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UBC Theses and Dissertations

Temperature and enzyme activity in poikilotherms : liver soluble NADP+-linked isocitrate dehydrogenase… Moon, Thomas William 1971

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TEMPERATURE AND ENZYME ACTIVITY IN POIKILOTHERMS: LIVER SOLUBLE NADP +-LINKED ISOCITRATE DEHYDROGENASE FROM TROUT by THOMAS WILLIAM MOON B.Sc. Oregon S t a t e U n i v e r s i t y , 1966 M.A* Oregon S t a t e U n i v e r s i t y , 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT o f ZOOLOGY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d : THE UNIVERSITY OF BRITISH COLUMBIA September 1971 In presenting th i s thes is in pa r t i a l fu l f i lment o f the requirements for r an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i l ab le for reference and study. I fur ther agree that permission for extensive copying of th i s thes i s for scholar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th i s thes i s fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada Date £&jk.QJd. ABSTRACT The e f f e c t o f t e m p e r a t u r e on t h e o x i d a t i v e - d e c a r b o x y l a t i o n o f i s o c i t r a t e by t h e s o l u b l e N A D P + - l i n k e d i s o c i t r a t e d e h ydrogenase (NADP-IDH, EC 1.1.1.42) from r a i n b o w t r o u t (Salmo g a i r d n e r i i ) l i v e r has been i n v e s t i g a t e d . A p a r t i c u l a r i n t e r e s t was g i v e n t h o s e p r o p e r t i e s of t h e enzyme w h i c h m i g h t h e l p t o e x p l a i n t h e t e m p e r a t u r e - i n d e p e n d e n t f u n c t i o n o f t h e K r e b s c y c l e and t h e l a r g e i n c r e a s e i n f a t t y a c i d s y n t h e s i s known t o o c c u r d u r i n g low t e m p e r a t u r e a c c l i m a t i o n . W i t h i n t h e t h e r m a l r a n g e e x p e r i e n c e d by r a i n b o w t r o u t , c o n t r o l o f c a t a l y s i s by t h i s enzyme i s t e m p e r a t u r e - i n d e p e n d e n t . A c c l i m a t i o n t o an a l t e r e d t h e r m a l r e g i m e i s accompanied by an i n c r e a s e i n t h e r e l a t i v e p r o p o r t i o n o f t h e s l o w e s t m i g r a t i n g i s o z y m e of l i v e r NADP-IDH on s t a r c h -g e l e l e c t r o p h o r e s i s . These c o l d - and warm-isozyme v a r i a n t s d i s p l a y 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 , and a l l o w f o r t e m p e r a t u r e - i n d e p e n d e n t m o d u l a t i o n of enzyme a c t i v i t y t h r o u g h t h e e n t i r e t h e r m a l r a n g e t h i s s p e c i e s i s l i k e l y t o e n c o u n t e r i n n a t u r e . Other t r o u t s p e c i e s , i n c l u d i n g t h e b r o o k ( S a l v e l i n u s f r o n t i n a l i s ) , l a k e (Salmo namaycush) and t h e i r h y b r i d , t h e s p l a k e t r o u t , were i n v e s t -i g a t e d f o r s i m i l a r r e s p o n s e s . The e l a b o r a t i o n of enzyme v a r i a n t s i n b r o o k and s p l a k e t r o u t a r e c o m p l e x l y r e g u l a t e d by t e m p e r a t u r e changes, b u t t h e l a k e t r o u t genome c o n t a i n s a s i n g l e gene c o d i n g f o r l i v e r NADP-IDH w h i c h i s n o t a f f e c t e d by t e m p e r a t u r e . C a t a l y s i s by t h e t r o u t l i v e r enzyme i s m o d u l a t e d n o t o n l y by temp-e r a t u r e , b u t a l s o ADP and^X-KGA. B o t h of t h e s e m e t a b o l i t e s a l t e r t h e Km o f D L - i s o c i t r a t e ; a t p h y s i o l o g i c a l ADP c o n c e n t r a t i o n s , t h e Km i s r e d u c e d as i t i s with^K-KGA below 0.05 mM, but a t h i g h e r 0(-KGA c o n c e n t -r a t i o n s i t i s m a r k e d l y i n c r e a s e d . These two c o n t r o l s s u g g e s t t h i s enzyme may be i m p o r t a n t f o r t h e K r e b s c y c l e o x i d a t i o n o f i s o c i t r a t e . The a v a i l a b i l i t y o f a p u r i f i e d NADP-IDH fr o m p i g h e a r t a l l o w e d a s t u d y o f t h e k i n e t i c p r o p e r t i e s o f homologous enzymes f r o m b o t h a p o i k i l o t h e r m and a homeotherm. Even though t h e m o l e c u l a r w e i g h t s , Ea v a l u e s , s u b s t r a t e , c o f a c t o r and i n h i b i t o r s p e c i f i c i t i e s a r e s i m i l a r , s u b t l e changes i n enzyme s t r u c t u r e and/or c o n f o r m a t i o n as i d e n t i f i e d by e l e c t r o p h o r e s i s , may r e s u l t i n t h e o b s e r v e d d i f f e r e n c e s i n t e m p e r a t u r e c h a r a c t e r i s t i c s . These a p p a r e n t a d a p t i v e enzyme r e s p o n s e s a r e of i m p o r t a n c e t o t h e r a i n b o w t r o u t w h i c h l i v e s i n a f l u c t u a t i n g t h e r m a l r e g i m e , b u t n o t t h e p i g w h i c h does n o t e x p e r i e n c e t h e s e changes. I n v i v o , t h e r e s p o n s e o f enzymes t o t e m p e r a t u r e f l u c t u a t i o n s may b e q u i t e d i f f e r e n t t o t h o s e seen i n v i t r o . The l o c u s ( i ) c o d i n g f o r r a i n b o w t r o u t l i v e r NADP-IDH was f o u n d t o c o n t a i n a l a r g e amount o f h e t e r o g e n e i t y ; i n f a c t , s e v e n d i s t i n c t p h e n o t y p e s were f o u n d t o c o -e x i s t i n one h a t c h e r y p o p u l a t i o n . The k i n e t i c s o f t h r e e o f t h e s e p h e n o t y p e s were i n v e s t i g a t e d and i t was f o u n d t h a t by i n c r e a s i n g t h e number o f s l o w m oving i s o z y m e s , an i n c r e a s e i n K m ( D L - i s o c i t ) a t h i g h a s s a y t e m p e r a t u r e s o c c u r s . T h i s s u g g e s t s t h a t i r r e s p e c t i v e o f changes i n t h e c e l l u l a r m i l i e u , i s o z y m a l c o n t e n t c a n d e t e r m i n e t h e Km-temperature r e s p o n s e . The d a t a f r o m t h i s s t u d y s u g g e s t t h a t changes i n e n z y m e - s u b s t r a t e a f f i n i t y w i t h t e m p e r a t u r e as a r e s u l t o f e i t h e r t h e t e m p e r a t u r e 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 and/or t h e i r g e n e t i c e x p r e s s i o n , a r e i m p o r t a n t i n c o n t r o l l i n g t h e c a t a l y t i c a c t i v i t y o f NADP-IDH fr o m t h e e u r y t h e r m a l r a i n b o w t r o u t . A l s o , u n l i k e t h e mammalian enzyme, t h e t r o u t l i v e r enzyme may be i m p o r t a n t i n b o t h f a t t y a c i d s y n t h e s i s and t h e K r e b s c y c l e o x i d a t i o n o f i s o c i t r a t e . i i i TABLE OF CONTENTS Page A b s t r a c t i L i s t o f T a b l e s v i L i s t o f F i g u r e s v i i i A c knowledgements x i C h a p t e r I : I n t r o d u c t i o n 1 Tempera t u r e and t h e b r a n c h p o i n t o f l i p o g e n e s i s and t h e K r e b s c y c l e 3 The N A D P + - l i n k e d i s o c i t r a t e d e h y d r o g e n a s e 7 C h a p t e r I I : M a t e r i a l s and Methods 13 F i s h and t h e a c c l i m a t i o n p r o c e s s 13 P r e p a r a t i o n o f NADP-IDH 14 A s s a y o f NADP-IDH a c t i v i t y 15 E s t i m a t e s o f p r o t e i n c o n t e n t 16 S t a r c h - g e l e l e c t r o p h o r e s i s 16 I s o e l e c t r i c f o c u s i n g o f NADP-IDH 17 D E A E - c e l l u l o s e c h r o m a t o g r a p h y 18 S u c r o s e g r a d i e n t u l t r a c e n t r i f u g a t i o n 18 G e l - f i l t r a t i o n o f NADP-IDH 19 C h a p t e r I I I : E f f e c t s o f t h e r m a l a c c l i m a t i o n on m u l t i p l e forms of t r o u t l i v e r NADP-IDH 20 I n t r o d u c t i o n 20 R e s u l t s 22 W i n t e r l a k e , s p l a k e and b r o o k t r o u t S-form IDH 22 S p r i n g l a k e , s p l a k e and b r o o k t r o u t S-form IDH 24 Summer s p l a k e and b r o o k t r o u t S-form IDH 26 IV Page D i s c u s s i o n 31 C h a p t e r IV: Temperature and enzyme a c t i v i t y i n p o i k i l o t h e r m s 34 I n t r o d u c t i o n 34 R e s u l t s and d i s c u s s i o n 36 NAD- v s NADP-IDH 36 Isozymes o f l i v e r NADP-IDH 36 E f f e c t s o f t e m p e r a t u r e on Vm 42 E f f e c t s o f t e m p e r a t u r e on Km 42 E f f e c t s o f t e m p e r a t u r e on t h e M i c h a e l i s c o n s t a n t s of Mg"1^ and M n + + 49 E f f e c t s o f t e m p e r a t u r e on t h e pH optimum 51 E f f e c t s o f o t h e r m e t a b o l i t e s 51 E f f e c t s o f t e m p e r a t u r e a c c l i m a t i o n on enzyme a c t i v i t i e s 58 C h a p t e r V: Compa r i s o n o f t h e p i g h e a r t and r a i n b o w t r o u t l i v e r NADP-IDH 59 I n t r o d u c t i o n 59 R e s u l t s 61 C h a r a c t e r i z a t i o n o f NADP-IDH 61 Compar i s o n o f k i n e t i c c o n s t a n t s 67 E f f e c t s o f i n h i b i t o r s 67 D i s c u s s i o n 76 C h a p t e r V I : E f f e c t s o f t e m p e r a t u r e on i n d i v i d u a l NADP-IDH phenotypes o f r a i n b o w t r o u t l i v e r 79 I n t r o d u c t i o n 79 R e s u l t s 81 Changes i n t h e c e l l u l a r m i l i e u 81 Page K i n e t i c s o f t r o u t l i v e r NADP-IDH i s o z y m e s 84 D i s c u s s i o n 89 C h a p t e r V I I : Summating remarks 93 C h a p t e r V I I I : L i t e r a t u r e c i t e d 102 V I L I S T OF TABLES T a b l e Page I I I , 1. F r e q u e n c y d i s t r i b u t i o n of t h e t h r e e S-form IDH isozyme p a t t e r n s from summer b r o o k t r o u t l i v e r 3 0 IV, 1. T i s s u e - s p e c i f i c NADP-IDH a c t i v i t i e s o f h i g h speed s u p e r n a t a n t f r o m r a i n b o w t r o u t 37 IV , 2. Q-J^ Q v a l u e s f o r t h e warm- and cold-enzyme v a r i a n t s a t v a r i o u s a s s a y t e m p e r a t u r e s 48 IV , 3. K m ( c a t i o n s ) f o r t h e c o l d - and warm-enzyme v a r i a n t s a t v a r i o u s a s s a y t e m p e r a t u r e s 5 0 IV , 4. The e f f e c t s o f v a r i o u s m e t a b o l i t e s on t h e a c t i v i t y o f t r o u t l i v e r NADP-IDH 5 3 IV , 5. The e f f e c t s o f ADP on t h e K m ( D L - i s o c i t ) o f t h e v cold-NADP-IDH v a r i a n t o f t r o u t l i v e r 5 6 V, 1. QIQ v a l u e s f o r t h e p i g h e a r t NADP-IDH a t v a r i o u s D L - i s o c i t r a t e c o n c e n t r a t i o n s 69 V, 2. The e f f e c t s o f c e r t a i n c i t r i c a c i d c y c l e i n t e r -m e d i a t e s on t h e a c t i v i t y o f p i g h e a r t NADP-IDH 7 0 V, 3. The e f f e c t s o f t e m p e r a t u r e on t h e K m ( D L - i s o c i t ) i n t h e p r e s e n c e o f 0.75 mM (C<-KGA) 7 3 V, 4. The e f f e c t s o f t e m p e r a t u r e on t h e Ki(°<-KGA) f o r p i g h e a r t and t r o u t l i v e r NADP-IDH 75 V I , 1. The r e l a t i v e d i s t r i b u t i o n o f i s o z y m i c forms o f t r o u t l i v e r NADP-IDH f r o m a h a t c h e r y p o p u l a t i o n 86 V I , 2. Q-J^ Q v a l u e s between 1 0 and 2 0 ° C f o r t h e A2 and A2,B2>C2 t r o u t l i v e r NADP-IDH a t v a r i o u s D L - i s o c i t r a t e c o n c e n t r a t i o n s 9 1 T a b l e V I I , 1. C o m p a r a t i v e p r o p e r t i e s o f r a i n b o w t r o u t l i v e r and p i g h e a r t NADP-IDH v i i i L I S T OF FIGURES F i g u r e Page I I I , 1. Composite e l e c t r o p h o r e t o g r a m o f w i n t e r l a k e , s p l a k e and b r o o k t r o u t l i v e r NADP-IDH 23 I I I , 2. R e s o l u t i o n o f s p r i n g a c c l i m a t e d b r o o k t r o u t l i v e r S-form IDH 25 I I I , 3. D i a g r a m m a t i c a l r e p r e s e n t a t i o n o f s p r i n g b r o o k t r o u t l i v e r S-form IDH 27 I I I , 4. D i a g r a m m a t i c a l r e p r e s e n t a t i o n o f S-form IDH is o z y m e s f r o m summer b r o o k t r o u t l i v e r 28 I I I , 5, a. Composite e l e c t r o p h o r e t o g r a m o f l i v e r S-form IDH f r o m t h e f a m i l y S a l m o n i d a e 32 b, D i a g r a m m a t i c a l r e p r e s e n t a t i o n o f p o s s i b l e l i v e r S-form IDH s u b u n i t t y p e s 32 IV, 1. S t a r c h - g e l e l e c t r o p h o r e t o g r a m o f t h e c o l d - and warm-enzyme v a r i a n t s w i t h d e n s i t o m e t e r t r a c e s 38 I V , 2. C o n c u r r e n t e l e c t r o f o c u s s e p a r a t i o n s o f t r o u t l i v e r c o l d - and warm-NADP-IDH enzyme v a r i a n t s 40 IV, 3. D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f NADP-IDH fr o m t r o u t l i v e r 41 IV, 4. A r r h e n i u s p l o t s o f t r o u t l i v e r NADP-IDH f r o m c o l d - and w a r m - a c c l i m a t e d a n i m a l s 43 I V , 5. D L - i s o c i t r a t e s a t u r a t i o n c u r v e s and L i n e w e a v e r -B u r k p l o t s a t d i f f e r e n t a s s a y t e m p e r a t u r e s f o r t h e c o l d - and w a r m - a c c l i m a t e d t r o u t l i v e r NADP-IDH 44 I V , 6. NADP + s a t u r a t i o n c u r v e s and L i n e w e a v e r - B u r k p l o t s a t d i f f e r e n t a s s a y t e m p e r a t u r e s 45 i x F i g u r e Page I V , 7. K m ( D L - i s o c i t r a t e ) f o r t h e c o l d - and warm-NADP-IDH v a r i a n t s f r o m t r o u t l i v e r a t d i f f e r e n t t e m p e r a t u r e s 47 IV , 8. R e l a t i v e NADP-IDH a c t i v i t i e s p l o t t e d a t two t e m p e r a t u r e s and v a r i o u s pH v a l u e s 52 IV, 9. The e f f e c t o f CX -KGA on t h e K m ( D L - i s o c i t ) f o r th e cold-NADP-IDH v a r i a n t 57 V, 1. S t a r c h - g e l e l e c t r o p h o r e s i s o f NADP-IDH f r o m p i g h e a r t and t r o u t l i v e r 62 V, 2. S u c r o s e d e n s i t y u l t r a c e n t r i fu*at i o n and Sephadex G-100 g e l f i l t r a t i o n o f t r o u t l i v e r and p i g h e a r t NADP-IDH 63 V, 3. S u c r o s e d e n s i t y u l t r a c e n t r i f u g a t i o n of t r o u t l i v e r NADP-IDH i n t h e p r e s e n c e o f s u b s t r a t e s , c o f a c t o r s and/pr i n h i b i t o r s 65 V, 4. A r r h e n i u s p l o t s o f t r o u t l i v e r and p i g h e a r t NADP-IDH 66 V, 5. The e f f e c t s o f t e m p e r a t u r e on D L - i s o c i t r a t e s a t u r a t i o n c u r v e s f o r p i g h e a r t NADP-IDH 68 V, 6, The e f f e c t s of°(-KGA i n h i b i t i o n on D L - i s o c i t r a t e s a t u r a t i o n c u r v e s f o r p i g h e a r t NADP-IDH 72 V I , 1. The e f f e c t s of c h a n g i n g s u b s t r a t e c o n c e n t r a t i o n s on t h e Km of t h e a l t e r n a t e s u b s t r a t e f o r t r o u t l i v e r NADP-IDH 82 V I , 2. The e f f e c t s o f enzyme p r o t e i n c o n c e n t r a t i o n upon t h e K m ( D L - i s o c i t ) o f t r o u t l i v e r NADP-IDH 83 V I , 3. E l e c t r o p h o r e t o g r a m o f s i x t r o u t l i v e r NADP-IDH phe n o t y p e s 85 F i g u r e Page V I , 4. The e f f e c t s of t i s s u e i s o z y m a l c o n t e n t on t h e K m ( D L - i s o c i t ) v s t e m p e r a t u r e r e l a t i o n s h i p f o r t r o u t l i v e r NADP-IDH 87 ACKNOWLEDGEMENTS I would l i k e t o t h a n k t h e members of my c o m m i t t e e , D r s . G. I . Drummond, J . E. P h i l l i p s and D. J . R a n d a l l f o r t h e i r comments c o n c e r n i n g t h i s t e x t . S p e c i a l t h a n k s goes t o my s u p e r v i s o r , Dr. P. W. Hochachka, whose comments and encouragement have a l l o w e d t h i s t h e s i s work t o come t h i s f a r , and who has made my g r a d u a t e t r a i n i n g s u c h a r e w a r d i n g a n d e x c i t i n g e x p e r i e n c e . A l s o , I would l i k e t o tha n k t h e o t h e r members o f Dr. Hochachka's g r o u p , i n c l u d i n g D r s . G. N . S o m e r o , J , B a l d w i n and H. B e h r i s c h f o r t h e i r c r i t i c i s m and h e l p w i t h t h i s s t u d y . T a r i q M u s t a f a , as a f e l l o w commrade i n arms, has h e l p e d t o make my work a t UBC i n t e r e s t i n g . I must t h a n k my p a r e n t s , w i f e and c h i l d f o r t h e i r m o r a l s u p p o r t t h r o u g h o u t my g r a d u a t e c a r e e r . And f i n a l l y , t h e F i s h e r i e s R e s e a r c h B o a r d o f Canada w h i c h has s u p p o r t e d b o t h m y s e l f a n d t h i s s t u d y t h r o u g h o u t . CHAPTER I: Introduction " t h e c o l d - b l o o d e d a n i m a l s u c c e s s f u l l y a d o p t i n g i n g e n i o u s mechanisms, f i r s t b i o c h e m i c a l , t h e n p h y s i o l o g i c a l , i n o r d e r t o adapt i t s h e a r t t o t h e v a r i a t i o n s o f i t s e n v i r o n m e n t ; t h e warm-blooded a n i m a l d i s c a r d i n g what i t s c o l d - b l o o d e d p r e d e c e s s o r has l a b o r i o u s l y b e a t e n o u t , i n v o k i n g t h e n e r v o u s system t o r e v e r s e t h e n o r m a l b i o c h e m i c a l r e l a t i o n s h i p and g a i n i n g a new freedom by a d a p t i n g , n o t i t s e l f t o t h e i n t e r n a l e n v i r o n m e n t , but t h e i n t e r n a l e n v i r o n m e n t t o i t s e l f . " B a r c r o f t (1934, p. 62) The i n i t i a l r e s p o n s e of c o l d - b l o o d e d o r p o i k i l o t h e r m i c a n i m a l s t o a change i n e n v i r o n m e n t a l t e m p e r a t u r e i s a r a p i d a l t e r a t i o n i n i t s p h y s i o l o g i c a l p r o c e s s e s . T h i s i n s t a n t a n e o u s o r immediate r e s p o n s e i s -f o l l o w e d by a s e r i e s of s l o w e r b i o c h e m i c a l and p h y s i o l o g i c a l changes i n t h e d i r e c t i o n o f t h e o r i g i n a l s t a t e r e s u l t i n g i n a new e q u i l i b r i u m c o n d i t i o n . The term used t o d e f i n e t h i s s l o w e r p r o c e s s i s a c c l i m a t i z - a t i o n , i f t h e p r o c e s s o c c u r s i n t h e o r g a n i s m ' s h a b i t a t , o r a c c l i m a t i o n , i f i t i i s i n d u c e d i n t h e l a b o r a t o r y . T h e rmal a c c l i m a t i z a t i o n o c c u r s s e a s o n a l l y i n a q u a t i c p o i k i l o t h e r m s r e s i d i n g i n t e m p e r a t e f r e s h w a t e r s t r e a m s . I t i s p o s s i b l e , however, t h a t t h e t h e r m a l s t r e s s w i l l be o f l o n g e r d u r a t i o n r e s u l t i n g i n e v o l u t i o n a r y  a d a p t a t i o n , a p r o c e s s r e q u i r i n g many g e n e r a t i o n s t o c o m p l e t e . I n a l l c a s e s , t h e r m a l c o m p e n s a t i o n , be i t i m m e d i a t e , a c c l i m a t i o n or e v o l u t i o n a r y a d a p t a t i o n , c o u n t e r a c t s t h e p o s s i b l e d e l e t e r i o u s e f f e c t s o f a f l u c t u a t i n g o r c o n s t a n t l y r e d u c e d t h e r m a l r e g i m e e n c o u n t e r e d i n N a t u r e by p o i k i l o -t h e r m i c a n i m a l s . E a r l y i n v e s t i g a t i o n s i n t o t h e p h y s i o l o g i c a l b a s i s of t h e r m a l compen-s a t i o n and p a r t i c u l a r l y a c c l i m a t i o n , d e a l t m a i n l y w i t h d e f i n i t i o n s o f t h e " s c o p e f o r a c t i v i t y " ( F r y , 1 9 4 7 ) , t h e r m a l l e t h a l l i m i t s ( B r e t t , 1 9 5 6 ) , 2 and o t h e r whole o r g a n i s m f u n c t i o n s ( P r o s s e r & Brown, 1961; B r e t t , 1970; F r y & Hochachka, 1970). I n r e c e n t y e a r s an i n t e r e s t has a r i s e n i n t h e c e l l u l a r mechanisms o f t h e s e changes. S u b s e q u e n t l y , t h e p r o c e s s of t h e r m a l a c c l i m a t i o n has been found t o b e accompanied by a f u n d a m e n t a l r e o r g a n i z a t i o n of c e l l u l a r m e t a b o l i s m ( H o c h a c h k a , 1967). These changes a r e e x c e e d i n g l y complex and have been r e v i e w e d r e c e n t l y by Hochachka & Somero (1971) and Somero & Hochachka ( 1 9 7 1 ) . 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 w i l l d e a l w i t h a s i n g l e a s p e c t o f t h i s p r o b l e m : t h e c o n t r o l of enzyme a c t i v i t y . What mechanisms a r e a v a i l a b l e t o a l l o w enzymes t o f u n c t i o n e f f i c i e n t l y i n t h e f a c e o f e n v i r o n m e n t a l t e m p e r a t u r e f l u c t u a t i o n s ? Has t h e p r o c e s s o f e v o l u t i o n t h r o u g h n a t u r a l s e l e c t i o n r e s u l t e d i n p o i k i l o t h e r m i c enzymes t h a t a r e f u n d a m e n t a l l y d i f f e r e n t f r o m t h e homologous homeothermic enzymes? That i s , can a d a p t a t i o n t o t e m p e r a t u r e be o b s e r v e d a t t h e enzyme l e v e l ? I s o c i t r a t e d ehydrogenase ( t h r e o - D s i s o c i t r a t e : NADP o x i d o r e d u c t a s e ( d e c a r b o x y l a t i n g ) , EC 1.1.1.42) from t h e l i v e r o f r a i n b o w t r o u t (Salmo  g a r d n e r i i ) was s e l e c t e d because o f i t s p o t e n t i a l f u n c t i o n i n b o t h t h e t r i c a r b o x y l i c a c i d c y c l e ( K r e b s c y c l e ) and l i p o g e n e s i s . 