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The biochemistry of the skipjack swimming musculature and its application to metabolic control in vertebrate… Guppy, Michael 1978

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THE BIOCHEMISTRY OF THE SKIPJACK SWIMMING MUSCULATURE AND ITS APPLICATION TO METAECIIC CONTROL IN VERTEERAT I WHITE MUSCLE. BY MICHAEL GOPPY E. Sc (Honours). # A u s t r a l i a n N a t i o n a l U n i v e r s i t y , Canberra, 1973. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept the t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF ERITISfi COLUMBIA AUGUST, 1978 © Michael Guppy, 1978 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h Co lumb ia , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5 i i ABSTRACT The tunas c o u l d be c a l l e d the • u l t i m a t e teleosts». They have a high percentage of muscle and a high percentage of that muscle i s red muscle, the muscles are kept at above ambient temperatures by a counter c u r r e n t heat exchanger and the r e s p i r a t o r y c a p a b i l i t i e s of these f i s h are a c c o r d i n g l y high. The b e h a v i o r a l c u l m i n a t i o n of the s e c h a r a c t e r i s t i c s i s manifested i n swimming speeds, which can be e x t r a o r d i n a r i l y h i g h , on a s u s t a i n e d , or a bu r s t b a s i s . One of the h o t t e s t and f a s t e s t tunas, the s k i p j a c k , was used i n a study to determine (1) when each muscle i s a c t i v e (2) when and where the muscle heat i s produced and (3) what the advantage of the hot musculature i s to t h e animal. Evidence from E. H. , h i s t o l o g i c a l , enzyme and me t a b o l i t e s t u d i e s suggest t h a t the red muscle i s q u a l i t a t i v e l y q u i t e t y p i c a l although i t s a e r o b i c c a p a c i t y i s somewhat above t h a t of other t e l e o s t red muscles. The white muscle has t r u e l y astounding anaerobic c a p a b i l i t i e s , but a l s o d i s p l a y s an a e r o b i c c a p a c i t y not u s u a l l y found i n t e l e o s t white muscle. Further examination of white muscle biochemical o r g a n i z a t i o n r e v e a l e d a GP c y c l e which balances redox d u r i n g the a e r o b i c c a t a b o l i s m of glycogen and/or glucose. Both LDH (the t e r m i n a l step i n anaerobic g l y c o l y s i s ) and GPDH (the cy t o p l a s m i c arm of the GP c y c l e ) a r e present i n white muscle i n high a c t i v i t i e s . Since these enzymes p o t e n t i a l l y compete f o r a common c o - s u b s t r a t e , NADH, a t i g h t c o n t r o l of these two enzymes seemed necessary to ensure mutual e x c l u s i v e a c t i v i t y . M e t a b o l i t e r e g u l a t o r s of both enzymes were found which by i i i a f f e c t i n g the a b i l i t y o f each enzyme to compete f o r NADH, channel carbon and hydrogen to l a c t a t e and C02 and H20 under anaerobic and a e r o b i c c o n d i t i o n s r e s p e c t i v e l y . , The e f f e c t of temperature on metabolism was i n v e s t i g a t e d and i t i s concluded t h a t the s t a b i l i t y r a t h e r than the absolute •set p o i n t ' of the body temperature i s the more important f e a t u r e . i v TABLE OF CONTENTS ABSTRACT ............ , .. ....... . ........ . . . i i LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x LIST OF FIGURES .... ....... ... ....... X ACKNOWLEDGEMENTS . x i i CHAPTER 1. GENERAL INTRODUCTION ........................... 1 CHAPTER 2. MATERIALS AND METHODS 13 Animals .................... ................. ,14 Fresh ...... ........................................ 14 Frozen ............................................. 14 Hi s t o c h e m i s t r y ....................................... . 15 Su c c i n a t e Dehydrogenase S t a i n i n g ................... 15 LDH S t a i n i n g , 15 General S t a i n i n g 16 E l e c t r o n Microscopy ...................................16 Enzyme P r e p a r a t i o n ................... ..... ... ........ . 17 Enzymes For P r o f i l e s Of Red And White Muscles ..... . 17 Enzymes For K i n e t i c C h a r a c t e r i z a t i o n ............... 17 Enzymes For G e l E l e c t r o p h o r e s i s . ................... 19 Enzyme Assays 20 Enzyme Assays For Muscle P r o f i l e s .................. 20 Enzyme Assays For K i n e t i c C h a r a c t e r i z a t i o n ......... 22 Starch Gels ........................................... 22 Met a b o l i t e s ........................................... 23 P r e p a r a t i o n Of T i s s u e .............................. 24 M e t a b o l i t e Assay Techniques ........................ 25 P r e p a r a t i o n Of Blood ............................... 26 Competition S t u d i e s ................................... 26 V P r o t e i n Determination ................................. 26 Temperature Measurements .............................. 27 CHAPTER 3. HISTOCHEMISTRY, ULTRASTRUCTURE AND ENZYME PROFILES OF THE SWIMMING MUSCULATURE .................. 28 INTRODUCTION ......... ................... .. . 29 Mammalian Muscle ..................................... . 29 U I t r a s t r u c t u r e .................................... * ,29 E l e c t r o p h y s i o l o g y S t u d i e s .......................... 30 Enzymes And Metabolism ............................. 30 F i s h M u s c l e ... «.«.»«.«.....«. .... ...... ............... 32 HES0LTS AND DISCUSS ION: PART 1. ORGANIZATION OF THE SKIPJACK MUSCULATURE AT THE MYOTOMAL, CELLULAR AND SUBCELLULAR LEV EL ....*.......... «: ..• . . . ....•........«» 35 General Observations ............. . •................... 35 His t o c h e m i s t r y 35 Two Basic F i b e r Types .............................. 35 E l e c t r o n Microscopy Of The Red Muscle ................. 37 F i b e r S t r u c t u r e .................................... 37 M i t o c h o n d r i a l Abundance 37 C a p i l l a r i t y 38 I n t r a c e l l u l a r Fat .................................. 38 E x e r c i s e d Red Muscle ............................... 38 E l e c t r o n Microscopy Of White Muscle ................... 39 Fine S t r u c t u r e : Overview ........................... 39 General F i b e r S t r u c t u r e ............................ 39 L i p i d , M i t o c h o n d r i a , And C a p i l l a r i t y ............... 40 Glycogen Storage ................................... 40 Glycogen D e p l e t i o n In E x e r c i s e 41 v i RESULTS AND DISCUSSION: PART 2. ENZYME PROFILES OF RED AND WHITE MUSCLE ............... . 44 CH8PTER 4. METAEOLITE LEVELS AND TEMPERATURES IN THE RED AND WHITE MUSCLES DURING REST AND WORK 57 INTRODUCTION 58 RESULTS ................... ... ................ ............ 61 Meta b o l i t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 1 Glycogen ........................................... 61 Glucose .................. .......................... 63 Glucose-6-phosphate 63 Fructose-6-phosphate 63 Fruc t o s e Diphosphate ............................*. . 64 Pyruvate ...........................................64 La c t a t e .... . ....................................... 64 GP ............................................... , 65 C i t r a t e And Malate 65 Adenylates And Creatine-phosphate .................. 66 Aniino Acids ................................... ..... 66 Met a b o l i t e R a t i o s .......... .......... .. ............... 67 Cross Over P l o t s ...................................... 69 Temperatures Of The Red And White Muscle .............. 70 DISCUSSION ............................................... 72 CHAPTER 5. BOLE OF DEHYDROGENASE COMPETITION IN METABOLIC REGULATION: THE CASE OF LACTATE AND A-GLYCEBOPHOSPHATE DEHYDROGENASE ..... ................... ....... .......... 101 INTRODUCTION .... 102 PART 1. FACTORS AFFECTING THE ACTIVITY OF PURIFIED LDH AND GPDH FROM SKIPJACK WHITE MUSCLE. 105 v i i R e s ults 105 Gel E l e c t r o p h o r e s i s ................................ 105 Pfl P r o f i l e s 106 Substrate A f f i n i t i e s .....106 AT .P Iniixbxtion * •« **• • • • # # • • • • • • • f • • • • * * * • • » '10 8 GP And Creatine-phosphate E f f e c t s 108 Dxscussxon * •«• ••*••••••••*•••••»•«••**• "TO9 PART 2. COMPETITION FOR NADH BETWEEN GPDH AND LDH: THE EFFECTS OF IZ02YME FORM AND MODULATOR CONCENTRATION ... 113 Re s u l t s ............................................... 113 LDH-creatine-phosphate I n t e r a c t i o n s ............. ... 113 H4 And M4 LDH Versus GPDH ..........................114 GPDH Product I n h i b i t i o n ..115 GP S e n s i t i v e And I n s e n s i t i v e GPDH's Versus H Type LDH . 116 D i s c u s s i o n .....................•.. .................117 CHAPTER 6. GENERAL DISCUSSION ......v.....................150 APPENDIX I. THE ALPHA-GLYCEROPHOSPHATE CYCLE IN SKIPJACK WHITE MUSCLE ........................... ... ............. 159 INTRODUCTION ............. ............... 160 M a t e r i a l s And Methods ................................. 161 P r e p a r a t i o n Of Mit o c h o n d r i a ........................ 161 R e s p i r a t o r y Measurements ...........................162 Spectrophotcmetric S t u d i e s Of Mitochondria .........163 R e s u l t s And D i s c u s s i o n ................................ 163 I s o l a t e d White Muscle Mitochondria ................. 163 Rec o n s t r u c t i o n Of The GP C y c l e ..................... 165 APPENDIX I I . THE IMPORTANCE OF WATER AND OXYGEN IN THE v i i i EVOLUTION OF HYDROGEN SHUTTLE MECHANISMS .............. 176 INTRODUCTION .177 The S h u t t l e s ........... .... ........................... 178 Di s c u s s i o n ...180 APPENDIX I I I PYRUVATE KINASE FUNCTIONS IN HOT AND COLD ORGANS OF TUNA ....... ........... ...................... 194 INTRODUCTION ............................ ............ .....195 MATERIALS AND METHODS .................................... 198 F i s h Samples ........................ .... .......... .198 P r e p a r a t i o n Of Pyruvate Kinase ..................... 198 Assays 199 E l e c t r o p h o r e s i s .200 RES ULTS 201 E l e c t r o p h o r e s i s ............... ...... . •....••....... 201 S p e c i f i c A c t i v i t i e s ................................201 E f f e c t Of PH ....................................... 201 A f f i n i t i e s For PEP And ADP ......................... 201 E f f e c t Of ATP ......................................202 E f f e c t Of FDP ...........,202 E f f e c t Of A l a n i n e ..................................203 Combined E f f e c t s Of FDP, ATP And Alanine ...........203 Q10 Values ..................................v... . ».203 DISCUSSION 205 REFERENCES CITED .........................................227 ABBREVIATIONS USED .............................. ,•'. ...... .256 i x LIST OF TABLES Table 3-1. Enzyme P r o f i l e s In Red And White Muscles* ..... 