n a c t i v e a t low and presumably p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s . S i m i l a r l y , a f a l l i n temperature from 20°C to 15°C leads to a 3 f o l d i n c r e a s e i n the Km of the e l e c t r i c e e l enzyme. T h i s decrease i n enzyme-substrate a f f i n i t y i s r e f l e c t e d i n a r e d u c t i o n o f the r a t e s o f ACh h y d r o l y s i s a t low s u b s t r a t e c o n c e n t r a t i o n a t temperatures below the minimum Km p o i n t (Table 4 ) . For Trematomus b o r c h g r e v i n k i , the amount of enzyme a v a i l a b l e was s u f f i c i e n t o n l y f o r Km determinations a t two temperatures. Lineweaver-Burk p l o t s o b t a i n e d a t 2°C and 10°C w i t h the crude b r a i n homogenate are presented i n F i g u r e 9. The e f f e c t o f assay temperature on Km, and on r e a c t i o n r a t e s a t s e v e r a l s u b s t r a t e c o n c e n t r a t i o n s , are g i v e n i n Table 5. As wi t h the t r o u t and e l e c t r i c e e l AChEs, the a f f i n i t y o f the Trematomus enzyme f o r ACh changes markedly w i t h temperature. The r e a c t i o n r a t e s g i v e n i n Table 5 show t h a t a t low s u b s t r a t e -4 c o n c e n t r a t i o n s (below 5 x 10 M ACh) there i s c o n s i d e r a b l e overcompensation f o r r e a c t i o n r a t e . For example, a t 1.5 x 1 0 - 4 M ACh the r a t e o f ACh h y d r o l y s i s a t 10°C i s o n l y 52 percent o f the r a t e a t 2°C. Again, t h i s ; e f f e c t i s of l i t t l e importance i n v i v o as Trematomus b o r c h g r e v i n k i does not i n h a b i t waters above 2°C, and d i e s w i t h i n 81 minutes a t 10°C (Somero and DeVries, 1967). 43 Figure 9. E f f e c t of Assay Temperature on the Km of ACh for Trematomus borchgrevinki AChE. Lineweaver-Burk Plots at 2°C (M ) and 10°C ( © ) . Standard assay i n 1 0 - 2 M t r i s - H C l buffer, pH 7 . 2 . Table 5. E f f e c t o f Temperature upon the Km and Rate o f ACh H y d r o l y s i s f o r B r a i n AChE from Trematomus  b o r c h g r e v i n k i Parameter Re a c t i o n r a t e ^nM ACh hydrolysed/ml/hr) 2°C 10°C ACh (M) 10" 3 46 91 5 x 10" 4 49 50 2.5 x 10" 4 39 26* -4 a a 1.5 x 10 31 16 d Km 1.5 x 10~ 4 4 x 10~ 3 a. Values o b t a i n e d by e x t r a p o l a t i o n from Lineweaver-Burk p l o t s . 4 5 C l e a r l y , more data must be o b t a i n e d b e f o r e the s i g n i f i c a n c e of Km-temperature r e l a t i o n s h i p s can be a s s e s s e d f o r AChE from such a remarkably stenothermal s p e c i e s . The r e l a t i o n s h i p s between change i n Km and r e a c t i o n r a t e , expressed as Q--, over temperature ranges above the temperature of minimum Km f o r the rainbow t r o u t , e l e c t r i c e e l and Trematomus AChEs are g i v e n i n Table 6. For the t r o u t and e l e c t r i c e e l AChEs a 2 to 3 f o l d change i n Km over 10°C w i l l m a i n t a i n an approximately c o n s t a n t r a t e of ACh h y d r o l y s i s a t s u b s t r a t e c o n c e n t r a t i o n s approaching the minimum Km v a l u e . c. R e l a t i o n s h i p between Thermally Induced Changes i n Km and S t r u c t u r a l Conformation of E l e c t r i c E e l A c e t y l c h o l i n e s t e r a s e Main (1969) has presented evidence i n d i c a t i n g t h a t the k i n e t i c p r o p e r t i e s of bovine e r y t h r o c y t e AChE are dependent upon t h e r m a l l y induced changes i n m o l e c u l a r a g g r e g a t i o n of the s o l u b i l i z e d enzyme. I f the Km-temperature r e l a t i o n s h i p s observed w i t h the s o l u b i l i z e d f i s h AChEs are dependent upon such a g g r e g a t i o n i t i s d i f f i c u l t to see how the Km-temperature e f f e c t c o u l d occur in v i v o where the enzyme forms a s t r u c t u r a l component of the n e u r a l membrane. Thus, the p o s s i b i l i t y e x i s t s t h a t the Km-temperature r e l a t i o n s h i p i s simply an a r t i f a c t o f the ir i v i t r o assay system. To examine t h i s q u e s t i o n , the e f f e c t of temperature upon the m o l e c u l a r conformation o f e l e c t r i c e e l AChE over the range where Km i s markedly a f f e c t e d by temperature was i n v e s t i g a t e d by sucrose g r a d i e n t c e n t r i f u g a t i o n and u l t r a v i o l e t d i f f e r e n c e s p e c t r o s c o p y . 46 Table 6. R e l a t i o n s h i p between Km Change and Q 1 Q o f the Rate of ACh H y d r o l y s i s a t Conce n t r a t i o n s o f ACh Approaching the Minimum Km f o r AChEs from Rainbow Trout, E l e c t r i c E e l and Trematomus b o r c h g r e v i n k i AChE source ACh (M) Temperature Q Km r a n g e ( C ) 10 2 ° c a c c l i m a t e d t r o u t 2.5 x 1 0 - 4 2 - 1 2 0.9 2.2 17°C a c c l i m a t e d . t r o u t 2.5 x 10 1 7 - 2 7 1.2 2.4 E l e c t r i c e e l 1 0 - 4 25 - 35 1.1 3.0 Trematomus borchcrrevinki 1.5 x 10" 4 2 - 10 0.4 26.8 Q-Lo v a l u e s were c a l c u l a t e d from the r e l a t i o n s h i p Q_0 = ( v l / v 2 ) l 0 / / t l t 2 where V1 and V 2 are r e a c t i o n r a t e s a t temperatures t-^ and t 2 r e s p e c t i v e l y 47 AChE from the e l e c t r i c organ of the e e l E l e c t r o p h o r u s  e l e c t r i c u s has a m o l e c u l a r weight of 260,000 and possess a s u b u n i t s t r u c t u r e of the 2tl2(± type (Leuzinger e_t .al, 1969) . Studies by Changeux (19 66) and G r a f i u s e t a l . (19 68) have shown t h a t the s e d i m e n t a t i o n behaviour of e l e c t r i c organ AChEs i s dependent upon the i o n i c environment, p o l y d i s p e r s i t y of the system i n c r e a s i n g a t i o n i c s t r e n g t h s below t h a t of 2 x 10 % MgClj. Sucrose g r a d i e n t c e n t r i f u g a t i o n p a t t e r n s o b t a i n e d a t 15°, 2 5° and 33°C f o r e l e c t r i c e e l AChE i n the presence of 2 x 10 M magnesium c h l o r i d e show a s i n g l e major component a c c o u n t i n g f o r the m a j o r i t y o f the t o t a l a c t i v i t y , w i t h some p o l y d i s p e r s i t y about t h i s f r a c t i o n (Figure 10). As the Km of AChE i s known to be dependent upon the i o n i c environment (Changeux, 1966) Km determinations were made O n o i n the c e n t r i f u g a t i o n medium a t 15 , 2 5 u and 33 C. The Lineweaver-Burk p l o t s i n F i g u r e 11 i n d i c a t e a s i g n i f i c a n t change i n the Km o f the enzyme w i t h temperature under these c o n d i t i o n s . In sucrose g r a d i e n t s t u d i e s of t h i s type s e d i m e n t a t i o n c o e f f i c i e n t s of p r o t e i n s are g e n e r a l l y determined by running known standards w i t h the t e s t sample. The method c o u l d not be used i n t h i s study as s e d i m e n t a t i o n behaviour of both the t e s t sample and the s t a n d a r d might be expected to change w i t h temperature. However, s i n c e the s e d i m e n t a t i o n c o e f f i c i e n t o f a p r o t e i n i s d i r e c t l y p r o p o r t i o n a l to the v i s c o s i t y o f the c e n t r i f u g a t i o n medium (see M a r t i n and Ames, 1961), and the v i s c o s i t y o f the sucrose s o l u t i o n i s e s s e n t i a l l y a l i n e a r f u n c t i o n of temperature over the temperature range i n v e s t i g a t e d , 48 Figure 10. Sucrose Gradient Sedimentation P r o f i l e s of E l e c t r i c Eel AChE at 15°C ( • ),. 