3 Temperature and t h e b r a n c h p o i n t o f l i p o g e n e s i s and t h e K r e b s c y c l e . C i t r a t e o c c u p i e s a m e t a b o l i c b r a n c h p o i n t o f c o n s i d e r a b l e i m p o r t a n c e , l e a d i n g n o t o n l y t o i t s f u r t h e r o x i d a t i o n and t h e e v e n t u a l p r o d u c t i o n o f ene r g y i n t h e fo r m o f ATP, but a l s o t o f a t t y a c i d s ( A t k i n s o n , 1966; 1968). A c c o r d i n g t o A t k i n s o n , c o m p e t i t i o n f o r c i t r a t e by A T P - c i t r a t e l y a s e and N A D + - l i n k e d i s o c i t r a t e d e h ydrogenase w i l l be r e g u l a t e d by t h e a d e n y l a t e e n e r g y c o n t r o l s y s tem. When ATP c o n c e n t r a t i o n s a r e h i g h , c i t r a t e t r a v e r s e s t h e m i t o c h o n d r i a l b a r r i e r f r e e l y and i s c o n v e r t e d by A T P - c i t r a t e l y a s e t o a c e t y l - C o A w h i c h s e r v e s as t h e p r e c u r s o r o f f a t t y a c i d s . As ADP o r AMP l e v e l s i n c r e a s e , A T P - c i t r a t e l y a s e i s s t r o n g l y i n h i b i t e d , a n d t h e N A D + - l i n k e d i s o c i t r a t e d e h ydrogenase i s a c t i v a t e d . T h i s mechanism a s s u r e s t h a t c i t r a t e and ATP w i l l be c o n v e r t e d t o s t o r a g e compounds o n l y when t h e l e v e l o f ATP i s h i g h . C o m p e t i t i o n f o r c i t r a t e by t h e s e enzymes may be an i m p o r t a n t mechanism t o e x p l a i n t h e o b s e r v e d e f f e c t s o f t e m p e r a t u r e on l i p o g e n e s i s and K r e b s c y c l e a c t i v i t y known t o o c c u r d u r i n g t h e r m a l a c c l i m a t i o n (Hochachka, 1968; Dean, 1969; N e w e l l & P y e , 19 7 1 ) . The e f f e c t s o f t h e r m a l a c c l i m a t i o n on t h e p a r t i c i p a t i o n o f t h e s e d i f f e r e n t m e t a b o l i c pathways i s dependent upon t h e t i s s u e examined (Hochachka, 1967). E k b e r g (1958) r e p o r t e d warm-adapted g i l l t i s s u e o f g o l d f i s h t o be more r e s i s t a n t t o 10 ^M c y a n i d e , b u t more s e n s t i v e t o i o d o a c e t a t e (5.4 x 1 0 - ^ M) when compared t o t h e same t i s s u e i n c o l d -a d a p t e d g o l d f i s h . I t was i m p l i e d t h a t i n t h e warm-adapted g i l l t i s s u e , e i t h e r t h e cy t o c h r o m e s y s t e m was a l t e r e d o r a n o t h e r o x i d a t i v e pathway l e s s s e n s i t i v e t o c y a n i d e was u t i l i z e d . S i m i l a r l y , c a r b o n f l o w t h r o u g h t h e p e n t o s e pathway was d i v e r t e d i n t h e s e warm-adapted f i s h . These o b e r s e r v a t i o n s c o u l d n o t be made f o r l i v e r o r b r a i n p r e p a r a t i o n s under 4 i d e n t i c a l c o n d i t i o n s . The r a t e o f oxygen c o n s u m p t i o n i n g o l d f i s h l i v e r homogenates was f ound by Kanungo & P r o s s e r (1959) t o be h i g h e r i n c o l d - a d a p t e d t h a n i n warm-adapted f i s h , a l t h o u g h t h e e f f i c i e n c y o f p h o s p h o r y l a t i o n ( as m e a sured by P/0 r a t i o s ) was d e c r e a s e d i n t h e c o l d . A number o f p o s s i b l e mechanisms l e a d i n g t o d e c r e a s e d P/0 r a t i o s i n t h e c o l d were d i s c u s s e d , i n c l u d i n g u n c o u p l i n g o f p h o s p h o r y l a t i o n , i n c r e a s i n g m i t o c h o n d r i a l ATPase a c t i v i t y , i n c r e a s i n g t h e " C a l o r i g e n i c s h u n t " f o r NADPH o x i d a t i o n v i a a c y t o c h r o m e c s y s t e m , o r an i n c r e a s e d a c t i v i t y of t h e p y r i d i n e n u c l e o t i d e t r a n s h y d r o g e n a s e . None o f t h e s e a l t e r n a t i v e s , however, were examined. T h e y c o n c l u d e d by s u g g e s t i n g t h a t m e t a b o l i c a d a p t a t i o n t o t e m p e r a t u r e i n g o l d f i s h o c c u r s by t h e q u a n t i t a t i v e change i n a c t i v i t i e s o f s e r v e r a l enzyme s y s t e m s . Oxygen c o n s u m p t i o n s t u d i e s , a t b e s t , a r e n o t a dequate t o i n d i c a t e s p e c i f i c s h i f t s i n m e t a b o l i c pathways d u r i n g t h e r m a l c o m p e n s a t i o n . I s o t o p i c i n c o r p o r a t i o n i n t o s p e c i f i c m e t a b o l i c i n t e r m e d i a t e s have l e a d t o some u n d e r s t a n d i n g o f t h e changes o c c u r r i n g a t t h i s t i m e . Hochachka & Hayes (1962) d e m o n s t r a t e d d i r e c t l y a s h i f t f a v o r i n g h i g h e r l e v e l s of p e n t o s e c y c l e p a r t i c i p a t i o n accompanying a c c l i m a t i o n t o low t e m p e r a t u r e s f r o m t h e p r e d o m i n a n t Embden-Meyerhof and K r e b s c y c l e c a r b o n f l o w n o r m a l l y o b s e r v e d i n t h e e a s t e r n b r o o k t r o u t , S a l v e l i n u s  f r o n t i n a l i s . These d a t a s u g g e s t e d an a l t e r e d a c e t a t e m e t a b o l i s m w i t h l o w t e m p e r a t u r e a d a p t a t i o n . A c e t a t e - l - ^ C m e t a b o l i s m has been examined by Dean (1969) i n b o t h m u s c l e and l i v e r t i s s u e from warm- and c o l d - a d a p t e d r a i n b o w t r o u t , Salmo  g a ' r d n e r i i . C o l d - a d a p t e d f i s h m u s c l e showed h i g h e r t u r n o v e r v a l u e s f o r b o t h l a b e l l e d a c e t a t e and p a l m i t a t e compared wJ :h warm-adapted t i s s u e , 5 a g a i n s u g g e s t i n g an a l t e r K r e b s c y c l e a c t i v i t y . L i v e r t i s s u e f r o m c o l d -a d a p t e d f i s h a l s o d e m o n s t r a t e d a h i g h e r o x i d a t i v e a c t i v i t y , a l t h o u g h a t an i n t e r m e d i a t e t e m p e r a t u r e (11.5°C), t h e r e was no d i f f e r e n c e between th e c o l d - and warm-adapted t i s s u e s . At t h i s t e m p e r a t u r e (11.5°C), t h e r e was no d i f f e r e n c e between t h e amount of a c e t a t e i n c o r p o r a t e d i n t o ^ 4 C 0 2 , b u t a marked d i f f e r e n c e i n i n c o r p o r a t i o n i n t o t o t a l l i p i d s . T h i s a g a i n s u g g e s t s a s h i f t f a v o r i n g l i p o g e n e s i s r e s u l t i n g f r o m t h e more e f f i c i e n t c o m p e t i t i o n by t h i s pathway f o r common m e t a b o l i t e s . These d a t a g e n e r a l l y c o n c l u d e t h a t c a r b o n f l o w t h r o u g h t h e K r e b s c y c l e i s more o r l e s s i n d e p e n d e n t o f t e m p e r a t u r e . Hochachka (1968) s u g g e s t s t h a t c i t r a t e and ATP r e d u c e t h e e f f e c t i v e n e s s o f t h e K r e b s c y c l e c o m p e t i n g f o r a c e t y l - C o A , r e s u l t i n g i n i n c r e a s e d c o n v e r s i o n t o l i p i d s . T h i s mechanism i s so e f f i c i e n t t h a t i n t h e e l e c t r i c o r g a n o f E l e c t r o p h o r u s e l e c t r i c u s , mM c o n c e n t r a t i o n s o f c i t r a t e a l l o w f o r a c e t a t e o x i d a t i o n t o p r e c e e d i n d e p e n d e n t o f t e m p e r a t u r e o v e r a 20°C r a n g e . Hochachka d i d not e s t a b l i s h t h e p r e c i s e mode o f a c t i o n o f ATP or c i t r a t e , b u t i t i s t h o u g h t t o be p r i m a r i l y on a c e t y l - C o A - c a r b o x y l a s e ( W a i t e & W a k i l , 1 9 6 2 ) , t h e c i t r a t e c l e a v a g e enzyme ( A T P - c i t r a t e l y a s e ) o r c i t r a t e s y n t h a s e (Hathaway & A t k i n s o n , 1 9 6 5 ) . C i t r a t e s y n t h a s e i n h i b i t i o n by ATP has been i n v e s t i g a t e d b y Hochachka & L e w i s (1970) and i s found t o be c o m p l e x l y r e g u l a t e d by pH and t e m p e r a t u r e , as was s u g g e s t e d e a r l i e r (Hochachka, 1968). The i n c r e a s e i n l i p o g e n e s i s o b s e r v e d d u r i n g i n c o r p o r a t i o n s t u d i e s r e s u l t s i n an a l t e r e d l i p i d e n v i r o n m e n t a t low t e m p e r a t u r e s . I n a l l c a s e s r e p o r t e d , t h e p r o p o r t i o n o f u n s a t u r a t e d t o s a t u r a t e d f a t t y a c i d s i n c r e a s e i n t h e c o l d . T h i s a l t e r e d l i p i d c o n t e n t has been found f o r g o l d f i s h b r a i n l i p i d s ( J o h n s t o n & R o o t s , 1 9 6 4 ) , m u s c l e and o r g a n l i p i d s o f r a i n b o w t r o u t ( K n i p p r a t h & Mead, 1 9 6 5 ) , t o t a l l i p i d s of m o s q u i t o - f i s h 6 a n d g u p p i e s ( K n i p p r a t h & Mead, 1 9 6 6 ) , g o l d f i s h i n t e s t i n a l l i p i d s (Kemp & S m i t h , 1 9 7 0 ) , and g o l d f i s h g i l l m i t o c h o n d r i a l l i p i d s ( A n d e r s o n , 1970; C a l d w e l l & V e r n b e r g , 1 9 7 0 ) . I t i s a l s o known t h a t v e r y s p e c i f i c l i p i d changes c a n o c c u r ( K n i p p r a t h & Mead, 1968; R o o t s , 1968; A n d e r s o n , 1 9 7 0 ) . R o o t s (1968) found t h a t s p e c i f i c p h o s p h o l i p i d s a r e a l t e r e d , p r o b a b l y as r e l a t e d t o n e r v e f u n c t i o n . Membrane l i p i d changes a r e e x t r e m e l y i m p o r t a n t due t o t h e number of enzymes a s s o c i a t e d w i t h them. S m i t h & Kemp (1969) have found t h a t membrane ATPase behaves d i f f e r e n t l y i n membranes c o n t a i n i n g d i f f e r e n t f a t t y a c i d s . T h i s t y p e o f d a t a may be e x p l a i n e d by a l t e r a t i o n i n t h e c o n f o r m a t i o n of t h e ATPase p r o t e i n r e s u l t i n g f r o m phase changes i n t h e membrane l i p i d component (Kumamoto, R a i s o n & L y o n s , 1 9 7 1 ) , as r e c e n t l y seen f o r t h e m i t o c h o n d r i a l s u c c i n a t e d e h y d r o g e n a s e and cy t o c h r o m e o x i d a s e systems ( R a i s o n , Lyons & Thomson, 1 9 7 1 ) . T r a n s i t i o n t e m p e r a t u r e s i n A r r h e n i u s p l o t s f o r t h e t r a n s p o r t o f l a c t o s e o r l a c t o s e a n a l o g s have been found f o r v a r i o u s u n s a t u r a t e d f a t t y a c i d a u x o t r o p h s o f E_. c o l i . The t y p e o f f a t t y a c i d s u p p l i e d i n t h e g r o w t h medium w i l l d e t e r m i n e t h e t r a n s i t i o n t e m p e r a t u r e ( S c h a i r e r & O v e r a t h , 1969; Fox, Law, T s u k a g o s h i & W i l s o n , 1970; O v e r a t h , ; S c h a i r e r & S t o f f e l , 1970; W i l s o n , Rose & Fox, 1 9 7 0 ) . E s f a h a n i , B a r n e s & W a k i l (1970) have r e p o r t e d an i n c r e a s e i n t r a n s - f a t t y a c i d i n c o r p o r a t i o n i n t o an E_. c o l i f a t t y a c i d a u x o t r o p h y a t 37°C compared t o 27°C; a t 27°C t h e r e was an i n c r e a s e i n c i s - f a t t y a c i d i n c o r p o r a t i o n o v e r 37°C, These changes i n t o t a l l i p i d s and s p e c i f i c membrane l i p i d s a r e e x t r e m e l y i m p o r t a n t t o t h e f i e l d of t h e r m a l a d a p t a t i o n s i n c e t h e y demon-s t r a t e u n e q u i v o c a l l y (1) t h a t t h e t e m p e r a t u r e range o f a g i v e n k i n d o f enzyme f u n c t i o n can be d e t e r m i n e d by t h e a s s o c i a t e d p h o s p h o l i p i d s , and 7 ( 2 ) t h a t t h e p h y s i c a l s t a t e of t h e membrane depends upon t e m p e r a t u r e ( E s f a h a n i , L i m b r i c k , K n u t t o n & W a k i l , 1 9 7 1 ) . I t had been assumed t h a t t h e s e membrane p r o p e r t i e s were c r i t i c a l t o t h e r m a l a d a p t a t i o n (Hochachka, 1967; Hochachka & Somero, 1 9 7 1 ) , but o n l y w i t h t h e work of Fox, W i l s o n , E s f a h a n i , . - W a k i l and o t h e r s have t h e s e s u g g e s t i o n s been given...an e x p e r i m e n t a l b a s i s . I n o r d e r f o r e x t r a m i t o c h o n d r i a l l i p o g e n e s i s t o c o n t i n u e , r e d u c i n g e q u i v a l e n t s i n t h e f o r m of NADPH a r e n e c e s s a r y ( W a k i l , T i t c h i e n e r & G i b s o n , 1 9 5 9 ) . One enzyme w h i c h has t h i s a c t i v i t y i s t h e N A D P + - l i n k e d i s o c i t r a t e d e h ydrogenase ( L o w e n s t e i n , 1961a,b; S a v a r d , Marsh & H o w e l l , 1963; Hanson & B a l l a r d , 1967; O'Hea & L e v e i l l e , 1968; Walker & B a i l e y , 1969; Bauman, Brown & D a v i s , 1970; F l i n t & Denton, 1 9 7 0 ) . The NADP" 1"-linked i s o c i t r a t e d e h y d r o g e n a s e . The p r e s e n c e o f NADP+-1inked i s o c i t r a t e d ehydrogenase (NADP-IDH) a c t i v i t y has been n o t e d f r o m most t i s s u e s o f v e r t e b r a t e s t h u s f a r examined ( P l a u t , 1963) and many m i c r o o r g a n i s m s (Hampton & Hanson, 1 9 6 9 ) . I n m i c r o o r g a n i s m s and p l a n t s , t h e p a r t i t i o n i n g o f i s o c i t r a t e between t h e K r e b s and g l y o x y l a t e c y c l e s o c c u r s t h r o u g h t h e c o n c e r t e d i n h i b i t i o n o f NADP-IDH by g l y o x y l a t e and o x a l o a c e t a t e ( O z a k i & S h i i o , 1 9 6 8 ) . The v e r t -e b r a t e enzyme i s l i k e w i s e s u b j e c t e d t o c o n c e r t e d i n h i b i t i o n by t h e s e m e t a b o l i t e s ( O z a k i & S h i i o , 1968; S h i i o & O z a k i , 1968; Hampton & Hanson, 1969; Marr & Weber, 1969a,b; C h a r l e s , 1970) even though t h e g l y o x y l a t e ' c y c l e i s a b s e n t ( M a h l e r & C h o r d e s , 1 9 6 6 ) . T h i s a p p a r e n t g e n e r a l p r o p e r t y o f a l l NADP-IDHs may r e s u l t f r o m t h e e f f e c t s o f g l y o x y l a t e and o x a l o a c e t a t e o n t h e a g g r e g a t i o n s t a t e o f t h e enzyme s u b u n i t s (Kemper & K a p l a n , 1 9 7 1 ) . T h i s enzyme i s under a d e n y l a t e c o n t r o l i n m i c r o o r g a n i s m s . Ones 8 r e a s o n f o r t h e a c c e p t a n c e of t h e N A D + - l i n k e d IDH enzyme b e i n g i m p o r t a n t i n i s o c i t r a t e o x i d a t i o n w i t h i n t h e m i t o c h o n d r i a i s t h a t t h e enzyme i s m o d u l a t e d by ADP o r AMP ( P l a u t & A o g a i c h i , 1968). P a r k e r & Weitzman (1970) i d e n t i f i e d two k i n e t i c a l l y , m o l e c u l a r l y and 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 NADP-IDH enzymes i n A c i n e t o b a c t e r l w o f f ; i s o z y m e I I ( t h e h i g h e r m o l e c u l a r w e i g h t s p e c i e s ) , i n t h e p r e s e n c e of 1 mM AMP o r ADP, i s a c t i v a t e d f i v e - f o l d and t w o - f o l d r e s p e c t i v e l y , w h i l e 1 mM ATP had e s s e n t i a l l y no e f f e c t . These r e s u l t s r e s e m b l e c l o s e l y t h o s e o b t a i n e d b y A t k i n s o n , Hathaway & S mith (1965) f o r t h e NAD-IDH from y e a s t . M arr & Weber have i d e n t i f i e d ATP as a n o n c o m p e t i t i v e i n h i b i t o r o f C r i t h i d i a f a s c i c u l a t a (a p r o t o z o a n ) NADP-IDH (1969a,c) and ADP and -ATP as i n h i b i t o r s of t h e same enzyme i n S a l m o n e l l a t y p h i m u r i u m (1968) . A l t h o u g h n o t c o n c l u s i v e , e n t r o p y and f r e e e n e r g y changes a r e c o n s i s t e n t w i t h p r o b a b l e p h y s i c a l changes i n t h e enzyme d u r i n g b i n d i n g o f t h e s e i n h i b i t o r s . H i g a s h i , Maruyama, O t a n i & Sakamoto (1965) a t t r i b u t e a d e n y l a t e i n h i b i t i o n of t h i s enzyme w i t h m e t a l i o n c h e l a t i o n . The o n l y e s t a b l i s h e d f u n c t i o n f o r metazoan NADP-IDH i s i n t h e p r o -d u c t i o n of r e d u c i n g e q u i v a l e n t s f o r l i p o g e n e s i s as p r e v i o u s l y m e n t i o n e d . T h e r e i s c o n s i d e r a b l e d i s a g r e e m e n t among a u t h o r s w i t h r e g a r d t o t h e r e l a t i v e c o n t r i b u t i o n of t h e NAD- and N A D P - l i n k e d IDH enzymes tow a r d s o x i d a t i o n of i s o c i t r a t e i n m i t o c h o n d r i a ( N i c h o l l s & G a r l a n d , 1 9 6 9 ) . However, w i t h t h e r e c o g n i t i o n of t h e a c t i v a t i o n of NAD-IDH by ADP (Chen & P l a u t , 1963) o r p h o s p h a t e ( G o e b e l l & K l i n g e n b e r g , 1 9 6 3 ) , t h e c o l d -l a b i l i t y of NAD-IDH ( P l a u t & A o g a i c h i , 1967) and t h e a c t i v a t i o n by m a l a t e o f i s o c i t r a t e p e r m e a t i o n i n t o r a t l i v e r m i t o c h o n d r i a ( C h a p p e l l & R o b i n s o n , 1 9 6 8 ) , t h e s i g n i f i c a n c e of t h e NAD-IDH enzyme i n mammalian m i t o c h o n d r i a l o x i d a t i v e m e t a b o l i s m i s w i d e l y a c c e p t e d , w h i l e t h e q u e s t i o n 9 o f p o t e n t i a l r o l e s f o r t h e NADP-IDH enzyme has been l e f t l a r g e l y u n c o n s i d -e r e d . I n f a c t , Colman, S z e t o & Cohen (1970) s u g g e s t t h e i m p o r t a n c e o f t h i s enzyme i s i n i t s f u n c t i o n a l s i m i l a r i t i e s w i t h NAD-IDH w h i c h w i l l a l l o w t h e c o m p a r a t i v e s t u d y of an a l l o s t e r i c y_s n o n - a l l o s t e r i c enzyme. I n l o w e r v e r t e b r a t e s , i n c l u d i n g f i s h , t h e f u n c t i o n o f t h i s enzyme i s unknown, b u t t h e l a c k o f an a c t i v e NAD-IDH i n t h e s e o r g a n i s m s ( C r a b t r e e & Newsholme, 1970) may put t h i s enzyme i n an u n i q u e p o s i t i o n . The enzyme has been p u r i f i e d f r o m a number o f m i c r o o r g a n i s m s , b u t t h e m a j o r s o u r c e has been t h e p i g h e a r t c y t o p l a s m ( P l a u t , 1 9 6 3 ) . Colman c(1968) and Colman, S z e t o & Cohen (1970) have i n v e s t i g a t e d t h e p r o p e r t i e s o f t h i s enzyme under a number o f c o n d i t i o n s and s u g g e s t t h a t i t i s a s i n g l e p o l y p e p t i d e c h a i n w i t h a m o l e c u l a r w e i g h t of 58,000. R e p o r t s of a s i m i l a r m o l e c u l a r w e i g h t a r e a v a i l a b l e (Moyle & D i x o n , 1 9 5 6 ) , a l t h o u g h M agar & R o b b i n s (1969) s u g g e s t t h e enzyme i s a d i m e r . The s u b u n i t s t r u c t u r e o f NADP-IDH fr o m p i g h e a r t has r e c e n t l y been f o u n d t o r e s u l t f r o m enzyme a g g r e g a t i o n and t h a t t h e e x t e n t of a g g r e g a t i o n c o n t r o l s t h e d i r e c t i o n o f enzyme c a t a l y s i s (Kemper & K a p l a n , 1 9 7 1 ) . The a c t i v e s p e c i e s c a t a l y z i n g t h e r e d u c t i o n o f NADP + i s a 30,000 m o l e c u l a r w e i g h t monomer; w h e r e a s , t h e a c t i v e s p e c i e s c a t a l y z i n g t h e o x i d a t i o n o f NADPH i s a 120,000 m o l e c u l a r w e i g h t t e t r a m e r . These c o n f l i c t i n g d a t a c o n c e r n i n g t h e s u b u n i t c o m p o s i t i o n o f t h i s p i g h e a r t enzyme have y e t t o be r e s o l v e d . The p i g l i v e r enzyme has been s t u d i e d by I l l i n g w o r t h & T i p t o n (1970) a n d i t s p r o p e r t i e s v a r y somewhat fr o m t h e h e a r t enzyme: a m o l e c u l a r w e i g h t 1.3 t i m e s t h a t r e p o r t e d f o r t h e p i g h e a r t enzyme, and a d i m e r s u b u n i t s t r u c t u r e have been a c c e p t e d . The d i m e r i c f o r m o f p i g l i v e r and mouse m u s c l e s o l u b l e NADP-IDH has been c o n f i r m e d by g e n e t i c a n a l y s e s ( H e n d e r s o n , 1965; M i n t z & B a k e r , 1967). I n b a c t e r i a , a s i m i l a r m o l e c u l a r 10 w e i g h t as t h a t found by I l l i n g w o r t h & T i p t o n has been r e p o r t e d (Chung & F r a n z e n , 1969; Howard & B e c k e r , 1970; B a r r e r a & J u r t s h u k , 1 9 7 1 ) . E a r l y work by L o w e n s t e i n & S m i t h (1962) and Baron & B e l l (1962) i n d i c a t e d t h a t NADP-IDH e x i s t e d as i m m u n o l o g i c a l l y d i s t i n c t enzymes, o r i s o z y m e s . These forms a r e found i n t h e s o l u b l e and m i t o c h o n d r i a l p o r t i o n o f h i g h e r v e r t e b r a t e c e l l s . U s i n g b r e e d i n g e x p e r i m e n t s between n o r m a l a n d mutant a l l e l e - c a r r y i n g m i c e s t r a i n s , Henderson (1965) found t h a t o n l y t h e s o l u b l e enzyme f r o m b o t h h e a r t and l i v e r a p p e a r e d as h e t e r o z y g o u s f o r m s . Such e v i d e n c e i n d i c a t e d t h a t t h e s o l u b l e and m i t o c h o n d r i a l IDH enzymes a r e n o t g e n e t i c a l l y l i n k e d , and t h a t t h e s o l u b l e f o r m i s a d i m e r . S i m i l a r e x p e r i m e n t s by M i n t z & Baker (1967) on m u s c l e NADP-IDH gave i d e n t i c a l c o n c l u s i o n s . H i g a s h i , Maruyama, O t a n i & Sakamoto (1965) s t u d i e d b e e f h e a r t s o l u b l e and m i t o c h o n d r i a l i s o z y m e s o f NADP-IDH and f o u n d t h a t each had s i m i l a r c a t a l y t i c p r o p e r t i e s , b u t t h e s o l u b l e enzyme was more s e n s i t i v e t o i n h i b i t o r s . M u l t i p l e forms o f NADP-IDH have a l s o been i n v e s t i g a t e d i n m i c r o -o r g a n i s m s . A s t r a i n o f E_. c o l i grown on a c e t a t e as i t s s o l e c a r b o n s o u r c e p r o d u c e s an enzyme v a r i a n t c o m p l e t e l y d i f f e r e n t t h a n the two p r o -d u c e d by t h e same o r g a n i s m w i t h g l u c o s e as a c a r b o n s o u r c e (Reeves, Brehmeyer & A j l , 1968a,b). T h i s s u g g e s t s t h a t NADP-IDH may be i n d u c i b l e i n t h e s e b a c t e r i a . S e l f & Weitzman (1970) found two i s o z y m e s o f A c i n e t o b a c t e r l w o f f by z o n a l c e n t r i f u g a t i o n w h i c h d i f f e r e d k i n e t i c a l l y , m o l e c u l a r l y and e l e c t r o p h o r e t i c a l l y . A l s o , T a i t (1970) has shown m i t o c h o n d r i a l and s o l