51 Table 4-1. 1976 M e t a b o l i t e C o n c e n t r a t i o n s . ................78 Table 4-2. Amino Acid C o n c e n t r a t i o n s . .................... 81 Table 4-3. 1977 M e t a b o l i t e C o n c e n t r a t i o n s . . . . . . . . . . . . . . . . 83 Table 4-4. Glycogen And L a c t a t e L e v e l s * 85 Table 4-5. Glucose E q u i v a l e n t s . .......................... 87 Tafcle 4-6. Energy Charge And Adenylate Pool S i z e . ........ 89 Table 4-7. M e t a b o l i t e Ratios For Enzyme Reactions. ....... 91 Table 4-8. Lacta t e / p y r u v a t e R a t i o s . ..................... . 93 Table 4-9. Muscle Temperatures. 95 Table 5-1. K i n e t i c Constants Of LDH And GPDH From White Muscle. ......................................••*.... ... 120 Table 5-2. I n h i b i t i o n Of LDH By 20 MM Creatine-phosphate. 122 Table 5-3. The A f f i n i t i e s For GP Of GPDHs From Various V e r t e b r a t e And I n v e r t e b r a t e Muscles. .................. 124 Table 5-4. Competition For NADH Ox i d a t i o n Between LDH And GPDH. ................................................. 126 Table 1-1. R e l a t i v e R e s p i r a t i o n Bate Of I s o l a t e d White Muscle Mitochondra. ................................... 170 Tafcle I I I - 1 . ATP I n h i b i t i o n Constants For The Red Muscle And Heart Pyruvate Kinases From The S k i p j a c k . .........211 Table I I I - 2 . The E f f e c t Of FDP On Pyruvate Kinase From The Heart And Red Muscle Of S k i p j a c k . .....213 Table I I I - 3 . The E f f e c t Of 0.1 MM FDP On The A c t i v i t y Of The Heart And Red Muscle Pyruvate Kinases ............. 215 Table I I I - 4 . Q10 Values Of Sk i p j a c k Red Muscle And Heart Pyruvate Kinases. , * 217 LIST OF FIGURES Fi g u r e 1-1. Cross S e c t i o n s Of F i s h 11 Figur e 3-1. A P l o t Of F i b e r Diameters For Red And White Muscle Versus Frequency. .............................. 53 Figure 3-2. His t o c h e m i s t r y Of The Red And S h i t e Muscle. .. 55 Figure 4-1. Cross Over P l o t ; Rest Verses Feeding. ........ 97 Fig u r e 4-2. Cross Over P l o t ; Rest Verses Burst. .......... 99 Figur e 5-1. S t a r c h Gel E l e c t r o p h o r e s i s Of S k i p j a c k Red And White Muscle LDH And GPDH. 128 Fig u r e 5-2. E f f e c t Of PH On S k i p j a c k White Muscle LDH And GPDH. .130 Figur e 5-3. E f f e c t Of The Co-su b s t r a t e On The Km Of Pyruvate And NADH Of S k i p j a c k White Muscle LDH. ....... 132 F i g u r e 5-4. The E f f e c t Of Temperature On The S u b s t r a t e A f f i n i t y Constants Of S k i p j a c k White Muscle LDH And GPDH. ......«..«..................'.......^.«........... .134 F i g u r e 5-5. The E f f e c t s Of PH On The Km Of Pyruvate And NADH Of Skipjack White Muscle LDH. ...136 Fi g u r e 5-6. The E f f e c t Of ATP On The A c t i v i t i e s Of LDH And GPDH From S k i p j a c k White Muscle.......................138 F i g u r e 5-7. The E f f e c t Cf GP On The A c t i v i t y Of GPDH From Skipjack White Muscle. ................................ 140 Figure 5-8. The E f f e c t Of Creatine-phosphate On The A c t i v i t y Of LDH From S k i p j a c k White Muscle. ........... 142 F i g u r e 5-9. The E f f e c t s Of PH And Creatine-phosphate On The Km Of Pyruvate Of S k i p j a c k White Muscle LDH. ...... 144 Figure 5-10. R e l a t i v e A c t i v i t i e s Of LDH And GPDH In A Crude 1:9 Supernatant. 146 x i F i g u r e 5-11. K i n e t i c s Of Creatine-phosphate I n h i b i t i o n Of Sk i p j a c k White Muscle LDH. ............................ 148 Figur e 1-1. Transport Of Reducing E q u i v a l e n t s Between C y t o s o l And Mito c h o n d r i a : The GP C y c l e and The Malate-a s p a r t a t e C y c l e . <.. ....... *.......... y.. ...... ........... 172 Figure 1-2. Reconstructed GP C y c l e In I s o l a t e d Mitochondria From S k i p j a c k White Muscle. .............. 174 Figur e I I - 1 . T r a n s p o r t Of Reducing E q u i v a l e n t s Between C y t o s o l And Mito c h o n d r i a : The GP C y c l e And Malate-cispciir'&ci^ b^ C yc 1© * • • *** * * •#«*•** ** • * * * * • •# • • • • * • •• • "IQ ^  Figure I I - 2 . Transport Of Reducing E q u i v a l e n t s Between C y t o s o l And Mito c h o n d r i a : The F a t t y A c i d S h u t t l e ......186 Figu r e I I - 3 . T r a n s p o r t Of Reducing E q u i v a l e n t s Between Cy t o s o l And Mito c h o n d r i a : The L a c t a t e - p y r u v a t e C y c l e . .188 Figur e I I - 4 . Mechanisms For The Generation Of NAD* From NADH In The Cytoplasm: Cytoplasmic NADH Oxidase. ...... 190 Figure I I - 5 , A And B. Mechanisms For The Generation Of NAD+ From NADH I n The Cytoplasm: A Cytoplasmic Ddi y dir ocj sn £i s s • • • • • • «• • •.. • * **.<•<* • • * * •«* • 192 Fig u r e I I I - 1 . R e l a t i v e A c t i v i t y Of Pyruvate Kinase Versus PH At 20OC And 40<>C.• ,-. ................................. 219 Figure I I I - 2 . Km(mM) Versus T°C: Sk i p j a c k Red Muscle And Heart Pyruvate Kinases. ........................221 Figure I I I - 3 . % Of C o n t r o l A c t i v i t y Versus ATP Conc e n t r a t i o n . .... ............ •.... .. •. ...............223 Figu r e I I I - 4 . % Of C o n t r o l A c t i v i t y Versus Alanine C o n c e n t r a t i o n . ......... .......... ...... ............... 225 x i i ACKNOWLEDGEMENTS I wish t o thank Peter Hochachka f o r i n v a l u a b l e help with both the t e c h n i c a l and academic as p e c t s o f t h i s t h e s i s . H is s c i e n t i f i c enthusiasm, which appears to be i n f i n i t e , was the g r e a t e s t help of a l l . I am very g r a t e f u l t o B i l l Hulbert who d i d a l l of the E.M. work and who was very experienced i n i n t e r p r e t i n g the r e s u l t s . My thanks a l s o go t o t h e people at the Kewalo Basin l a b o r a t o r y i n Hawaii, e s p e c i a l l y t o Andy Dizon, Hichard B r i l l and S h c j i . 1 CHAPTER J . , GENERAL. INTjR0DJ2GJfIGI 2 The tunas (Scombridae) are represented by 13 s p e c i e s weighing from 1 to 500 Kg and i n h a b i t i n g waters ra n g i n g from 5° C - 32°C. Some s p e c i e s are r e s t r i c t e d t o c e r t a i n waters whereas ethers undergo e x t e n s i v e m i g r a t i o n s . The tunas are mostly s u r f a c e feeding f i s h whose prey are f a s t swimming s c h o o l f i s h and f r e q u e n t l y squid. Tunas are of i n t e r e s t to the metabolic biochemist f o r three reasons, each of which I w i l l d i s c u s s i n t u r n . F i r s t l y , the tunas provide i d e a l m a t e r i a l f o r the study of the r o l e s of r e d and white muscle, a s u b j e c t which has held the i n t e r e s t of p h y s i o l o g i s t s and bi o c h e m i s t s f o r at l e a s t 20 years. The tunas have a r e l a t i v e l y l a r q e amount of muscle (Bene, i n prep.), a high p r o p o r t i o n o f t h i s muscle i s r e d , and red and white f i b r e s a re t o t a l l y d i s c r e e t a l l o w i n g homogeneous sampling ( F i g u r e 1-1). The r e l a t i v e p o s i t i o n s of the red muscle masses i n the a r r a y o f scombrids encompass s e v e r a l a l t e r n a t i v e s . There i s an i n c r e a s i n g trend toward red muscle i n t e r n a l i z a t i o n from §cembercmerous ( S e e r f i s h e s ) , t o Sarda (bonito) and f i n a l l y i n Euthynnus pelamis (tuna). where the red muscle i s contiguous with the v e r t e b r a l column. E. pelamis i s an example of r e l a t i v e l y complete i n t e r n a l i z a t i o n , where l i t t l e or no red muscle i s found at the l a t e r a l s u r f a c e o f the swimming musculature. The Thunnus s p e c i e s have f u r t h e r developed the i n t e r n a l i z a t i o n phenomenon i n s e v e r a l v a r i a t i o n s . The t r o p i c a l cr warm water i n h a b i t a n t s ( Thunnus t o n j ^ o l j Thunnus a t l a n t i c u s and Thunnus alJbacares ) have a continuous, r e l a t i v e l y i n t e r n a l i z e d r e d muscle c o n n e c t i n g the l a t e r a l s u r f a c e with the 3 v e r t e b r a l column and d o r s a l a o r t a - post c a r d i n a l v e i n complex., The b l u e f i n s p e c i e s which i n h a b i t broad ranges i n the temperate oceans and p o l a r seas have e i t h e r a more i n t e r m e d i a t e red muscle placement ( Thunnus maccoyii ) or, as i n the two Thunnus thynnus s u b s p e c i e s , the red muscle i s completely i n t e r n a l i z e d , with no l a t e r a l s u r f a c e c o n t i g u i t y (Sharp, 197 8 ) . The r e d muscle i n the tunas o c c u p i e s about 2058 of the t o t a l musculature, although the p r o p o r t i o n of red muscle to t o t a l musculature v a r i e s along the l e n g t h of the f i s h and v a r i e s between s p e c i e s (Sharp, 1978; Stevens e t . a l . , 1974 ). T h i s f i g u r e of 20% i s a r e l a t i v e l y high percentage when compared to other f i s h . The percentage of red muscle i n the m a j o r i t y of f i s h " s t e a k s " i s between 1 and 10 although the percentage can r i s e t o 36 at the p o s t e r i o r end of some s p e c i e s (Mosse and Hudson, 1977). The body of the tunas c o n s i s t s of 60-70% muscle, which i s a r e l a t i v e l y high p r o p o r t i o n (Bone, i n prep.). Secondly, the tunas have warm muscles, which r a i s e s the q u e s t i o n s : when, where and how i s the heat produced and what e f f e c t does i t have on the muscles* metabolism? The r a t e of heat t r a n s f e r between blood and water across the g i l l s of f i s h i s approximately ten times that of oxygen t r a n s f e r ( i n terms of h a l f - t i m e of e g u i l i b r a t i o n ) ( C a r e y e t . a l . , 1971), Thus i t i s evident t h a t one " c o s t " of oxygenating the blood w i l l be near complete e g u i l i b r a t i o n of blood temperature to that of the ambient water.,Fishes with normal c i r c u l a t i o n p a t t e r n s t h e r e f o r e , are always p e r f u s i n g the t i s s u e s with blood a t ambient water temperature. The c i r c u l a t i o n i n the tunas i s such t h a t heat i s not l o s t from the muscle. Heat l o s s i s prevented 4 by a c o u n t e r - c u r r e n t exchange system, or r e t e . T h i s r e t e i s a network of a r t e r i o l e s and venules i n c l o s e p r o x i m i t y . Heat coming cut of the muscle i n the venous blood, i s l o s t to the a r t e r i a l blood before i t reaches the g i l l s (Figure 1-1). The r e t e i n the tunas i s organized i n one of two ways. In some s p e c i e s , the c e n t r a l c i r c u l a t i o n i s much reduced and a cutaneous v e i n - a r t e r y system runs along the s i d e of the body a t the l e v e l of the l a t e r a l l i n e . The myotomal