25° C ( © ) and 33°C ( A ) . Experimental conditions are given i n the methods section. A c t i v i t i e s are expressed r e l a t i v e to the f r a c t i o n with the highest a c t i v i t y i n each experiment. I 1 1 1 I - I 0 5 10 15 20 25 30 Fraction Number TOP E f f e c t of Temperature on the Km of ACh for E l e c t r i c Eel AChE Assayed i n the Centrif ugation Medium.' Linweaver-Burk Plots at 15°C (II), 25°C ( © ) and 33°C ( A ) . i t was argued t h a t i f the s e d i m e n t a t i o n c o e f f i c i e n t of the p r o t e i n does not change s i g n i f i c a n t l y w i t h temperature, p l o t s o f sedimentation, d i s t a n c e versus temperature, o r v i s c o s i t y , s h o u l d y i e l d a s t r a i g h t l i n e f o r a standard s e t of experimental c o n d i t i o n s . Marked changes i n p r o t e i n conformation or m o l e c u l a r a g g r e g a t i o n w i t h temperature would be expected to a l t e r the s e d i m e n t a t i o n behaviour o f the enzyme and, u n l e s s the s e d i m e n t a t i o n c o e f f i c i e n t changed i n a l i n e a r f a s h i o n w i t h temperature, p l o t s of sedimentation d i s t a n c e a g a i n s t temperature would be n o n - l i n e a r . The p l o t of s e d i m e n t a t i o n d i s t a n c e (as a f u n c t i o n o f f r a c t i o n number) a g a i n s t temperature f o r the e l e c t r i c e e l AChE i s shown i n F i g u r e 12, and i s c l e a r l y l i n e a r . One i s l e f t to conclude t h a t e i t h e r , (a) no s t r u c t u r a l change l a r g e enough to be d e t e c t e d by the method has o c c u r r e d or, (b) changes may have o c c u r r e d i n such a way t h a t the sedimentation c o e f f i c i e n t of the enzyme a l t e r e d i n a l i n e a r f a s h i o n w i t h temperature over a temperature range where the Km temperature r e l a t i o n s h i p shows a sharp change. Thus, although the Km changes may be accompanied by s m a l l c o n f o r m a t i o n a l changes i n the enzyme, the l a r g e Km d i f f e r e n c e between 15° and 25°C cannot be a t t r i b u t e d to such major changes as a l t e r a t i o n s i n m o l e c u l a r a g g r e g a t i o n or d i s s o c i a t i o n of s u b u n i t s . S i m i l a r c o n c l u s i o n s can be drawn from the u l t r a v i o l e t d i f f e r e n c e s p e c t r a p l o t t e d i n F i g u r e 13. Although changes presumably r e s u l t i n g from c o n f o r m a t i o n a l a l t e r a t i o n s i n the enzyme occur i n the u l t r a v i o l e t s p e c t r a w i t h changing temperature, the a c t u a l absorbance changes are s m a l l i n comparison to those accompanying such events as p r o t e i n d e n a t u r a t i o n . 51 Fi g u r e 12. E f f e c t of Temperature upon the Sedimentation Behaviour of E l e c t r i c E e l AChE. Each p o i n t r e p r e s e n t s the mean of three d e t e r m i n a t i o n s . The e r r o r bars i n d i c a t e the range about t h i s mean, and are i n the order of - 0.5 f r a c t i o n s (approximately t 0.08 m l ) . The f r a c t i o n number i s expressed as a f u n c t i o n of t o t a l f r a c t i o n number to compensate f o r v a r i a t i o n i n the number of f r a c t i o n s c o l l e c t e d i n each group of experiments (27 to 32 f r a c t i o n s ) . Experimental c o n d i t i o n s are gi v e n i n the methods s e c t i o n . in _ l I 1 — K o o o o s u o i p o j j JO * O N p + o i / 0 | S | U O | p D J j 52 F i g u r e 13. U l t r a v i o l e t D i f f e r e n c e Spectra of E l e c t r i c E e l AChE as a F u n c t i o n o f Temperature. Spectra are p l o t t e d r e l a t i v e to the spectrum o b t a i n e d a t 2 5°C. Wavelength (mu) 53 5. Thermal Accorniaodation, Thermal A c c l i m a t i o n , and E v o l u t i o n a r y  A d a p t a t i o n to Temperature f o r A c e t y l c h o l i n e s t e r a s e from  the Nervous System of F i s h a. Thermal Accommodation The b a s i s f o r the o b s e r v a t i o n t h a t a t low and probably-p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s the enzyme r e a c t i o n may remain r e l a t i v e l y u n a f f e c t e d by temperature over a p a r t i c u l a r temperature range appears to l i e i n the Km-temperature r e l a t i o n s h i p s shown i n Fi g u r e 14. For the rainbow t r o u t and e l e c t r i c e e l AChE's, a 2 to 3 f o l d change i n Km over 10°C a t temperatures above the minimum Km p o i n t w i l l m a i n t a i n an approximately c o n s t a n t r a t e of ACh h y d r o l y s i s (Table 6). With the Trematomus enzyme i n s u f f i c i e n t data were a v a i l a b l e to determine i f r a t e accommodation and t h e r m a l l y induced Km changes occur over temperature ranges normally encountered by the animal. The minimum temperature f o r thermal accommodation of r e a c t i o n r a t e i s s e t by the temperature a t the minimum Km. A t temperatures below t h i s p o i n t the r e a c t i o n r a t e f a l l s r a p i d l y (Table 4) as both the enzyme-substrate a f f i n i t y and the thermal energy of the system decrease. In the t r o u t AChE system the r e a c t i o n r a t e a t low s u b s t r a t e c o n c e n t r a t i o n s i s maintained a t a r e l a t i v e l y c o n s t a n t l e v e l to temperatures beyond the upper thermal t o l e r a n c e (about 2 5°C) o f the animal. Thus, i f the c e n t r a l nervous system i s the l i m i t i n g system i n s e t t i n g the temperature range of the organism, the r a t e of ACh h y d r o l y s i s by AChE i s probably not an important f a c t o r i n s e t t i n g the upper thermal l i m i t . 