u b l e i s o z y m e s i n Paramecium a u r e l i a w h i c h a r e n o t g e n e t i c a l l y l i n k e d . Some e l e c t r o p h o r e t i c i n v e s t i g a t i o n s have been c a r r i e d out on t h e enzyme f r o m s a l m o n i d f i s h e s . W o l f , E n g e l & F a u s t (1970) o b s e r v e d p o l y -11 m o r p h i c forms of t h e s o l u b l e l i v e r NADP-IDH of t h e r a i n b o w t r o u t Salmo  i r i d e u s , r e s u l t s s u g g e s t i n g o n l y a s i n g l e l o c u s f o r the enzyme e x i s t e d . However, t h e h e a r t m u s c l e m i t o c h o n d r i a l NADP-IDH c o u l d be shown t o be d e r i v e d f r o m two d i f f e r e n t gene l o c i , a l w a y s g i v i n g t h r e e 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 i s o z y m e s i n a l l i n d i v i d u a l s . The l i v e r o f t h i s f i s h c o n t a i n e d n o m i t o c h o n d r i a l NADP-IDH, a l t h o u g h t h e h e a r t showed b o t h forms o f t h e , enzyme. The a u t h o r s c o n c l u d e d t h a t t h e l o c u s c o d i n g f o r t h e h e a r t m i t o c h o n d r i a l NADP-IDH has undergone c o m p l e t e d i p l o i d i z a t i o n t o two s e p a r a t e l o c i ; however, t h e l i v e r s o l u b l e NADP-IDH i s s t i l l s u b j e c t e d t o ; t e t r a s o m i c i n h e r i t a n c e . T h i s d i p l o i d i z a t i o n mechanism of p h y l o g e n e t i c a l l y t e t r a p l o i d o r g a n i s m s has been d i s c u s s e d by Ohno ( 1 9 7 0 ) . O t h e r t e t r a p l o i d f i s h e s have a l s o been s t u d i e d . Q u i r o z - G u t i e r r e z & Ohno (1970) found t h a t t h e t e t r a p l o i d g o l d f i s h and c a r p a r e b o t h endowed w i t h two gene l o c i f o r t h e s o l u b l e NADP-IDH i n l i v e r and h e a r t . I n b o t h s t u d i e s , t h e enzyme was found t o be a d i m e r . L i n , Schipmann, K i t t r e l l & Ohno (1969) have found t h a t a t l e a s t 5 0% o f a w i l d p o p u l a t i o n o f L a k e E r i e g o l d f i s h a r e h e t e r o z y g o u s a t t h e s o r b i t o l d ehydrogenase l o c u s . I t has been s u g g e s t e d by Q u i r o z - G u t i e r r e z 6 Ohno (1970) t h a t an i d e n t i c a l s i t u a t i o n e x i s t s w i t h r e g a r d t o t h e s o l u b l e IDH l o c u s o f t h i s ^ a m e f i s h p o p u l a t i o n . A p o l l u t e d e n v i r o n m e n t , i t was f e l t , may s t r o n g l y s e l e c t f o r h e t e r o z y g o s i t y a t a number of gene l o c i , i n c l u d i n g t h a t c o d i n g f o r s o l u b l e IDH. However, no o t h e r p a r a m e t e r , s u c h as f o r example t e m p e r a t u r e , was i m p l i c a t e d i n t h e m a i n t e n a n c e o f h e t e r o z y g o s i t y a t t h i s l o c u s . The a d v a n t a g e of u s i n g t h e r a i n b o w t r o u t Salmo g a i r d n e r i i as an e x p e r i m e n t a l o r g a n i s m r e l a t e s t o i t s a p p a r e n t t e t r a p l o i d e v o l u t i o n , a t o p i c d i s c u s s e d i n Ch, I I I o f t h i s t h e s i s . The t e t r a s o m i c l o c u s c o n s i s t s o f 12 i n i t i a l l y f o u r i d e n t i c a l a l l e l e s i n w h i c h t h e chance of m u t a t i o n i s g r e a t e r t h a n i n a d i p l o i d o r g a n i s m s i n c e each a l l e l e i s s u b j e c t t o l e s s s e l e c t i v e p r e s s u r e (Ohno, 1970; W o l f , E n g e l & F a u s t , 1 9 7 0 ) . T h i s i n c r e a s e i n g e n e t i c m a t e r i a l i s t h e b a s i s f o r n a t u r a l s e l e c t i o n . H a l d a n e (1955) and Mayr (.1963) have emphasized t h a t g e o g r a p h i c a l and t e m p o r a l changes i n t h e e n v i r o n m e n t c o u l d p l a c e a premium on m e t a b o l i c f l e x i b i l i t y . As a r e s u l t , t h e l a r g e amount of p o t e n t i a l gene p r o d u c t s ( i . e . , p r o t e i n s ) a v a i l a b l e i n a t e t r a p l o i d o r g a n i s m s u c h as t h e r a i n b o w t r o u t , may be o f an a d a p t i v e s i g n i f i c a n c e . W i t h t h e s e c o n s i d e r a t i o n s i n mind, a s t u d y was i n i t i a t e d t o e x a m i n e t h e p r o p e r t i e s and p a r t i c u l a r l y t h e t h e r m a l b e h a v i o u r o f NADP-IDH f r o m t h e l i v e r o f t h e r a i n b o w t r o u t , Salmo g a i r d n e r i i , a h i g h l y e u r y t h e r m a l s p e c i e s o f t h e f a m i l y S a l m o n i d a e . CHAPTER I I : M a t e r i a l s and Methods 13 MATERIALS AND METHODS F i s h and t h e a c c l i m a t i o n p r o c e s s . Rainbow t r o u t (Salmo g a i r d n e r i ) were p u r c h a s e d f r o m t h e Sun V a l l e y T r o u t Farm, M i s s i o n , B.C. f o r a l l e x p e r i m e n t s . P r i o r , t o a c c l i m a t i o n , t h e t r o u t were s t o r e d i n a l a r g e o u t s i d e h o l d i n g t a n k s u p p l i e d w i t h a c o n s t a n t f l o w o f s u b t e r r a n e a n w a t e r and f e d t w i c e w e e k l y w i t h New Age F i s h p e l l e t s ( M o o r e - C l a r k Co., S a l t L ake C i t y , U t a h ) . These f i s h were u s e d as a s o u r c e o f f r e s h t i s s u e when r e q u i r e d . A l s o , t r i p s were made t o t h e t r o u t f a r m i n o r d e r t o e x c i s e a l a r g e number o f t r o u t l i v e r s t o b e used as a s o u r c e o f f r o z e n t i s s u e . No d i f f e r e n c e s c o u l d be d e t e c t e d b e t w e e n t h e isozyme p a t t e r n s i n t h e f r e s h o r t h e f r o z e n l i v e r t i s s u e , so t h e f r o z e n t i s s u e was used p r e f e r e n t i a l l y . A c c l i m a t i o n e x p e r i m e n t s were c a r r i e d out on r a i n b o w t r o u t d u r i n g e a r l y f a l l o f 1968 and 1969. I t has been o b s e r v e d t h a t w a r m - a c c l i m a t i o n i s e x t r e m e l y d i f f i c u l t i n l a t e f a l l o r w i n t e r ( B a l d w i n & Hochachka, 1 9 7 0 ) . Two n o n - c i r c u l a t i n g 150 g a l s t a i n l e s s - s t e e l t a n k s were used t o a c c l i m a t e a p p r o x i m a t e l y 12 f i s h a t a t i m e under a c o n s t a n t 14 h r l i g h t - 10 h r d a r k p h o t o r e g i m e . One t a n k was m a i n t a i n e d a t 18°C (+ 2°C) and t h e o t h e r a t 2°C (+ 0.5°C). A p p r o x i m a t e l y o n e - t h i r d o f t h e w a t e r i n each t a n k was exc h a n g e d d a i l y , and f e e d was g i v e n f o u r - t i m e s w e e k l y . The p e r i o d o f a c c l i m a t i o n was a t l e a s t f o u r weeks i n l e n g t h . These t e m p e r a t u r e s a r e c o n s i s t e n t w i t h f l u c t u a t i o n s seen i n many Wes t e r n C a n a d i a n w a t e r s . A l l i n d i v i d u a l s o f b r o o k - ( S a l v e l i n u s f r o n t i n a l i s ) , l a k e - (Salmo  n amaycush), and s p l a k e t r o u t used i n t h e s e e x p e r i m e n t s were k i n d l y d o n a t e d by Dr. F . E . J . F r y , U n i v e r s i t y o f T o r o n o t o , Canada, t o whom I am g r e a t l y i n d e b t e d . 14 A c c l i m a t i o n o f t h e b r o o k - , l a k e - and s p l a k e t r o u t was c a r r i e d out f o r a t l e a s t one month a t t h e Maple L a b o r a t o r i e s , T o r o n o t o , O n t a r i o , by a number o f Dr. F r y ' s c o - w o r k e r s . L a r g e 100 g a l c o n c r e t e t a n k s s u p p l i e d b y a c o n s t a n t t e m p e r a t u r e w a t e r s o u r c e were used i n a l l a c c l i m a t i o n s t u d i e s . These t a n k s were m a i n t a i n e d on a 12 h r l i g h t - 12 h r d a r k photo r e g i m e no m a t t e r what t i m e o f y e a r t h e f i s h were a c c l i m a t e d . The f i s h w e r e a l l a p p r o x i m a t e l y f i v e i n c h e s l o n g and two y e a r s o f age, and w h i l e a c c l i m a t i n g we're f e d f i s h p e l l e t s , d a i l y . A t l e a s t f i f t y f i s h c o u l d be a c c l i m a t e d i n each t a n k . P r e p a r a t i o n o f NADP-IDH. A l l r a i n b o w t r o u t t i s s u e s were homogenized i n f o u r volumes o f i c e c o l d b u f f e r c o n s i s t i n g o f 100 mM t r i s , 2 mM EDTA, 2 mM g l u t a t h i o n e ( r e d u c e d ) , 0.25 M s u c r o s e , t i t r a t e d t o pH 7.0 w i t h 1 M HC1, w i t h an O m n i - M i x e r ( S o r v a l l ) a t medium speeds f o r s h o r t t i m e i n t e r v a l s . T h i s p r e p a r a t i o n was c e n t r i f i i g e d a t 40,000g f o r 30 m i n ( S o r v a l l , RC-2B) and f u r t h e r a t 105,000g f o r 1 h r ( S p i n c o , 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 , M o d e l L ) . A l l f u r t h e r p r e p a r a t i v e s t e p s were c a r r i e d out i n an i c e b a t h . T h e enzyme was p u r i f i e d by (NH^^SO^ f r a c t i o n a t i o n between 35 and 60% s a t u r a t i o n . The f i n a l e x t r a c t was r e s u s p e n d e d i n a m i n i m a l q u a n t i t y o f t h e above b u f f e r , minus s u c r o s e , and d i a l y z e d a g a i n s t t h e same b u f f e r f o r two h o u r s t o e l i m i n a t e s a l t s ( t h i s p r o c e d u r e t e n d s t o r e d u c e t h e enzyme a c t i v i t y , so i t i s n o t recommended t o i n c r e a s e t h e d i a l y s i s p e r i o d ) . T h i s enzyme p r e p a r a t i o n i s s t a b l e f o r a t l e a s t one month when s t o r e d a t -30°C. P r e p a r a t i o n o f l i v e r s f r o m b r o o k , l a k e and s p l a k e t r o u t depended u p o n t h e t i m e o f y e a r c o l l e c t e d . A l l w i n t e r a n i m a l s were s a c r i f i c e d , l i v e r s 15 removed and q u i c k l y f r o z e n as a group. S p r i n g a n i m a l s were i n i t i a l l y f r o z e n i n l i q u i d n i t r o g e n and a t a l a t e r t i m e , t h e l i v e r s were i n d i v i d u a l l y r emoved. The l i v e r enzyme f r o m i n d i v i d u a l summer a n i m a l s was p r e p a r e d a n d s u b j e c t e d t o e l e c t r o p h o r e s i s b e f o r e and a f t e r f r e e z i n g o f t h e t i s s u e s . No d i f f e r e n c e s i n e l e c t r o p h o r e t i c m o b i l i t y c o u l d be d e t e c t e d between f r e s h l y p r e p a r e d and f r o z e n s a m p l e s . I n a l l c a s e s , t h e l i v e r t i s s u e s were homogenized i n f o u r volumes of 5 0 mM t r i s c o n t a i n i n g 0.25 M s u c r o s e and t i t r a t e d t o pH 7.2 w i t h 1 M HC1, a c c o r d i n g t o t h e method of de Duve, e t a l . ( 1 9 5 5 ) . S i n c e no u n i q u e m i t o c h o n d r i a l NADP-IDH p a t t e r n c o u l d be d e t e c t e d , t h e s u p e r n a t a n t r e m a i n i n g a f t e r a 30 m i n c e n t r i f u g a t i o n a t 27,000g was used as a s o u r c e o f enzyme f o r e l e c t r o p h o r e s i s . P i g h e a r t s o l u b l e NADP-IDH i s a v a i l a b l e c o m m e r c i a l l y f r o m Sigma C h e m i c a l Co. ( S t . L o u i s , M i s s o u r i , No. 1-2002) i n a 50% g l y c e r o l s o l u t i o n . T h i s p r e p a r a t i o n was used as a s o u r c e o f t h e enzyme f o r column c h r o m a t o -g r a p h y and e l e c t r o p h o r e s i s f o l l o w i n g p r o p e r d i l u t i o n w i t h 50mM t r i s - H C l b u f f e r , pH 7.3. A s s a y o f NADP-IDH a c t i v i t y . NADP-IDH c a t a l y s e s t h e r e v e r s i b l e o x i d a t i v e d e c a r b o x y l a t i o n o f t h r e o -D g i s o c i t r a t e a c c o r d i n g t o t h i s r e a c t i o n sequence w h i c h proceeds t h r o u g h a n u n s t a b l e i n t e r m e d i a t e (Ochoa, 1948) : E + t h r e o - D i s o c i t r a t e + NADP + s E * ( o x a l o s u c c i n a t e ) + NADPH + H+ \ Me4"*" o r Mn4"4" I C( - k e t o g l u t a r a t e + C 0 9 + E 16 T h e s p e c i f i c a b s o r b a n c y o f NAI(E)H a t 340 nm (E34Q) can be used as a means of f o l l o w i n g t h i s r e a c t i o n . A l l a s s a y s were c a r r i e d out on e i t h e r a Unicam SP 800 or SP 1800 s p e c t r o p h o t o m e t e r (Pye Unicam, L t d . , Cambridge, E n g l a n d ) . The b a s i c r e a c t i o n m i x t u r e c o n t a i n e d 50 mM t r i s - H C l b u f f e r , pH 8.0 ( t e m p e r a t u r e a d j u s t e d a c c o r d i n g t o Sigma T e c h n i c a l B u l l e t i n No. 106B, 1 9 6 7 ) , 1 mM MgCl2» 0.15 mM NADP +, v a r i o u s D L - i s o c i t r a t e c o n c e n t r a t i o n s , a n d enzyme, added l a s t i n a t o t a l volume of 2.0 m l . A l l c h e m i c a l s were p u r c h a s e d f r o m Sigma C h e m i c a l Co., S t . L o u i s , M i s s o u r i . Ln a l l c a s e s , i s o c i t r a t e c o n c e n t r a t i o n s a r e g i v e n as D L - i s o c i t r a t e , even though o n l y 5 0% o f t h i s i s t h e e n z y m a t i c a l l y a c t i v e t h r e o - D s f o r m ( P l a u t , 1 9 6 3 ) . C u v e t t e t e m p e r a t u r e s were a c c u r a t e l y c o n t r o l l e d by t h e use o f a c i r c u l a t i n g w a t e r b a t h (Lauda B r i n k m a n , K-2/R) c o u p l e d t o t h e c u v e t t e h o l d e r . E s t i m a t e s o f p r o t e i n c o n t e n t . The p r o t e i n c o n t e n t o f a l l enzyme s o l u t i o n s was d e t e r m i n e d by t h e method o f Lowry, Rosebrough, F a r r & R a n d a l ( 1 9 5 1 ) . A l l samples were d i l u t e d w i t h w a t e r t o a p p r o x i m a t e l y 50 yugm/ml p r o t e i n , and compared w i t h a s t a n d a r d c u r v e d e t e r m i n e d f r o m 0 t o 100 }jgm/ml b o v i n e serum albumen p r o t e i n . S t a r c h - g e l e l e c t r o p h o r e s i s . H o r i z o n t a l s t a r c h - g e l e l e c t r o p h o r e s i s was c a r r i e d out a c c o r d i n g t o S m i t h i e s (1955) u s i n g a c i t r a t e - p h o s p h a t e b u f f e r s y s t e m a t pH 7.0 ( t a n k b u f f e r was 9 mM c i t r i c a c i d and 90 mM d i b a s i c sodium p h o s p h a t e ; g e l b u f f e r i s a 2 0 - f o l d d i l u t i o n o f t h i s t a n k b u f f e r ) . A l t e r n a t e b u f f e r s y s t e m s were employed, b u t r e s o l u t i o n was found t o be t h e b e s t w i t h t h e c i t r a t e - p h o s p h a t e s y stem. S u p e r n a t a n t samples were a p p l i e d t o t h r e e p i e c e s o f 5 mm s q u a r e Whatman No. 1 f i l t e r p a p e r , and two t h i c k n e s s e s of t h i s same paper were used as a b r i d g e between t h e e l e c t r o d e t a n k s and t h e g e l . Adequate s e p a r a t i o n o f i s o z y m e s was o b t a i n e d u s i n g a 13% s t a r c h - g e l ( H y d r o l y s e d , Connaught M e d i c a l R e s e a r c h L a b o r a t o r i e s , T o r o n t o , O n t a i r o ) and e l e c t r o p h o r e s i n g a t 200 V ( a p p r o x i m a t e l y 20mA) f o r 17 h r a t 4°C. The g e l s were s t a i n e d f o r NADP-IDH a c t i v i t y u s i n g a m o d i f i e d h i s t o c h e m i c a l s t a i n d e v e l o p e d by H u n t e r & M a r k e r t ( 1 9 5 7 ) . The s t a i n i n g s o l u t i o n c o n t a i n e d 62 mgm D L - i s o c i t r a t e , 15 mgm NADP +, 15 mgm n i t r o b l u e t e t r a z o l i u m , a p p r o x i m a t e l y 5 ^ugm p h e n a z i n e m e t h o s u l f a t e , and 2.5 m l o f 20 mM M g C l 2 s o l u t i o n i n 50 ml of 0.1 M t r i s - H C l b u f f e r , pH 7.0. A l l c h e m i c a l s were o b t a i n e d f r o m Sigma C h e m i c a l Co. S t a i n i n g was c a r r i e d o u t on s l i c e d g e l s i n t h e d a r k a t room t e m p e r a t u r e f o r 2 h r . Numerous NADP-IDH is o z y m e p a t t e r n s (zymograms) were scanned u s i n g a r e c o r d i n g m i c r o d e n s i t o m e t e r ( J o y c e , L o e b l , B u r l i n g t o n , M a s s . ) . T h i s method can be u s e d t o q u a n t i f y t h e d i s t r i b u t i o n o f i s o z y m i c bands. The a r e a under each peak i s d e t e r m i n e d and e x p r e s s e d as a p e r c e n t of t h e e n t i r e a r e a . I s o e l e c t r i c f o c u s i n g of NADP-IDH. E l e c t r o f o c u s i n g o f t h e r a i n b o w t r o u t l i v e r enzyme was c a r r i e d o u t a c c o r d i n g t o t h e method o f Haglund (1967) t o e s t i m a t e p i v a l u e s f o r e a c h l i v e r i s o z y m e . The b e s t r e s o l u t i o n was a t t a i n e d u s i n g a pH 3-10 g r a d i e n t (LKB A m p h o l i n e 8 1 4 1 ) , r u n a t 300 V f o r 32 h r . L onger e l e c t r o f o c u s i n g as w e l l as h i g h e r v o l t a g e s gave l o w e r enzyme r e c o v e r y even though t h e column was m a i n t a i n e d a t 5°C t h r o u g h o u t t h e e x p e r i m e n t . 18 Each 2 ml f r a c t i o n was c o l l e c t e d u s i n g a LKB u l t r o r a c f r a c t i o n c o l l e c t o r and a s s a y e d f o r NADP-IDH a c t i v i t y by t h e s t a n d a r d method. The i s o e l e c t r i c p o i n t of each isozyme peak was e s t i m a t e d f r o m t h e pH p r o f i l e . D E A E - c e l l u l o s e c hromatography. The enzyme was p r e p a r e d as above, e x c e p t t h a t t h e h o m o g e n i z a t i o n b u f f e r was 1 mM t r i s + 1 mM EDTA + 1 mM ^ - m e r c a p t o e t h a n o l + 0.25 M s u c r o s e , t i t r a t e d t o pH 7.5 w i t h 1 M HC1. The 35-60% ( N H 4 ) 2 S 0 4 p r e c i p i t a t e d f r a c t i o n was r e s u s p e n d e d i n a m i n i m a l q u a n t i t y o f t h e above b u f f e r ( m i n u s s u c r o s e ) and d i a l y z e d f o r 12 h r a g a i n s t e i g h t l i t e r s o f t h e same b u f f e r . The d i a l y z e d sample was a p p l i e d t o a column(2.0 x 25 cm) of D E A E - c e l l u l o s e (Whatman, M i c r o g r a n u l a r ) p r e v i o u s l y e q u i l i b r a t e d t o 4°C w i t h t h e same b u f f e r . A t l e a s t two column volumes of t h e i n i t i a l b u f f e r w e r e n e c e s s a r y t o wash out a l l n o n - a d h e r i n g p r o t e i n p r i o r t o e l u t i o n o f t h e NADP--IDH i s o z y m e s , The enzyme was e l u t e d w i t h a l i n e a r g r a d i e n t i n w h i c h t h e t r i s c o n c e n t r a t i o n was i n c r e a s e d t o 0.3 M. Each 2 ml f r a c t i o n was c o l l e c t e d and a s s a y e d f o r NADP-IDH a c t i v i t y as above. The g r a d i e n t was d e t e r m i n e d by c o n d u c t i v i t y measurements o f e l u t e d samples a n d compared t o t h o s e o f s t a n d a r d N a C l s o l u t i o n s . S u c r o s e g r a d i e n t c e n t r i f u g a t i o n . S u c r o s e g r a d i e n t c e n t r i f u g a t i o n was u n d e r t a k e n i n o r d e r t o compare t h e m o l e c u l a r w e i g h t s o f t h e NADP-IDH fr o m r a i n b o w t r o u t and t h e p u r i f i e d p i g h e a r t enzyme, as w e l l as t o d e t e r m i n e i f t h e i s o z y m e s o f t h e t r o u t enzyme had i d e n t i c a l w e i g h t s . Samples were r u n u s i n g a S p i n c o 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 e q u i p p e d w i t h a SW 39 r o t o r . The 5 t o 20% g r a d i e n t s were p r e p a r e d w i t h a d u a l chamber g r a d i e n t maker i n 100 mM t r i s 19 b u f f e r c o n t a i n i n g 1 mM ^-mercaptoethanol and 2 mM EDTA, t i t r a t e d w i t h 1 M HC1 t o pH 7.0. A 0.2 ml enzyme sample was l a y e r e d onto the top o f each g r a d i e n t and run f o r t e n hours at 35,000 RPM and 0°C. Drops were c o l l e c t e d by g r a v i t y from a hole punched i n the bottom of each tube and assayed f o r NADP-IDH a c t i v i t y u s i n g the standard assay. Gradient d e n s i t i e s 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 readings. G e l - f i l t r a t i o n o f NADP-IDH. Sephadex G-100 (Pharmacia, Uppsala, Sweden) g e l - f i l t r a t i o n was c a r r i e d out as another method t o determine comparable s i z e o f the rainbow t r o u t l i v e r isozymes. A 1 x 32 cm column was employed w i t h a V Q of approximately 6.5 ml as determined by dye d i l u t i o n u s i n g Dextran Blue 2000 (Pharmacia). The p r e s w o l l e n g e l was packed under g r a v i t y , and e q u i l i b r a t e d at k°C w i t h 50 mM t r i s b u f f e r w i t h 2 mM ^-mercaptoethanol t i t r a t e d t o pH 7-2 w i t h 1 M HC1. A 1 ml sample o f enzyme was a p p l i e d t o the top of the column, and the a c t i v i t y was e l u t e d w i t h the same b u f f e r . One ml f r a c t i o n s were c o l l e c t e d w i t h a LKB u l t r o r a c f r a c t i o n c o l l e c t o r and assayed f o r NADP-IDH a c t i v i t y u s i n g the standard assay. CHAPTER I I I : E f f e c t s o f Thermal A c c l i m a t i o n on M u l t i p l e Forms o f t h e L i v e r S o l u b l e N A D P + - L i n k e d I s o c i t r a t e Dehydrogenase i n t h e F a m i l y S a l m o n i d a e 20 INTRODUCTION The e x i s t e n c e o f a m i t o c h o n d r i a l (M-IDH) and a s u p e r n a t a n t (S-IDH) N A D P + - s p e c i f i c i s o c i t r a t e dehydrogenae (IDH) (EC 1.1.1.42) has been d e m o n s t r a t e d i n m i c e s t r a i n s ( H e n d e r s o n , 1965; M i n t z & B a k e r , 1 9 6 7 ) , i n Paramecium a u r e l i a ( T a i t , 1970) and i n s m e l t , c a r p and g o l d f i s h ( Q u i r o z - G u t i e r r e z & Ohno, 1 9 7 0 ) . M u l t i p l e forms o f t h i s enzyme a r e a l s o known i n a number of b a c t e r i a ( R e e v e s , Brehmeyer & A j l , 1968; S e l f & Weitzman, 1 9 7 0 ) . I n a l l c a s e s t h u s f a r examined, t h e s e m u l t i p l e f o r m s d i f f e r i n k i n e t i c , m o l e c u l a r and e l e c t