branches of these cutaneous v e s s e l s enter, and e x i t from the muscles, and form a l a t e r a l r e t e . The r e t e mainly serves the red muscle although the white muscle i s s u p p l i e d as w e l l . There are two s e t s of cutaneous v e s s e l s i n these tuna ( b l u e f i n , bigeye, a l b a c o r e and y e l l o w f i n ) and thus a r e t e on both the d o r s a l and v e n t r a l s i d e of the red muscle mass (Carey g t . a l . , 1971). In the r e s t of the tunas which have been examined ( T. t o n g o i l . T. a t l a n t i c u s . i * pelamis. T. a l b a c a r e s and the b l a c k s k i p j a c k ) a c e n t r a l r e t e i s present (Carey e_£.a;l., 1971; Stevens e£. a l . t 1974; Graham, 1973; Sharp, pers comm). The c i r c u l a t i o n i s of the t y p i c a l t e l e o s t type (except f o r the presence o f only one p o s t - c a r d i n a l vein) and i n each segment, the d o r s a l a o r t a and the post-c a r d i n a l v e i n branch i n t o s m a l l v e s s e l s and form a l a r g e three d imensional r e t e , j u s t below the s p i n a l c o r d . T h i s r e t e s e r v e s both the red and the white muscles, but again the major mass of the s t r u c t u r e i s concerned with red muscle due simply to the lower v a s c u l a r i t y of the white muscle. In the black s k i p j a c k and the s k i p j a c k , the d o r s a l aorta t r a v e l s i n s i d e the p o s t c a r d i n a l v e i n i n the r e t e r e g i o n and thus f u r t h e r maximises heat exchange before the g i l l s (Graham, 1973; Stevens e t . a 1., 5 1974). These f i s h s t i l l r e t a i n the cutaneous r e t e , but i t s f u n c t i o n has been superceded by the c e n t r a l r e t e ; thus the former has a t r o p h i e d . The outcome o f these r e t e systems i s warm s k e l e t a l muscles which can be up to 15°C above ambient temperature. The m a j o r i t y of muscle temperature s t u d i e s have i n v o l v e d the s k i p j a c k tuna ( c e n t r a l rete) and b l u e f i n tuna ( l a t e r a l r e t e ) . The deep r e d muscle i s always the h o t t e s t t i s s u e r e a c h i n g 12°C above ambient i n the b l u e f i n (Carey §t.ai., 1971; Carey and Lawson, 1973) and 9°C above ambient i n the s k i p j a c k (Stevens and Fry, 1971). The white muscle temperature i n these f i s h i s a l s o above ambient temperature. The temperature of the white muscle i n both the s k i p j a c k and the b l u e f i n i s as high as the red at the j u n c t i o n o f the r e d and white muscles and decreases with d i s t a n c e from the red muscle. Subcutaneous muscle temperatures (both r e d and white) are c o n s i d e r a b l y lower than deep r e d or deep white muscle temperatures (Carey et;.al. . 1971; Hulbert and B r i l l , pers. comm.). A v a r i e t y of other tunas have a l s o been sampled and excess (of ambient) muscle temperatures range from 2°C - 13°C (Carey s t . a l . , 1971). A l l data on the b l u e f i n tuna was c o l l e c t e d i n the f i e l d so ac c u r a t e a c t i v i t y temperature r e l a t i o n s h i p s i n t h i s f i s h are not known. Data on s k i p j a c k tuna however (which can be held i n c a p t i v i t y ) suggest t h a t : 1. the h i g h e s t muscle temperatures are a t t a i n e d during the fe e d i n g f r e n z y which i s high speed s u s t a i n e d swimming (Stevens and Fry, 1971) . 2. excess temperature o f t h e muscles of s k i p j a c k tuna 6 decreases a f t e r capture (Stevens and Fry, 1971). 3. a c t i v i t y d u r i n g capture does r a i s e the muscle temperature, but not to pre-capture l e v e l s (Stevens and Fry, 1971; Dizon and B r i l l , pers comm ). Temperatures of other parts of the body such as the b r a i n , eye and i n t e s t i n e are a l s o above ambient temperature and have been shown t o be served by r e t e s (Carey e t . a l . , 1971; Stevens and F r y , 1971). The heart of the tunas i s o b v i o u s l y not served by a re t e and i s presumably at near-ambient temperature. T h i r d l y , the tunas are unusually f a s t swimmers, which r a i s e s the g u e s t i o n o f the muscles' c o n t r i b u t i o n to swimming speeds and the p o s s i b l e metabolic s p e c i a l i z a t i o n f o r i n c r e a s e d power output. The u s u a l v e l o c i t y term used with regards to f i s h swimming i s lengths/second. Bainbridge (1958) has shown t h a t the swimming speeds of d i f f e r e n t s p e c i e s of f i s h can be compared using t h i s term. F a i r l y wide agreement e x i s t s that a c r u i s i n g speed of 2 -3 L/second can be maintained f o r an hour or more by most f i s h e s and values f o r bu r s t swimming range from 6 - 2 0 L/second (B a i n b r i d g e , 1958; Wardle, 1975). The higher burst speeds (above) were measured over extremely s h o r t p e r i o d s ( 0 - 5 seconds) and one was a c t u a l l y measured duri n g a rush preceding a leap out of the water (Bainbridge, 1958). I t i s d i f f i c u l t to measure the swimming speeds of tunas due to innumerable d i f f i c u l t i e s such as high oxygen reguirements, extreme f r a g i l i t y of the f i s h and the d i f f i c u l t i e s of c o n s t r u c t i n g an apparatus which w i l l measure high swimming speeds o f t h i s r e l a t i v e l y l a r g e f i s h . However, the evidence t h a t i s a v a i l a b l e suggests t h a t both c r u i s i n g and 7 bu r s t speeds are comparably high. S k i p j a c k tuna can swim at 8 knots f o r over and hour (Commercial F i s h e r i e s Review, 1969), and a s k i p j a c k has been t r a c k e d at 6 L/second f o r over an hour (Dizon and B r i l l , 1978). Burst swimming speeds f o r tunas are high and are s u s t a i n e d f o r r e l a t i v e l y l o n g p e r i o d s . H a l t e r s e t . a l , , (1961) measured v e l o c i t i e s of 21 L/second f o r y e l l o w f i n over 1 5 - 2 0 second p e r i o d s , and s k i p j a c k have been r e p e a t e d l y timed at 15-25 L/second and over (Dizon and B r i l l , p e r s . Ccmm; Yuen, 1966). The evidence f o r high swimming speeds i s f a r from overwhelming, but simple o b s e r v a t i o n was a l l t h a t was necessary to convince me t h a t a t l e a s t s k i p j a c k tuna e x h i b i t s u s t a i n e d and burst swimming speeds which are o b v i o u s l y i n excess o f t h a t shown by other f i s h e s . , With these three c h a r a c t e r i s t i c s , the tunas o f f e r the metabolic biochemist the o p p o r t u n i t y to study an organism i n which t h e r e are a v a r i e t y of i n t e r r e l a t e d problems. The q u e s t i o n s which are immediately obvious a r e : 1. fit the purely d e s c r i p t i v e l e v e l , how many f i b r e types i s the s k i p j a c k musculature composed of and how do these types f i t i n t o the usual red-white muscle c l a s s i f i c a t i o n ? 2. How i s c o n t r i b u t i o n t o p r o p u l s i o n p a r t i t i o n e d between the d i f f e r e n t muscle types i . e . at what swimming v e l o c i t y does each muscle type c o n t r i b u t e power and under what c o n d i t i o n s i s the metabolism of each muscle type a e r o b i c and anaerobic? 3. Which muscle(s) produce(s) the heat, under what c o n d i t i o n s i s the heat produced i n each muscle and what are the metabolic sources of heat? 4. Are t h e r e a d a p t a t i o n s at the molecular l e v e l i n the 8 muscles' metabolism which enable the tunas to swim at such high v e l o c i t i e s f o r such l o n g p e r i o d s ? 5. What i s the advantage to the animal of the high temperature musculature? I have t r i e d t o r e s o l v e these, and other q u e s t i o n s which arose d u r i n g my study, u s i n g the s k i p j a c k tuna ( ffuthunnus £«l§S»is ). The reasons f o r choosing the s k i p j a c k are a conpromise between convenience and r e p r e s e n t a t i o n . The s k i p j a c k tuna are r e a d i l y a v a i l a b l e , e i t h e r as f r e s h l y f r o z e n m a t e r i a l , or as l i v e animals. The f r o z e n samples are a v a i l a b l e from F i s h e r i e s c e n t e r s i n San Diego and Honolulu. L i v e animals are kept i n Honolulu {the only place i n the world which keeps l i v e tuna) where c o n d i t i o n s allow a wide range o f l i v e animal experiments. The s k i p j a c k h a r d l y f a l l s s h o r t as a t r u e r e p r e s e n t a t i v e of the tunas. I t i s not the h o t t e s t , nor the most thermoregulatory tuna, n e i t h e r i s i t the b i g g e s t ; but i t s muscle temperature excesses are only a few degrees below those of the b l u e f i n , i t s r e d muscle i s completely i n t e r n a l i s e d and i n terms of lengths/second, the swimming speeds o f the s k i p j a c k are never matched. I t i s a l s o , due to the h o l d i n g f a c i l i t i e s at Honolulu, a s c i e n t i f i c a l l y w e l l known member o f the tuna group and thus b e h a v i o r a l , anatomical and p h y s i o l o g i c a l data are a v a i l a b l e t o support b i o c h e m i c a l s t u d i e s . , There are ap p a r e n t l y t h r e e d i s t i n c t p o p u l a t i o n s of s k i p j a c k tuna. Young f i s h are found i n food abundant shallow waters over the c o n t i n e n t a l s h e l v e s . The a d u l t s migrate to the c e n t r a l p a c i f i c to breed and then presumably t h e r e i s a mig r a t i o n of the young back to shallow waters. The f i s h found 9 o f f the c o a s t s of the Hawaiian c h a i n are a mixture of the three p o p u l a t i o n s (Inter-American T r o p i c a l Tuna Commission bimonthly r e p o r t , M a r c h - A p r i l , 1 9 7 7 ; Sharp, per s , coma.). There i s l i t t l e known about t h e i r d i u r n a l behavior. They do make f o r a y s i n t o the depths a l l day, presumably t o feed, and have been t r a c k e d down to 1 9 5 meters (Yuen, 1 9 6 6 ) . At n i g h t they hug the upper l a y e r of water. F i s h i n g a c t i v i t i e s i n d i c a t e a major AH and PH c y c l e of " w i l l i n g n e s s to b i t e " , but no more i s known about f e e d i n g i n c i d e n c e . The "hot spot" i n the s k i p j a c k i s next t o the s p i n a l chord under the l e a d i n g edge of the main d o r s a l f i n . The temperature decreases as one moves l a t e r a l l y , a n t e r i o r l y and p o s t e r i o r l y away from t h i s p o s i t i o n (Graham, 1 9 7 5 ; Dizon and B r i l l , pers. C O M . ) . The r e t e i s about 9 7 % e f f i c i e n t i n exchanging heat ( N e i l l e t . a l . , 1 9 7 6 ) . Bed muscle e f f e c t s 2 7 - 8 4 % of i t s heat exchange a c r o s s the g i l l s whereas t h i s f i g u r e i s 0 - 3 5 % f o r the white muscle which l o s e s most o f i t s heat a c r o s