54 Figure 14 . E f f e c t of Assay Temperature on the Km of ACh for AChEs from Rainbow Trout, E l e c t r i c Eel and Trematomus borchgrevinki. E3 2°C acclimated trout © 17°C acclimated trout A e l e c t r i c e e l O Trematomus borchgrevinki Temperature (°C) 55 I t i s p o s s i b l e , however, t h a t enzyme c h a r a c t e r i s t i c s o t h e r than r e a c t i o n r a t e are a f f e c t e d by the t h e r m a l l y induced Km changes, and t h a t these f a c t o r s are i n v o l v e d i n s e t t i n g thermal l i m i t s f o r the enzyme system i n v i v o . One mechanism which may be important i n t h i s r e s p e c t i s the a b i l i t y o f s m a l l f l u c t u a t i o n s i n s u b s t r a t e c o n c e n t r a t i o n to r e g u l a t e enzyme a c t i v i t y . The e f f e c t of t h e r m a l l y induced Km changes upon t h i s form of enzyme r e g u l a t i o n i s demonstrated i n the s u b s t r a t e s a t u r a t i o n p l o t s f o r e l e c t r i c e e l AChE shown i n F i g u r e 15. At 2 5 0C, the temperature a t which the Km reaches a minimum v a l u e , s m a l l f l u c t u a t i o n s i n AChE c o n c e n t r a t i o n about t h i s minimum Km value (0.98 x 10~ 4) can g r e a t l y a f f e c t the r a t e o f enzyme a c t i v i t y , a c t i n g e s s e n t i a l l y as an a l l - o r - n o t h i n g s w i t c h . As the Km value i n c r e a s e s a t temperatures above and below 25°C, much g r e a t e r changes i n ACh c o n c e n t r a t i o n are r e q u i r e d to achieve the same e f f e c t . A s i m i l a r s i t u a t i o n i s observed w i t h the Trematomus and rainbow t r o u t AChEs (Figures 16 and 17). Thus, as the Km o f the enzyme i n c r e a s e s a t temperatures above the minimum Km po i n t , the advantages to be o b t a i n e d from r a t e s t a b i l i z a t i o n may be o f f s e t by l o s s o f c o n t r o l over the r e a c t i o n . A t temperatures below the minimum Km p o i n t , i n c r e a s i n g Km i s accompanied by a decrease i n both r e a c t i o n r a t e and c o n t r o l over the r e a c t i o n . i f t h i s form of c o n t r o l i s important i n the AChE system, then both enzyme r e g u l a t i o n and r a t e accommodation may be important i n s e t t i n g thermal l i m i t s f o r the enzyme system j_n v i v o . As d i s c u s s e d p r e v i o u s l y , many i n v e s t i g a t o r s have attempted to c o r r e l a t e the upper thermal t o l e r a n c e or organisms w i t h the t h e r m o s t a b i l i t i e s o f enzymes 56 Figure 15. ACh Saturation Curves of E l e c t r i c Eel ACh at 15° (<§), 25° ( B ) , and 40°C ( A ) . Relative Activity 57 Figure 16. ACh Saturation Curves of Trematomus borchgrevinki AChE at 2° ( H ) and 10°C ( ® ). 100 ACh] (mM) 5 8 F i g u r e 17. ACh S a t u r a t i o n Curves of 2°C a c c l i m a t e d Rainbow Trout AChE a t 0° ( EH ) , 2° ( © ), 12° ( A ) and 18°C ( O ). 8 0 59 from p o s s i b l y l i m i t i n g p h y s i o l o g i c a l systems. The Km-temperature r e l a t i o n s h i p s observed w i t h the f i s h AChE's provide an a l t e r n a t e mechanism by which enzymes may be t h e r m a l l y i n a c t i v a t e d i n v i v o i n the absence of p r o t e i n d e n a t u r a t i o n . b. Thermal A c c l i m a t i o n ( i ) Adjustment of the thermal accommodation range In the t r o u t b r a i n AChE system adjustment of the thermal accommodation range f o l l o w i n g thermal a c c l i m a t i o n i s a c h i e v e d by r e g u l a t i n g the r e l a t i v e p r o p o r t i o n s of the two enzyme types i n response to changing environmental temperature. I t i s proposed t h a t when the environmental temperature i s a l t e r e d to a range where one form of the enzyme can no longer t h e r m a l l y accommodate f o r r e a c t i o n r a t e , or where r e g u l a t i o n of c a t a l y t i c a c t i v i t y i s l o s t , a second form i s produced f o r which the Km-temperature r e l a t i o n s h i p i s b e t t e r s u i t e d f o r c o n t r o l of these f u n c t i o n s . The two enzymes occur s i n g l y a t thermal extremes (2° and 17°C) and together a t i n t e r m e d i a t e temperatures where the r e l a t i v e amounts of each enzyme v a r i e s w i t h the a c c l i m a t i o n temperature. The p r o d u c t i o n of enzyme v a r i a n t s w i t h a l t e r e d and a p p a r e n t l y a d a p t i v e changes i n the Km-temperature curve f o l l o w i n g changes i n environmental temperature has a l s o been observed w i t h t r o u t pyruvate kinases (Somero and Hochachka, 1968), l a c t a t e dehydrogenases (Hochachka and Somero, 1968), i s o c i t r a t e dehydrogenases (Moon, p e r s o n a l communeiation) and c i t r a t e synthases (Hochachka and Lewis, 1970). 60 ( i i ) Rate compensation o f AChE a c t i v i t y An i n t e r e s t i n g f e a t u r e o f the two rainbow t r o u t AChE enzymes i s t h a t the minimum Km va l u e s are s i m i l a r although they occur a t w i d e l y d i f f e r e n t temperatures. Thus, a t low and presumably p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s the r e a c t i o n c a t a l y s e d by the 'c o l d ' enzyme a t 2°C w i l l proceed a t a slower r a t e than the r e a c t i o n c a t a l y s e d by an equal amount of the 'warm' enzyme a t 17°C i f the two enzymes have s i m i l a r turnover numbers. There are a number of f a c t o r s which may a c t to r a i s e the r a t e o f ACh h y d r o l y s i s i n the c o l d a c c l i m a t e d s t a t e . Hickman + + e t a l (19 64) observed changes i n b r a i n Na , K and CI l e v e l s o o i n rainbow t r o u t t r a n s f e r r e d from 16 to 6 C. A study o f the e f f e c t s o f s a l t s on b r a i n AChE from t r o u t a c c l i m a t e d to 2°C (Table 7) shows t h a t a t h i g h ACh c o n c e n t r a t i o n s i n c r e a s i n g i o n i c s t r e n g t h leads to a marked i n c r e a s e i n both Km and maximum v e l o c i t y of ACh h y d r o l y s i s . A s i m i l a r r e l a t i o n s h i p has been r e p o r t e d f o r AChE from the e l e c t r i c organ o f Torpedo  marmorata (Changeux, 1966). However, a t lower ACh c o n c e n t r a t i o n s the r a t e o f the r e a c t i o n g e n e r a l l y decreases as i o n i c s t r e n g t h i s i n c r e a s e d . Thus, the r a t e o f ACh h y d r o l y s i s i n v i v o may be modulated by changes i n the i o n i c environment t h a t occur d u r i n g the thermal a c c l i m a t i o n p r o c e s s . An i n c r e a s e i n i n t r a c e l l u l a r and b l o o d pH on lowering environmental temperature (about 0.014 pH u n i t s per °C) has been observed i n s e v e r a l poikolotherms (Rahn, 1965; Reeves and Wilson, 1969). With the 2°C t r o u t AChE, a f a l l i n temperature 61 Table 7. E f f e c t of S a l t s on the Km and Rate of H y d r o l y s i s of ACh by AChE from 2°C A c c l i m a t e d Rainbow Trout S a l t Km x 10" 4 r e l a t i v e a c t i v i t y 3 ACh (M) 2.5 x 10" 4 5 x 10" 4 s a t u r a t -i n g 10" 3M NaCl 3.3 0.36 0.58 0.96 5 x 10" 3M NaCl 4.1 0.35 0.58 1.06 10~ 2M NaCl 7.7 0.31 0.61 1.50 10" 2M KC1 4.9 0.35 0.61 1.20 2 x 10~ 3M MgCl 2 4.4 0.45 0.70 1.33 5 x 10~ 3M MgCl^ 7.0 0.32 0.62 1.42 C o n t r o l 2.7 0.44 0.63 1.00 Standard assay i n 10"" 2 M t r i s - H C l b u f f e r , pH 7.2 ( c o n t r o l ) a t 2°C. a. A c t i v i t i e s are expressed r e l a t i v e to the a c t i v i t y of the c o n t r o l a t s a t u r a t i n g ACh l e v e l s 62 from 17° to 2°C c o u l d r e s u l t i n an i n c r e a s e i n the r a t e of ACh h y d r o l y s i s i f the r e l a t i o n s h i p between pH and a c t i v i t y (Figure 4) h o l d s a t p h y s i o l o g i c a l l e v e l s of s u b s t r a t e . I t was c o n s i d e r e d p o s s i b l e t h a t r a t e compensation might be a