r o p h o r e t i c p r o p e r t i e s , a l t h o u g h t h e i r f u n c t i o n a l s i g n i f i c a n c e has n o t been c l a r i f i e d . I n t h o s e o r g a n i s m s where t h e S-form IDH has been e x t e n s i v e l y s t u d i e d , t h e enzyme a p p e a r s as a d i m e r , w i t h a m o l e c u l a r w e i g h t o f a p p r o x i m a t e l y 75,000 ( I l l i n g w o r t h & T i p t o n , 1970; Howard & B e c k e r , 1 9 7 0 ) . G e n e t i c a n a l y s i s c a r r i e d out u s i n g m i c e s t r a i n s ( M i n t z & B a k e r , 1967; Henderson, 1965) and s m e l t , a d i p l o i d f i s h ( Q u i r o z - G u t i e r r e z & Ohno, 1 9 7 0 j , s u g g e s t t h a t t h e enzyme i s s p e c i f i e d by a s i n g l e gene l o c u s . M u l t i p l i c i t y o f p r o t e i n s t r u c t u r e has been found t o o c c u r f r e q u e n t l y i n S a l m o n i d f i s h e s . T h i s group of f i s h e s a r e b e l i e v e d t o be t e t r a p l o i d , m a i n l y f r o m t h e f i n d i n g by Ohno, Wolf & A t k i n (1968) t h a t t h e s e a n i m a l s m a i n t a i n about t w i c e as much D N A / c e l l as f o u n d i n most v e r t e b r a t e s . Enzymes such as m a l a t e dehydrogenase ( B a i l e y , Cocks & W i l s o n , 1 9 6 9 ) , l a c t a t e d ehydrogenase (Massaro & M a r k e r t , 1 9 6 8 ) , e n o l a s e ( T s u y u k i & Wold, 1 9 6 4 ) , a l d o l a s e ( L e b h e r z & R u t t e r , 1 9 6 9 ) , and c r e a t i n e k i n a s e ( E p p e n b e r g e r , S c h o l l & U r s p r u n g , 1971) a r e known t o o c c u r as m u l t i p l e enzyme s y s t e m s , s u p p o r t i n g t h i s t e t r a p l o i d h y p o t h e s i s . Work 21 m e n t i o n e d by Q u i r o z - G u t i e r r e z & Ohno ( 1 9 7 0 ) , but not r e p o r t e d , and by W o l f , E n g e l & F a u s t (1970) on Salmo i r i d e u s i n d i c a t e t h a t t h e gene l o c i c o d i n g f o r t h e h e a r t M-IDH has a l s o been d u p l i c a t e d , a l t h o u g h t h e l i v e r S-IDH a p p a r e n t l y has n o t . Carp and g o l d f i s h , a l s o b e l i e v e d t o be t e t r a p l o i d f i s h , were shown t o be endowed w i t h two s e p a r a t e gene l o c i f o r S-form IDH ( Q u i r o z -G u t i e r r e z & Ohno, 1970). A p a r t of t h e e v i d e n c e i n f a v o u r of t h i s h y p o t h e s i s i s t h a t t h e p r e d i c t e d f r e q u e n c y o f p h e n o t y p i c e x p r e s s i o n of t h e enzyme v a r i a n t was i n f a c t o b s e r v e d i n a w i l d p o p u l a t i o n o f g o l d f i s h . I n t h i s s t u d y , as i n o t h e r s of i t s t y p e , t h e i m p l i c i t a s s u m p t i o n i s t h a t e x t r i n s i c f a c t o r s have no i n f l u e n c e on t h e e x p r e s s i o n of t h e s e g e n e s . P r e v i o u s s t u d i e s o f r a i n b o w t r o u t S-IDH (Moon & Hochachka, 1971) and o t h e r i s o z y m e systems i n f i s h (Hochachka, 1965; B a l d w i n & H o c h a c h k a , 1970; Hochachka & Somero, 1971) i n d i c a t e t h a t t h e e x p r e s s i o n o f d i f f e r e n t enzyme v a r i a n t s can depend upon s u c h e n v i r o n m e n t a l p a r a m e t e r s a s s e a s o n and t h e r m a l a c c l i m a t i o n . To g a i n f u r t h e r i n s i g h t i n t o t h i s p r o b l e m , f o u r members of t h e S a l m o n i d a e f a m i l y a r e examined f o r m u l t i p l e S-form IDH i s o z y m e s . Based upon t h e a s s u m p t i o n t h a t t h e l i v e r S-form IDH i s a d i m e r , t h e IDH of l a k e t r o u t (Salmo namaycush) i s assembled f r o m a s i n g l e s u b u n i t t y p e whose e x p r e s s i o n i s t e m p e r a t u r e i n d e p e n d e n t ; t h e IDHs o f b r o o k t r o u t ( S a l v e l i n u s f r o n t i n a l i s ) a r e formed by d i m e r a s s e m b l y f r o m s u b u n i t t y p e s whose d i s t r i b u t i o n i s t e m p e r a t u r e dependent; a s s e m b l y of t h e i s o z y m e s i n s p l a k e t r o u t r e q u i r e s a t l e a s t two s u b u n i t t y p e s , t h e e x p r e s s i o n o f w h i c h i s c o m p l e x l y a f f e c t e d by t e m p e r a t u r e . RESULTS 22 W i n t e r L a k e , S p l a k e and B r o o k T r o u t S-form IDH. P o o l e d samples of w i n t e r b r o o k t r o u t y i e l d a p a t t e r n of f i v e a n o d a l l y m o v i n g i s o z y m e s ( P i g . I l l , I D ) . T h i s p a t t e r n i s c o n s i s t e n t w i t h t h e i d e a o f h e t e r o z y g o s i t y a t one of t h e two gene l o c i as s u g g e s t e d by Q u i r o z -G u t i e r r e z & Ohno (1970) f o r g o l d f i s h . S i n c e i n d i v i d u a l s were n o t a s s a y e d , i t i s i m p o s s i b l e t o d e t e r m i n e whether o r n o t p o l y m o r p h i c .specimens a r e seen i n t h i s group. T h e r e a r e no d e t e c t a b l e e f f e c t s of- a c c l i m a t i o n s e e n i n t h e s e w i n t e r b r o o k t r o u t . W i n t e r s p l a k e t r o u t d e m o n s t r a t e an e x c e e d i n g l y complex a c c l i m a t i o n p a t t e r n . F i g . I l l , 1 shows t h a t t h e r e has been a s h i f t i n t h e i s o z y m e p a t t e r n t o w a r d s t h o s e forms w i t h l o w e r m o b i l i t y a t t h e extreme a c c l i m a t i o n t e m p e r a t u r e s (4° and 17°C, p a n e l s B and E ) . The t h r e e bands seen i n t h e 9°C t r o u t c o r r e s p o n d t o t h e t h r e e f a s t e s t moving a n o d a l bands i n t h e b r o o k t r o u t , and t h e 4°C bands show h o m o l o g i e s w i t h t h e t h r e e s l o w e r m i g r a t i n g b r o o k t r o u t bands (see F i g . I l l , 1 ) . The 17°C p a t t e r n o v e r l a p s o n l y i t s most a n o d a l band w i t h t h e b r o o k t r o u t p a t t e r n . T h i s i s u n e x p e c t e d s i n c e t h e s p l a k e i s a b r o o k t r o u t - l a k e t r o u t h y b r i d c r o s s , a n d n e i t h e r o f t h e p a r e n t a l s p e c i e s e x p r e s s e s t h i s s l o w e s t moving band ( o r s u b u n i t ) . Whether t h i s 17°C i s o z y m e p a t t e r n i s due t o g e n e t i c o r i p h y s i c a l c h a r a c t e r i s t i c s o f t h e enzyme i s unknown, a l t h o u g h numerous e l e c t r o p h o r e s i s r u n s o v e r l o n g t i m e p e r i o d s show t h e same s e p a r a t i o n . The i n t e r e s t i n g f e a t u r e o f t h e l a k e t r o u t S-form IDH f r o m l i v e r , i s t h a t genome d u p l i c a t i o n , i f i t d i d o c c u r , d i d n o t r e s u l t i n t h e p r o d u c t i o n o f d i f f e r e n t e l e c t r o p h o r e t i c forms as i n t h e o t h e r s a l m o n i d s s t u d i e d h e r e ( F i g . I l l , 1 A ) . S t u d i e s by W o l f , E n g e l & F a u s t (1970) s u g g e s t 2 3 F i g . I l l , 1. Composite s t a r c h - g e l e l e c t r o p h o r e t o g r a m o f w i n t e r a c c l i m a t e d l a k e , s p l a k e and b r o o k t r o u t l i v e r S-form IDH. 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 : 17 h r a t 15 mA and 200 V, i n p h o s p h a t e - c i t r a t e b u f f e r , pH 7.0. G e l t e m p e r a t u r e c o n s t a n t a t 5-6°C. Anode a t bottom o f a l l e l e c t r o p h o r e t o g r a m s , O r i g i n marked by 0. A. 9°C-acclimated l a k e t r o u t ; B. 4°C-acclimated s p l a k e t r o u t ; C. 9°C-acclimated s p l a k e t r o u t ; D. 9°C-acclimated b r o o k t r o u t ; and E. 17°C-acclimated s p l a k e t r o u t . o A B C D E 24 t h a t t h e l i v e r S-form IDH l o c u s of Salmo i r i d e u s s i m i l a r l y has n o t u n d e r g o n e d u p l i c a t i o n . Lake and r a i n b o w t r o u t l i v e r LDHs e x h i b i t i d e n t i c a l s i n g l e band p a t t e r n s (Hochachka, 1966). A g a i n , as i n b r o o k t r o u t , t h e r e i s a p p a r e n t l y no a c c l i m a t i o n e f f e c t , w i t h t h e i s o z y m e p a t t e r n b e i n g the same a t 4° and 17°C. I n summary, gene d u p l i c a t i o n r e s u l t i n g i n v a r i a n t enzyme forms h a s a p p a r e n t l y o c c u r r e d i n b r o o k t r o u t l i v e r S-form IDH, but n o t i n . l a k e t r o u t . S i n c e s p l a k e t r o u t i s a h y b r i d , t h i s c h a r a c t e r i s t i c p a t t e r n s e e n i n t h e b r o o k t r o u t i s c a r r i e d o v e r w i t h t h e a d d i t i o n o f a non-homologous s u b u n i t t y p e i n t h e c a s e o f t h e 17°C s p l a k e t r o u t . O n l y i n t h e c a s e o f s p l a k e t r o u t l i v e r S-form IDH i s t h e r e a s h i f t i n p a t t e r n w i t h t e m p e r a t u r e a c c l i m a t i o n , a l t h o u g h t h e g e n e t i c b a s i s f o r t h i s i s n o t u n d e r s t o o d . I t may be, however, t h a t t h e 17°C p a t t e r n seen i n s p l a k e t r o u t r e p r e s e n t s a m u t a t e d l o c u s d e r i v e d f r o m t h e b r o o k t r o u t , w h i c h i s n o t e x p r e s s e d e x c e p t under p r o p e r c o n d i t i o n s of s e a s o n and t e m p e r a t u r e . S p r i n g L a k e , S p l a k e and B r o o k T r o u t S-form IDH. L a ke t r o u t a c c l i m a t e d t o 4° and 17°C i n t h e s p r i n g e x h i b i t i d e n t i c a l e l e c t r o p h o r e t i c p a t t e r n s , and, i n f a c t , t h e s e p a t t e r n s a r e unchanged f r o m t h e w i n t e r g roup. A g a i n , o n l y a s i n g l e enzyme f o r m i s p r e s e n t . S p l a k e t r o u t a c c l i m a t e d under i d e n t i c a l c o n d i t i o n s a l s o e x h i b i t s i m i l a r s p r i n g p a t t e r n s , but t h e s e a r e u n l i k e t h o s e seen i n w i n t e r f i s h . The s p r i n g p a t t e r n i s i d e n t i c a l t o t h a t seen a t 9°C d u r i n g t h e w i n t e r . The s p r i n g b r o o k t r o u t a c c l i m a t e d t o 4° and 17°C d e m o n s t r a t e d a t l e a s t f o u r d i f f e r e n t e l e c t r o p h o r e t i c p a t t e r n s ( F i g . I l l , 2) based on i n d i v i d u a l s a mples. S t a t i s t i c a l i n t e r p r e t a t i o n of t h e s e d a t a was 25 F i g . I l l , 2. R e s o l u t i o n o f s p r i n g a c c l i m a t e d b r o o k t r o u t l i v e r S-form IDH on s t a r c h - g e l 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 c o n d i t i o n s as i n F i g . I l l , 1. Examples o f f o u r e l e c t r o p h o r e t i c p a t t e r n s a r e : B r - 1 i n c o l u m n C, D and G; B r - 2 a i n A and F; B r - 3 i n E and H; and Br-4 i n B. 26 d i f f i c u l t because of t h e s m a l l sample s i z e ( o n l y e i g h t f i s h i n each a c c l i m a t i o n g r o u p ) . The dominant p a t t e r n r e m a i n e d t h a t seen i n w i n t e r b r o o k t r o u t £five i s o z y m e s ) and i s c a l l e d B r - 1 ; t h i s group c o n s i s t e d o f 6 of t h e 16 specimens u s e d . The second most abundant p a t t e r n ( B r - 2 , F i g . I l l , 2) made up f i v e o f t h e 16 s p e c i m e n s . B r - 3 c o n s i s t e d o f 3 o f 16, an d was a v a r i a n t o f p a t t e r n B r - 1 w i t h o u t the e x p r e s s i o n o f t h e most a n o d a l homodimer. Br-4 ( o c c u r r i n g o n l y 2 o f 16 t i m e s ) c o n s i s t s o f a s i x - i s o z y m e p a t t e r n , a l s o s i m i l a r t o B r - 1 . F i g . I l l , 3 i s a d i a g r a m m a t i c r e p r e s e n t a t i o n o f t h e s e f o u r d i s t i n c t b a n d i n g p a t t e r n s , u s i n g a n o m e n c l a t u r e s i m i l a r t o t h a t o f Q u i r o z -G u t i e r r e z & Ohno ( 1 9 7 0 ) . I t can be seen t h a t t h e p a t t e r n s a r e c o n s i s t e n t w i t h t h e e x i s t e n c e o f a t l e a s t f o u r homodimers (AA, a a , BB and bb) p l u s t h e numerous h y b r i d forms o f each homodimer. Summer S p l a k e and B r o o k T r o u t S-form IDH. S i n c e no u n u s u a l isozyme p a t t e r n s were seen i n l a k e t r o u t d u r i n g t h e w i n t e r and s p r i n g t e s t i n g , no work was c a r r i e d out on t h e s e t r o u t d u r i n g t h e summer. A l s o , l a k e t r o u t a r e e x t r e m e l y h a r d t o warm a c c l i m a t e ( P e t e r I h s s e n , p e r s o n a l c o m m u n i c a t i o n ) a p p a r e n t l y due t o t h e i r l o n g a n c e s t r a l h i s t o r y a t t h e b o t t o m of l a k e s where t e m p e r a t u r e s a r e v e r y l o w . A l s o , no d i f f e r e n c e s i n i s o z y m i c p a t t e r n s were d e t e c t e d i n summer s p l a k e as compared t o t h o s e r u n i n t h e s p r i n g , so t h e y w i l l n o t be d e a l t w i t h f u r t h e r . A g a i n , i n d i v i d u a l b r o o k t r o u t showed v a r i a n t S-form IDH p a t t e r n s , a l t h o u g h t h e number of v a r i a n t s was r e d u c e d f r o m s p r i n g . U n l i k e t h e s p r i n g e x p e r i m e n t s , a l a r g e sample s i z e was c o l l e c t e d a l l o w i n g f o r a s t a t i s t i c a l t r e a t m e n t o f t h e d a t a . F i g . I l l , 4 r e p r e s e n t s a d i a g r a m m a t i c a l 27 Fig. I l l , 3. Diagrammatical representation of spring acclimated brook trout l i v e r S-form isozyme patterns seen i n F i g . I l l , 2. < < < o o < jQ l_ m < < o ro i a GO CD CD o CM I CD <t a o o < < U OQ CD CD 28 Fig. I l l , 4. A diagrammatical representation of S-form IDH isozymes from summer acclimated brook trout l i v e r observed after starch-gel e l e c t r o -phoresis. Only r e l a t i v e positions of the bands are shown i n the three d i s t i n c t patterns. representation of the three patterns, with the r e l a t i v e staining i n t e n s i t i e s indicated. In Table 1, the frequency d i s t r i b u t i o n of these three variant patterns i s given, correlating both temperature acclimation i n one case, and sex i n the other. According to X^ values, the frequency d i s t r i b u t i o n between the 4° and 17°C acclimated groups i s s i g n i f i c a n t l y d i f f e r e n t , with the 17°C group having more of the Br-2 pattern and the 4°C group more of the Br-1 pattern. There i s l i t t l e difference between the sexes as far as the frequency of bands i s concerned. 30 T a b l e I I I , 1. F r e q u e n c y d i s t r i b u t i o n o f t h e t h r e e S-form IDH isozyme p a t t e r n s f r o m summer a c c l i m a t e d b r o o k ' t r o u t l i v e r , w i t h C h i s q u a r e c a l c u l a t i o n s . B r - 1 Br-2 B r - 3 T o t a l X 2 - V a l u e A c c l i m a t i o n t e m p e r a t u r e 4°C 13 5 5 23 17°C 14 24 5 43 Sex M a l e 16 11 3 30 Female 11 16 7 34 28.9 6.5 0.005(2) = 10,6 31 DISCUSSION The r e s u l t s o f t h e s e e l e c t r o p h o r e t i c e x p e r i m e n t s t e n d t o s u p p o r t t h e o b s e r v a t i o n s o f Ohno e_t a l _ . (1968) as t o t h e t e t r a p l o i d n a t u r e o f s a l m o n i d f i s h e s . As can be seen i n t h e c o m p o s i t e e l e c t r o -p h o r e t o g r a m ( F i g . I l l , 5a) and t h e accompanying d i a g r a m m a t i c a l r e p r e s e n -t a t i o n o f t h e t y p i c a l f o u r t r o u t p a t t e r n s ( F i g . I l l , 5 b ) , a l l r e s u l t s c a n be e x p l a i n e d i f two s e p a r a t e gene l o c i f o r S-form IDH a r e m a i n t a i n e d i n t h e s a l m o n i d genome ( Q u i r o z - G u t i e r r e z & Ohno, 1970) as i n h e a r t M-form IDH ( W o l f , E n g e l & F a u s t , 1 9 7 0 ) . The s p l a k e and r a i n b o w t r o u t l i v e r S-form IDH show a t w o - s u b u n i t p a t t e r n , whereas f o u r s u b u n i t s i n t h e b r o o k t r o u t a r e n o t u n u s u a l ( s p r i n g e x p e r i m e n t , F i g . I l l , 2 ) . The l a k e t r o u t i s u n i q u e i n p r o d u c i n g o n l y a s i n g l e s u b u n i t t y p e , a f i n d i n g s i m i l a r t o t h a t o f W o l f , E n g e l &. F a u s t (1970) f o r l i v e r S-form IDH i n Salmo i r i d e u s . The g e n e t i c model o f Q u i r o z - G u t i e r r e z & Ohno (1970) i s c o m p a t i b l e w i t h t h e t y p i c a l b r o o k p a t t e r n seen i n F i g . I l l , 5a,b, i . e . , two gene l o c i , w h i c h a r e h e t e r o z y g o u s i n one o r t h e o t h e r a l l e l e . H owever, s u c h a model c a n n o t e x p l a i n t h e r e s u l t s seen i n t h e s p r i n g e x p e r i m e n t , n o r f o r t h a t m a t t e r , i n t h e 17°C-acclimated summer b r o o k t r o u t . T e n t a t i v e l y , we s u g g e s t t h a t t h e b r o o k t r o u t l i v e r S-form IDH c o n s i s t s o f f o u r b a s i c s u b u n i t s ( a l t h o u g h n o t a l l f i s h w i l l e x p r e s s them a l l a t any one t i m e ) , whose e x p r e s s i o n i s i n d u c e d by t e m p e r a t u r e c h a n g e o v e r l o n g t i m e p e r i o d s ( i . e . , a c c l i m a t i o n ) . This would e x p l a i n t h e d i f f e r e n c e s seen i n t h e f r e q u e n c y d i s t r i b u t i o n d a t a g i v e n i n T a b l e I I I , 1 between t h e 4° and 17°C-acclimated b r o o k t r o u t . Such a s h i f t i n i s o z y m e p a t t e r n has been seen i n r a i n b o w t r o u t l i v e r S-form IDH F i g . I l l , 5a. Composite e l e c t r o p h o r e t o g r a m o f l i v e r S-form IDH fr o m t h e f a m i l y S a l m o n i d a e on s t a r c h - g e l . 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 as i n F i g . I l l , 1. Lake t r o u t , A and H. T y p i c a l s p l a k e t r o u t 2°C p a t t e r n , B. E q u a l amounts o f w i n t e r a c c l i m a t e d 9° and 17°C s p l a k e t r o u t , C. 2oc- " a c c l i m a t e d r a i n b o w t r o u t , D. 17°C-acclimated r a i n b o w t r o u t , E. Dominant b r o o k t r o u t p a t t e r n s , F and G. b. D i a g r a m m a t i c a l r e p r e s e n t a t i o n o f p o s s i b l e l i v e r S-form IDH s u b u n i t t y p e s f r o m t h e f a m i l y S a l m o n i d a e o b s e r v e d f r o m v a r i o u s s t a r c h - g e l e l e c t r o p h o r e t o g r a m s . A B C D E F G H A A A'A Ba • m Brook Splake Lake Rainbow 33 (Moon & Hochachka, 1971) and 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 ( B a l d w i n & H o c h a c h k a , 1 9 7 0 ) , a l t h o u g h t h e s e s ystems have f e w e r i s o z y m i c f o r m s . S p l a k e t r o u t , a b r o o k t r o u t - l a k e t r o u t h y b r i d , i s complex o n l y i n t h e w i n t e r a c c l i m a t e d a n i m a l s ( F i g . I l l , 1 ) . I n a l l o t h e r s e a s o n s , n o q u a l i t a t i v e a c c l i m a t i o n phenomenon i s a p p a r e n t , and t h e i s o z y m e s y s t e m c a n be d e f i n e d as a s i m p l e two homodimer system. The w i n t e r p a t t e r n i s u n u s u a l , i n t h a t i t r e q u i r e s t h e e x p r e s s i o n of a n o t h e r s u b u n i t n o t p r e s e n t i n t h e l i v e r IDH f r o m e i t h e r p a r e n t a l s t o c k . B r e e d i n g e x p e r -i m e n t s a r e n e c e s s a r y t o d e t e r m i n e what p o s s i b l y c o n t r o l s t h e e x p r e s s i o n o f t h i s s l o w e s t m i g r a t i n g s u b u n i t i n t h e s p l a k e t r o u t l i v e r . As d e m o n s t r a t e d i n t h e w i n t e r e x p e r i m e n t s , t h e s e s u b u n i t s a r e p r e s e n t , but t h e i r e x p r e s s i o n a p p e a r s t o be t i g h t l y r e g u l a t e d . E c o l o g i c a l l y t h e l a k e t r o u t i s u n i q u e among t h e s a l m o n i d f i s h e s s t u d i e d i n b e i n g a d a p t e d t o t h e b e n t h i c e n v i r o n m e n t w h i c h i n deep l a k e s o f t e n d i s p l a y s n e a r c o n s t a n c y of t e m p e r a t u r e , s a l i n i t y , p r e s s u r e , l i g h t , e t c . And, i n d e e d , o f t h e s p e c i e s examined i n t h i s s t u d y , i t i s known t o be so s t e n o t h e r m a l t h a t i t i s d i f f i c u l t t o warm a c c l i m a t e . The o c c u r r e n c e o f a s i n g l e S-form IDH i n t h i s s p e c i e s i s c o n s i s t e n t w i t h t h e i d e a t h a t o r g a n i s m s w h i c h have e v o l v e d i n s t a b l e e n v i r o n m e n t s show l i t t l e enzyme h e t e r o g e n e i t y . I n c o n t r a s t , i n s p e c i e s s u c h as t h e r a i n b o w and b r o o k t r o u t , b o t h of w h i c h a r e d i s t i n c t l y e u r y t h e r m a l , s e l e c t i o n may f a v o u r enzyme h e t e r o g e n e i t y i n o r d e r t o m a i n t a i n g r e a t e r p h y s i o l o g i c a l f l e x i b i l i t y . CHAPTER IV: Temperature and Enzyme A c t i v i t y i n Poikilotherms: I s o c i t r a t e Dehydrogenases i n the Rainbow Trout Liver 34 INTRODUCTION The r e l a t i v e r o l e s of t h e N A D - l i n k e d i s o c i t r a t e d e h y d r o g e n a s e (NAD-IDH, EC 1.1.1.41) and t h e NADP-1inked IDH (NADP-IDH, EC 1.1.1.42) i n t h e m e t a b o l i s m of most o r g a n i s m s a r e unknown. L a r g e l y on t h e b a s i s of t i s s u e and i n t r a c e l l u l a r d i s t r i b u t i o n , G o e b e l l & K l i n g e n b e r g (1963; 1964) p r o p o s e d t h a t o n l y t h e NAD-IDH was o p e r a t i v e i n t h e K r e b s c y c l e , and t h i s p r o p o s a l seems t o be s u p p o r t e d by r e c e n t s t u d i e s ( s e e , f o r e x a m p l e , N i c h o l l s & G a r l a n d , 1 9 6 9 ) . However, i n a c o m p r e h e n s i v e s u r v e y o f v e r t e b r a t e m u s c l e t y p e s , C r a b t r e e & Newsholme (1970) p o i n t out t h a t NADP-IDH a c t i v i t i e s exceed NAD-IDH a c t i v i t i e s by some 10-100 t i m e s i n mammalian t i s s u e s and by up t o 300 t i m e s i n f i s h e s . D u r i n g i n i t i a l p h a s e s of t h i s s t u d y , I c o n f i r m e d t h e o b s e r v a t i o n s of C r a b t r e e & Newsholme (1970) . I t h e r e f o r e i n i t i a t e d d e t a i l e d s t u d i e s of t h e k i n e t i c p r o p e r t i e s o f t h e NADP-IDHs i n f i s h t i s s u e s i n o r d e r t o g a i n some f u r t h e r i n s i g h t i n t o t h e p o s s i b l e f u n c t i o n a l s i g n i f i c a n c e of t h e s e enzymes i n t h e m e t a b o l i s m o f v e r t e b r a t e t i s s u e s . I n mammals, NADP-IDH o c c u r s i n m u l t i m o l e c u l a r forms w i t h 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 s u p e r n a t a n t and m i t o c h o n d r i a l i s o z y m e s ( H e n d e r s o n , 1965) . R e c e n t l y , t h e s u p e r n a t a n t NADP-IDH has a l s o been r e p o r t e d t o o c c u r i n i s o z y m i c f orms i n b a c t e r i a (Reeves, Brehmeyer & A j l , 1968; S e l f & Weitzman, 1 9 7 0 ) , i n t h e c a r p and i n t h e t r o u t , b o t h of w h i c h a r e t h o u g h t t o be t e t r a p l o i d ( Q u i r o z - G u t i e r r e z & Ohno, 1970; W o l f , E n g e l & F a u s t , 1970) . P r e v i o u s work f r o m t h i s l a b o r a t o r y ( s e e Hochachka & Somero, 1971) i n d i c a t e t h a t i s o z y m i c changes may be c o r r e l a t e d w i t h changes i n a c c l i m a t -35 i z a t i o n temperature and that these changes may be adaptive f o r the s u r v i v a l of the f i s h . For t h i s reason, and because NADPH produced by IDH may be u t i l i z e d during the increased l i p o g e n e s i s that occurs i n f i s h e s at low temperatures (Knipprath & Mead, 1968; Dean, 1969), I was p a r t i c u l a r l y i n t e r e s t e d i n the r e l a t i o n s h i p of t h i s enzyme a c t i v i t y t o the a c c l i m a t i z a t i o n s t a t e of the organism. The r e s u l t s are c o n s i s t e n t w i t h the thermal i n d u c t i o n of NADP-IDH v a r i a n t s which are w e l l s u i t e d f o r f u n c t i o n at t h e i r r e s p e c t i v e a c c l i m a t i z a t i o n temperatures. A p r e l i m i n a r y r e p o r t of t h i s work was presented r e c e n t l y (Moon, 1970). RESULTS AND DISCUSSION NAD- v e r s u s NADP-IDH. The NAD-IDH does n o t appear t o be p r e s e n t i n r a i n b o w t r o u t t i s s u e s . Enzyme a c t i v i t y c o u l d n o t be d e t e c t e d u s i n g t h e methods o f i s o l a t i o n a n d a s s a y d e s c r i b e d by Chen & P l a u t ( 1 9 6 3 ) , n o r a f t e r a t t e m p t s a t s t a b i l i z a t i o n o f t h e enzyme w i t h s u b s t r a t e , c o f a c t o r , c a t i o n s (Mg"*"*" o r M n ^ ) o r a known p o s i t i v e m o d u l a t o r (AMP o r ADP) . G l u t a t h i o n e and - m e r c a p t o e t h a n o l were a l s o w i t h o u t e f f e c t . V a r i o u s t i s s u e s ( l i v e r , g i l l , b r a i n , h e a r t and s k e l e t a l m u s c l e ) known t o d i f f e r i n o x i d a t i v e p o t e n t i a l were examined. I n no c a s e was t h e r e any s i g n i f i c a n t NAD-IDH a c t i v i t y m e a s u r a b l e , a l t h o u g h t h e a c t i v i t y o f t h e NADP-IDH enzyme was a l w a y s r e l a t i v e l y h i g h ( e s t i m a t e s of NADP-IDH a c t i v i t i e s a r e g i v e n i n T a b l e I V , 1 ) . These r e s u l t s a r e i n c l o s e agreement w i t h t h o s e o f C r a b t r e e & Newsholme ( 1 9 7 0 ) , and s u g g e s t e i t h e r (1) t h a t t h e NAD-IDH d o e s n o t o c c u r i n f i s h t i s s u e s , o r (2) t h a t i t i s h i g h l y u n s t a b l e and i t s a c t i v i t y i n homogenized t i s s u e p r e p a r a t i o n s i s no measure o f i t s a c t i v i t y in. v i v o . Isozymes of l i v e r NADP-IDH. I n p r e p a r a t i o n s of r a i n b o w t r o u t l i v e r , t h r e e bands of NADP-IDH a c t i v i t y a r e t y p i c a l l y s t a i n e d i n b o t h warm-(17°C) and cold-(2°C) a c c l i m a t e d t r o u t , b u t t h e d i s t r i b u t i o n o f t h e s e bands i s somewhat d i f f e r e n t . E s t i m a t e s made fr o m d e n s i t o m e t e r t r a c e s ( s e e F i g . I V , 1) i n d i c a t e a 5 - f o l d i n c r e a s e i n t h e r e l a t i v e amount o f t h e low m o b i l i t y i s o z y m e i n t h e c o l d - a c c l i m a t e d t r o u t , whereas t h e warm p a t t e r n shows a p p r o x i m a t e l y 37 T a b l e I V , 1, T i s s u e - s p e c i f i c NADP-IDH a c t i v i t i e s o f h i g h speed (35,000g) s u p e r n a t a n t f r o m r a i n b o w t r o u t . I s o l a t i o n and a s s a y as i n M a t e r i a l s and Methods. Temperature o f a s s a y : 25°C. T i s s u e limol NADPH/min/g wet wt. b r a i n 2.86 l i v e r 4.76 g i l l 1.58 h e a r t 90.50 s k e l e t a l m u s c l e 0.30 38 Fig. IV, 1. Starch-gel electrophoretograms of the cold- and warm-enzyme variants with the corresponding densitometer traces. Electro-phoresis run at 4°C for 17 hr at 200 V and 20 mA using a c i t r a t e / phosphate buffer system, pH 7.0. 39 9 0% of a l l a c t i v i t y i n the f a s t e s t moving band. If a system of two sub-u n i t s , aggregating into three types of dimers i s assumed, the data are co n s i s t e n t with two gene l o c i coding f o r S-form IDH, a f i n d i n g i n agree-ment with that proposed for g o l d f i s h (Quiroz-Gutierrez & Ohno, 1970) and trout heart M-form IDH (Wolf, Engel & Faust, 1970). The rainbow trout l i v e r NADP-IDH i s also i n t e r e s t i n g i n that the homodimers appear to be stained more intensely than does the heterodimer (see F i g . IV, 1). This suggests e i t h e r (1) that each band represents more than a s i n g l e isozyme; or (2) that assembly of polypeptide subunits i s non-random. Unlike mammalian NADP-IDH (Henderson, 1965), rainbow trout l i v e r does not d i s p l a y an unique mitochondrial enzyme form. Similar r e s u l t s have been reported by Wolf, Engel & Faust (1970) for l i v e r from Salmo  i r i d e u s , but not heart where d i s t i n c t mitochondrial forms are present. When the enzyme i s i s o l a t e d from c a r e f u l l y prepared trout l i v e r mito-chondria, the Km for substrate i s s i m i l a r to values obtained for super-natant IDH (Moon, unpublished observations). Similar r e s u l t s are a v a i l a b l e for beef heart NADP-IDH (Higashi, Maruyama, Otani & Sakamoto, 1965). This suggests contamination of the mitochondrial p e l l e t with the S-form IDH. The r e s u l t s of el e c t r o f o c u s i n g are consistent with the existence o f m u l t i p l e forms of l i v e r IDH ( F i g . IV, 2). However, the recovery of enzyme a c t i v i t y i s quite low, so that only shoulders are v i s i b l e at the p I of the minor enzyme components. The r e s u l t s of DEAE-cellulose chromatography ( F i g . IV, 3) are unequivocal i n demonstrating three isozymic forms i n l i v e r of cold-adapted f i s h . As i n starch-gel e l e c t r o p h o r e s i s , 4 0T Fig. IV, 2. Concurrent electrofocus separations on a pH 3-10 Ampholyte gradient of the cold (upper panel) and warm (lower panel) enzyme variants. Run at 5°C for 32 hr at 300 V. Fractions were assayed at pH 8.0, 50mM t r i s - H C l buffer, 1 mM DL-isocitrate, 1 mM MgCl 2, and 0.15 mM NADP+. 41 F i g . I V , 3. D E A E - c e l l u l o s e c h r o m a t o g r a p h y o f t h e cold-enzyme v a r i a n t f r o m r a i n b o w t r o u t l i v e r . NADP-IDH i s o z y m e s were e l u t e d f r o m a 2.0 x 25 cm column w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n s ( f r o m 1.0 t o 300 mM t r i s ) . I n i t i a l b u f f e r c o n s i s t e d o f 1.0 mM t r i s + 1 . 0 mM EDTA + 1.0 mM & - m e r c a p t o e t h a n o l , t i t r a t e d t o pH 7.1 ( a t 5°C) w i t h 1 M HC1. i4.0 ih A o o 120 130 140 150 160 170 180 190 200 210 Fraction Number 3.0 E 2.05 1.0 42 the h e t e r o d i m e r ( m i d d l e peak i n F i g . I V , 3) o c c u r s i n r e d u c e d a c t i v i t y . T he m a j o r peak i n F i g . I V , 3 c o r r e s p o n d s t o t h e f a s t - m o v i n g band i n F i g . IV , 1. The e x i s t e n c e of t h e s e i s o z y m e s of l i v e r NADP-IDH l e d t o a k i n e t i c s t u d y of t h e c o l d - and warm-enzyme f o r m s . E f f e c t s o f t e m p e r a t u r e on Vm. One method o f i n c r e a s i n g t h e c a t a l y t i c e f f i c i e n c y o f enzymes w h i c h work a t l o w e n v i r o n m e n t a l t e m p e r a t u r e s i s t o d e c r e a s e t h e e n e r g y o f a c t i v a t i o n n e c e s s a r y f o r t h e f o r m a t i o n of t h e e n z y m e - s u b s t r a t e complex. Such an i d e a has been s u g g e s t e d by Vroman & Brown ( 1 9 6 3 ) , a l t h o u g h i n enzymes examined i n t h i s l a b o r a t o r y no g e n e r a l r e l a t i o n s h i p b e t w e e n e n v i r o n m e n t a l t e m p e r a t u r e and Ea has been c o n f i r m e d (Hochachka & Somero, 1 9 7 1 ) . A r r h e n i u s v a l u e s (Ea) of 1 8 k c a l / m o l f o r b o t h t h e warm-and cold-enzyme v a r i a n t s were d e t e r m i n e d f r o m a p l o t o f l o g Vopt v s 1/T ( F i g . IV, 4 ) . E f f e c t s o f t e m p e r a t u r e on Km. F i g . I V , 5 shows s u b s t r a t e s a t u r a t i o n c u r v e s o f D L - i s o c i t r a t e a t d i f f e r e n t a s s a y t e m p e r a t u r e s and t h e i r L i n e w e a v e r - B u r k t r e a t m e n t s . I n t h e c a s e of t h e warm-enzyme c u r v e s , t h e a c t i v i t y a t 17°C ( t h e t e m p e r a t u r e a t w h i c h t h e f i s h were a c c l i m a t i z e d ) i s as h i g h as t h e a c t i v i t y a t 25°C when examined 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 . e . , l e s s t h a n 10 yuM DL-i s o c i t r a t e ) . A s i m i l a r f i n d i n g i s s u g g e s t e d f r o m F i g . I V , 6 w h i c h p l o t s N A D P + s a t u r a t i o n c u r v e s a t d i f f e r e n t t e m p e r a t u r e s . A g a i n , a c t i v i t y i s as h i g h a t 15° and 17°C as i t i s a t 25°C. T h i s has been found i n a number o f enzymes when s u b s t r a t e s a t u r a t i o n c u r v e s a r e p l o t t e d a t d i f f e r e n t temp-e r a t u r e s , i n c l u d i n g p h o s p h o f r u c t o k i n a s e f r o m g o l d f i s h ( F r e e d , 1 9 6 9 ) , A3 F i g . I V , 4. A r r h e n i u s p l o t s o f t r o u t l i v e r NADP-IDH fr o m c o l d - ( c l o s e d c i r c l e s ) and warm- (open c i r c l e s ) a c c l i m a t e d a n i m a l s . A s s a y c a r r i e d out a t pH 8.0, 50 mM t r i s - H C l b u f f e r , i n t h e p r e s e n c e o f s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n s . Ea d e t e r m i n e d f r o m t h e s l o p e o f t h e l i n e . 44 F i g . IV, 5. D L - i s o c i t r a t e saturation curves and Lineweaver-Burk p l o t s at d i f f e r e n t assay temperatures f o r the c o l d - (lower panel) and warm-(upper panel) acclimated rainbow trout NADP-IDH. Assay c a r r i e d out at pH 8.0, 50 mM t r i s - H C l buffer (temperature adjusted), 0.15 mM NADP+, and 1.0 mM MgCl~. Equal enzyme qu a n t i t i e s added i n a l l cases. 45 F i g . I V , 6. NADP s a t u r a t i o n c u r v e s and L i n e w e a v e r - B u r k p l o t s a t d i f f e r e n t a s s a y t e m p e r a t u r e s f o r t h e c o l d - ( l o w e r p a n e l ) and warm-( u p p e r p a n e l ) a c c l i m a t e d r a i n b o w t r o u t NADP-IDH. A s s a y c a r r i e d out a t pH 8.0, 50 mM t r i s - H C l b u f f e r ( t e m p e r a t u r e a d j u s t e d ) , 1.0 mM DL-i s o c i t r a t e and 1.0 mM MgC^. E q u a l enzyme q u a n t i t i e s added i n a l l c a s e s . 46 f r u c t o s e diphosphatase from l u n g f i s h l i v e r (Behrisch & Hochachka, 1969) and trout brain a c e t y l c h o l i n e s t e r a s e (Baldwin & Hochachka, 1970). When the Michaelis constant, Km, derived from Lineweaver-Burk p l o t s ( F ig. I V , 5 and 6), i s plotted as a function of assay temperature, complex curves are found ( F i g . I V , 7). At the upper b i o l o g i c a l temper-at u r e extreme, Km v a r i e s d i r e c t l y with temperature, approaching a minimal Km (maximal apparent a f f i n i t y ) within the temperature range at which a c c l i m a t i z a t i o n occurs. This i s p a r t i c u l a r l y marked f o r the Km of EXD-i s o c i t r a t e ( F i g . I V , 7), but the r e l a t i o n s h i p i s not so extreme i n the case of the cofactor, NADP+ (see F i g . I V , 6). Similar complex Km vs temperature curves have been seen for other enzymes from several d i f f e r e n t t i s s u e s of aquatic organisms (Hochachka & Somero, 1971; Somero & Hochachka, 1971). The s i g n i f i c a n c e of t h i s upper s h i f t i n Km at high b i o l o g i c a l temperatures has been previously discussed (Baldwin & Hochachka, 1970; Hochachka & Somero, 1971). By increasing the apparent Km at these high temperatures, the thermal e f f e c t s on v e l o c i t y are reduced, thereby l e a v i n g the re a c t i o n r e l a t i v e l y temperature-independent. The upswing seen at the lower b i o l o g i c a l temperature range for the warm-enzyme ( F i g . I V , 7) i s i n t e r e s t i n g and may be coupled with the high Ea value seen i n t h i s region f or t h i s enzyme va r i a n t ( F i g . I V , 4). Table I V , 2, showing Q I Q values for both enzymes, demonstrates that f o r the warm-variant between 5 and 10°C, Q-^ Q increases with decreasing substrate ( D L - i s o c i t r a t e ) concentrations because Km increases and thermal energy decreases ( F i g . I V , 7). These two e f f e c t s , high Km and high Q^Q (at p h y s i o l o g i c a l concentrations of substrate) probably set a lower thermal l i m i t for 47 F i g . IV, 7. Km of D L - i s o c i t r a t e f o r the c o l d - (open squares) and warm-(open c i r c l e s ) NADP-IDH enzyme v a r i a n t s from rainbow t r o u t l i v e r as a f u n c t i o n of assay temperature. Km was estimated from Lineweaver-Burk p l o t s i n F i g . IV, 5. Table IV, 2. Q^o values for the warm- and cold-enzyme variants at various DL-isocitrate concentrations. Q J Q ; Temperature range 5-10°C 15-20°C DL-isocitrate C-enzyme W-enzyme C-enzyme W-enzyme 1.0 mM 4.0 5.14 2.92 3.55 0.1 mM 4.0 4.0 2.65 3.47 0.075mM 3.44 4.0 2.34 3.62 0.05 mM 3.41 7.1 2.0 2.56 4>-0 0 \ c o n t r o l l e d c a t a l y t i c f u n c t i o n by t h i s warm-enzyme v a r i a n t . I n F i g . I V , 7 i t i s e v i d e n t t h a t t h e m i n i m a l Km v a l u e s f o r t h e two enzyme p r e p a r a t i o n s a r e s i m i l a r . I n consequence, t h e r a t e o f c a t a l y s i s b y t h e cold-enzyme a t 2°C ( m i n i m a l Km f o r t h i s form) w i l l be much l o w e r t h a n t h e r a t e c a t a l y z e d by t h e w a r m - v a r i a n t a t 17°C ( m i n i m a l Km f o r t h e warm-enzyme). - I f a c c l i m a t i z a t i o n i s t o l e a d t o s i g n i f i c a n t r a t e c o m p e n s a t i o n a t any g i v e n s u b s t r a t e c o n c e n t r a t i o n ( H a z e l & P r o s s e r , 1 9 7 0 ) , t h e Km of t h e cold-enzyme v a r i a n t would have t o be much l o w e r t h a n f o r t h e warm-form. S i n c e t h i s i s n o t t h e c a s e , we have c o n s i d e r e d t h e p o s s i b l e r o l e s o f c a t i o n s , o t h e r m e t a b o l i t e s , pH and enzyme c o n c e n t -r a t i o n i n a f f e c t i n g NADP-IDH a c t i v i t y d u r i n g t h e t h e r m a l a c c l i m a t i z a t i o n p r o c e s s . E f f e c t o f t e m p e r a t u r e on t h e M i c h a e l i s c o n s t a n t s o f Mg"1""*" and Mn As i n t h e c a s e w i t h NADP-IDH f r o m o t h e r o r g a n i s m s ( P l a u t , 1 9 6 3 ) , t h e i s o z y m e s f r o m t r o u t l i v e r d i s p l a y an a b s o l u t e r e q u i r e m e n t f o r a I | 4-(-d i v a l e n t c a t i o n , w h i c h can be f u l f i l l e d by e i t h e r Mg o r Mn . I t i s I | e v i d e n t f r o m T a b l e I V , 3 t h a t t h e Ka v a l u e s f o r Mg a r e e s s e n t i a l l y t e m p e r a t u r e i n d e p e n d e n t . C a l c u l a t e d Ka v a l u e s a r e i n t h e ra n g e o f 3.3-3.8 x 10" 5M f o r t h e c o l d - v a r i a n t and 2.3-2.6 x 10" 5M f o r t h e warm-v a r i a n t ( T a b l e I V , 3 ) . A l t h o u g h t h e Ka o f Mn has n o t been c a l c u l a t e d f o r t h e w a r m - v a r i a n t , t h a t f o r t h e c o l d - v a r i a n t i s f i v e t i m e s s m a l l e r t h a n t h e Ka o f Mg"*""*" ( T a b l e IV, 3 ) . S i n c e d i s t r i b u t i o n and c o n c e n t r a t i o n o f c a t i o n s i s t h o u g h t t o be a d j u s t e d d u r i n g t h e r m a l a c c l i m a t i z a t i o n i n f i s h e s (Hickman, McNabb, N e l s o n , v a n Breeman & C o m f o r t , 1964; H o u s t o n , Madden & DeW i l d e , 1 9 7 0 ) , i t i s p o s s i b l e t h a t a p p r o p r i a t e a d j u s t m e n t s i n 50 T a b l e IV, 3. Ka ( c a t i o n s ) f o r t h e c o l d - and warm-enzyme v a r i a n t s a t v a r i o u s a s s a y t e m p e r a t u r e s . Ka ( c a t i o n s ) M g ^ x 10 5M Mn 4 4" x 10 5M Temperat u r e C-enzyme W-enzyme C-enzyme 5°C 3.3 2.5 6.7 10°C 3.5 2.6 6.4 15°C 3.3 2.5 20°C 3.8 2.6 6.4 25°C 3.3 2.3 " 6.5 30°C 6.4 51 Mg or Mn c o n c e n t r a t i o n s i n l i v e r d u r i n g t h e r m a l a c c l i m a t i z a t i o n c o u l d l e a d t o some c o m p e n s a t i o n of NADP-IDH a c t i v i t y . E f f e c t o f t e m p e r a t u r e on t h e pH optimum. F i g . I V , 8 i s a p l o t o f t h e r e l a t i v e IDH a c t i v i t y o f b o t h enzyme p r e p a r a t i o n s as a f u n c t i o n o f pH and t e m p e r a t u r e , and d e m o n s t r a t e s t y p i c a l pH p r o f i l e s f o r NADP-IDH ( H i g a s h i , e t a l . , 1 9 6 5 ) . I n t h e c a s e o f t r o u t l i v e r IDH, b o t h forms d i s p l a y b r o a d pH o p t i m a i n t h e pH range 7.0 t o 9.0. An i n t e r e s t i n g f e a t u r e o f t h e s e c u r v e s i s t h a t as t h e t e m p e r a t u r e i s d e c r e a s e d f r o m 25° t o 15°C, t h e pH optimum f o r t h e c o l d -enzyme i s ex t e n d e d t o w a r d s l o w e r pH v a l u e s . Work by Rahn (1965) and R e e v e s & W i l s o n (1969) has shown t h a t t h e i n t r a c e l l u l a r and b l o o d pH o f f i s h e s i s i n c r e a s e d as t e m p e r a t u r e d e c r e a s e s . F o r t h e c o l d - f o r m o f t r o u t l i v e r IDH, t h e pH c h a r a c t e r i s t i c s may e x a g g r a t e t h e e f f e c t s seen by Rahn and c o w o r k e r s , t h u s i n c r e a s i n g t h e c a t a l y t i c r a t e s a t t h i s l o w t e m p e r a t u r e . T h i s e f f e c t , u n l i k e t h e Km-temperature r e l a t i o n s h i p , would t h e r m a l l y s t a b i l i z e t h e r e a c t i o n i r r e s p e c t i v e o f s u b s t r a t e c o n c e n t r a t i o n s . E f f e c t s o f o t h e r m e t a b o l i t e s . S i n c e a p a r t o f t h e argument f a v o u r i n g NAD-IDH f u n c t i o n i n t h e K r e b s c y c l e i s based on i t s r e s p o n s i v e n e s s t o r e g u l a t o r y m e t a b o l i t e s s u c h as t h e a d e n y l a t e s ( N i c h o l l s & G a r l a n d , 1 9 6 9 ) , a s y s t e m a t i c s e a r c h f o r compounds w h i c h may s e r v e a r e g u l a t o r y r o l e i n t h e c a s e o f t r o u t l i v e r NADP-IDH was i n i t i a t e d . T a b l e IV,4 g i v e s a l i s t o f t h e v a r i o u s m e t a b o l i t e s w h i c h were t e s t e d a g a i n s t t r o u t l i v e r NADP-IDH a t two d i f f e r e n t c o n c e n t -r a t i o n s o f s u b s t r a t e . The a p p a r e n t i n h i b i t i o n o f t h e enzyme by t h e F i g . I V , 8. R e l a t i v e NADP-IDH a c t i v i t i e s p l o t t e d a t two t e m p e r a t u r e s and v a r i o u s pH v a l u e s f o r t h e c o l d - (open s q u a r e s ) and w a r m - ( c l o s e d c i r c l e s ) enzyme v a r i a n t s f r o m r a i n b o w t r o u t l i v e r . A s s a y c a r r i e d o ut w i t h 50 mM t r i s - H C l b u f f e r ( t e m p e r a t u r e a d j u s t e d ) , 1.0 mM D L - i s o c i t r a t e , 0.15 mM NADP +, 1.0 mM M g C l 2 and e q u a l enzyme q u a n t i t i e s . Relative IDH Activity - o T a b l e I V , 4. The e f f e c t s o f v a r i o u s m e t a b o l i t e s on t h e a c t i v i t y o f NADP-IDH fr o m r a i n b o w t r o u t l i v e r . A c t i v i t y e x p r e s s e d as p e r c e n t c c o n t r o l i n absence o f m e t a b o l i t e . M e t a b o l i t e D L - i s o c i t r a t e c o n c e n t r a t i o n 1 mM 0.1 mM g l y o x y l a t e — 5 mM 1 mM .5 mM 100.0 101.0 104.5 96.0 96.0 106.0 o x a l o a c e t a t e — 5 mM 1 mM .5 mM g l y o x y l a t e + o x a l o a c e t a t e — 1 mM(each) c i t r i c a c i d — 5 mM 1 mM .5 mM g l u t a m i c a c i d — 5 mM 1 mM .5 mM o\ - k e t o g l u t a r a t e — 5 m M 1 mM .5 mM p y r u v a t e — 5 mM 1 mM .5 mM f r u c t o s e d i p h o s p h a t e — 5 m M 1 mM .5 mM p h o s p h o e n o l p y r u v a t e — 5 mM 1 mM .5 mM NAD+—5mM 1 mM .5 mM 100.0 102.5 104.5 42.0 100.0 100.0 101.0 102.5 100.0 102.5 55.5 80.0 86.5 91.0 91.0 95.5 95.5 91.0 82.0 66.7 82.0 78.0 95.5 91.0 95.5 92.5 92.5 92.5 0.0 100.0 100.0 100.0 88.5 96.0 106.0 0.0 34.6 50.0 92.5 96.0 92.5 77.0 80.5 80.5 57.5 88.5 88.5 115.0 100.0 104.0 54 T a b l e I V , 4. C o n t i n u e d NADH— 0.25 mM 84.5 81.0 0.1 mM 91.0 100.0 0.05 mM 91.0 92.5 A T P — 1 mM 94.5 62.5 .5 mM 96.5 94.0 ADP--5 mM 100.0 65.5 1 mM 101.0 94.0 .5 mM 96.0 100.0 AMP—1 mM .5 mM 100.0 94.5 115.0 106.0 55 l | a d e n y l a t e s , p a r t i c u l a r l y ATP, i s due e n t i r e l y t o c h e l a t i o n o f Mg ; a t h i g h Mg c o n c e n t r a t i o n s , no p o i t i v e o r n e g a t i v e a d e n y l a t e e f f e c t s a r e s e e n , u n l i k e t h e c a s e i n some b a c t e r i a (Hampton & Hanson, 1969; P a r k e r & Weitzman, 1970) and p r o t o z o a (Marr & Weber, 1969a,b). However, a t h i g h Mg c o n c e n t r a t i o n s , p h y s i o l o g i c a l c o n c e n t r a t i o n s o f ADP t e n d t o d e c r e a s e t h e Km of D L - i s o c i t r a t e . T a b l e I V , 5 s u g g e s t s t h a t a t low ADP (0.4 mM and l e s s ) , t h e Km of t h e cold-enzyme v a r i a n t a t 10°C i s d e c r e a s e d up t o t h r e e - f o l d , whereas h i g h e r c o n c e n t r a t i o n s a r e n o t as e f f e c t i v e . Such an e f f e c t i s known f o r t h e NAD-IDH fr o m a n i m a l t i s s u e s (Chen & P l a u t , 1 9 6 3 ) , b u t has n o t been r e p o r t e d f o r t h e NADP-IDH enzyme. A l s o , F i g . IV, 9 d e m o n s t r a t e s t h a t f o r t h e cold-enzyme v a r i a n t , &• - k e t o g l u t a r a t e (°(-KGA) i s a c o m p e t i t i v e i n h i b i t o r o f NADP-IDH, i n c r e a s i n g t h e Km o f D L - i s o c i t r a t e by a f a c t o r o f 2.5 a t 1 mM and 10 a t 5 mM °( -KGA. These c o n c e n t r a t i o n s o f -KGA appear t o be i n t h e p h y s i o l -o g i c a l r a n g e ( W i l l i a m s o n , S c h o l z & B r o w n i n g , 1 9 6 9 ) . T h i s i n h i b i t i o n o f NADP-IDH by ^  -KGA i s of p a r t i c u l a r i n t e r e s t s i n c e i n v i v o , c o n t r o l o f t r o u t l i v e r IDH c o u l d be c o o r d i n a t e d w i t h a d e n y l a t e c o n t r o l o f c i t r a t e s y n t h a s e (Hochachka & L e w i s , 1 9 7 0 ) . I n v i v o under most c i r c u m s t a n c e s , h i g h ^ -KGA c o n c e n t r a t i o n s would be synonymous w i t h h i g h ATP c o n c e n t r a t i o n s . Hence, i n h i b i t i o n o f t h e c i t r a t e s y n t h a s e c o u l d be a c h i e v e d by h i g h ATP c o n c e n t r a t i o n s i n c o n c e r t w i t h N A D P - I D H i n h i b i t i o n by h i g h C^-KGA c o n c e n t r a t i o n s . T r o u t l i v e r NADP-IDH i s a l s o i n h i b i t e d by t h e c o n c e r t e d a c t i o n o f g l y o x y l a t e and o x a l o a c e t a t e ( T a b l e I V , 4 ) . T h i s i n h i b i t i o n has a l s o been o b s e r v e d i n b a c t e r i a (Hampton & Hanson, 1969; O z a k i & S h i i o , 1968) and by Mar r & Weber (1969c) i n p r o t o z o a . The s i g n i f i c a n c e o f t h i s a c t i o n i s not known i n h i g h e r a n i m a l s . 