s the s k i n . (This i s compared t o 9 5 % f o r a l l muscles i n t y p i c a l t e l e o s t s ) . warming and c o o l i n g experiments y i e l d no evidence f o r p h y s i o l o g i c a l thermoregulation i n s k i p j a c k although s k i p j a c k tuna e g u i l i b r a t e with water temperature 6 0 % as r a p i d l y as t y p i c a l t e l e o s t s ( N e i l l e t . a l . , 1 9 7 6 ) . Skipjack do not vary a c t i v i t y i n a way d e t e c t a b l y r e l a t e d to temperature ( N e i l l e t . a l . , 1 9 7 6 ) . Metabolism of f r e e swimming s k i p j a c k i s 3 - 5 times t h a t f o r non-scombrid f i s h e s of the same s i z e ( N e i l l e t . a l . , 1 9 7 6 ) and s e v e r a l f e a t u r e s of the c i r c u l a t o r y and r e s p i r a t o r y p h y s i o l o g y are c o n s i s t e n t with the s k i p j a c k ' s high metabolic r a t e (Stevens, 1 9 7 2 ) . , 10 O l t r a s t r u c t u r a l , h i s t o c h e m i c a l and enzyme p r o f i l e s t u d i e s (Chapter 3) demonstrate t h a t the s k i p j a c k musculature i s composed of only two major (red and white) muscle types. Information on the white muscle shows i t t o be a t i s s u e with a s u r p r i s i n g l y high (even f o r white muscle) anaerobic p o t e n t i a l . , E q u a l l y s u r p r i s i n g i s the a e r o b i c c a p a b i l i t y (based on carbohydrate fuel) which i s demonstrated i n the white muscle by these s t u d i e s . M e t a b o l i t e and temperature s t u d i e s (Chapter 4) support the evidence presented i n Chapter 3. The red muscle c o n t r i b u t e s a e r o b i c a l l y t o a l l swimming s t a t e s , while the white muscle demonstrates an i n t e n s e anaerobic metabolism d u r i n g b u r s t swimming and an a e r o b i c c o n t r i b u t i o n to steady s t a t e swimming. The white muscle has an a c t i v e GP c y c l e (Appendix I) and thus an important aspect of metabolic c o n t r o l i s the comp e t i t i o n f o r NADH between GPDH and LDH. The a b i l i t y of these twc enzymes t o compete f o r NADH can be perturbed by a v a r i e t y of c o n d i t i o n s (Chapter 5) a l l o w i n g carbon flow to proceed along an a e r o b i c or anaerobic route depending upon the t i s s u e oxygen l e v e l . 11 F i g u r e 1-1. Cross s e c t i o n s of f i s h . Cross s e c t i o n s of (a) a c l a s s i c a l " c o l d " t e l e o s t ; (b) the b l u e f i n tuna, showing the l a t e r a l r e t e , and (c) the s k i p j a c k tuna, showing the c e n t r a l r e t e . CHAPTER 2. MATERIALS AND SETHGDS 14 ANIMALS Fresh Fresh t i s s u e samples of s k i p j a c k were obtained from f i s h being held i n tanks a t the N a t i o n a l Marine F i s h e r i e s Center, Honolulu, Hawaii. Samples were only taken from f i s h which had been i n c a p t i v i t y f o r l e s s than three days. For c e r t a i n comparative s t u d i e s honey bees (Apis melifera) were c o l l e c t e d from h i v e s around Vancouver and white l a b o r a t o r y r a t s (Rattus no£l®ai£JS§) a n ^ rainbow t r o u t (SaJ.mo g a i r d n e r i ) were obtained from h o l d i n g f a c i l i t i e s at the U n i v e r s i t y o f B r i t i s h Columbia. T u r t l e s (Pseudomas s c r i p t a ) were purchased from NASCO Co L t d , Fo r t A t k i n s o n , Wisconsin and were kept a t 10°C. Frozen Frozen t i s s u e samples of s k i p j a c k tuna were obtained from the I n t e r American T r o p i c a l Tuna Commission i n San Diego. The tuna were caught by oceanographic v e s s e l s , immediately f r o z e n and t r a n s f e r e d to the l a b o r a t o r y a t d r y i c e temperatures. Again f o r comparative purposes, f r o z e n t i s s u e samples of Amazon f i s h e s (Osteoglossum b i c i r r h o s u m . flrapaima g i g a s . H o p l i a s m a l a t a r i c a s and R o p i e r y t h r i n u s u n i t a e n i a t u s ) were obtained from the Alpha H e l i x e x p e d i t i o n t o t h e Amazon River (Randall and Hochachka, 1978) . These f i s h were f r o z e n a f t e r capture and t r a n s f e r e d to the l a b o r a t o r y on dry i c e . Heddell s e a l heart was obtained from the A n t a r c t i c and was s i m i l a r l y f r o z e n a f t e r 15 e x t r a c t i o n and t r a n s f e r r e d t o t h e l a b o r a t o r y on dry i c e . HISTOCHEMISTRY Muscle pieces f o r h i s t o c h e m i c a l analyses were e x c i s e d from f r e s h s k i p j a c k , immersed f o r one minute i n a bath of isopentane cooled with l i g u i d n i t r o g e n and then placed d i r e c t l y i n l i g u i d n i t r o g e n . The t i s s u e blocks were s e c t i o n e d i n a c r y o s t a t at -20°C. S e c t i o n s (10 micron) were picked up on g l a s s s l i d e s and allowed t o dry 2-3 minutes before s t a i n i n g with haemotoxylin-e o s i n or pr o c e s s i n g s p e c i f i c a l l y f o r e i t h e r LDH, (see Ab b r e v i a t i o n s used) myosin ATPase or SDH. The s l i d e s were then mounted i n 15% g e l a t i n . S uccinate Dehydrogenase S t a i n i n g S l i d e s were incubated at room temperature i n 0.05M sodium phosphate b u f f e r , pH 7.6, 0.05M sodium s u c c i n a t e and 1 mg/ml Mitro blue t e t r a z o l i u m i n a f i n a l volume of 50 mis. S l i d e s were then washed i n s a l i n e (4% NaCl), f i x e d i n 10% formal s a l i n e f o r 10 minutes, washed i n d i s t i l l e d water, taken through an et h a n o l s e r i e s and mounted. The s i t e of enzyme a c t i v i t y was evidenced by b l u e - p u r p l e diformazan d e p o s i t s . C o n t r o l s l i d e s were processed i n the absence o f s u c c i n a t e and showed no s t a i n i n g . LDH S t a i n i n g S l i d e s were incubated i n a s u b s t r a t e s o l u t i o n c o n t a i n g 2g l i g u i d D L - l a c t a t e , 0.22g UAD+, G.22g N i t r o Blue T e t r a z o l i u m and 0.1 M Na cyanide i n 50 ml of 0.06 M Na-phosphate b u f f e r . S l i d e s 16 were then r i n s e d i n d i s t i l l e d water, taken through an ethanol s e r i e s and mounted. S i t e s o f enzyme a c t i v i t y were evidenced by the c h a r a c t e r i s t i c blue purple diformazan d e p o s i t s . C o n t r o l s l i d e s were i n c u b a t e d i n the absence of D l - l a c t a t e and produced no s t a i n i n g . , General S t a i n i n g S l i d e s were placed d i r e c t l y i n t o haemotoxylin, r i n s e d i n running tapwater and then placed i n e o s i n then r i n s e d again i n tapwater. S l i d e s were then put through a graded e t h a n o l s e r i e s and mounted. ELECTRON MICBOSCOPY Muscle t i s s u e was c o l l e c t e d from f r e s h f i s h and f i x e d i n 3% g l u t a r a l d e h y d e and 100 mM sodium phosphate b u f f e r , pB 7,4, c o n t a i n i n g 400 mM s u c r o s e , f o r 1.5 hours followed by washing with sodium phosphate b u f f e r and p o s t - f i x a t i o n i n 1,5% osmium t e t r o x i d e . The muscle pieces were subsequently dehydrated i n a graded ethanol s e r i e s and embedded i n Epon 812 ac c o r d i n g to L u f t (1961). T h i n s e c t i o n s were cut using g l a s s k n i v e s f i t t e d to a P o r t e r Blum MT-1 ultramicrotome, n e g a t i v e l y s t a i n e d with Uranyl acetate (Watson, 1958) and l e a d c i t r a t e (Reynolds, 1963) and viewed with a Z e i s s EM-10. 17 ENZYME PREPARATION Enzymes For P r o f i l e s Of Rgd And White. Muscles T i s s u e s were e x c i s e d from f r e s h l y k i l l e d f i s h and homogenised with a c o l y t r o n PCU-2-110 i n 19 volumes of 50 mM i m i d a z o l e b u f f e r , pH 7.0 c o n t a i n i n g 20 mM MgS04, 200 mM KC1 and 1 mM EDTA. Hercapto e t h a n o l (20 mM) was added f o r the p r e p a r a t i o n of hexokinase and PFK. The w e l l s t i r r e d homogenates were spun at 12000g f o r 20 minutes and the supernatants used f o r the enzyme assays., Enzymes For K i n e t i c • Cha r a c t e r i z a t i o n - -S k i p j a c k white muscle GPDH was prepared from f r o z e n muscle samples. T i s s u e s were e x c i s e d , b l o t t e d , weighed and homogenised i n 10 mM T r i s , 2 mM EDTA, 20mM mercapto-ethanol, pH 7.5, using a S o r v a l cmnimixer f o r t h r e e 20 second p e r i o d s . The homogenate was c e n t r i f u g e d at 12000g f o r 15 minutes. The supernatant was adjusted t o 80% ammonium su l p h a t e , spun a t 12000g f o r 15 minutes and the p e l l e t r e d i s s o l v e d i n a s m a l l amount of 50 mM T r i s , 20 mM mercapto-ethanol, pH 7,4, and put on a 2,6 x 100 cm Sephadex G-100 column. The a c t i v e f r a c t i o n s were pooled, concentrated by 80% ammonium s u l p h a t e and d i a l y z e d f o r 45 minutes twice a g a i n s t 10 mM i m i d a z o l e b u f f e r , 20 mM mercapto-e t h a n o l , pH 7.0, and loaded onto a 1.5 x 5 cm agarose-hexane-18 n i c o t i n a m i d e adenine d i n u c l e o t i d e (NAD*) column, The NAD* column was washed with b u f f e r to remove any unbound enzyme and the enzyme was e l u t e d with a 100 ml 0-300 mi! K C l g r a d i e n t . These simple s t e p s led to h i g h l y p u r i f i e d enzyme p r e p a r a t i o n s . A c t i v e f r a c t i o n s obtained from the a f f i n i t y chromatography were pooled and s t o r e d i n 80S ammonium sulph a t e , 20 mM mercapto-ethanol on i c e i n a 5°C c o l d room. The p r e p a r a t i o n s were s t a b l e f o r at l e a s t one month. GPDH from t u r t l e white muscle and t r o u t e p a x i a l muscle (predominately white muscle) was prepared from f r e s h animals using the same procedure as d e s c r i b e d above f o r s k i p j a c k white muscle GPDH. Sk i p j a c k white muscle LDH was prepared frcm f r o z e n muscle samples. The muscle was cut i n t o s m a l l pieces and homogenised i n a S o r v a l l omnimixer f o r one minute i n 20 volumes of 10 mM i m i d a z o l e b u f f e r , 20 mM o e r c a p t o - e t h a n o l , and 1 mM EDTA, pH 6.5. The homogenate was spun at 12000g f o r 20 minutes and the supernatant was mixed with a batch of c e l l u l o s e phosphate e q u i l i b r a t e d with the homogenising b u f f e r . A l l of the a c t i v i t y bound to the c e l l u l o s e phosphate and was brought o f f between 250 and 400 mM KCl with a 2- to 3- f o l d p u r i f i c a t i o n . The enzyme was concentrated by b r i n g i n g the s o l u t i o n t o 80% s a t u r a t i o n with ammonium s u l p h a t e , s p i n n i n g at 12000g f o r 20 minutes and r e d i s s o l v i n g the p e l l e t i n homogenising b u f f e r . The concentrated enzyme was then a p p l i e d to an oxamate column. In the presence o f O i l mM NADH, about 80% of the a c t i v i t y was bound to the a f f i n i t y matrix. The column was washed s e v e r a l times with homogenising b u f f e r and then the