c h i e v e d through an i n c r e a s e i n the t o t a l amount of enzyme present i n the c o l d a c c l i m a t i o n s t a t e , as proposed by Baslow and N i g r e l l i (1964) to account f o r r a t e compensation of b r a i n c h o l i n e s t e r a s e a c t i v i t y i n t h e r m a l l y a c c l i m a t e d k i l l i f i s h . However, e s t i m a t i o n s of the s p e c i f i c a c t i v i t y of AChE : from t r o u t a c c l i m a t e d to 2° and 17°C f o r 35 days (Table 8) f a i l e d to show any s i g n i f i c a n t d i f f e r e n c e . On the o t h e r hand, i t can be argued t h a t i t may not be necessary to m a i n t a i n the same r a t e of ACh h y d r o l y s i s i n the warm and c o l d a c c l i m a t e d . s t a t e s because of a l t e r e d ACh c o n c e n t r a t i o n s , or changes i n p r o p e r t i e s of n e u r a l membranes which may a f f e c t c h o l i n e r g i c mechanisms. Although no i n f o r m a t i o n i s a v a i l a b l e on a l t e r e d b r a i n ACh l e v e l s i n f i s h , a number of i n v e s t i g a t o r s have r e p o r t e d s i g n i f i c a n t changes i n membrane s t r u c t u r e f o l l o w i n g thermal a c c l i m a t i o n . Johnston and Roots (1964) found a t r e n d towards i n c r e a s e d u n s a t u r a t i o n of the t o t a l b r a i n f a t t y a c i d s w i t h d e c r e a s i n g a c c l i m a t i o n temperature i n the g o l d f i s h , and i n a l a t e r study (Roots, 1968) t h i s same t r e n d was e s t a b l i s h e d f o r the b r a i n p h o p h o l i p i d s , p a r t i c u l a r l y c h o l i n e and ethanolamine g l y c e r o p h o s p h o t i d e s . These two p h o s p h o l i p i d types account f o r over 60 percent o f the t o t a l l i p i d i n r a t b r a i n s y n a p t i c plasma membranes (Cotman e t a l , 1969). I t i s g e n e r a l l y agreed t h a t 63 Table 8. S p e c i f i c A c t i v i t i e s o f B r a i n AChE from Rainbow Trout A c c l i m a t e d to 2° and 17°C f o r 35 days S p e c i f i c a c t i v i t y 3 A c c l i m a t i o n Number of A s s a y temperature (°C) temperature ( C) f i s h assayed 18 10 2 2 12 10.1 ± 0.1 8.6 ± 0.3 6.2 ± 0.3 17 11 10.1 ± 0.3 8.4 ± 0.1 6.2 ± 0.1 Whole b r a i n homogenates (100 mg brain/ml) were assayed i n 10~ 2 M t r i s - H C l b u f f e r , pH 7.2, w i t h 2 x 10~ 3 M ACh as s u b s t r a t e . a. S p e c i f i c a c t i v i t i e s are expressed asyu-M ACh h y d r o l y z e d per mg p r o t e i n per hour. ± v a l u e s i n d i c a t e the range about the mean. the l i p i d c o n s t i t u e n t s of b i o l o g i c a l membranes p l a y a c r i t i c a l r o l e i n r e g u l a t i o n of i o n i c p e r m e a b i l i t y , although most evidence f o r t h i s has been somewhat i n d i r e c t . For example, W a t l i n g t o n and Harland (1969) have found a c o r r e l a t i o n between the p h o s p h o l i p i d content of f r o g s k i n s and p r o p e r t i e s o f the N a + and C l ~ t r a n s p o r t systems while other workers have reached s i m i l a r c o n c l u s i o n s from s t u d i e s w i t h a r t i f i c i a l membranes (Fast, 1967; Tobias e_t a l , 1962), and from the b i n d i n g of ions to v a r i o u s p h o s p h o l i p i d s p e c i e s (Vanatta, 1969; Papahadjopoulos, 1968). Thus, there i s c o n s i d e r a b l e support f o r the s u g g e s t i o n by Roots (19 68) t h a t changes i n the l i p i d component of n e u r a l membranes d u r i n g thermal a c c l i m a t i o n may be necessary f o r the maintenance o f membrane p e r m e a b i l i t y c h a r a c t e r i s t i c s u n d e r l y i n g nerve co n d u c t i o n . A l t e r a t i o n i n the i o n i c composition i n t i s s u e s from f i s h exposed to r a p i d changes i n temperature may r e f l e c t t h e r m a l l y induced changes i n membrane p e r m e a b i l i t y t h a t can not be compensated f o r by the l i p i d s p e c i e s p r e s e n t . Continued maintenance a t the a l t e r e d temperature g e n e r a l l y leads to a g r a d u a l s t a b i l i z a t i o n of the i o n i c balance over a time course s i m i l a r to t h a t f o l l o w e d by changes i n t i s s u e l i p i d s (Hickman e t a l . 1964) . I t should be s t r e s s e d t h a t p h o s p h o l i p i d s do not occur f r e e i n n e u r a l membranes, but r a t h e r as l i p o p r o t e i n complexes p o s s i b l y i n c o r p o r a t i n g AChE. This may be o f some importance when c o n s i d e r i n g the e f f e c t s of thermal a c c l i m a t i o n upon AChE, as i t has been found i n o t h e r membrane systems t h a t enzyme a c t i v i t y can be a l t e r e d by removal o f the p h o s p h o l i p i d component. For example, phospholipds are e s s e n t i a l f o r r e s p i r a t o r y a c t i v i t y i n mitochondria, ( F l e i s c h e r e t a_l, 1962) and form an a b s o l u t e requirement f o r such m i t o c h o n d r i a l enzymes as .cytochrome oxidase ( T z a g o l o f f and MacLennan, 1965) , f _-hydroxy-butyric dehydrogenase (Sekuzu e t a l , 1963) and c y t i d i n e diphosphocholine t r a n s f e r a s e (Fiscus and Schneider, 1965), and f o r membrane bound Na-K ATPase (Tanaka and S t r i c k l a n d , 1965). Thus the p o s s i b i l i t y e x i s t s t h a t changes i n membrane l i p i d s f o l l o w i n g thermal a c c l i m a t i o n might have some e f f e c t upon AChE a c t i v i t y . With the rainbow t r o u t AChEs, comparisons made between crude homogenates and l i p i d f r e e butanol-acetone e x t r a c t s of b r a i n from warm and c o l d a c c l i m a t e d f i s h f a i l e d to show any changes i n s p e c i f i c a c t i v i t y t h a t c o u l d be a t t r i b u t e d to the l i p i d component. One p o i n t t h a t should not be o v e r l o o k e d i n c o n s i d e r i n g the need f o r r a t e compensation d u r i n g thermal a c c l i m a t i o n i s the r e l a t i o n s h i p between in v i v o enzyme and s u b s t r a t e c o n c e n t r a t i o n s . I f AChE was present i n g r e a t excess i n the warm a c c l i m a t e d s t a t e then a r e d u c t i o n i n enzyme a c t i v i t y f o l l o w i n g a drop i n environmental temperature might have l i t t l e p h y s i o l o g i c a l e f f e c t i f i t was s t i l l p o s s i b l e to m a i n t a i n the r a t e of ACh h y d r o l y s i s above c r i t i c a l l e v e l s w i t h a l a r g e c o n c e n t r a t i o n of c a t a l y t i c a l l y l e s s e f f i c i e n t enzyme. I t i s d i f f i c u l t to determine enzyme and s u b s t r a t e c o n c e n t r a t i o n s w i t h i n such d i s c r e t e systems as mitochondria w i t h any degree of a ccuracy. With b r a i n AChE these d i f f i c u l t i e s are compounded by l a c k of p r e c i s e knowledge as to the a c t i o n of the enzyme i n the c e n t r a l nervous system. I f i t i s assumed 66 t h a t c h o l i n e r g i c mechanisms i n the c e n t r a l nervous system and neuromuscular j u n c t i o n are e s s e n t i a l l y s i m i l a r , some estimate can be made o f the l e v e l a t which ACh h y d r o l y s i s must be main-t a i n e d f o r the t r a n s m i s s i o n of nerve impulses. Namba and Grob (1969) have c a l c u l a t e d t h a t the p o s t s y n a p t i c membrane from r a t g i n t e r c o s t a l muscle c o n t a i n s 10 molecules o f AChE, and t h a t one _g synapse h y d r o l y s e s 5 x 10 molecules of ACh per m i l l i s e c o n d . 