56 T a b l e IV, 5. The e f f e c t s o f ADP on t h e Km of D L - i s o c i t r a t e i n t h e c o l d - e n z y m e v a r i a n t o f r a i n b o w t r o u t l i v e r NADP-IDH. As s a y e d a t 10°C i t h 0.15 mM NADP+, 1 mM M g C l 2 and 50 mM t r i s - H C l b u f f e r , pH 8.0. w ADP c o n c e n t r a t i o n K m ( D L - i s o c i t r a t e ) x 10^M 0 4.8 0.1 mM 2.8 0.4 mM 1.57 0.8 mM 2.58 57 F i g . I V , 9. The e f f e c t o f 0(-KGA on t h e Km o f D L - i s o c i t r a t e f o r t h e cold-NADP-IDH enzyme v a r i a n t f r o m r a i n b o w t r o u t l i v e r a s s a y e d a t 10°C. A s s a y c a r r i e d out a t pH 8.0 (50 mM t r i s - H C l b u f f e r , t e m p e r a t u r e a d j u s t e d ) w i t h 0.15 mM NADP + and 1.0 mM M g C l 2 --20 -15 -10 -5 0 5 10 15 20 [DL-lsocit]xl05M 58 E f f e c t s o f t e m p e r a t u r e a c c l i m a t i z a t i o n on enzyme a c t i v i t i e s . One g e n e r a l k i n d o f mechanism w h i c h has been p r o p o s e d p r e v i o u s l y f o r a c h i e v i n g r a t e c o m p e n s a t i o n d u r i n g t h e r m a l a c c l i m a t i z a t i o n i n v o l v e s changes i n t h e s t e a d y s t a t e a c t i v i t i e s o f enzymes. Thus i t i s known t h a t t o t a l 6 - p h o s p h o g l u c o n a t e dehydrogenase and a l d o l a s e a c t i v i t i e s ( Jankowsky, 1 9 6 8 ) , t o t a l p h o s p h o f r u c t o k i n a s e a c t i v i t i e s ( F r e e d , 1969) a n d t o t a l l a c t a t e d ehydrogenase a c t i v i t i e s (Hochachka, 1965) appear t o b e i n c r e a s e d d u r i n g c o l d - a c c l i m a t i z a t i o n . A l t h o u g h we have n o t examined t h i s p r o b l e m i n t h e c a s e o f t r o u t l i v e r NADP-IDH i n d e t a i l , i t i s e v i d e n t f r o m F i g . I V , 5 t h a t t o t a l NADP-IDH a c t i v i t i e s i n l i v e r p r e p -a r a t i o n s f r o m b o t h c o l d - and w a r m - a c c l i m a t i z e d t r o u t seen t o be about . t h e same. I f t h e r e i s any a c c l i m a t i z a t i o n e f f e c t i t i s t o s l i g h t l y r educe t h e a c t i v i t y of t h e enzyme d u r i n g c o l d - a c c l i m a t i z a t i o n . And i n d e e d as we have argued above, t h e two b a s i c forms o f t h i s IDH appear t o be b e s t s u i t e d f o r f u n c t i o n a t t h e i r r e s p e c t i v e t h e r m a l r a n g e s . A_ p r i o r i i t i s c l e a r t h a t t h e p r o d u c t i o n o f more of t h e warm-enzyme v a r i a n t d u r i n g c o l d - a c c l i m a t i z a t i o n ( o r v i c e v e r s a ) would n o t make sen s e b i o l o g i c a l l y . As i n t h e c a s e o f t r o u t m u s c l e p y r u v a t e k i n a s e (Somero, 1969) and 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 ( B a l d w i n & Hochachka, 1 9 7 0 ) , a more f u n c t i o n a l s o l u t i o n t o t h e p r o b l e m o f low t e m p e r a t u r e a p p e a r s t o be t o i n c r e a s e t h e r e l a t i v e s t e a d y s t a t e c o n c e n t r a t i o n s o f an enzyme v a r i a n t x<rhich i s b e t t e r a d a p t e d f o r c o n t r o l l e d f u n c t i o n a t t h e low t e m p e r a t u r e s e n c o u n t e r e d by t h e o r g a n i s m i n N a t u r e . CHAPTER V: C o m p a r i s o n o f t h e S o l u b l e N A D P + - L i n k e d I s o c i t r a t e Dehydrogenase of Rainbow T r o u t L i v e r w i t h t h e P u r i f i e d P i g H e a r t Enzyme 59 INTRODUCTION The changes i n e n z y m e - s u b s t r a t e a f f i n i t y w h i c h have been found t o o c c u r i n t h e s o l u b l e r a i n b o w t r o u t l i v e r NADP-IDH a r e i n an a d a p t i v e d i r e c t i o n (Ch. I V ) ; i . e . , by m a i n t a i n i n g a c o n s t a n t Km o v e r t h e t h e r m a l r a n g e t h e f i s h i s l i k e l y t o e n c o u n t e r i n n a t u r e , c o n t r o l o f c a t a l y s i s by t h i s enzyme i s e s s e n t i a l l y t e m p e r a t u r e - i n d e p e n d e n t , and by i n c r e a s i n g Km a t h i g h e r t e m p e r a t u r e s , t h e e f f e c t s o f t e m p e r a t u r e i n c r e a s e s a r e mini m i z e d ' . I s t h i s p r o p e r t y a g e n e r a l f e a t u r e o f a l l s o l u b l e NADP-IDH enzymes, o r a l t e r n a t i v e l y i s i t a p a r t i c u l a r f e a t u r e o f t h e r a i n b o w t r o u t enzyme? I t has been assumed (Hochachka & Somero, 1971) t h a t p r o p e r t i e s u n i q u e t o p o i k i l o t h e r m i c enzymes a r e r e s p o n s i b l e f o r t h i s k i n e t i c b e h a v i o u r , s i n c e as w i t h o t h e r p h e n o t y p i c c h a r a c t e r s , enzymes a r e t h o u g h t t o be shaped by t h e e v o l u t i o n a r y p r o c e s s ( A t k i n s o n , 1 9 6 8 ) . Few c o m p a r a t i v e s t u d i e s a r e a v a i l a b l e w i t h r e g a r d s t o t h e p h y s i c a l and c h e m i c a l c h a r a c t e r i s t i c s ofriomologous enzymes f r o m p o i k i l o t h e r m i c and homeo t h e r m i c t i s s u e s . When t h e y a r e , t e m p e r a t u r e i s u s u a l l y c o n s i d e r e d o f s e c o n d a r y i m p o r t a n c e ( A s s a f & G r a v e s , 1969; K a l o u s t i a n & K a p l a n , 1 9 6 9 ) . Most mammalian t i s s u e s have been f o u n d t o c o n t a i n a s o l u b l e NADP-IDH, b u t a h i g h l y p u r i f i e d s o u r c e o f t h i s enzyme has o n l y been p r e p a r e d f r o m p i g h e a r t ( P l a u t , 1 9 6 3 ) . I n r e c e n t y e a r s , Colman and c o w o r k e r s have r e p o r t e d t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f t h i s p u r i f i e d enzyme (Co l m a n , 1968; Colman, S z e t o & Cohen, 1 9 7 0 ) . I t was of i n t e r e s t t o compare t h e p r o p e r t i e s o f t h i s enzyme a v a i l a b l e on a c o m m e r c i a l b a s i s w i t h t h e r a i n b o w t r o u t enzyme i n o r d e r t o b e g i n t o answer t h e q u e s t i o n , o f c h e m i c a l s i m i l a r i t y v s t r u e a d a p t a t i o n . 60 The res u l t s suggest that even though the two enzymes are sim i l a r i n molecular weights and response to i n h i b i t o r s , they d i f f e r i n e l e c t r o -phoretic m o b i l i t i e s and response of a f f i n i t y constants to temperature changes. I t therefore appears that the changes seen i n the control of the NADP-IDH enzyme from rainbow trout l i v e r are an outcome of the s e l e c t i v e process. 61 RESULTS C h a r a c t e r i z a t i o n of NADP-IDH. 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 p i g h e a r t and r a i n b o w t r o u t l i v e r NADP-IDH d i f f e r s i g n i f i c a n t l y ( F i g . V, 1 ) . T r o u t l i v e r NADP-IDH m i g r a t e s a n o d a l l y as t h r e e d i s t i n c t i s o z y m e s , whereas t h e p i g h e a r t enzyme moves c a t h o d a l l y as a s i n g l e s t a i n i n g band of a c t i v i t y . T h i s d i f f e r e n c e i n m o b i l i t y can be a t t r i b u t e d t o t h e b a s i c n a t u r e of t h e p i g h e a r t enzyme r e s u l t i n g f r o m i t s amide c o n t e n t (Colman, 1 9 6 8 ) . O t h e r w o r k e r s have r e p o r t e d l i t t l e o r no e l e c t r o p h o r e t i c m o b i l i t y between pH 5,6 and 8.5 and an i s o e l e c t r i c p o i n t o f 7.8 ( P l a u t , 1 9 6 3 ) . I n c o n t r a s t , t h e p i o f t h e t r o u t l i v e r enzymes a r e b e l o w 7.0 ( F i g . I V , 2) and, t h e r e -f o r e , l e s s b a s i c i n s t r u c t u r e , The IDHs f r o m two b a c t e r i a , B_. s t e a r o - t h e m o p h i l u s (Howard & B e c k e r , 1970) and A, v i n e l a n d i i (Chung & F r a n z e n , 1 9 6 9 ) , a r e s i m i l a r i l y more a c i d i c and m i g r a t e a n o d a l l y d u r i n g e l e c t r o p h o r e s i s a s compared t o t h e p i g h e a r t enzyme. I n c o n t r a s t t o e l e c t r o p h o r e t i c m o b i l i t y , t h e d i f f e r e n c e s i n m o l e c u l a r weight a r e s m a l l . Based on a number o f p h y s i c a l c r i t e r i a , Colman and c o -w o r k e r s (1968; 1970) have d e t e r m i n e d t h e m o l e c u l a r w e i g h t of t h e p i g h e a r t enzyme as 58,000, and s u g g e s t e d i t c o n s i s t s of a s i n g l e p o l y p e p t i d e c h a i n . However, t h e enzyme has has been found t o a g g r e g a t e (Kemper & K a p l a n , 1 9 7 1 ) , so i t s a c t u a l s u b u n i t s t r u c t u r e i s s t i l l i n d o u b t . A l t h o u g h F i g . V, 2 does no t a l l o w an a c c u r a t e a s s e s m e n t , i t i s a p p a r e n t f r o m b o t h Sephadex G-100 g e l f i l t r a t i o n and u l t r a c e n t r i f u g a t i o n , t h a t t h e t r o u t l i v e r enzyme i s s l i g h t l y l a r g e r t h a n t h e 58,000 v a l u e r e p o r t e d f o r t h e p i g h e a r t enzyme, I l l i n g w o r t h & T i p t o n (1970) have r e p o r t e d t h e m o l e c u l a r w e i g h t o f 62 Fig , V, 1. Starch-gel electrophoresis of the soluble NADP-IDH from 2°C-acclimated rainbow trout l i v e r and pig heart. Run for 20 hr at 2 00V (approximately 20 mA, 5°C) using a citrate/phosphate buffer system, pH 7.0, origin Rainbow Trout Liver Pig Heart 63 F i g , V, 2, S u c r o s e d e n s i t y u l t r a c e n t r i f u g a t i o n ( r i g h t p a n e l ) and Sephadex G-100 g e l f i l t r a t i o n ( l e f t p a n e l ) o f t h e s o l u b l e NADP-IDH enzyme f r o m 2°C-acclimated r a i n b o w t r o u t l i v e r (R.T. l i v e r ) and p i g h e a r t (P.- h e a r t ) . E x p e r i m e n t a l p r o t o c o l can be f o u n d i n M a t e r i a l s a n d Methods. Elution Volume (ml) Ultracentrifugation t*- P Heart © 1.345 1.350 1.355 Refractive Index 64 p i g l i v e r NADP-IDH as 76,000 and t h a t t h e enzyme i s a dimer ( s u b u n i t m o l e c u l a r w e i g h t of 36,000). The m o l e c u l a r w e i g h t r e p o r t e d f o r b a c t e r i a l NADP-IDH i s s i m i l a r t o t h i s v a l u e o r exceeds i t (Chung & F r a n z e n , 1969; B a r r e r a & J u r t s h u k , 1970; Howard & B e c k e r , 1 9 7 0 ) , s u g g e s t i n g t h a t t h e t r o u t enzyme may be i n t h i s same r a n g e . E l e c t r o p h o r e t i c d a t a i n d i c a t e t h a t t h e t r o u t enzyme i s a dimer ( s e e Ch. I V ) , w h i c h i s c o n s i s t e n t w i t h i t s h i g h e r m o l e c u l a r w e i g h t . R e c e n t l y , Kemper & K a p l a n (1971) have s u g g e s t e d t h a t t h e p i g h e a r t NADP-IDH a g g r e g a t e s , and t h e d e g r e e o f a g g r e g a t i o n i s d e t e r m i n e d by s u b s t r a t e and c o f a c t o r b i n d i n g . I n an a t t e m p t t o v a r i f y i f t h e r a i n b o w t r o u t enzyme behaves i n an i d e n t i c a l manner, u l t r a c e n t r i f u g a t i o n e x p e r i m e n t s i n t h e p r e s e n c e of v a r i o u s s u b s t r a t e , c o f a c t o r s , and/or i n h i b i t o r s w e r e i n i t i a t e d . F i g . V, 3 shows t h a t i f a g g r e g a t i o n does o c c u r , i t r e s u l t s i n a s m a l l m o b i l i t y change under t h e p r e s e n t e x p e r i m e n t a l c o n d i t i o n s . E l e c t r o p h o r e t i c e x p e r i m e n t s u s i n g a s i m i l a r s e t of c o n d i t i o n s a g a i n d i d n o t s u g g e s t changes i n s u b u n i t s t r u c t u r e . P i g h e a r t and t r o u t l i v e r NADP-IDH d e m o n s t r a t e d i f f e r e n t temper-a t u r e d e p e n d e n c i e s . The Ea o r a c t i v a t i o n e nergy f o r t h e p i g h e a r t enzyme i s a p p r o x i m a t e l y 5 k c a l / m o l e g r e a t e r t h a n t h a t seen f o r t h e t r o u t enzyme ( F i g . V, 4 ) . C a t a l y t i c e f f i c i e n c y can n o t be e x t r a p o l a t e d f r o m t h i s d a t a s i n c e a d d i t i o n a l thermodynamic p a r a m e t e r s a r e i n v o l v e d ( W r 6 b l e w s k i & G r e g o r y , 1961). I t i s i n t e r e s t i n g t o n o t e t h a t b o t h enzymes obey t h e A r r h e n i u s l a w ( i . e . , a l i n e a r r e s p o n s e of l o g r e a c t i o n r a t e w i t h r e s p e c t t o t e m p e r a t u r e ) w e l l beyond t h e i r r e s p e c t i v e t h e r m a l r a n g e , a g a i n s u g g e s t i n g t h a t enzyme a c t i v i t y a t s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n s can n o t be c o r r e l a t e d w i t h h a b i t a t p r e f e r r e n c e (Hochachka & Somero, 1971). 6 5 F i g . V, 3. S u c r o s e d e n s i t y u l t r a c e n t r i f u g a t i o n o f t h e l i v e r s o l u b l e NADP-IDH fr o m 2°C-acclimated r a i n b o w t r o u t , i n t h e p r e s e n c e o f s u b s t r a t e s , c o f a c t o r s , and/or i n h i b i t o r s . The 5-20% s u c r o s e g r a d i e n t s w e r e r u n a t 40,000 RPM f o r 8 h r (0°C) u s i n g a SW 50.1 r o t o r ( S p i n c o M o d e l 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 ) . Code f o r s y m b o l s : O no added components; © 1 x 1 0 " 5 M NADP +, 2 x 1 0 " 5 M D L - i s o c i t r a t e , 1 x 1 0 " 5 M M g C l 2 ; • 2 x 1 0 " 5 M D L - i s o c i t r a t e , 1 x 1 0 ~ 5 M M g C l 2 ; H 1 x 1 0 " 5 M NADP +; A 2 x 1 0 " 5 M D L J i s o c i t r a t e , 5 x 1 0 ~ 3 M OC -KGA. B o t h IDH a c t i v i t y and f r a c t i o n number a r e p l o t t e d as p e r c e n t of t o t a l . 66 F i g . V, 4. A r r h e n i u s p l o t s o f t h e s o l u b l e NADP-IDH fr o m 2°C-acclimated r a i n b o w t r o u t l i v e r and p i g h e a r t . A s s a y e d i n t h e p r e s e n c e o f 50 mM t r i s - H C l b u f f e r (pH 8.0 f o r t r o u t and pH 7.5 f o r p i g h e a r t NADP-IDH), 1.0 mM D L - i s o c i t r a t e , 0.15 mM NADP +, and 1.0 mM MgCl2- Ea v a l u e s c a l c u l a t e d f r o m t h e s l o p e o f t h e l i n e . 30 1 1 — ' ' ' — i — i — • 50 40 30 20 10 0 Temp. (°C) 310 330 350 370 I09/T CK"1) 67 Comparison of k i n e t i c constants, At the higher temperature range, trout l i v e r and pig heart NADP-IDH demonstrate s i m i l a r Km(isocit) vs_ temperature responses; i . e . , a temperature decrease r e s u l t s i n increased enzyme-isocitrate a f f i n i t y . However, u n l i k e the trout enzyme, the pig heart enzyme does not l e v e l o f f , but continues to increase enzyme-isocitrate a f f i n i t i e s at a l l temp-e r a t u r e s assayed (Pig. V, 5). Q 1 Q values c a l c u l a t e d from F i g , V,5 tend to decrease as D L - i s o c i t r a t e concentrations decrease (Table V, 1) over a l l temperature ranges. This thermal 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 i s a necessary outcome of a decreasing Km with decreasing temperature response. At the high temperature i n t e r y a l (40^-45°C, T a b l e V, 1), Q^g values are g r e a t l y reduced. The value of the Km of D L - i s o c i t r a t e c a l c u l a t e d from Lineweaver-Burk p l o t s of F i g . V, 5, are approximately l O ^ f o l d higher than those p r e v i o u s l y reported f o r t h i s enzyme (Moyle, 1956; Plaut, 1963). Commercial sources of t h i s enzyme are s t a b i l i z e d with g l y c e r o l which may account f o r t h i s a l t e r e d a f f i n i t y constant. E f f e c t s of i n h i b i t o r s . The most potent i n h i b i t o r of trout l i v e r NADP-IDH as well as other v e r t e b r a t e and b a c t e r i a l IDHs, i s the concerted i n h i b i t i o n by oxaloacetate and glyoxylate. Table V, 2 shows that even though separately oxaloacetate and glyoxylate do not show a s i g n i f i c a n t i n h i b i t i o n , together at low su b s t r a t e concentrations the enzyme i s completely i n h i b i t e d . This i n h i b i t i o n i s i n the concentration range reported for the trout l i v e r enzyme (Table. IV, 4) and other IDHs (Ozaki & S h i i o , 1968; Shiio & Ozaki, 1968; Marr & Weber, 1969c), Although the p h y s i o l o g i c a l importance of t h i s i n h i b i t i o n i s not known i n vertebrates which lack a glyoxylate c y c l e , i t may be 68 F i g . V, 5. The e f f e c t s o f t e m p e r a t u r e on D L - i s o c i t r a t e s a t u r a t i o n c u r v e s f o r t h e p u r i f i e d NADP-IDH f r o m p i g h e a r t . A s s a y e d w i t h 50 mM t r i s - H C l b u f f e r (pH 7.5, t e m p e r a t u r e a d j u s t e d ) , 0.15 mM NADP +, and 1.0 mM MgCl2> The K m ( D L - i s o c i t ) was c a l c u l a t e d f r o m L i n e w e a v e r - B u r k p l o t s . Specific Activity (ymols NADPHAnin/mg protein) ro & cn GO o F3 Km(S)xl08M o — ro CM 4^ CJI 8 O i i Table V, 1. Q^Q values for pig heart NADP-IDH at various DL-isocitrate concentrations. Values determined from substrate saturation curves i n Fig. V, 5. Q 1 0 : Temperature Range (°C) [DL-isocitrate] mM 20-30° 30-35° 35-40° 40-45° 1.0 3.76 3.34 2.90 1.74 0.1 3.76 2.75 3.10 1,93 0.05 3.84 2.27 2.85 1.90 0.025 3.50 2.35 2.60 1.87 O N 70 Table V, 2. The effects of certain c i t r i c acid cycle intermediates on the a c t i v i t y of pig heart NADP-IDH. Values are given as per cent of the a c t i v i t y i n the absence of the pa r t i c u l a r metabolite. The assay contained 50 mM t r i s - H C l buffer, pH 7.5, 0.15 mM NADP+, 1.0 mM MgCl 2, D L - i s o c i t r a t e as specifi e d , and was i n i t i a t e d by the addition of 0.08 mg of the p u r i f i e d enzyme. The cuvette temperature was 25°C, Metabolite 1.0 mM DL-isocitrate 0.04 mM DL-isocitrate C{ -KGA--5.0 mM 71.0 10.3 1.0 mM 87.0 27.6 0.5 mM 100.0 44.8 oxaloacetate—1.0 mM 100.0 86.2 glyoxylate-—1.0 mM 100.0 100.0 (oxaloacetate + glyoxylate)—1,0 mM 51.6 0.0 71 -t r e l a t e d t o t h e s a t e o f IDH a g g r e g a t i o n (Kemper & K a p l a n , 1 9 7 1 ) . As was r e p o r t e d p r e v i o u s l y ( F i g . IV, 9 ) , IDH from t r o u t l i v e r i s m a r k e d l y i n h i b i t e d by C A . - k e t o g l u t a r a t e (°(-KGA) . The p i g h e a r t enzyme shows a s i m i l a r b e h a v i o u r i n t h e p r e s e n c e of t h i s m e t a b o l i t e ( T a b l e V, 2) even a t l o w i n h i b i t o r c o n c e n t r a t i o n s . -KGA i s a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t t o D L - i s o c i t r a t e as seen from F i g . V, 6, but u n l i k e t r o u t l i v e r IDH, t h e Km o f D L - i s o c i t r a t e i n c r e a s e s as a l i n e a r f u n c t i o n o f t h e - K G A c o n c e n t r a t i o n ( i n s e r t , F i g , V, 6 ) . I n t h e c a s e o f t r o u t IDH ( F i g . IV, 9 ) , a t low CX -KGA c o n c e n t r a t i o n s (0.5 mM and l e s s ) , t h e Km of D L - i s o c i t r a t e r e m a i n s c o n s t a n t o r d e c r e a s e s s l i g h t l y , and o n l y a t h i g h X^ ^KGA c o n c e n t r a t i o n s does t h e Km i n c r e a s e d r a s t i c a l l y . The d i f f e r e n c e s i n Km r e s p o n s e ( 1 2 - f o l d f o r t r o u t l i v e r and 1 9 - f o l d f o r p i g h e a r t NADP-IDH.at 5 mM -KGA) would s u g g e s t t h a t C>{-KGA i s a more e f f e c t i v e i n h i b i t o r o f t h e p i g h e a r t enzyme. When t h e Km of D L - i s o c i t r a t e i s d e t e r m i n e d i n t h e p r e s e n c e o f 0,75 mM Q>i -KGA, Km f o r b o t h t h e t r o u t l i v e r and p i g h e a r t enzyme i s t e m p e r a t u r e i n d e p e n d e n t ( T a b l e V, 3 ) , T h i s i s a d i r e c t e f f e c t o f t h e c o m p e t i t i v e b i n d i n g o f C\-KGA t o t h e D L - i s o c i t r a t e s i t e , s i n c e b o t h R m ( i s o c i t ) and K i ( 0 ( - K G A ) a r e c o m p l e x l y e f f e c t e d by t e m p e r a t u r e ( T a b l e V, 4 ) . The change i n t h e Km o f D L - i s o c i t r a t e when compared i n t h e p r e s e n c e an d absence o f CX.