enzyme was e l u t e d 19 with a s m a l l amount of homogenising b u f f e r c o n t a i n i n g 500 mM KCl, pH 8.5. The r e s u l t i n g enzyme was a 15- to 20- f o l d p u r i f i c a t i o n and was s t a b l e i n the e l u t i o n b u f f e r , p l u s 20 mM MgSG4, pH 7.0, f o r at l e a s t 2 weeks. GPDH from honey bee f l i g h t muscle was prepared as f o r the GPDH from s k i p j a c k white muscle except that the NAD* column was del e t e d . The r e s u l t i n g p r e p a r a t i o n contained no measurable LDH. LDH from t u r t l e white muscle, r a t b r a i n and Amazon f i s h muscle was prepared by homogenizing the t i s s u e s i n approximately 5 volumes of 50 mM i m i d a z o l e b u f f e r , pH 7.0, c o n t a i n i n g 2 mM EDTA. The homogenate was spun at 12000g f o r 20 minutes and the supernatant used f o r the assays of LDH. H4 and M4 LDH from r a b b i t muscle and beef heart r e s p e c t i v e l y was purchased from Sigma Chemical Co; any contaminants were l e s s than 0.01% of the LDH a c t i v i t y , t h e r e was no GPDH a c t i v i t y and the isozyme form was 99% homogeneous. GPDH from r a b b i t muscle was o b t a i n e d from Sigma Chemical Co; any contaminants were l e s s than 0.01% of the GPDH a c t i v i t y and t h e r e was nc measurable LDH a c t i v i t y . Enzymes For G e l E l e c t r o p h o r e s i s . Enzyme p r e p a r a t i o n s from f r o z e n t i s s u e were crude supernatants d i s s o l v e d 1:4 i n e l e c t r o d e b u f f e r . Pure enzyme p r e p a r a t i o n s i n 80% ammonium sulphate were spun down and the p e l l e t r e d i z z o l v e d i n e l e c t r o d e b u f f e r . 20 ENZYME ASSAYS Enzyme a c t i v i t i e s were monitered i n 1 ml c u v e t t e s (1 cm l i g h t path) u s i n g a Unicam SP 1800 r e c o r d i n g spectrophotometer. The r e a c t i o n c u v e t t e s were held i n c e l l h o l d e r s t h e r m a l l y e q u i l i b r a t e d with a constant temperature bath and c i r c u l a t o r . The r a t e was determined by the decrease i n absorbance of NADH at 340 nm ( i n the case of c i t r a t e s y n t h e t a s e , the r a t e was determined by the i n c r e a s e i n absorbance of DT NB at 412 nm). Enzyme Assays For Muscle P r o f i l e s A l l assays were done i n 50 mM i m i d a z o l e b u f f e r , 10 mM MqCl2 and 100 mM KCl. NADH (0.15 mM) was used i n a l l assays except phosphorylase, hexokinase, phosphoglucomutase, phosphoglucoseisomerase and c r e a t i n e phosphokinase, i n which 0.4 mM NADP was used. Phosphorylase: pH 7.0, 2 mg glycogen/ml (emitted f o r c o n t r o l ) , 4uM glucose-1,6-diphosphate, 2 mM AMP and excess phosphoglucomutase and G6PDH. , Hexokinase: pH 7.5, glucose 1 mM, ATP 1 mM (omitted f o r co n t r o l ) and excess G6PDH. Phosphoglucomutase: pH 7.5, 4.0 mM glucose-1-phosphate(omitted f o r c o n t r o l ) , 0.02 mM glucose-1,6-diphosphate, and excess G6PDH. Phosphoglucoseisomerase: pH 7.5, 1.5 mM f r u c t o s e - 6 -phesphate(omitted f o r c o n t r o l ) and excess G6PDH. Glucose-6-phosphate dehydrogenase: pH 7.6, 1 mM glucose-6-phosphate(omitted f o r c o n t r o l ) . Phosphofructokinase: pH 8.0, 2 mM ATP, 5 mM f r u c t o s e - 6 -21 phosphate(omitted f o r c o n t r o l ) , excess GPDH, a l d o l a s e , and tr i o s e p h o s p h a t e isomerase. A l d o l a s e : pH 7,5, 0.4 mM fru c t o s e - 1 , 6 - d i p h c s p h a t e ( o m i t t e d f o r c o n t r o l ) , and an excess of GPDH and triosephosphate isomerase. Triosephosphate isomerase: pH 7.5 6 aM glyc e r a l d e h y d e phosphate (emitted f o r c c n t r o l ) , and excess GPDH.,• Phosphoglycerate k i n a s e : pH 7.5, 5 mM ATP, 10 mM glycerate-3-phosphate(omitted f o r c o n t r o l ) and excess glyceraldehyde-3-phosphate dehydrogenase., Enolase: 1 mM ADP, 1 mM 2-phosphoglyceric a c i d (omitted f o r c o n t r o l ) and excess PK and LDH. Pyruvate kinase: pH 6.5, 5 mM ADP, 5 mM phosphoenol pyruvate(omitted f o r c o n t r o l ) and excess LDH. L a c t a t e dehydrogenase: pH 6.5, 10 mM pyruvate(omitted f o r c o n t r o l ) . GPDH: pH 7,0, 2.0 mM dihydroxyacetone phosphate(omitted f o r c o n t r o l ) . C i t r a t e synthase: pH 8 . 0 ( T r i s b u f f e r ) , 0.1 mM DTNB, 0.3 mM a c e t y l CoA (emitted f o r c o n t r o l ) , 0.5 mM o x a l o a c e t a t e , Glutamate dehydrogenase: pH 7.3, 250 mM ammonium s u l f a t e , 1 mM ADP, 10 mM a - k e t o g l u t a r a t e (omitted f o r c c n t r o l ) . Malate dehydrogenase: pH 7.5, 0.5 mM o x a l o a c e t a t e ( o m i t t e d f o r c o n t r o l ) . Glutamate/oxaloacetate transaminase: pH 7.5, 40 mM a s p a r t a t e , 10 mM a - k e t o g l u t a r a t e (omitted f o r c o n t r o l ) , 0.1 mM pyridoxal-5-phosphate and excess malate dehydrogenase. Glutamate/pyruvate transaminase: pH 7,5, 20 mM a l a n i n e , 10 22 mM a - k e t o g l u t a r a t e (emitted f o r c o n t r o l ) , 0.1 mM p y r i d o x a l - 5 -phosphate and excess LDH. Adenylate kinase: pH 7.5, 2 mM AMP(omitted f o r c o n t r o l ) , 5 mM ATP, 5 mM phosphoenolpyruvate, and excess PK and LDH. C r e a t i n e phosphokinase: pH 7.5, 10 mM c r e a t i n e phosphate, 1 mM ADP, 4 mM g l u c o s e , 2 mM AMP(to i n h i b i t myokinase) and excess hexokinase and G6PDH. , Enzyme Assays For Kin e t i c C h a r a c t e r i z a t-io n The assays used f o r k i n e t i c c h a r a c t e r i z a t i o n d i f f e r e d from those used f o r the muscle p r o f i l e s (above) only i n t h a t a p u r i f i e d enzyme p r e p a r a t i o n was used and pH, temperature, s u b s t r a t e , product and modulator c o n c e n t r a t i o n s were v a r i e d . Values f o r the Michaelis-Menten constants (Km) were determined by double r e c i p r o c a l p l o t s of 1 / v e l o c i t y versus 1/substrate c o n c e n t r a t i o n . , I n h i b i t i o n c o n s t a n t s (Ki) were obtained from Dixon p l o t s of 1 / v e l o c i t y versus i n h i b i t o r c o n c e n t r a t i o n a t var y 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 . Values obtained were reproducable t o w i t h i n +or -15%. STABCH GELS Starch g e l e l e c t r o p h o r e s i s was done on 13% s t a r c h . g e l s at 5°C f o r 14 hours (LDH) and 9 hours (GPDH) with a c u r r e n t of 25 MA. The e l e c t r o d e b u f f e r was 50 mM phosphate c i t r a t e b u f f e r , pH 5.9 (LDH) or 6.3 (GPDH). The g e l b u f f e r was a 1:20 d i l u t i o n o f the e l e c t r o d e b u f f e r . Both LDH and GPDH ran towards the cathode. S t a i n i n g at 25°C was accomplished by o v e r l a y i n g the g e l s with f i l t e r paper soaked i n 1 mM NAD*, 0.1 mM phenazine 23 methcsulphate, 1 mM N i t r o Blue T e t r a z o l i u m and 50 mM GP or l a c t a t e i n 50 mM T r i s b u f f e r , pB 8.0. ,Control g e l s were o v e r l a i d with f i l t e r paper soaked i n the i n c u b a t i o n medium minus GP or l a c t a t e and showed no a c t i v i t y . METABOLITES Muscle and blood samples were taken from f i s h t h a t were performing at one o f t h r e e d i f f e r e n t l e v e l s of a c t i v i t y , termed r e s t i n g , b u r s t swimming, or steady s t a t e swimming..Samples from r e s t i n g tuna were o b t a i n e d from animals swimming l a p s i n c i r c u l a r pools at approximately 1-2 body lengths/second. For tuna, t h i s i s the o n l y approximation to b a s a l metabolism that e x i s t s , s i n c e , u n l i k e many t e l e o s t s with swimbladders, the s k i p j a c k tuna l o s e hydrodynamic e q u i l i b r i u m and f a l l through the water column i f t h e i r c r u i s i n g speed decreases below about 1.2 lengths/second (Dizon, pers. Comm.) . In terms of metabolite c o n c e n t r a t i o n s t h e r e f o r e , we are d e a l i n g with a working e q u i l i b r i u m system not d i r e c t l y comparable t o " r e s t i n g " mammalian muscles. Burst swimming f o r p e r i o d s of 7-10 minutes was obtained by a t t a t c h i n g a l i n e and hook to the lower mandible and r e l e a s i n g the tuna " o n - l i n e " i n t o a c i r c u l a r tank, 5 meters i n diameter, 1 meter deep. Under these c o n d i t i o n s , b u r s t s o f up to 20 lengths/second can be achieved (Dizon, pers. Comm.). Samples from tuna i n high v e l o c i t y , steady s t a t e swimming a s s o c i a t e d with f e e d i n g f r e n z i e s were obtained through the c o o p e r a t i o n of l o c a l fishermen. These f i s h were hooked a t sea at the s t e r n o f the boat and immediately hauled on board f o r 24 sampling. Sampling time was about 15 seconds l o n g e r than f o r th€ other two experimental groups ( r e s t i n g and b u r s t swimming). P r e p a r a t i o n Of T i s s u e F i s h were netted and a 2 cm t h i c k steak was q u i c k l y cut from the area immediately a n t e r i o r t o the l e a d i n g edge of the d o r s a l f i n . In the 1976 experiments (see Chapter 4 ) , s m a l l pieces of red and white muscle ( l e s s than 1 gram) were e x c i s e d from the steak and immediately f r o z e n i n l i q u i d n i t r o g e n ; t h i s procedure took l e s s than 30 seconds. In the 1977 experiments ( see chapter 5 ) , the steak (which was l e s s than 1 cm t h i c k ) was f r e e z e clamped with Hollenburger tongs c o o l e d i n l i q u i d n i t r o g e n ; t h i s procedure a l s o took about 20 seconds. .Some of the f r o z e n t i s s u e was s e t a s i d e f o r glycogen assays; the r e s t was powdered i n a mortar and p e s t l e , p r e v i o u s l y c o o l e d by l i q u i d n i t r o g e n and then an a l i q u o t of the powdered t i s s u e was placed i n t o a g l a s s tube (cooled i n a dry i c e - e t h a n o l b a t h ) . Immediately before and a f t e r the a d d i t i o n of the powder, the tube was weighed t o the nearest m i l l i g r a m . Approximately 4 p a r t s (by weight) of 8% (v/v) HC104 i n 40% (v/v) e t h a n o l was added to the c o l d powder, mixed q u i c k l y with a s p a t u l a and homogenized f o r 1 minute, a t dry i c e e t h a n o l temperatures, with a p o l y t r o n PCU-2-110.,The r e s u l t i n q supernatant was c e n t r i f u g e d at 25000g f o r 10 minutes t o produce supernatant "a"; the p r e c i p i t a t e remaining i n the homogenizing tube was r e -homogenized i n two volumes of 6% (v/v) HC104. T h i s homoqenate was added to supernatant "a" and spun at 25000g f o r 10 minutes.,. The "second" supernatant was adjusted t o pH 6.0 by the slow 25 a d d i t i o n of 3M K2C03 c o n t a i n i n g 0.5 M t r i e t h a n o l a m i n e base, and then spun at 25000g f o r 10 minutes to remove the p r e c i p i t a t e d KC104, The f i n a l supernatant was measured t o the nearest 0.1 ml and s t o r e d at -20°C. M e t a b o l i t e Ass ay. Techniques A l l m e t a b o l i t e s were measured e n z y m a t i c a l l y and were based on the absorbance of the p y r i d i n e n u c l e o t i d e s at 340 nm. Assays were c a r r i e d out on a Dnicam SP 1800 dual-beam spectrophotometer connected t o a s t r i p - c h a r t r e c o r d e r . ATP and g l u c o s e -6- phosphate were determined by the method of Lamprecht and T r a u t s c h o l d (1974); ADP, AMP, pyruvate, a-glycerophosphate, c i t r a t e and malate, by the method of Lowry and Passonneau (1972); fructose-6-phosphate and f r u c t o s e diphosphate by the method of Racker (1974); l a c t a t e by the method o f Sigma B u l l e t i n #826; a - k e t o g l u t a r a t e by the method of Eergmeyer and Bernt (1974); c r e a t i n e and creatine-phosphate by the methods of Bernt e t a l . (1974) and Lamprecht e t a l (1974); and glucose by the method of Bergmeyer et a l (1974). Glyceraldehyde-3-phosphate and dihydroxyacetone phosphate Here determined by the s e q u e n t i a l a d d i t i o n of GPDH and t r i o s e phosphate isomerase. Glycogen was i s o l a t e d and hydrolysed by the method of O s t e r b e r g , (1929) and assayed using the Sigma k i t # 510 (Sigma Chemical Co. S t . L o u i s ) . For amino a c i d d e t e r m i n a t i o n , the p e r c h l o r i c a c i d e x t r a c t s were f i l t e r e d through m i l l i p o r e f i l t e r s (0.45um) and a p p l i e d to a Beckman 118C amino a c i d a n a l y s e r . The samples were e l u t e d with sodium c i t r a t e b u f f e r s (pH 3.25, 0.2N; pH 4.12, 0.4N; pH 6.4, 1. ON) . 