7 They estimate t h a t 10 molecules o f ACh are r e l e a s e d i n t o the s y n a p t i c space per a c t i o n p o t e n t i a l , a q u a n t i t y of s u b s t r a t e which c o u l d be h y d r o l y s e d w i t h i n 0.02 m i l l i s e c o n d s by the e s t i m a t e d a v a i l a b l e AChE. I f the time taken f o r ACh to c r o s s the s y n a p t i c space, and the d u r a t i o n o f t r a n s m i t t e r a c t i o n a t the post s y n a p t i c membrane were known, i t would be p o s s i b l e to make some 7 estimate of the amount of AChE r e q u i r e d to h y d r o l y s e 10 molecules of ACh w i t h i n the time a v a i l a b l e . E c c l e s (1957) s t a t e s t h a t the l a t e n c y p e r i o d of s y n a p t i c t r a n s m i s s i o n as measured from the time of a r r i v a l o f a p r e s y n a p t i c impulse to the time o f post-s y n a p t i c response i s g e n e r a l l y i n ' t h e order of 0.3 to 0.5 m i l l i -seconds. However, the d u r a t i o n of t r a n s m i t t e r a c t i o n i s c o n s i d e r a b l y longer, i n the o r d e r o f 50 m i l l i s e c o n d s f o r the ACh mediated t r a n s m i s s i o n a t the Renshaw c e l l synapse. C a l c u l a t i o n s based upon the r a t e of f r e e d i f f u s i o n of ACh i n d i c a t e d t h a t ACh l e v e l s should become n e g l i g i b l e a t the p o s t -s y n a p t i c membrane w i t h i n one m i l l i s e c o n d o f r e l e a s e , and i t was concluded from t h i s t h a t some b a r r i e r to f r e e d i f f u s i o n e x i s t s i n the s y n a p t i c space ( E c c l e s , 1957). Using 0.5 to 50 m i l l i -seconds as extreme valu e s f o r the time a v a i l a b l e f o r ACh h y d r o l y s i s , one a r r i v e s a t an e s t i m a t e d 2 5 to 2 500 f o l d f u n c t i o n a l excess o f AChE. C l e a r l y t h i s i s a rough c a l c u l a t i o n a t best, but i t does i n d i c a t e t h a t p o s t s y n a p t i c AChE may never 67 be s a t u r a t e d w i t h s u b s t r a t e but r a t h e r , i s exposed to ACh c o n c e n t r a t i o n s a t or below the Km of the enzyme. S i m i l a r c o n c l u s i o n s have been a r r i v e d a t f o r m i t o c h o n d r i a l enzyme systems (Srere, 1968; Vegotsky and F r i e d e n , 1958). These estimates serve to u n d e r l i n e the importance of enzyme-substrate a f f i n i t i e s i n the r e g u l a t i o n of enzyme a c t i v i t i e s i n v i v o . There i s some experimental data which c o u l d be i n t e r p r e t e d as evidence of a f u n c t i o n a l excess of AChE i n the c e n t r a l nervous system. For example, Glow and Rose (1966) have shown i n b i o a s s a y s of r a t b r a i n t h a t ACh l e v e l s do not i n c r e a s e s i g n i f i c a n t l y u n t i l AChE a c t i v i t y has been reduced below 40 to 50 percent of normal v a l u e s . A sharp drop i n neve cond u c t i o n a l s o occurs a t t h i s l e v e l of AChE i n h i b i t i o n (Wilson and Cohen, 1963). I f there i s a f u n c t i o n a l excess of AChE i n the t r o u t c e n t r a l nervous system there would seem l i t t l e p o i n t i n s t a b i l i z i n g r e a c t i o n r a t e s d u r i n g temperature f l u c t u a t i o n s . However, the adaptive advantages i n h e r e n t i n the r e l a t i o n s h i p s between h a b i t a t temperature, minimum Km v a l u e s , probably p h y s i o l o g i c a l ACh c o n c e n t r a t i o n s , t h e r m a l l y induced Km changes and s t a b i l i z a t i o n of r e a c t i o n r a t e s f o r the t r o u t AChEs, seem too c o m p e l l i n g to be d i s m i s s e d as simply c o i n c i d e n t a l . From the data a t hand, i t i s proposed t h a t while thermal accommodation of r e a c t i o n r a t e i s necessary i n both the c o l d and warm a c c l i m a t i o n s t a t e s i t may not be necessary to m a i n t a i n the same r a t e s of ACh h y d r o l y s i s a t d i f f e r e n t a c c l i m a t i o n temperatures. In t h i s s i t u a t i o n , s e l e c t i o n f o r a p a r t i c u l a r minimum Km value may be determined by i n v i v o s u b s t r a t e 68 c o n c e n t r a t i o n s , f o r i t i s o n l y when Km v a l u e s are a t or below p h y s i o l o g i c a l s u b s t r a t e l e v e l s t h a t s m a l l f l u c t u a t i o n s i n s u b s t r a t e c o n c e n t r a t i o n can e f f e c t i v e l y r e g u l a t e enzyme a c t i v i t y . c. E v o l u t i o n a r y a d a p t a t i o n to temperature (i ) Adjustment o f the thermal accommodation range The a d a p t a t i o n of AChE f u n c t i o n to temperature i n s p e c i e s i n h a b i t i n g d i f f e r e n t thermal environments appears to be based upon s e l e c t i o n f o r a Km-temperature r e l a t i o n s h i p t h a t w i l l a l l o w thermal accommodation f o r r e a c t i o n r a t e over the temperature range normally e x p e r i e n c e d by the s p e c i e s . As suggested f o r the t r o u t enzymes, s e l e c t i o n of a p a r t i c u l a r range of Km v a l u e s may be determined p r i m a r i l y by p h y s i o l o g i c a l ACh c o n c e n t r a t i o n . I f the Km-temperature r e l a t i o n s h i p i s dependent upon t h e r m a l l y induced c o n f o r m a t i o n a l changes i n the enzyme molecule, s h i f t s i n the Km-temperature curve d u r i n g s p e c i a t i o n can be r e a d i l y i n t e r p r e t e d i n terms o f changes i n conformation r e s u l t i n g from the gradual accumulation of amino a c i d r e p l a c e -ments. C l e a r l y i t would be an advantage a t t h i s p o i n t to present amino a c i d sequence data to support the view t h a t the t r o u t and e l e c t r i c e e l AChEs are i n f a c t homologous enzymes which have d i v e r g e d from a common a n c e s t r a l gene. In the absence of such i n f o r m a t i o n , however, the b e s t t h a t can be done i s to l i s t s i m i l a r i t i e s between the AChEs which are a t l e a s t compatible w i t h t h i s h y p o t h e s i s of a common o r i g i n . 1. Both the t r o u t and e l e c t r i c e e l AChEs have mol e c u l a r weights of about 260,000, and are probably s i m i l a r i n net charge and c o n f i g u r a t i o n as judged from e l e c t r o p h o r e s i s on acrylamide g e l s . While by no means c o n c l u s i v e , t h i s data i s c o n s i s t e n t w i t h the p r o p o s i t i o n t h a t both the t r o u t and e e l enzymes have s i m i l a r s u b u n i t s t r u c t u r e s . 