-KGA, however, i s d i f f e r e n t . P i g h e a r t IDH shows a f o u r -fco s i x - f o l d i n c r e a s e i n Km (compare v a l u e s f r o m T a b l e V, 3 w i t h t h o s e of F i g . V, 5 ) , whereas t h e t r o u t l i v e r shows a maximum t w o - f o l d i n c r e a s e a t low t e m p e r a t u r e s and n e a r l y no change above 15°C (compare T a b l e V, 3 w i t h F i g . I V , 7 ) . T h i s l a r g e d i f f e r e n c e a g a i n s u g g e s t s t h e NADP-IDH f r o m p i g h e a r t i s more t i g h t l y c o n t r o l l e d by (X -KGA t h a n i s t h e t r o u t enzyme. T h i s i n h i b i t i o n of IDH by CX-KGA has n o t been r e p o r t e d f o r any 7 2 Fig. V, 6. The effects of <p( -KGA i n h i b i t i o n on DL-isocitrate saturation curves for the p u r i f i e d NADP-IDH from pig heart. Assayed with 5 0 mM t r i s - H C l buffer, pH 7.5, 0.15 mM NADP+ and 1.0 mM MgCl 2. Cuvette temperature was 25°C. Specific Activity (jimols NADPH/rnin/mg protein) 73 T a b l e V, 3. The e f f e c t s o f t e m p e r a t u r e on t h e Km of D L - i s o c i t r a t e i n t h e p r e s e n c e o f 0.75 mM^-KGA.- The Km v a l u e s were c a l c u l a t e d f r o m L i n e w e a v e r -B u r k p l o t s u s i n g 0.15 mM NADP +, 1.0 mM M g C l 2 and 50 mM t r i s - H C l b u f f e r , t e m p e r a t u r e a d j u s t e d (pH 8.0 for.2°C-trout l i v e r and pH 7.5 f o r p i g h e a r t NADP-IDH). K m ( D L - i s o c i t r a t e ) x 10 3M Temp. °C P i g h e a r t IDH T r o u t l i v e r IDH 1.0 6.5 5.0 6.5 . 10.0 ' 6.5 15,0 6.5 20.0 12.5 6.5 25.0 12.5 6.5 30,0 12.5 35.0 12.5 40,0 12.5 45.0 12.5 o t h e r NADP-IDH. I t has been s u g g e s t e d (Hochachka & Somero, 1971) t h a t i n o r d e r f o r i n h i b i t o r s t o be e q u a l l y e f f e c t i v e a t a l l e n v i r o n m e n t a l t e m p e r a t u r e s , t h e K i o f t h e i n h i b i t o r s h o u l d be r e l a t i v e l y t e m p e r a t u r e i n d e p e n d e n t . T a b l e V, 4 i s a l i s t o f K i ^ - K G A ) v a l u e s - f o r b o t h p i g h e a r t and t r o u t l i v e r NADP-IDH. These v a l u e s a r e i n t h e r a n g e r e p o r t e d by W i l l i a m s o n , S c h o l z & B r o w n i n g (1969) forC^-KGA i n mammalian t i s s u e s . The t r e n d i s t o w a r d s a n i n c r e a s e d K i a t t h e extreme h a b i t a t t e m p e r a t u r e f o r b o t h enzymes, w i t h n - v a l u e s ( r e p r e s e n t i n g t h e number o f s i t e s a t w h i c h t h e m e t a b o l i t e may b i n d ( A t k i n s o n , 1966) a p p r o a c h i n g 1.0 i n a l l c a s e s e x c e p t a t l o w t e m p e r a t u r e s f o r t h e t r o u t l i v e r enzyme. A t t e m p e r a t u r e s below 5°C, o( -KGA i s more e f f e c t i v e as an i n h i b i t o r of t r o u t l i v e r NADP-IDH s i n c e b o t h t h e n - v a l u e i n c r e a s e s and K i d e c r e a s e s . T h i s would s u g g e s t i n t h e a b s e n c e o f o t h e r c o n t r o l f a c t o r s , t h e ^ { - K G A and t e m p e r a t u r e d e c r e a s e s may t i g h t l y r e g u l a t e c a t a l y s i s by t h e t r o u t enzyme under t h e s e c o n d i t i o n s . S i n c e t h e K i r e p r e s e n t s a b i n d i n g c o n s t a n t ( D i x o n & Webb, 1 9 6 4 ) , t h e c h a n g e i n K i w i t h t e m p e r a t u r e s h o u l d and does f o l l o w t h e same p a t t e r n a s does t h e Km of D L - i s o c i t r a t e . T h i s i n t u r n i n d i c a t e s t h a t t h e Km of D L - i s o c i t r a t e s u p p l i e s a good a p p r o x i m a t i o n o f e n z y m e - s u b s t r a t e a f f i n i t y . 75 T a b l e V, 4. The e f f e c t s o f t e m p e r a t u r e on t h e K i ( c ^ - K G A ) f o r NADP-IDH f r o m p i g h e a r t and 2°C-acclimated t r o u t l i v e r . K i v a l u e s c a l c u l a t e d f r o m H i l l p l o t s i n p r e s e n c e o f 0.06 mM D L - i s o c i t r a t e , 0.15 mM NADP +, 1.0 mM M g C l 2 and 50 mM t r i s - H C l , t e m p e r a t u r e a d j u s t e d (pH 8.0 f o r 2oc-t r o u t l i v e r and pH 7.5 f o r p i g h e a r t NADP-IDH). N - v a l u e s c a l c u l a t e d f r o m t h e s l o p e o f t h e H i l l p l o t l i n e and g i v e n i n b r a c k e t s f o l l o w i n g K i v a l u e s . Ki(<*.-KGA) x 10 4M Temp. °C P i g h e a r t IDH T r o u t l i v e r IDH 3.0 6.8(2.1) 5,0 4.5(3.2) 10.0 6.0(1.3) 15,0 — 8.1(.77) 20.0 7.0(0.9) 25.0 6,4(1.0) 30,0 7.3(1.0) 35.0 7.3(1.0) 40.0 9.5(1.0) 45,0 9.2(1.0) 76 DISCUSSION It i s apparent that the control of c a t a l y s i s by NADP-IDH from pig heart and trout l i v e r d i f f e r s . Values of a c t i v a t i o n energies and molecular weights, although not i d e n t i c a l , do not d i f f e r s i g n i f i c a n t l y . A l s o , the magnitude of i n h i b i t i o n byO^-KGA and oxaloacetate + glyoxylate are s i m i l a r , suggesting these s p e c i f i c properties allow for IDH to function as an oxidoreductase of i s o c i t r a t e and are to be expected wherever the enzyme i s found, Similar findings have been demonstrated f o r certain pressure responses at saturating substrate concentrations for fructose-diphosphatase and pyruvate kinase (Hochachka, Schneider & Moon, 1971; Moon, Mustafa & Hochachka, 1971), and probably are related t o the s t e r i c placement of substrates and cofactors. However, the response of the Km of DL-isocitrate to temperature d i f f e r s as does the magnitude of the response i n the presence of the i n h i b i t o r ^-KGA. L i t t l e work has been reported regarding the effects of temperature on the a f f i n i t y constants of other vertebrate enzymes. The Km of fructose-diphosphate for rabbit muscle aldolase exhibits a complex U-shaped curve when determined at temperatures between 5 and 5 0°C (Lehrer & Barker, 1970), a res u l t which mimics the response of the binding constant. These results suggested to Lehrer & Barker that changes i n the i o n i z a t i o n and solvation of the substrate or i n h i b i t o r were responsible for the temperature e f f e c t s , An equally v a l i d interpretation might assume the same direct effects on the enzyme conformation. Cowey (1967) found a 15-fold increase i n the Km of D-glyceraldehyde-3-phosphate for D-glyceraldehyde-3-phosphate dehydrogenase i n rabbit muscle between 5 and 35°C. When these data were compared with the homologous l o b s t e r and cod f i s h enzymes, i n c r e a s e s o f l e s s t h a n 1 0 - f o l d w e r e n o t e d between t h e same t e m p e r a t u r e s . The Km o f p h o s p h o e n o l p y r u v a t e f o r r a t m u s c l e p y r u v a t e k i n a s e v a r i e s by l e s s t h a n a f a c t o r o f two be t w e e n 10 and 40°C, u n l i k e t h e r e s p o n s e seen i n p o i k i l o t h e r m i c p y r u v a t e k i n a s e s (Somero & Hochachka, 1 9 6 8 ) . No g e n e r a l i z e d p a t t e r n can be d e r i v e d f r o m t h e s e mammalian s t u d i e s . However, f o r NADP-IDH, i t i s a p p a r e n t t h a t m o d u l a t i o n o f enzyme a c t i v i t i e s a r e d i f f e r e n t , d e p e n d i n g upon t h e o r g a n i s m examined. I n t h e p i g h e a r t enzyme, l i t t l e o r no s e l e c t i v e p r e s s u r e would be a p p l i e d t o t h e temper-a t u r e c h a r a c t e r i s t i c s o f t h e Km; i n s t e a d , t h e t h e r m a l p r o p e r t i e s o b s e r v e d a r e m e r e l y t h e e x p r e s s i o n o f t h e c h e m i s t r y o f t h e r e a c t i o n , F o r t r o u t l i v e r NADP-IDH, t h e m a i n t e n c e o f a c o n s t a n t Km f o r e f f i c i e n t m o d u l a t i o n a t l o w s u b s t r a t e c o n c e n t r a t i o n s a t n o r m a l b i o l o g i c a l t e m p e r a t u r e s , and t h e r e d u c t i o n o f Qio by i n c r e a s i n g t h e Km a t t h e h i g h t e m p e r a t u r e s i s u n d e r s t r i c t s e l e c t i v e p r e s s u r e s . These d i f f e r e n c e s c a l l f o r d i f f e r e n t e v o l u t i o n a r y s t r a t e g i e s w h i c h may be an outcome o f t h e i n c r e a s e d p o t e n t i a l f o r gene e x p r e s s i o n i n t h e t e t r a p l o i d f i s h (Ch. V I ) as w e l l as t h e i r t h e r m a l h i s t o r y . A n o t h e r a s p e c t of c a t a l y t i c c o n t r o l by NADP-IDH fr o m p i g h e a r t and t r o u t l i v e r i s t h e d i f f e r e n c e i n t h e Km o f D L - i s o c i t r a t e i n t h e p r e s e n c e o f °(-KGA. Even though t h e Km iat.emperature i n d e p e n d e n t i n t h e p r e s e n c e o f t h e i n h i b i t o r ( T a b l e V, 3 ) , t h e K i of 0(-KGA i s t e m p e r a t u r e dependent, w h i c h i s p a r t i c u l a r l y marked a t t h e l o w t e m p e r a t u r e s ( T a b l e V, 4 ) . T h i s may s u g g e s t t h a t ^ -KGA i s an i m p o r t a n t c o n t r o l l i n g m e t a b o l i t e a t t h i s e n v i r o n m e n t a l t e m p e r a t u r e (5°C), a l t h o u g h o t h e r f a c t o r s s u c h as pH o r i o n c o n c e n t r a t i o n may r e v e r s e the e f f e c t i v e n e s s o f t h i s i n h i b i t o r as has b e e n seen f o r AMP m o d u l a t i o n o f salmon f r u c t o s e - d i p h o s p h a t a s e ( B e h r i s c h , 78 1 9 6 9 ) . The b i n d i n g s i t e f o r b o t h t h e s u b s t r a t e , i s o c i t r a t e and t h e i n h i b i t o r , CX -KGA, a r e p r o b a b l y s i m i l a r f o r b o t h NADP-IDHs examined. E s t i m a t e s o f a c t i v i t y a t s a t u r a t i n g s u b s t r a t e c o n c e n t r a t i o n s and i n h i b i t i o n s t u d i e s s u p p o r t t h i s c o n c l u s i o n . However, t h e t e r t i a r y s t r u c t u r e s u n d o u b t e d l y d i f f e r . O p p o s i t e 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 a t pH 7.0, h i g h n - v a l u e s f o r enzyme-C\-KGA a f f i n i t i e s f o r t h e t r o u t l i v e r enzyme, and a l t e r e d t e m p e r a t u r e - K m r e s p o n s e s , a l l s u b s t a n t i a t e t h i s p r o p o s a l . The t r o u t enzyme has e v o l v e d a p r o t e i n s t r u c t u r e w h i c h i s more f l e x i b l e p o s s i b l y a s a r e s u l t o f a l t e r a t i o n i n t h e exposed amino a c i d s , but p r o b a b l y not b y a change i n t h e a c t i v e s i t e . T h i s i d e a o f a l t e r a t i o n o f t h e e x t e r i o r a m ino a c i d s and c o n s e r v a t i o n o f a c t i v e s i t e g r o u p s has been found t o be a g e n e r a l r u l e when p r o t e i n sequences a r e i n v e s t i g a t e d ( H i l l , e t a l . , 1 9 6 9 ) . These a l t e r a t i o n s i n p r i m a r y sequence may be r e s p o n s i b l e f o r f u n c t i o n a l a d a p t a t i o n o f t h e enzyme t o e n v i r o n m e n t a l p a r a m e t e r s such as t e m p e r a t u r e o r h y d r o s t a t i c p r e s s u r e . CHAPTER VI; The Effects of Temperature on the Kinetic Properties of Various Isozymes of the Liver Soluble NADP+-Linked I s o c i t r a t e Dehydrogenase from Rainbow Trout 79 INTRODUCTION P o i k i l o t h e r m i c enzymes appear t o be e v o l u t i o n a r i l y t a i l o r e d t h r o u g h n a t u r a l s e l e c t i o n f o r t h e r m a l l y i n d e p e n d e n t f u n c t i o n . K i n e t i c s t u d i e s o n a number o f s u c h enzymes, p a r t i c u l a r l y f r o m t h e t e m p e r a t e r a i n b o w t r o u t , have d e m o n s t r a t e d complex U-shaped c u r v e s when t h e a p p a r e n t Km i s p l o t t e d a g a i n s t t e m p e r a t u r e . T h i s work has r e c e n t l y been r e v i e w e d b y Hochachka & Somero (1971) and Somero & Hochachka ( 1 9 7 1 ) . A t t h e u p p e r b i o l o g i c a l t h e r m a l r a n g e , t e m p e r a t u r e d e c r e a s e s a c t a n a l o g o u s l y t o p o s i t i v e m o d u l a t o r s by i n c r e a s i n g e n z y m e - s u b s t r a t e (ES) a f f i n i t y . N e a r t h e l o w e r r a n g e , t h e o p p o s i t e i s t r u e ; t e m p e r a t u r e d e c r e a s e s r e s u l t i n E S - a f f i n i t y d e c r e a s e s . Between t h e s e two e x t r e m e s , w h i c h n o r m a l l y c o i n c i d e s w i t h t h e a n i m a l ' s h a b i t a t t e m p e r a t u r e , E S - a f f i n i t y r e m a i n s c o n s t a n t , t h u s a s s u r i n g e f f i c i e n t c o n t r o l of c a t a 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 found i n t r a c e l l u l a r l y , T here a r e t h r e e g e n e r a l mechanisms a v a i l a b l e t o t h e p o i k i l o t h e r m f o r a c h i e v i n g t h e r m a l l y - c o m p e n s a t e d enzyme c a t a l y s i s : (1) a s i n g l e enzyme s u i t e d f o r f u n c t i o n o v e r t h e e n t i r e b i o l o g i c a l r a n g e ; (2) e x p r e s s i o n of i s o z y m e s w i t h d i f f e r e n t t h e r m a l " p r e f e r e n c e s " ; o r (3) a s i n g l e enzyme w i t h an a d j u s t m e n t i n t h e c e l l u l a r m i l i e u . A p r i o r i i t a p p e a r e d t h a t each of t h e s e mechanisms a r e e q u a l l y l i k e l y t o o c c u r , a l t h o u g h i n p r e v i o u s work (Hochachka & Somero, 1971; Ch. I V , t h i s t h e s i s ) t h e l a t t e r have been emphasized. However, an adequate t e s t o f t h e s e a l t e r n a t i v e s f o r any g i v e n enzyme sys t e m i s n o t y e t a v a i l a b l e . I n a p r e l i m i n a r y e x a m i n a t i o n , a h a t c h e r y grown p o p u l a t i o n of r a i n b o w t r o u t , Salmo g a i r d n e r i i , was found t o c o n t a i n a l a r g e amount of h e t e r o -g e n e i t y a t t h e gene l o c u s ( i ) c o d i n g f o r t h e l i v e r s o l u b l e NADP-IDH. I t 80 was of i n t e r e s t to determine whether or not a l l i n d i v i d u a l s , i r r e s p e c t i v e o f t h e i r NADP-IDH isozymal content, showed i d e n t i c a l IDH k i n e t i c pro-p e r t i e s . In t h i s way, i t may be possible to suggest whether the presence o f isozymes, which are p a r t i c u l a r l y numerous i n these t e t r a p l o i d f i s h , may r e s u l t i n the unusual k i n e t i c behaviour exhibited by t h i s rainbow trout enzyme system as previously reported (Ch. IV). The r e s u l t s suggest that although changes i n the c e l l u l a r m i l i e u may a l t e r the Km-temperature response, a l t e r a t i o n i n t i s s u e isozyme content are of more probable s i g n i f i c a n c e . By increasing the number o f the slowest migrating isozymic forms, the Km-temperature response tends to increase at high assay temperatures. Also, i n d i v i d u a l s which maintain a s i n g l e s t a i n i n g band of the l i v e r soluble NADP-IDH show temperature-independent E S - a f f i n i t y , a response which would appear to be s e l e c t i v e l y disadvantageous i n eurythermal species such as the r ainbow tr o u t . RESULTS 8.1 Changes i n the c e l l u l a r m i l i e u . One possible mechanism leading to an a l t e r a t i o n i n the apparent Km-temperature response i s a change i n the c e l l u l a r m i l i e u . F i g. VI, 1, suggests that by changing either the NADP+ or DL-isocitrate concentration independent of one another, the Km for the alternate substrate at 15°C w i l l be reduced to minimal values reported for the enzyme system from pooled 2°C-acclimated rainbow trout l i v e r s (see F i g , IV, 7). In either case, the response i s again a complex function of temperature; at 15°C, the Km vs substrate curve i s U-shaped, whereas at 3°C the relationship-i s l i n e a r at a l l measured substrate concentrations, As has been previously shown (Ch, IV), the fluctuations i n the Km of NADP+ are not as great as seen for the Km of DL-isocitrate, This res u l t suggest that a minimal value of Km for substrate can be obtained irrespective of temperature, and t h i s value may approximate the true i n t r a c e l l u l a r i s o c i t r a t e and NADP* l e v e l s . Changes i n metabolite concentrations are known to occur during altered tissue metabolism (Williamson, Herczeg, Coles & Cheung, 1967) and i n some cases are known to result i n modified a f f i n i t y constants (Gumaa, McLean & Greenbaum, 1971), Theoretically, changing the enzyme concentration may likewise modify the Km-temperature response. However, Fig. VI, 2 shows that although Vmax i s l i n e a r when plotted against enzyme-protein concentration, the Km i s invariant. This mechanism i s probably of l i t t l e importance since changes i n NADP-IDH a c t i v i t i e s during acclimation are minimal (Ch. IV). 82 Fig. VI, 1. The effects of changing substrate concentrations on the Km of the alternate substrate for the l i v e r soluble NADP-IDH from 2°C~-acclimated rainbow trout. In the s o l i d symbols ( @ 15°C and H 3°C assay temperatures), the Km(DL-isocit) i s determined at a number of NADP+ concentrations; the open symbols ( O 15°C and G 3°C assay temperatures), the Km(NADP+) i s determined at a number of DL-isocit-r a t e concentrations. 83 Fig. VI, 2, The effects of enzyme protein concentration upon the DL-i s o c i t r a t e saturation curves of the l i v e r soluble NADP-IDH from 2°C-acclimated rainbow trout. Assayed at 10°C with 50 mM t r i s - H C l , pH 8.0, 0.15 mM NADP+ and 1.0 mM MgCl 2. The insert i s a plot of Vopt (•) and Km(DL-isocit) CO) at three enzyme protein concentrations. Velocity i s given i n absorbancy un i t s . II A h oocP*o"° o jj—O 2 0 X f I I ' I ' I H I •1 -2 - 3 - 4 - 5 " 1 0 M(DL-isocitrate) X 1 0 3 84 Kinetics of trout l i v e r NADP-IDH isozymes. Heterogeneity at the l i v e r NADP-IDH loc u s ( i ) has been observed and F i g . VI, 3 i s a composite electrophoretogram of f i v e out of the s i x isozymic patterns expressed i n the trout hatchery population. The three-(A2,B-2,C2) and one-band (A 2) phenotypes occur i n the greatest abundance during both l a t e winter and l a t e spring (Table VI, 1). I t i s assumed that trout NADP-IDH i s si m i l a r to b a c t e r i a l and vertebrates IDHs i n being a dimer (e.g., Henderson, 1965; Howard & Becker, 1970; Quiroz-Gutierrez & Ohno, 1970), so there are at least four possible subunit types. The absence of the expression of the B and C subunits as i n d i v i d u a l s would suggest, (1) the A subunit i s the pr i m i t i v e or ancestral subunit, (2) epigenetic control of gene expression of these l o c i as has been i d e n t i f i e d for the LDH system (Whitt, 1970; Rosenberg, 1971), and/or (3) that only under the extremes of summer acclimatization w i l l these subunits be expressed. The three isozymic patterns used i n the k i n e t i c analysis are seen i n F ig. VI, 4 together with a composite plot of the Km of DL-isocitrate v s temperature re l a t i o n s h i p . I t i s apparent that the A2-NADP-IDH from spring rainbow trout l i v e r , whether as a crude high speed supernatant or a p a r t i a l l y p u r i f i e d preparation ( i . e . , (NH^^SO^ p e r c i p i t a t i o n followed by d i a l y s i s ) , shows temperature independent k i n e t i c behaviour. Unlike the A 2, as the number of B and C subunits increase, an upswing i n  Km at the high temperatures i s noted. Therefore, at 25°C, the A2 ,B2 ,C2 isozymes show a three-fold increase i n Km over the value at 10°C. With only the A2>AB,B2 isozyme system, the upswing i s less pronounced and begins at 15°C instead of 10°C. This observation makes evident that f o r the NADP-IDH from trout l i v e r , at l e a s t , the p a r t i c u l a r Km-temperature 85 Fig. VI, 3. Electrophoretogram of f i v e out of the s i x observed l i v e r soluble NADP-IDH phenotypes i n the Sun Valley rainbow trout hatchery population. Run for 20 hr at 200 V (approximately 20 mA, 5°C) using a citrate/phosphate buffer system, pH 7.0. 1. A2,AB,B2,BC,C2; 2. A 2; 3. A 2,B 2,C 2; 4. A'2,A'A,A2,AB,B2,BC,C2; 5. A2',A'A,A2. 