26 Preparat.iQ.rj Of Blood Blood drawn by c a r d i a c puncture was added to an egual volume of 15% (v/v) HC104 and then spun a t 25000a f o r 10 minutes. The supernatant from t h i s s p i n was then t r e a t e d as was the "second" supernatant above. COMPETITION STUDIES A. To determine the e f f e c t of GP upon d i f f e r e n t forms of GPDH, i d e n t i c a l a c t i v i t i e s (as determined a t 0.1 mM pyruvate or DH1P and 0.1 mM NADH, pH 7.0 at 25°C) o f H4 LDH and e i t h e r r a b b i t muscle or honey bee GPDH were put i n t o a c u v e t t e a t 25 nC c o n t a i n i n g 100 mM i m i d a z o l e , pH 7.0, 0.1 mM pyruvate , 0 . 1 mM DHAP and 0.1 mM NADH; the i n c u b a t i o n was done i n the presence and absence of 2.0 mM GP. The NADH was monitered at 340 nm u n t i l the r e a c t i o n was complete, at which time, 1 ml a l i g u o t s were added to 1 ml of 15% HC104. The r e s u l t i n g mixture was n e u t r a l i s e d and c e n t r i f u g e d and the a c i d e x t r a c t used f o r the determination o f pyruvate and DHAP. B. To determine the e f f e c t of creatine-phosphate upon d e f f e r e n t forms of LDH, r a b b i t muscle GPDH was incubated as above with e i t h e r H4 or H4 LDH i n the presence and absence o f 20 mM c r e a t i n e phosphate. PROTEIN DETERMINATION P r o t e i n c o n c e n t r a t i o n s were determined s p e c t r o p h o t o m e t r i c a l l y at 280 and 260 nm u s i n g the formula below (Layne, 1957): mg protein/ml = 1.75(0D 280) - 0.74(OD 260). 27 TEMPERATURE MEASUREMENTS Temperature measurements were taken with Y e l l o w s p r i n g s Instrument (YSI) temperature probes (22 guage) which were l i n k e d to a YSI Telethermometer. accurate t o 0.1°C. Temperature measurements with these probes were taken i n deep red muscle (next t o the s p i n a l chord) and i n deep white muscle (halfway between the red muscle and the d o r s a l edge of the f i s h ) unless otherwise s t a t e d . Temperature measurements were taken a t the l e v e l of the p e c t o r a l f i n s unless otherwise s t a t e d . Temperature tags came from Jim R o c h e l l e and C h a r l e s Coutant, Oakridge N a t i o n a l L a b o r a t o r i e s and were pulse modulated temperature telemetary tags. 28 CHAPTER 3. HIS TOC HEM IS TBJ, ULTJASTB2CT08J AjND ENZYME PBOEILES OF T.HE SfllMMIMQ flPSCULATUBB 29 INTJODjgCTIOJ On the b a s i s of c o l o u r , v e r t e b r a t e s t r i a t e d muscle can be d i f f e r e n t i a t e d i n t o a t l e a s t two types, red and white. The red f i b e r s owe t h e i r c o l o u r to the presence of a f i n e c a p i l l a r y net which surrounds each muscle, to the presence o f myoglobin and to the presence of mitochondria and cytochromes (Love, 1970). The s t r u c t u r a l , m e t a b o l i c and f u n c t i o n a l d i f f e r e n c e s between these d i f f e r e n t f i b e r s has been r i g o r o u s l y i n v e s t i g a t e d i n mammalian muscles and much of the terminology a r i s e s from these mammalian muscle s t u d i e s . I t i s t h e r e f o r e expedient t e f i r s t l y review the mammalian muscle l i t e r a t u r e . MA-MMALIAN MUSCLE The nomenclature i n v o l v e d with the c l a s s i f i c a t i o n of mammalian muscles can become r a t h e r complicated. Romanul (1964), proposed a scheme i n c l u d i n g three major groups and a t o t a l o f e i g h t sub-groups. For the purposes of t h i s t h e s i s however, a d i s c u s s i o n of the t h r e e major groups, red, white and i n t e r m e d i a t e , w i l l s u f f i c e . The c h a r a c t e r i s t i c s of these t h r e e muscle types can be separated i n t o three groups: u l t r a s t r u c t u r e , e l e c t r o p h y s i o l o g y and enzymes and metabolism. U I t r a s t r u ct are The white f i b e r s have the l a r g e s t diameter, being about twice the s i z e of the red, and the i n t e r m e d i a t e f i b r e s are i n t e r m e d i a t e i n s i z e . In g e n e r a l , m i t o c h o n d r i a l content i s 30 i n v e r s e l y r e l a t e d to the diameter of the f i b e r s . The mitochondria i n red f i b e r s are present i n i n t e r f i b r i l l a r rows and i n p a i r s a t the I band (filamentous mitochondria) and l a r g e s p h e r i c a l mitochondria aggregate at the periphery of the f i b e r . The mitochondria of white muscle are almost e n t i r e l y f i l a m e n t o u s p a i r s at the I band. Intermediate f i b e r s have many of the red muscle c h a r a c t e r i s t i c s , but p e r i p h e r a l and i n t e r f i b r i l l a r aggregations are l e s s conspicuous. Z l i n e s are t h i n n e s t i n white f i b e r s and t h i c k e s t i n red and t h e r e are c l e a r u l t r a s t r u c t u r a l d i f f e r e n c e s between the three f i b e r types at t h e i r neuromuscular j u n c t i o n s (Gauthier, 1970; Hess, 1970). The development of the T system i s r e l a t e d to the s i z e of the f i b e r and to i t s speed of c o n t r a c t i o n . T h u s the l a r g e r the f i b e r , and the f a s t e r the f i b e r (assuming the same s i z e ) , the more ex t e n s i v e i s the T system (Peachey, 1970). E l e c t r o physiology St ud i e s The c o n t r a c t i o n time of the white muscle i s about 10 msec, t h a t of the i n t e r m e d i a t e i s 38 and t h a t of the red i s 18. Thus the red and white f i b e r s i n the mammals are c l a s s i f i e d as f a s t t w i t c h and the i n t e r m e d i a t e i s c l a s s i f i e d as slow t w i t c h ( C l o s e , 1967; Edgerton and Simpson, 1969; Barnard e t . a , l . , 1971). Enzymes And Metabolism The time honoured method f o r d i s t i n g u i s h i n g f i b e r t y p e s i s h i s t o c h e m i s t r y . T h i s t e c h n i q u e , coupled with a c t u a l enzyme assays c l e a r l y demonstrates the presence of at l e a s t three 31 f i b e r t y p es i n mammalian s k e l e t a l muscle. Using r e p r e s e n t a t i v e enzymes from g l y c o l y s i s , B - o x i d a t i o n and the Krebs c y c l e , the a c t i v i t i e s of m y o f i b r i l l a r ATPase, hexokinase a c t i v i t i e s , cytochrome c o n c e n t r a t i o n s , myoglobin c o n c e n t r a t i o n s , oxygen uptake, l i p i d s t o r e s and m i t o c h o n d r i a l r e s p i r a t o r y a c t i v i t y , the metabolic p o t e n t i a l of the three f i b e r types appears to be as f o l l o w s . 1.,The red f i b e r has a high a e r o b i c c a p a c i t y , a moderate to high anaerobic c a p a c i t y and high ATPase a c t i v i t y . ( A T P a s e a c t i v i t y has been c o r r e l a t e d with speed of c o n t r a c t i o n , (Barany, 1967)). 2. The i n t e r m e d i a t e f i b e r has a high a e r o b i c , low anaerobic c a p a c i t y and a low ATPase a c t i v i t y . 3. The white f i b e r has a low o x i d a t i v e and high* anaerobic c a p a c i t y , and high ATPase a c t i v i t y , (Peter e t . a l . , 1972; Barnard e t . a l . , 1971; Khan, 1976; Edgerton and Simpson, 1969; .Beatty and Bocek, 1970; George, 1962; Pande and Blanchaer, 1971; P e t t e and Dolken, 1975; Peter e t . a l . , 1968)., U n f o r t u n a t e l y , there i s one d i s t u r b i n g f a c t i n the whole scheme, i . e . red muscle demonstrates as much or more glycogen than does white muscle (Baldwin e t . a l , , 1973; Beitman et.aJL., 1973; Essen and Henriksson, 1974; Beatty and Bocek, 1970). Resu l t s of i n v i v o and i n v i t r o experiments are i n keeping with these metabolic p o t e n t i a l s . Numerous experiments have shown t h a t the red muscle i s an endurance muscle which uses predominately f a t as a f u e l f o r a e r o b i c o x i d a t i o n and t h a t white muscle i s a carbohydrate burning, more anaerobic muscle which i s a c t i v e during i n t e n s e muscular a c t i v i t y ( T e r j u n g , 1976; 32 Baldwin e t . a l . , 1973; Reitman e t . a l . . 