2. The AChEs d i s p l a y s i m i l a r s u b s t r a t e and i n h i b i t o r s p e c i f i c i t i e s and p H - a c t i v i t y r e l a t i o n s h i p s . The curved A r r h e n i u s p l o t s f o r the enzymes have e s s e n t i a l l y the same form, approaching v e r y low e n e r g i e s of a c t i v a t i o n a t h i g h e r temperatures. I t can be argued from these p r o p e r t i e s t h a t both the a c t i v e s i t e s and the mechanisms of enzyme h y d r o l y s i s are probably s i m i l a r i n the t r o u t and e e l AChEs. ( i i ) E v o l u t i o n o f the rainbow t r o u t b r a i n AChE complex While the accumulation o f amino a c i d s u b s t i t u t i o n s w i l l account f o r changes i n the p r o p e r t i e s of a s i n g l e enzyme, f u r t h e r mechanisms must be proposed to e x p l a i n the presence of the two AChE v a r i a n t s i n the t r o u t CNS. One e x p l a n a t i o n i s t h a t the two t r o u t enzymes arose by gene d u p l i c a t i o n , and t h a t they subsequently d i v e r g e d through amino a c i d replacements. S e l e c t i o n of d i f f e r e n t Km-temperature r e l a t i o n s h i p s f o r the two enzymes would be based upon advantages a c c r u i n g to i n d i v i d u a l s who c o u l d extend t h e i r thermal ranges. Gene d u p l i c a t i o n , f o l l o w e d by the independent e v o l u t i o n a r y f a t e s of the d u p l i c a t e s , has been i m p l i c a t e d i n the o r i g i n a l divergence of many s t r u c t u r a l l y homologous p r o t e i n s (Smithies e t a l , 1962; Ingram, 1963; Rutter, 1964; Cohen and M i l s t e i n , 1967; Augustinsson, 1968; Watts, 1968; Watts and Watts, 1969). In salmonids a t l e a s t f o u r d u p l i c a t e d g e n e t i c l o c i are known; A and B l a c t a t e dehydrogenases (Holmes and Markert, 1969; Massaro and Markert, 1968), enolase (Cory and Wold, 1966), and supernatant malate dehydrogenase ( B a i l e y e t a l , 1969), and r e c e n t l y m u l t i p l e forms of s e v e r a l enzymes i n a d d i t i o n to AChE have been found i n the rainbow t r o u t (Hochachka and Somero, 1968; Somero and Hochachka, 1968; Somero, 1969; Hochachka and Lewis, 1970). In f a c t , i t appears l i k e l y t h a t d u p l i c a t i o n of the e n t i r e genome has o c c u r r e d i n these f i s h as salmonids possess about twice as much DNA per nucleus and approximately twice as many chromosomes as other f i s h i n the same order (Ohno and A t k i n , 1966; Ohno e t a l , 1968; Hinegardner, 1969). Thus there i s c o n s i d e r a b l e support f o r the view t h a t the two t r o u t AChEs arose f o l l o w i n g d u p l i c a t i o n of an a n c e s t r a l gene. An a l t e r n a t i v e h y p o t h e s i s i s t h a t the two t r o u t AChEs may have d i v e r g e d from a s i n g l e m o l e c u l a r type i n the absence of gene d u p l i c a t i o n , f o l l o w i n g the r e p r o d u c t i v e i s o l a t i o n of two p o p u l a t i o n s t h a t were exposed to d i f f e r e n t thermal regimes. I n t e r b r e e d i n g between the p o p u l a t i o n s a t a l a t e r time might have l e d to the presence o f the two d i v e r g e n t enzymes w i t h i n the one i n d i v i d u a l . I f c o n d i t i o n s were such as to favour t h i s genotype i t might have been e s t a b l i s h e d i n the p o p u l a t i o n , e v e n t u a l l y becoming the dominant form. Massaro and Markert (1968) have i n f a c t suggested t h a t the t e t r a p l o i d salmonids may have a r i s e n from h y b r i d s formed between a n c e s t r a l d i p l o i d s p e c i e s t h a t possessed d i f f e r e n t a l l e l e s f o r a number of enzymes. There i s experimental evidence to support the view t h a t h y b r i d i z a t i o n among t r o u t can l e a d to the i n c o r p o r a t i o n of enzyme v a r i a n t s from both parents w i t h i n the h y b r i d . For example, Bouck and B a l l (1968) found t h a t s p e c k l e d trout-brown t r o u t F, h y b r i d s possessed a t l e a s t 27 e l e c t r o p h o r e t i c a l l y 71 d i s t i n c t l a c t a t e dehydrogenases, w h i l s t o n l y 15 c o u l d be det e c t e d i n each of the p a r e n t a l s t o c k s . Goldberg has d e s c r i b e d a s i m i l a r s i t u a t i o n w i t h splake, the s p e c k l e d t r o u t -lake t r o u t h y b r i d , and was able to show t h a t the l a c t a t e dehydrogenase p r o f i l e i n splake was maintained through subsequent genera t i o n s (Goldberg, 1966; Goldberg e t a l , 1967). An i n v e s t i g a t i o n of temperature t o l e r a n c e i n t r o u t h y b r i d s i s b e i n g conducted by Ihssen a t the U n i v e r s i t y of Toronto (Ihssen, p e r s o n a l communication). Speckled t r o u t and lake t r o u t have d i f f e r e n t upper thermal t o l e r a n c e s . The h y b r i d , splake, has a temperature r o l e r a n c e l y i n g between the two p a r e n t a l s p e c i e s , and a range of thermal t o l e r a n c e s have been found i n c r o s s e s between d i f f e r e n t generations o f speckled, lake and splake t r o u t b y b r i d s . I t was c o n s i d e r e d of p a r t i c u l a r i n t e r e s t with r e p s e c t to the e v o l u t i o n of the rainbow t r o u t AChE system to study the i n h e r i t a n c e o f AChEs i n these f i s h . The r e s u l t s o b t a i n e d f o r acrylamide g e l d i s c e l e c t r o p h o r e s i s of b r a i n homogenates from groups of s p e c k l e d t r o u t , lake t r o u t and splake P h y b r i d s a c c l i m a t e d to 4 ° , 9° and 20°C are shown i n F i g u r e 1 8 , and can be summarised as f o l l o w s : A f t e r a c c l i m a t i o n to 4 ° c s p e c k l e d and lake t r o u t each show one AChE band, whereas splake has one major AChE band and two minor bands. F o l l o w i n g a c c l i m a t i o n to 9°C, s p e c k l e d and lake t r o u t each give two AChE bands, while splake has two major bands and two minor bands. A f t e r 14 days a t 20°C, s p e c k l e d t r o u t show a s i n g l e AChE band, w h i l e splake has one major band and one minor band o f AChE a c t i v i t y . U n f o r t u n a t e l y , 20°C lake 72 F i g u r e 18. R e s o l u t i o n of B r a i n AChEs from Speckled Trout, Lake Trout and Splake by Acrylamide Gel D i s c E l e c t r o p h o r e s i s . S p e c i f i c AChE bands were i d e n t i f i e d by i n h i b i t i o n w i t h 284C51 and e s e r i n e . The diagram shows o n l y the number o f bands r e s o l v e d and does not i n d i c a t e the r e l a t i v e m i g r a t i o n r a t e s of the f r a c t i o n s , a p r o p e r t y which can o n l y be a c c u r a t e l y determined by running mixtures w i t h i n the one g e l . The e l e c t r o p h o r e t i c c o n d i t i o n s and s t a i n i n g technique are g i v e n i n the methods s e c t i o n . A c c l i m a t i o n p e r i o d s were as f o l l o w s : 4°C f i s h f o r 6 weeks, 9°C f i s h f o r 6 weeks, and 20°C f i s h f o r 2 weeks. 