86 Table VI, 1. The r e l a t i v e d i s t r i b u t i o n (as per cent of tot a l ) of isozymic forms of the l i v e r soluble NADP-IDH from a hatchery population of rainbow trout (S. g a i r d n e r i i ) . Distributions determined from the presence or absence of sta i n on starch-gel electrophoresis. Winter f i s h were co l l e c t e d and assayed on March 24, 1971 and spring f i s h May 31, 1971, Total number of f i s h i n each group was f i f t y i n d i v i d u a l s . Pattern Winter Spring 27.0 20.0 A 2 ,B2,C2 41.7 42.5 A2,AB,B2 8,3 12.5 A2,AB,B2,BC,C2 6.3 15.0 A21,A'A,A2 10.4 2.5 A2',A'A,A2,AB,B2,BC,C2 6.3 7.5 87 Fig. VI, 4. The effects of the tissue isozymal content on the Km (DL-isocit) vs temperature relationship for the l i v e r soluble NADP-IDH of spring rainbow trout. Assayed i n 50 mM t r i s - H C l buffer (pH 8.0, temperature adjusted), 0.15 mMNADP+ and 1.0 mM MgCl2» The curve for A2-NADP-IDH i s given for both the crude (O) and p a r t i a l l y p u r i f i e d (®) preparations. Electrophoretic i n s e r t : (1) A2,B2,C25 (2) A2,AB,B2J (3) p a r t i a l l y p u r i f i e d A2; and (4) crude A2. 88 curve obtained i s determined by the isozymal composition of the tissues, not by an adjustment i n the c e l l u l a r m i l i e u . 89 DISCUSSION The evolution of the family Salmonidae by the process of t e t r a -p l o i d i z a t i o n i s well documented (see Ohno, Wolf & Atkin, 1968; Ohno, 1970) and multiple gene l o c i coding for a large number of isozymes of p a r t i c u l a r enzymes attest to t h i s theory (e.g., Massaro & Markert, 1968; B a i l e y , Cocks & Wilson, 1969; Engle, Hof & Wolf, 1970; Wolf, Engel & Faust, 1970). Duplication of the NADP-IDH locus i n salmonids i s complex, since d i f f e r e n t tissues do not necessarily show the same rate of d r i f t i n g apart of the copied l o c i r e s u l t i n g i n the establishment of a new gene locus i n the population (Wolf, Engel & Faust, 1970). S almo g a i r d n e r i i l i v e r soluble NADP-IDH does not exhibit d i p l o i d i z a t i o n s ince a single a c t i v i t y band for t h i s enzyme i s seen i n some members of a hatchery population (Fig. VI, 1). I t i s interesting to note, however, that nearly three-quarters of the population maintain some polymorphism at t h i s gene locus, suggesting a selective advantage may be conferred upon these i n d i v i d u a l s . Polymorphic enzyme systems have been investigated i n a number of n a t u r a l populations, and the r e s u l t s are as numerous as the enzymes themselves. Heterozygote advantage i s maintained by temperature at the serum esterase locus i n the fresh water f i s h Catastomus c l a r k i i (Koehn, 1 969), but the k i n e t i c differences between variant heart LDHs are minimal i n geographically separated populations of the frog Rana pipiens, a l l of which maintain s p e c i f i c temperature preferences.(Levy & Salthe, 1971) . It i s d i f f i c u l t i n t h i s case to define the advantage of maintain-i n g polymorphic l i v e r soluble NADP-IDH. However, i t i s possible that the individuals containing only a single a c t i v i t y band, and therefore, 9 0 presumably homozygous at the IDH locus, can not tolerate extreme temper-ature changes, and c e r t a i n l y temperature compensation by Km modulation i s not possible. Table VI, 2 shows that the Q 1 Q between 1 0 and 20°C for the A 2 and A2>B2,C2 isozymal systems d i f f e r . Since the Km i s invariant, the temper-ature coeficient i s increased above that found for the A2,B2>C2 system near Km concentrations of substrates. As substrate concentrations approach i n vivo conditions, QIQ values for A2,B2,C2-NADP-IDH would furth e r decrease, res u l t i n g i n an even greater difference between the two enzyme variants. Thus, those individuals which are heterozygous at t h i s l o c u s ( i ) may be conferred a selective advantage over those that are homozygous. A sim i l a r argument has been made at the low temperature range, where both Km and thermal energies are changing, r e s u l t i n g i n extremely high Q-^ Q values (Hochachka & Somero, 1 9 7 1 ) . I t has been reported (Ch. IV) that i n cold-acclimation, the amount of the slowest migrating l i v e r soluble NADP-IDH isozymes increase, again indicating the importance of the temperature-isozyme re l a t i o n s h i p . Lake trout populations have been observed to maintain a single s t a i n i n g a c t i v i t y band of l i v e r soluble NADP-IDH (Ch. I l l ) and LDH (Hochachka, 1 9 6 6 ) . This f i s h i s known to be stenothermal (Peter Ihssen, personal communication), l i v i n g at the bottom of deep lakes where environmental fluctuations are minimal. The genotype showing t h i s i d e n t i c a l s i n g l e a c t i v i t y band for l i v e r NADP-IDH i n rainbow trout may have resided a t sometime i n thei r previous history i n a sim i l a r constant environment, and processes of natural selection may not have had the necessary time t o eliminate t h i s phenotypic pattern from the hatchery population where i t appears to be at a disadvantage. Table VI, 2. Q 1 Q values between 10 and 20°C for the A 2 and A2,B2,C, l i v e r soluble NADP-IDH isozyme systems from spring rainbow trout at various DL-isocitrate concentrations. Assays carried out with 1 mM MgCl 2, 0.15 mM NADP+ and 50 mM tr i s - H C l buffer, t i t r a t e d to pH 8.0 a t the respective temperatures. Q l 0 (10-20°C) [DL-isocitrate] mM A 2 A 2,B 2,C 2 0.1 3.6 3.8 0.05 3.4 3.3 0.025 4.2 2.8 Alterations i n substrate or cofactor concentrations may also be responsible for the complex k i n e t i c properties of this enzyme. From the r e s u l t s reported here, i t i s apparent that at 15°C, levels of NADP+ can determine the Km of DL-isocitrate; the minimal Km value i s f d e n t i c a l t o the lowest value at 3°C. By a l t e r i n g the NADP+ concentration at 3°C, however, no change i n Km occurs, suggesting that a s p e c i f i c enzyme may have a ch a r a c t e r i s t i c substrate a f f i n i t y constant which allows for maximal enzyme effectiveness. Temperature and the presence of non-phy s i o l o g i c a l substrate concentrations may a l t e r the binding of substrate t o enzyme by putting the enzyme into unfavorable conformation, thereby reducing i t s e f f i c i e n c y . These metastable enzyme conformations may be of importance to the enzyme i n vivo (Nickerson & Day, 1969; Ibsen, S c h i l l e r & Haas, 1971; I k a i & Tanford, 1971). The importance of these experiments i s that the complex k i n e t i c behaviour of certain poikilothermic enzymes need not be s p e c i f i c a l l y r e l a t e d to changes i n the primary structure of the enzyme. Instead, a l t e r a t i o n s i n the genetic make-up and the substrate/cofactor concent-r a t i o n s must be considered as possible mechanisms. These parameters are known to change i n natural populations and may be used as a sensitive measure of k i n e t i c changes i n enzyme function. CHAPTER VII: Summating Remarks 93 These studies on rainbow trout l i v e r NADP-IDH have suggested a number of conclusions concerning both the control of i s o c i t r a t e oxidation and possible mechanisms of enzyme adaptation to fluctuations i n environmental temperatures. To conclude t h i s study, a number of these w i l l be discussed as well as questions i n i t i a t i n g further experimentation. Homologous enzymes can show similar physical and c a t a l y t i c properties as a result of the strong selective pressure for the main-tanence of the st r u c t u r a l i n t r e g r i t y of the active s i t e . According to Lipscomb (1971), the active s i t e usually i s i n a l o c a l i z e d depressed area of the enzyme and not involved i n molecular interactions. Active s i t e analysis has so predisposed biochemists for the l a s t decade that i t has only recently been recognized that s t r u c t u r a l alterations of the entire molecule are equally important. For example, the f i l l i n g of the active s i t e cavity by the substrate of carboxypeptidase A results i n l o c a l i z e d movement of s p e c i f i c groups which i s amplified many fo l d throughout the entire molecule (Lipscomb, 1971). These d i s t o r t i o n s of enzyme by substrate complexing, re s u l t s i n a decrease i n the energy of a c t i v a t i o n , a theory used by Pauling i n 1948 to explain the c a t a l y t i c e f f i c i e n c y of enzymes. These events appear to be so basic, and the c a t a l y t i c s i t e so conservative, that active s i t e analysis, per se, may not give us any new information concerning the unique nature of p o i k i l o -thermic enzymes. In f a c t , Table VII, 1 indicates t h i s very conclusion. Each parameter l i s t e d on t h i s table for rainbow trout l i v e r NADP-IDH has a near mirror image i n the pig heart enzyme, except electrophoretic mobility. Molecular weights are s i m i l a r , although not i d e n t i c a l , as are the Km of DL-isocit-Table VII, 1. Comparative properties of rainbow trout l i v e r and p i g heart NADP-IDH, Approximate m.w. Electrophoretic mobility at pH 7,0 Ea value Km(DL-isocit) at ambient temperatures Cation requirement <^  -KGA in h i b i t o n ADP a c t i v a t i o n OXA + glyoxylate i n h i b i t i o n (1 mM) Trout Liver NADP-IDH > 60,000 anodally 19 Kcal/mole 2.5 x 10"5M(10°C) absolute present present complete Pig Heart NADP-IDH 60,000 cathodally 25.Kcal/mole 3,2 x 10~5M(37 absolute present complete 95 r a t e and Ea values. The substrate, cofactor and i n h i b i t o r s p e c i f i c i t i e s are i d e n t i c a l . Chung & Franzen (1970) have studied NADP +-binding and H + release from b a c t e r i a l (A. v i n e l a n d i i ) and mammalian (pig heart) NADP-IDH and found that they are i d e n t i c a l i n both cases. These properties can be termed general enzyme c h a r a c t e r i s t i c s ; that i s , the invariant properties which d i f f e r e n t i a t e IDH from other enzymes. Undoubtedly, there are strong selective pressures for t h e i r maintanence, and i f through mutations they are altered, natural selection w i l l take i t s t o l l . These types of mutations according to Ohno (1970) are termed forbidden  mutations since they are always selected against. Tolerable mutations, whether neutral or favored, can accumulate i n other parts of the amino acid sequence by missense or samesense mutations. F i t c h (1966) found that 40% of a l l missense mutations re s u l t i n changing the net charge of the polypeptide chain specified by that gene locus. I t i s not surprising, therefore, to find a difference i n electrophoretic m o b i l i t i e s of homologous enzymes from widely separated species. These subtle changes i n primary sequence may be responsible for the unique properties of poikilothermic enzymes. The hypothesis that the primary sequence of a protein determines i t s conformation i n a given environment has been proven i n many cases (Epstein, Goldberger & Anfinsen, 1963). However, i n vivo folding of a polypeptide chain could lead to a stable active conformation at an energy minimum, which need not necessarily be the thermodynamically most stable enzyme con-formation (Nickerson & Day, 1969). An example of such a "metastable" but active enzyme species, has been found for chicken mitochondrial malate dehydrogenase ( K i t t o , Wassarman & Kaplan, 1966). This increased conformational f l e x i b i l i t y , persumably under strong p o s i t i v e selective 96 pressures (Alexandrov, 1969), may be responsible for the altered f u n c t i o n a l properties of rainbow trout l i v e r NADP-IDH compared to the p i g heart enzyme. As has been argued by Hochachka & Somero (1971), the adaptive s i g n i f i c a n c e of the Km-temperature response resides i n two points; (1) at the thermal range encountered by the organism, a constant Km f o r temperature-independent control of c a t a l y s i s i s favored, and (2). a t the extreme temperatures, a reduction i n Q-^Q at low substrate concentrations minimizing the increase i n temperature i s favored. Trout l i v e r NADP-IDH, as seen i n Ch. IV, exhibits t h i s same response as seen for a large number of other enzymes from t h i s species (Somero • & Hochachka, 1971). I t i s apparent that selective pressures maintain t h i s adaptive response only when necessary; i n pig heart NADP-IDH, Km decreases d i r e c t l y with temperature, so that control of c a t a l y s i s i s not temperature-independent at any assay temperature. Other c o n t r o l l i n g parameters, such as the degree of product i n h i b i t i o n , also f a l l into t h i s area of enzyme properties which are under the control of natural se l e c t i o n , and w i l l vary depending upon the organism's environment and physiology. Before t h i s theoretical treatment can have experimental basis, homologous enzymes from poikilothermic and homeothermic tissues must be examined wit h the methods presently available to biochemists (Lipscomb, 1971). One postulated mechanism for the o r i g i n of metazoans, vertebrates and f i n a l l y mammals from u n i c e l l u l a r organisms i s evolution through t e t r a p l o i d i z a t i o n (Ohno, 1970). The duplication of the entire genome r e s u l t s i n the required redundancy needed for the creation of new fu n c t i o n a l genes. Many f i s h of the family Salmonidae, including the rainbow and other trout examined i n t h i s study (see Ch. I l l ) , are 97 autotetraploid, which explains the previously mentioned large number of mu l t i p l e enzyme forms as well as a DNA content per c e l l twice that of most vertebrates. If evolution from t h i s t e t r a p l o i d stage i s to occur, the disomic state must eventually be re-established by functional d i v e r s i f i c a t i o n of the four o r i g i n a l homologues, so that one o r i g i n a l linkage group i s s p l i t into two separate linkage groups. This process of d i p l o i d i z a t i o n i s occurring at di f f e r e n t rates not only i n the family Salmonidae, but also at s p e c i f i c gene l o c i within a single species (Ohno, 1970). Rainbow trout l i v e r soluble NADP-IDH appears to be subject to tetrasomic inheritance; that i s , d i p l o i d i z a t i o n of t h i s locus i s not complete as has been found by Wolf, Engel & Faust (1970) i n J3. i r i d e u s . The evidence for t h i s conclusion i s the existence of various isozymal phenotypes found i n i n d i v i d u a l hatchery trout, as reported i n Ch. VI. Brook trout are i n a s i m i l a r process of d i p l o i d i z a t i o n , but both splake and lake trout ( i f t e t r a p l o i d i z a t i o n r e a l l y occurred i n t h i s species) have disomic inheritance, with the occurrence of a single locus i n lake and two l o c i i n splake trout l i v e r NADP-IDH. The splake trout phenotype i s unique i n i t s response to temperature; here, three a c t i v i t y bands are always seen, only the r e l a t i v e m o b i l i t i e s are altered. Such an a l t e r a t i o n may be sim i l a r to the conformational isozymes seen for chicken mitochondrial MDH ( K i t t o , Wassarman & Kaplan, 1966), but furthe r study of t h i s isozyme system i s necessary. Acclimation to a new thermal regime has been found to a l t e r the expression of trout l i v e r NADP-IDH i n a number of species (Ch. I l l and IV). The increase i n the r e l a t i v e amount of the slowest migrating a c t i v i t y band i n the rainbow trout enzyme a l t e r s the Km-temperature 98 response of 2°C- vs 17°C-acclimated f i s h . During certain seasons, the 17°C-acclimated brook trout enzyme shows a simi l a r increase i n the number of NADP-IDH isozymes, The splake trout i s d i f f e r e n t i n that temperature either a l t e r s the enzyme already present, as suggested above, or that the expression of an e n t i r e l y new set of isozymes occur. In wild populations of Lake Erie g o l d f i s h , NADP-IDH heterogeneity has been correlated with the high levels of p o l l u t i o n , so that increased f l e x i b i l i t y w i l l be s e l e c t i v e l y advantageous (Quiroz-Gutierrez & Ohno, 1970). Thermal fluctuations can also put a premium on enzyme hetero-geneity, as seen from these re s u l t s i n dir e c t support of the theoreties of Haldane (1955) and Mayr (1963). Lake trout, however, being extremely s tenothermal have not been forced to maintain large amounts of enzyme heterogeneity. Taken together, the k i n e t i c results and the extent of heterogeneity a t the NADP-IDH locus(i) suggest either that t h i s enzyme i s evolving at a rapid rate, or that selective pressures to maintain one s p e c i f i c phenotype are not great. Studies on hatchery populations of trout can not d i f f e r e n t i a t e between these alternatives. The most important point here, however, i s that the i n vivo alterations i n the genotypic expression of these isozymes, irrespective of c e l l u l a r m ilieu changes, can determine the k i n e t i c response of NADP-IDH to temperature. This suggests the importance of the Km-temperature response to the i n vivo operation of t h i s enzyme. As mentioned i n the introduction, the role of NADP-IDH i n the c e l l u l a r metabolism of vertebrates (except for ruminants) i s unknown. NAD-IDH apparently functions i n the Krebs cycle oxidation of i s o c i t r a t e 99 (Nic h o l l s & Garland, 1969), and glucose-6-phosphate dehydrogenase of the pentose shunt and "malic" enzyme provide s u f f i c i e n t reducing equivalents f o r f a t t y acid synthesis ( F l a t t & B a l l , 1964; Wise & B a l l , 1964; Katz & Rognstad, 1966). The rainbow trout l i v e r enzyme may play a more important role i n metabolism than seen i n other vertebrates for three reasons: (1) the apparent absence of detectable NAD-IDH a c t i v i t y ; (2) the observed c o n t r o l properties of the enzyme; and (3) the requirement for increased f a t t y acid synthesis during thermal acclimation. Gumbman & Tappel (1962) investigated the Krebs cycle of f i s h t i s s u e s , but did not report the presence of a NAD-IDH, only the NADP-dependent enzyme. Further studies are i n order to completely eliminate the p o s s i b i l i t y of t h i s enzyme a c t i v i t y i n f i s h mitochondria, but present evidence i s a l l negative (Ch. IV; Crabtree & Newsholme, 1970). Modulation of the Km of DL-isocitrate by ADP and -KGA (Ch. IV) both suggest the functioning of t h i s enzyme when adenylate charge i s low; i . e . , during periods when ATP production i s required (Atkinson, 1968). Recent studies on the homologous enzyme from Alaskan king crab (Moon, unpublished data) tissues suggest NADPH i s a potent i n h i b i t o r of NADP-IDH, as i s 0( -KGA, again implying that high energy l e v e l s i n h i b i t t h i s enzyme. A sim i l a r observation of NADPH i n h i b i t i o n of A. v i n e l a n d i i NADP-IDH has been reported by Franzen,. Wichen & Chung (1971). A NADP-IDH enzyme with these c h a r a c t e r i s t i c s would function well within the Krebs cycle (Atkinson, 1968); i n fa c t , similar reasoning has been used i n assigning t h i s function to the NAD-IDH i n mammalian mitochondria ( N i c h o l l s & Garland, 1969). Ratios of NADPH/NADP within the cytoplasm are extremely high, 100 approaching 1000 (Krebs & Veech, 1967), Even at ra t i o s of 1:1, king crab IDH (Moon, unpublished data), "malic" enzyme (Behrisch, unpublished data) and glucose-6-phosphate dehydrogenase (Hochachka, unpublished data) are 30-50% i n h i b i t e d . Lipogenesis, per se, must control the a c t i v i t y of the NADPH producing enzymes by de- i n h i b i t i o n through a l t e r -i n g the NADPH/NADP r a t i o s . These cytoplasmically located enzymes would be under extremely r i g i d control and could function only when lipogenetic a c t i v i t y i s high. In the absence of further evidence, i t i s d i f f i c u l t to unequivocally assign a s p e c i f i c i n vivo function to NADP-IDH i n rainbow trout l i v e r . The k i n e t i c data presented here would suggest i t functions mainly i n the Krebs cycle oxidation of i s o c i t r a t e , while i t may have an additional p o t e n t i a l rol e i n lipogenesis. The question remains as to what mechanisms allow for the altered expression and k i n e t i c properties of trout l i v e r NADP-IDH following acclimation to a new thermal regime. For instance, does temperature have a dir e c t c e l l u l a r effect or does i t function coincidently with photoperiod? Recent studies on the altered expression of lactate dehydrogenase isozymes with changing photoperiod (Massaro & Markert, 1971) has an interesting s i m i l a r i t y to some of these data. This i n i t i a t i o n response i s followed by a systemic response, undoubtedly mediated through the action of hormones and/or the nervous system (Somero & Hochachka, 1971), A series of biochemical responses, culminating i n enzyme properties si m i l a r to those reported here, produce a new steady state d i f f e r e n t from that found before the acclimation process began. The gap between i n i t i a t i o n and culmination of the process i s 101 wide, although studies by Lehmann (1970a,b) on the transitory changes i n enzyme a c t i v i t i e s are a start i n the right d i r e c t i o n . However, a t o t a l approach to t h i s t r a n s i t i o n period thus far has not been i n i t i a t e d . CHAPTER VII I : Literature Cited 102 Alexandrov, V. Ya. 1969. Conformational f l e x a b i l i t y of proteins, their resistance to proteinases and temperature conditions of l i f e . Curr. Mod. B i o l . 3_:9-19. Anderson, T. R. 1970. 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