1973; H o l l o s z y and Booth, 1976; G o l l n i c k e t . a l . , 1S74). The in t e r m e d i a t e f i b e r s appear to behave i n an i n t e r m e d i a t e f a s h i o n (Reitaan e t . a l . , 1973). There i s presumed to be c o n s i d e r a b l e o v e r l a p p between the two extreme f i b e r types as both types appear capable of a e r o b i c and anaerobic metabolism using both l i p i d s and carbohydrates as a f u e l (Pande and Blanchaer, 1971; Reitman e t . a l . , 1973; Baldwin e t . a l . , 1973). Blanchaer(1964) suggests t h a t white muscle may have the components of a GP c y c l e t o f a c i l i t a t e redox balance during aerobic carbohydrate metabolism. FISH MUSCLE The morphology of f i s h s t r i a t e d muscle d i f f e r s i n one major r e s p e c t to mammalian s t r i a t e d muscle./In many of the muscle f i b e r s of f i s h , the f i b r i l s are not even approximately c y l i n d r i c a l , but are f l a t or s h e e t l i k e (Nakajima, 1969). The p e r i p h e r a l f i b r i l s tend to be more f l a t t e n e d than the c e n t r a l ones; thus the sa r c o p l a s m i c r e t i c u l u m (SR) and T systems are c o n s t r a i n e d t o a planar arrangement except near the cente r of the f i b e r (Peachey, 1970). Other u l t r a s t r u c t u r a l d i f f e n c e s between f i s h and mammalian muscles i n c l u d e sarcomere l e n g t h , i n n e r v a t i o n and the SR and T systems. The sarcomere length of the white muscles i n the c o a l f i s h are s l i g h t l y longer than that of the red and both are r e l a t i v e l y s h o r t compared with those of the mouse (Patterson and Goldspink, 1972; Goldspink, 1968). C o a l f i s h white muscle i s m u l t i p l y i n n e r v a t e d ( P a t t e r s o n and Goldspink, 1972) which i s i n c o n t r a s t t o mammalian white muscle (Hess, 1970). F i s h red muscle i s d i s t i n g u i s h a b l e from o t h e r 33 v e r t e b r a t e red muscle i n having a h i g h l y organised SB and T system (Patterson and Goldspink, 1972; K i l a n s k i , 1967; Peachey, 1970), i n f a c t there i s no s i g n i f i c a n t d i f f e r e n c e i n the % volume of the SB and T systems of the two f i b e r s i n the c o a l f i s h (Patterson and Goldspink, 1972). Apart from these d i f f e r e n c e s , the p a t t e r n i s s i m i l a r t o the mammalian one. The diameter of the white f i b e r s i s l a r g e r , the e l e c t r i c a l a c t i v i t y of the two f i b e r types d i f f e r s q u a l i t a t i v e l y , the i n n e r v a t i o n of the red f i b e r s i s m u l t i p l e , and the width of the Z l i n e i s g r e a t e r i n the red than i n the white. The red f i b e r s are r i c h e r i n c a p i l l a r i e s , myoglobin c o n c e n t r a t i o n , l i p i d and glycogen d e p o s i t s , o x i d a t i v e enzymes ( i n c l u d i n g l i p a s e ) and mitochondria, and the white f i b e r s show more g l y c o l y t i c enzyme a c t i v i t y and m y o f i b r i l l a r ATPase a c t i v i t y (Bone, 1966; P a t t e r s o n and Goldspink, 1972; Johnston e.t.aJL. , 1977; George, 1962; Takeuchi, 1959; P r i t c h a r d e t . a l . , 1971; P a t t e r s o n e j t . a l . , 1974; Piosse and Hudson, 1977; L i n e t . a l . , 1974). Again r e d muscle i s thought t o be a c t i v e during low to moderate a e r o b i c swimming. Oxygen uptake of red muscle i s g r e a t e r than t h a t of white, and e l e c t r o p h y s i o l o g i c a l s t u d i e s show t h a t the red muscle only i s a c t i v e a t the lower swimming speeds (Bone, 1966; Johnston e t . a l . , 1977; L i n e t . a l . , 1974). White muscle appears to provide the p r o p u l s i o n at above s u s t a i n e d swimming speeds., E l e c t r o p h y s i o l o g i c a l s t u d i e s again support t h i s (Bone, 1966; Johnston e t . a l . , 1977) and glycogen d e p l e t i o n and l a c t a t e accumulation can be shown to occur i n f i s h white muscle o n l y at the higher swimming speeds ( P r i t c h a r d e t . a l , , 1971; Bone, 1966). As i n the mammalian system, i t i s suggested t h a t there 34 i s c o n s i d e r a b l e o v e r l a p p i n g of f u n c t i o n between the two major muscle types. F i s h red muscle has the components of g l y c o l y s i s , glycogen d e p o s i t s and produces l a c t a t e a t high swimming speeds. Hhite muscle has a c t i v e Krebs c y c l e components, l i p i d d e p o s i t s and e x t r a c e l l u l a r l i p a s e (George, 1962; Johnston e t . a l . , 1977; P r i t c h a r d e t . a l . , 1971; Bone, 1966). The s o - c a l l e d i n t e r m e d i a t e f i b e r s i n f i s h myotomes have been r e p o r t e d i n s e v e r a l s t u d i e s and can occupy 10% of the myotome (Davidson e t . a l . , 1976). Information on them however, i s both sparse and c o n t r a d i c t o r y . Bone (1966) c l a s s i f i e s these f i b e r s i n the d o g f i s h with the white f i b e r s as they have the same p a t t e r n of i n n e r v a t i o n , the same s c a t t e r e d d i s t r i b u t i o n o f n u c l e i and low c o n c e n t r a t i o n s of l i p i d . These "pink" f i b e r s however, are i n t e r m e d i a t e i n s i z e between the r e d and the white. In the carp the i n t e r m e d i a t e f i b e r s are r e c r u i t e d a t i ntermediate swimming speeds, have i n t e r m e d i a t e ATPase and o x i d a t i v e enzyme a c t i v i t i e s , high PK and LDH a c t i v i t y and white muscle type myosin (Johnston gt.aj,. , 1977). P a t t e r s o n e t . a l . , (1975) f i n d the i n t e r m e d i a t e f i b e r s i n f i v e s p e c i e s o f f i s h t o be " i n t e r m e d i a t e " between th e red and white f i b e r s on the b a s i s of glycogen and l i p i d s t o r e s and SDH, GPDH and phosphorylase a c t i v i t i e s . The f i s h i n t e r m e d i a t e f i b e r appears t o be more l i k e the white f i b e r than the red f i b e r , which i s not the case i n mammalian muscles.. 35 IJSOLTS AND DISCUSSION: PART 1.. OjRGANIMIION OF THE SKJPJAC*-JIJSCULATURE AT THE MYOTOMAL. CELLULAR AND SUjBjCELLULAR LEVEL GENERAL OBSERVATIONS In the s k i p j a c k , a s m a l l wedge of ^ e d 1 muscle occurs i n the a n t e r i o r h a l f o f the myotcmal mass i n a s u p e r f i c i a l l a t e r a l p o s i t i o n . T h i s wedge becomes homogeneous with the white muscle i n the p o s t e r i o r h a l f o f the f i s h {Figure 3-1). However, the bulk of the s o - c a l l e d deep-red muscle, i s i n t e r n a l i z e d and l i e s a djacent t o the v e r t e b r a l column (Chapter 1). Despite these c o m p l e x i t i e s , myomeres c o n s i s t c f both red and white muscle f i b e r s . The boundary between the two i s sometimes very sharp, but i n t e r d i g i t a t i o n s are a l s o evident (Figure 3-2). HISTOCHEMISTRY Two B a s i c F i b e r Ty_£es Although some s u b t l e v a r i a t i o n s i n f i b e r type can be d i s t i n g u i s h e d a t the E.M. l e v e l , these v a r i a t i o n s do not appear to be f u n c t i o n - r e l a t e d , and by three h i s t o c h e m i c a l t e s t s , the s k i p j a c k myotome c o n s i s t s of only two b a s i c f i b e r t y p e s . , Succ i n a t e dehydrogenase a c t i v i t y as an index o f o x i d a t i v e c a p a c i t y , should be h i g h e s t i n f a s t - t w i t c h r e d , i n t e r m e d i a t e i n slew-twitch red and lowest i n f a s t - t w i t c h white (see 36 Introduction).,When a p p l i e d t o tuna myotomal muscle, however, only two s t a i n i n g p a t t e r n s are observed f o r SDH, with red muscle s t a i n i n g very d a r k l y , white muscle s t a i n i n g l i g h t l y . By c a r e f u l l y monitoring the SDH r e a c t i o n , i t can be shown th a t r e d and white muscle d i s p l a y only one f i b e r type by t h i s c r i t e r i o n ; furthermore, i n both muscles, the bulk of the SDH a c t i v i t y appears i n p e r i p h e r a l r a t h e r than i n m y o f i b r i l l a r p o s i t i o n s (Figure 3-2), The l a t e r a l wedge s t a i n s as red muscle i n the a n t e r i o r h a l f of the f i s h and as white muscle i n the p o s t e r i o r h a l f . Although LDH a c t i v i t y normally may be taken as an index of anaerobic metabolism, the L D H - s c e c i f i c s t a i n depends upon the back r e a c t i o n ( i . e . l a c t a t e o x i d a t i o n t o p y r u v a t e ) ; thus, l i k e SDH, the LDH s t a i n r e a c t i o n can be used as an i n d i c a t i o n of o x i d a t i v e c a p a c i t y . When a p p l i e d t o the s k i p j a c k myotome, only two s t a i n i n g p a t t e r n s are observed, r e d muscle s t a i n i n g very d a r k l y , white muscle s t a i n i n g l i g h t l y (Figure 3-2). Again the wedge c o n s i s t s of r e d and white f i b e r s i n the a n t e r i o r and p o s t e r i o r h a l f of the f i s h r e s p e c t i v e l y . L i p i d s p e c i f i c s t a i n i n g with sudan black again r e s u l t s i n only two forms with the wedge c h a r a c t e r i s t i c s being as they were f o r the SDH and LDH s t a i n s (Figure 3-2) . The sudan black s e c t i o n s a l s o demonstrate the i n t e r d i g i t a t i o n s which can occur between red and white muscle and d i f f e r e n t i a l d e p o s i t i o n or u t i l i z a t i o n c f l i p i d i n the red muscle (Figure 3-2). T h i s l a t t e r o b s e r v a t i o n does not re p r e s e n t d i f f e r e n t f i b e r types as (1) the p a t t e r n appears to be completely random, (2) i t i s not supported by d i f f e r e n t i a l SDH or LDH s t a i n i n g and (3) there are 37 no s t r u c t u r a l d i f f e r e n c e s between f a t loaded and f a t depleted red f i b e r s at the E. M. L e v e l . ELECTRON MICROSCOPY OF THE RED MUSCLE The u l t r a s t r u c t u r e o f the red and white muscle, although d e s c r i b e d i n d e t a i l by Hulb e r t , Guppy and Hochachka (in p r e s s ) , i s c o n s i d e r e d necessary f o r the l o g i c a l development of t h i s t h e s i s , and so i s d e s c r i b e d below i n summary form. F i b e r S t r u c t u r e In terms of s i z e , s k i p j a c k red muscle sarcomeres are about 1. 7um long. The f i b e r s are s u b s t a n t i a l l y s m a l l e r than i n white muscle, being between 12-53 um i n diameter; an i n d i c a t i o n o f the s i z e v a r i a t i o n i s evident i n F i g u r e 3-1, The sarcoplasmic r e t i c u l u m i s f a i r l y w e l l developed, a f e a t u r e common i n t e l e o s t , but not mammalian red muscle (see I n t r o d u c t i o n ) . , M i t o c h o n d r i a l abundance Red f i b e r s are extremely r i c h i n mitochondria; up to 355£ of the c r o s s s e c t i o n a l area c o n s i s t s of mitochondria, a value t h a t i s higher than p r e v i o u s l y observed f o r other t e l e o s t red muscle (Hulbert and Moon, 1978; P a t t e r s o n and Goldspink, 1972), but which c o r r e l a t e s w e l l with m i t o c h o n d r i a l abundance i n Sccmber, (Bone. 1978). Mitochondria are u s u a l l y more abundant i n p e r i p h e r a l r e g i o n s adjacent t o c a p i l l a r i e s . , 38 C a g i l l a r j t x L i k e mammalian red muscle, s k i p j a c k red muscle i s h i g h l y v a s c u l a r . Each f i b e r i s surrounded by 4-12 c a p i l l a r i e s which i s a high value compared to any other t e l e o s t ( M o s s e , 1978; Boddeke e t . a l . , 1959). I n t r a c e l l u l a r Fat The abundance of mitochondria plus the numerous c a p i l l a r i e s imply a h i g h l y oxygen dependent metabolism i n s k i p j a c k red muscle. Not s u r p r i s i n g l y , s k i p j a c k red muscle a l s o c o n t a i n s l a r g e amounts of i n t r a c e l l u l a r t r i g l y c e r i d e . These t r i g l y c e r i d e d r o p l e t s may be m y o f i b r i l l a r i n p o s i t i o n , u s u a l l y l o c a t e d near mitochondria, or they may be found p e r i p h e r a l l y . As a l r e a d y mentioned with r e f e r e n c e t o Figure 3-1, there i s d i f f e r e n t i a l u t i l i z a t i o n (or d e p o s i t i o n ? ) by d i f f e r e n t red muscle f i b e r s ; thus o f t e n there are f i b e r s loaded with l i p i d d r o p l e t s adjacent t o f i b e r s with the d r o p l e t s t o t a l l y depleted. E x e r c i s e d Bed Muscle No u l t r a s t r u c t u r a l changes are observed i n red muscle f i n e s t r u c t u r e f o l l o w i n g severe e x e r c i s e ('burst swimming*). What i s n o t a b l e , however, i s a l a r g e d e p l e t i o n of glycogen g r a n u l e s and of i n t r a c e l l u l a r l i p i d d r o p l e t s . 