4 C acclimated trout speckled lake splake 9°C acclimated trout speckled lake splake 2 0 ° C acclimated trout speckled splake 73 t r o u t b r a i n s were not o b t a i n e d . As o n l y a s m a l l number of f i s h were a v a i l a b l e i t was not p o s s i b l e to compare the e l e c t r o p h o r e t i c m o b i l i t i e s of the d i f f e r e n t AChEs by running mixtures of b r a i n homogenates, or to k i n e t i c a l l y c h a r a c t e r i z e the enzymes. Thus, i t i s not known i f t h i s i n c r e a s e i n the number of AChE types present i n splake i n v o l v e s a simple summation of the p a r e n t a l types, breakdown of the thermal s w i t c h i n g mechanism, or p o s s i b l y the f o r m a t i o n of h y b r i d molecules c o n t a i n i n g p o l y p e p t i d e chains from each p a r e n t a l AChE. However, i t i s apparent from the r e s u l t s o b t a i n e d t h a t i n t e r b r e e d i n g between t r o u t s p e c i e s which are normally r e p r o d u c t i v e l y i s o l a t e d i n nature can l e a d to an i n c r e a s e i n the number of AChE enzymes present i n the h y b r i d . F u r t h e r , the presence of s i m i l a r t h e r m a l l y i n d u c i b l e AChE systems i n rainbow, s p e c k l e d and lake t r o u t leads one to suggest t h a t the o r i g i n a l i n c o r p o r a t i o n of m u l t i p l e AChE types i n t o one s p e c i e s o c c u r r e d p r i o r to the e v o l u t i o n a r y divergence of these three t r o u t . ( i i i ) R e g u l a t i o n of the composition of the t r o u t b r a i n AChE complex d u r i n g thermal a c c l i m a t i o n While such events as gene d u p l i c a t i o n and h y b r i d i z a t i o n may u n d e r l y the presence of m u l t i p l e forms of AChE w i t h i n one s p e c i e s , the development of some form of t h e r m a l l y c o n t r o l l e d s w i t c h i n g mechanism must be p o s t u l a t e d to e x p l a i n changes i n enzyme p r o f i l e d u r i n g thermal a c c l i m a t i o n of t r o u t . A t present i t i s not known i f these changes r e s u l t from a l t e r e d r a t e s of enzyme s y n t h e s i s , enzyme degredation, o r both, or p o s s i b l y even m o d i f i c a t i o n of the gene products f o l l o w i n g 74 s y n t h e s i s , thus any d i s c u s s i o n of r e g u l a t i o n of the t r o u t AChE complex must be s p e c u l a t i v e . Even so, i t would seem o f value, a t t h i s p o i n t - to o u t l i n e p o s s i b l e c o n t r o l mechanisms i f o n l y to i n d i c a t e important areas f o r f u t u r e i n v e s t i g a t i o n . Although the e f f e c t s of temperature upon t o t a l p r o t e i n s y n t h e s i s have been s t u d i e d i n a v a r i e t y of p o i k i l o t h e r m s (Mews, 19 57; Jankawsky, 1960; Das and Prosser, 1967; Dean and B e r l i n , 1969; Haschemeyer, 1969) and probable t h e r m a l l y r a t e l i m i t i n g steps have been i d e n t i f i e d (Haschemeyer, 1969), i n f o r m a t i o n r e l a t i n g to the r a t e s of s y n t h e s i s and d e g r a d a t i o n of s p e c i f i c p r o t e i n s d u r i n g thermal a c c l i m a t i o n i s c o m pletely l a c k i n g . I f the amounts of each AChE enzyme present a t p a r t i c u l a r a c c l i m a t i o n temperatures i n t r o u t are determined by r e g u l a t i o n of p r o t e i n s y n t h e s i s , one can p o s t u l a t e t h a t changes i n the p r o p e r t i e s of r e g u l a t o r and o p e r a t o r genes may have accompanied the divergence of the s t r u c t u r a l genes, thereby r e g u l a t i n g enzyme s y n t h e s i s a t the l e v e l o f t r a n s c r i p t i o n . R e g u l a t i o n c o u l d a l s o be a c h i e v e d through t h e r m a l l y induced changes a t numerous steps subsequent to messenger RNA p r o d u c t i o n . With r e f e r e n c e to the t r o u t system, Somero (person communication) has found c l e a r d i f f e r e n c e s i n the m e l t i n g p r o f i l e s of ribosomal p r e p a r a t i o n s from the l i v e r s of warm and c o l d a c c l i m a t e d rainbow t r o u t . These d i f f e r e n c e s may r e f l e c t changes i n ribosomal s t r u c t u r e and f u n c t i o n d u r i n g thermal a c c l i m a t i o n . Other p o s s i b i l i t i e s i n c l u d e the modulation of such enzymes as the amino a c y l t r a n s f e r a s e s which appear to be a r a t e l i m i t i n g s t e p 75 i n p r o t e i n s y n t h e s i s f o l l o w i n g the t r a n s f e r o f warm a c c l i m a t e d f i s h to lower temperatures (Haschemeyer, 1969). A l t e r n a t e l y , the a c t i v a t i o n and i n a c t i v a t i o n of preformed p r o t e i n through m o d i f i c a t i o n o f amino a c i d s i d e groups, a d d i t i o n and removal o f amino a c i d s , o r changes i n subuni t composition c o u l d be important methods of r e g u l a t i o n . Thermal s w i t c h i n g o f the 'warm' and 'c o l d ' AChEs i n enzyme mediated mechanisms might be a c h i e v e d d i r e c t l y through Km-temperature r e l a t i o n s h i p s s i m i l a r to those observed w i t h the AChEs, or i n d i r e c t l y by such f a c t o r s as t h e r m a l l y induced changes i n hormone balance. The i n f l u e n c e of hormones i n c o n t r o l l i n g both s y n t h e s i s and a c t i v i t y of enzymes i s w e l l e s t a b l i s h e d (Metzenberg e_t a l , 1961; McKearns, 1963; Varner and Ramchandra, 1964; Kim e t a l , 1966; Oki e t a l , 1966; Tomkins e t a l , 1969). I t i s hoped t h a t these t h e r m a l l y c o n t r o l e d enzyme complexes i n p o i k i l o t h e r m s w i l l p r ovide a v a l u a b l e experimental system f o r s t u d i e s of the r e g u l a t i o n o f gene e x p r e s s i o n i n v e r t e b r a t e s . 76 SUMMARY The e f f e c t s of temperature upon AChE from the nervous system of f i s h were i n v e s t i g a t e d w i t h the o b j e c t of i n t e r p r e t i n g thermal accommodation, thermal a c c l i m a t i o n and e v o l u t i o n a r y a d a p t a t i o n to temperature a t the l e v e l of enzyme f u n c t i o n . A t probable p h y s i o l o g i c a l ACh c o n c e n t r a t i o n s , the r a t e of ACh h y d r o l y s i s by AChE can remain r e l a t i v e l y u n a f f e c t e d by assay temperature throughout a temperature range c o r r e s p o n d i n g to t h a t e x p e r i e n c e d by the animal i n nature. P l o t s o f Km versus temperature f o r AChE enzymes from rainbow t r o u t and e l e c t r i c e e l y i e l d U shaped curves w i t h minimum Km v a l u e s o c c u r r i h g r a t temperatures c l o s e to the h a b i t a t temperatures. Studies u t i l i z i n g sucrose g r a d i e n t c e n t r i f u g a t i o n and u l t r a v i o l e t d i f f e r e n c e s p e c t r a suggest t h a t changes i n Km w i t h temperature probably r e s u l t from s m a l l a l t e r a t i o n s i n enzyme conformation, and t h a t sharp breaks i n the Km-temperature curve are not a s s o c i a t e d w i t h changes i n m o l e c u l a r a g g r e g a t i o n or the d i s s o c i a t i o n of s u b u n i t s . E n ergies of a c t i v a t i o n f o r the t r o u t and e l e c t r i c e e l enzymes decrease w i t h i n c r e a s i n g temperature and show no c l e a r r e l a t i o n s h i p w i t h h a b i t a t temperature. I t i s concluded t h a t the observed thermal accommodation of AChE r e a c t i o n r a t e i s a c h i e v e d through changes i n enzyme s u b s t r a t e a f f i n i t y w i t h temperature. A r r h e n i u s p l o t s of l o g Vdptversus 1/T f o r the rainbow t r o u t and e e l AChEs continue to i n c r e a s e throughout the temperature range e x p e r i e n c e d by the animals, i n d i c a t i n g t h a t 77 thermal d e n a t u r a t i o n i s probably not a f a c t o r i n s e t t i n g upper l i m i t s f o r thermal accommodation. A s i m i l a r c o n c l u s i o n was reached f o r AChE from the A n t a r c t i c f i s h Trematomus b o r c h g r e v i n k i . I t i s proposed t h a t the Km-temperature r e l a t i o n s h i p may be important i n s e t t i n g both upper and lower l i m i t s f o r thermal accommodation of AChE a c t i v i t y through an i n t e r a c t i o n between ra t e s t a b i l i z a t i o n and the maintenance o f enzyme r e g u l a t i o n . Thermal a c c l i m a t i o n of AChE a c t i v i t y i n the rainbow t r o u t , and probably i n s p e c k l e d and lake t r o u t , i s a c h i e v e d by r e g u l a t i n g the r e l a t i v e p r o p o r t i o n s o f two AChE v a r i a n t s d i s p l a y i n g d i f f e r e n t Km-temperature r e l a t i o n s h i p s . When the environmental temperature i s a l t e r e d t o a range where one form of the enzyme can no longer t h e r m a l l y accommodate f o r r e a c t i o n r a t e , or where r e g u l a t i o n of c a t a l y t i c a c t i v i t y i s l o s t , a second form i s produced f o r which the Km-temperature r e l a t i o n s h i p i s b e t t e r s u i t e d f o r c o n t r o l of these f u n c t i o n s . In the case of the rainbow t r o u t , both AChE v a r i a n t s have s i m i l a r minimum Km v a l u e s although these minima occur a t markedly d i f f e r e n t temperatures. Thus a t probable p h y s i o l o g i c a l s u b s t r a t e c o n c e n t r a t i o n s the r e a c t i o n c a t a l y s e d by the 'cold' enzyme a t 2°C w i l l proceed a t a slower r a t e than the r e a c t i o n c a t a l y s e d by the 'warm' enzyme a t 17°C, u n l e s s o t h e r f a c t o r s a s s o c i a t e d w i t h the c o l d a c c l i m a t i o n process a c t to i n c r e a s e enzyme a c t i v i t y . With t h i s i n mind, the e f f e c t s of i o n i c environment, pH and membrane l i p i d s upon AChE a c t i v i t y were c o n s i d e r e d . I t was found t h a t w h ile changes i n both i o n i c environmnet and pH may be of importance, the presence o f membrane l i p i d s i n the enzyme e x t r a c t had no s i g n i f i c a n t e f f e c t upon AChE a c t i v i t y - Determinations of s p e c i f i c a c t i v i t y f o r b r a i n AChE from c o l d (2°C) and warm (17°C) a c c l i m a t e d t r o u t i n d i c a t e d t h a t r a t e compensation i s not a c h i e v e d through a l t e r a t i o n s i n the t o t a l amount of AChE present. I t i s concluded t h a t although thermal accommodation of r e a c t i o n r a t e i s probably necessary i n both the c o l d and warm a c c l i m a t i o n s t a t e s i t may not be necessary to m a i n t a i n the same r a t e s of AChE h y d r o l y s i s a t d i f f e r e n t a c c l i m a t i o n temperatures. The e v o l u t i o n a r y a d a p t a t i o n of AChE f u n c t i o n to temperature i n s p e c i e s i n h a b i t i n g d i f f e r e n t thermal environments appears to be based upon s e l e c t i o n f o r a _Km-temperature r e l a t i o n s h i p t h a t w i l l a l l o w thermal accommodation of r e a c t i o n r a t e over the temperature range normally encountered by the s p e c i e s . S h i f t s i n the Km-temperature curve d u r i n g s p e c i a t i o n are i n t e r p r e t e d i n terms o f changes i n enzyme conformation f o l l o w i n g the gradual accumulation of amino a c i d s u b s t i t u t i o n s . P h y s i c a l and k i n e t i c evidence i s presented which, while not c o n c l u s i v e , i s c o n s i s t e n t w i t h the view t h a t rainbow t r o u t and e l e c t r i c e e l AChEs were d e r i v e d from-, common a n c e s t r a l gene. P o s s i b l e mechanisms by which two AChE enzymes c o u l d be i n c o r p o r a t e d i n t o the t r o u t c e n t r a l nervous system were a l s o c o n s i d e r e d . While there i s c o n s i d e r a b l e evidence i m p l i c a t i n g gene d u p l i c a t i o n i n t h i s process, an a l t e r n a t i v e h y p o t h e s i s i n v o l v i n g h y b r i d i z a t i o n between f i s h p o p u l a t i o n s was a l s o suggested. T h i s theory was examined wi t h t r o u t i n t e r s p e c i e s h y b r i d s , and i t was observed t h a t c r o s s e s between t r o u t s p e c i e s which are normally r e p r o d u c t i v e l y i s o l a t e d c o u l d r e s u l t i n an 79 i n c r e a s e d number of AChE enzymes present i n the h y b r i d . F u r t h e r , the presence of s i m i l a r AChE complexes i n rainbow t r o u t , s p e c k l e d and lake t r o u t i n d i c a t e d t h a t the o r i g i n a l i n c o r p o r a t i o n of m u l t i p l e AChEs i n t o one s p e c i e s probably o c c u r r e d p r i o r to the e v o l u t i o n a r y divergence of these t r o u t . I t i s concluded from t h i s study t h a t thermal accommodation, thermal a c c l i m a t i o n and e v o l u t i o n a r y a d a p t a t i o n to temperature as d i s p l a y e d by many p h y s i o l o g i c a l systems i n p o i k i l o t h e r m s can be observed and i n t e r p r e t e d a t the l e v e l of enzyme f u n c t i o n . 80 ABBREVIATIONS ACh - a c e t y l c h o l i n e AChE - a c e t y l c h o l i n e s t e r a s e ChAc - c h o l i n e a c e t y l t r a n s f e r a s e Ea - energy of a c t i v a t i o n °K - degrees a b s o l u t e Km - M i c h a e l i s c o n s t a n t &0.D. - change i n o p t i c a l d e n s i t y T - ab s o l u t e temperature t r i s - tris(hydroxymethyl)aminomethane Vmax - maximum v e l o c i t y Vopt - optimum v e l o c i t y 284C51 - dimethobromide of 1:5 - d i ( p - N - a l l y l - N - m e t h y l -aminophenyl)-pentan-3-one LITERATURE CITED 81-Alexandrove, V. Y. 1969. 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