39 ELECTRON MICROSCOPY OF WHITE MUSCLE Iia« S t r u c t u r e : Overview The f i n e s t r u c t u r e o f s k i p j a c k white muscle d i f f e r s i n s e v e r a l aspects from the g e n e r a l i z e d concept of t e l e o s t white muscle. F i r s t l y , s k i p j a c k white muscle c o n t a i n s more glycogen than does red muscle ( t h i s i s g u a n t i f i e d i n Chapter 4). Secondly, glycogen granules are o f t e n sequestered away i n membrane bound s t r u c t u r e s , termed glycogen bodies, and l o c a t e d p r i m a r i l y i n i n t e r s t i t i a l r e g i o n s . T h i r d l y , i n t r a c e l l u l a r l i p i d d r o p l e t s are p e r i o d i c a l l y observed. F o u r t h l y , s k i p j a c k white muscle i s we l l perfused, f o r c a p i l l a r y d e n s i t y i s high compared to other t e l e o s t s . F i n a l l y , s k i p j a c k white muscle c o n t a i n s a s u b s t a n t i a l abundance of mitochondria,. General F i b e r SJtructujce The b a s i c ' c o n t r a c t i l e machinery* ( i . e . the sarcomere f i n e s t r u c t u r e ) of s k i p j a c k white muscle i s s i m i l a r to t h a t of other t e l e o s t s . In terms of the c o n t r a c t i l e p r o t e i n composition, the r a t i o of actin/myosin i s 6, as i s t y p i c a l l y observed elsewhere. In s i z e , the s k i p j a c k white muscle sarcomere i s 1.6-1,7 um l o n g , w e l l w i t h i n the range of white muscle f i b e r s i n other t e l e o s t s (Hulbert and Moon, 1978) . Compared to r e d f i b e r s , white muscle f i b e r s are l a r g e , and show a s u r p r i s i n g range of 40 diameters (Figure 3-1) . A h i g h l y complex sar c o p l a s m i c r e t i c u l u m i s c l e a r l y e v i dent, Li£i^# M i t o c h o n d r i a , And C a p i l l a r i t y S k i p j a c k white muscle co n t a i n s s m a l l amounts of i n t r a c e l l u l a r l i p i d . Even i f such l i p i d d r o p l e t s are r a r e (at l e a s t o n e / f i b e r ) they are abundant compared to a f i s h such as the e e l where l i p i d d r o p l e t s may be found i n ev e r y f i f t h f i b e r (Hulbert, pers, comm.). T h e i r occurence i n white muscle i s supported by the h i s t o c h e m i c a l s t u d i e s (Figure 3-2), About 2.3%; of the c r o s s s e c t i o n a l area i s occupied by mitochondria, compared t o 0.1% i n e e l white muscle (Bulbert and Moon, 1978) and 1,156 i n c o a l f i s h white muscle (Patterson and Goldspink, 1972). The mitochondria are a t l e a s t 50% p e r i p h e r a l . The occurence of unusually high amounts o f mitochondria imply the need f o r an e f f e c t i v e oxygen supply system. C a p i l l a r i e s i n s k i p j a c k white muscle approach 1 c a p i l l a r y / f i b e r , again a value t h a t i s up t o 1 0 - f o l d higher than t h a t of other t e l e o s t s (Boddeke e t . a l . , 1959; Mosse, 1978). Although an e f f e c t i v e oxygen d e l i v e r y system i s an absolute n e c e s s i t y i f l i p i d i s to be c a t a b o l i z e d , E.M. S t u d i e s leave no doubt as t o the f a r g r e a t e r importance of glycogen as a carbon and energy source f o r s k i p j a c k white muscle., Glycogen Storage--Glycogen depots i n s k i p j a c k white muscle are unusual i n two regards; i n abundance and i n storage mechanisms. Glycogen 41 i s extremely abundant, f a r more so than i n most t e l e o s t white muscle which t y p i c a l l y s t o r e s o n l y s m a l l amounts of glycogen (Walker and Johansen, 1977; Johnston and Goldspink, 1977). In f a c t , s k i p j a c k white muscle c l e a r l y s t o r e s higher c o n c e n t r a t i o n s of glycogen than does red muscle, i n t h i s f e a t u r e resembling the mammalian, r a t h e r than the t e l e o s t , c o n d i t i o n . White muscle glycogen i s s t o r e d e i t h e r as t y p i c a l B-p a r t i c l e s t h a t are found both i n m y o f i b r i l l a r and p e r i p h e r a l p o s i t i o n s , or i n d i s t i n c t glycogen-membrane a s s o c i a t i o n s termed glycogen bodies (Hochachka and H u l b e r t , 1978). Glycogen D e p l e t i o n In E x e r c i s e The p a r t i c i p a t i o n of glycogen i n white muscle metabolism can be c o n v e n i e n t l y demonstrated by sampling muscle f o l l o w i n g b u r s t s of swimming. Thus, f o l l o w i n g such severe e x e r c i s e , i n t e r f i b r i l l a r glycogen granules are almost completely u t i l i z e d , exposing the h i g h l y i n t r i c a t e , and e x t e n s i v e s a r c o p l a s m i c r e t i c u l u m that t y p i f i e s white m u s c l e . . S i m i l a r l y , p e r i p h e r a l and i n t e r s t i t i a l glycogen granules are s t r o n g l y depleted. And perhaps most i n t r i g u i n g of a l l , even glycogen bodies s u s t a i n a potent m o b i l i z a t i o n of glycogen l e a v i n g glycogen bodies c o n t a i n i n g only the weakly absorbing matrix. From a l l the above data, i t appears that the s k i p j a c k myotome i s formed from two main types of muscle f i b e r s , one red and one white, presumably corr e s p o n d i n g t o slow-twitch o x i d a t i v e and f a s t - t w i t c h g l y c o l y t i c f i b e r s of other v e r t e b r a t e s . In other f i s h e s , t h i s arrangement has been 42 i n t e r p r e t e d as a two-geared system {see I n t r o d u c t i o n ) . at low c r u i s i n g speeds, only the red f i b e r s are thought to be used, c o n t r a c t i n g at t h e i r r e l a t i v e l y low o p t i m a l v e l o c i t y ; the f a s t f i b e r s are a c t i v a t e d only d u r i n g b u r s t swimming. The f a s t f i b e r s have a higher o p t i m a l c o n t r a c t i o n v e l o c i t y and t h e r e f o r e allow f i s h e s to • s h i f t i n t o higher gear* and p r o p e l themselves more r a p i d l y without a great l o s s i n thermodynamic e f f i c i e n c y (Goldspink, 1977). In t h i s view, low gear f u n c t i o n i s a e r o b i c ; high gear f u n c t i o n i s anaerobic. Low gear f u n c t i o n i n s k i p j a c k red muscle c o u l d be primed by glycogen or f a t c a t a b o l i s m , while •high gear' f u n c t i o n o f white muscle would depend upon glycogen. Fine s t r u c t u r e s t u d i e s of red muscle i n d i c a t e a e r o b i c metabolic machinery surrounded by ample l i p i d and glycogen. White muscle, by c o n t r a s t , has f a r fewer mitochondria, but i s packed f u l l of glycogen granules and moreover i s well endowed with glycogen bodies; both sources of glycogen could be u t i l i z e d f o r anaerobic metabolism. Thus, the two gear system c l e a r l y c ould work; but does i t ? Obviously the white muscle of the s k i p j a c k has a high anaerobic p o t e n t i a l , i n terms of s u b s t r a t e (glycogen) f o r g l y c o l y s i s . But i t i s a l s o apparent t h a t t h i s muscle has a e r o b i c c a p a b i l i t i e s i n excess of any other t e l e o s t white muscle p r i v i o u s l y s t u d i e d . T h i s paradox i s r e a d i l y overcome i f i t i s assumed that i n s k i p j a c k , the simple two-gear system i s augmented by an o v e r l a p p i n g of red and white muscle f u n c t i o n s . The o v e r l a p i s l a r g e l y metabolic r a t h e r than mechanical and r e q u i r e s t h a t at l e a s t some (perhaps i n i t i a l ) white muscle work be supported by a e r o b i c c a t a b o l i s m . I f such an o v e r l a p occurred 43 i t would e x p l a i n the u l t r a s t r u c t u r a l i n d i c a t i o n s of a s i g n i f i c a n t a e r o b i c c a p a c i t y i n s k i p j a c k white muscle. These i n i t i a l s t u d i e s provide only a h i n t of t h i s p o s s i b l e o v e r l a p p i n g f u n c t i o n . However, as t h i s t h e s i s progresses, these u l t r a s t r u c t u r a l s t u d i e s are confirmed by more evidence, and i t tu r n s out that the white muscle i n the s k i p j a c k tuna does indeed play a s i g n i f i c a n t r o l e i n power output based on a e r o b i c metabolism. RESULTS AND DISCUSSION: PAST 2. ENZYME PROFILES OF RED ASP-WHITE MUSCLE To a t t a t c h any s i g n i f i c a n c e to measured i n v i t r o enzyme assays, one must show t h a t these measurements i n some way r e f l e c t the i n v i v o metabolic o r g a n i s a t i o n of the t i s s u e i n q u e s t i o n . T h i s r e f l e c t i o n i s one which i s c o n s t a n t l y assumed i n metabolic s t u d i e s but to my knowlege has never been d i s c u s s e d , although Newsholme and S t a r t (1973), Crabtree and Newsholme (1972) and Sugden and Newsholme (1975), mention some of the problems which may be i n v o l v e d . I b e l i e v e t h e r e can be a d e f i n i t e c o r r e l a t i o n between i n v i v o and i n v i t r o v a l u e s , but, mostly f o r my own peace of mind, I w i l l b r i e f l y e x p l a i n why there may be unseen dangers i n always assuming t h a t t h i s i s the case. The r a t e of c o n v e r s i o n of s u b s t r a t e to product i n an enzyme r e a c t i o n (assuming constant environment such as pH etc) i s dependent upon the frequency of c o l l i s i o n s between s u b s t r a t e molecules and the enzyme*s a c t i v e s i t e s and thus on the c o n c e n t r a t i o n s of s u b s t r a t e and enzyme. The r a t e of c o n v e r s i o n i s a l s o dependent upon the turnover number of the enzyme (only when the enzyme i s the l i m i t i n q f a c t o r i n the r e a c t i o n ) . Thus i f a r e a c t i o n i s measured at s a t u r a t i n g s u b s t r a t e l e v e l s (which i t u s u a l l y i s i n enzyme p r o f i l e s t u d i e s ) the r a t e w i l l be a f f e c t e d by the c o n c e n t r a t i o n of enzyme, and the turnover number. The l a t t e r c h a r a c t e r i s t i c may have no An v i v o conseguence i f the enzyme i s not the l i m i t i n g f a c t o r . The c o n c e n t r a t i o n of enzyme, however w i l l have an e f f e c t at a l l s u b s t r a t e l e v e l s . Maximal r a t e s t h e r e f o r e , can imply 45 d i f f e r e n c e s which would disappear under i n v i v o , n o n - s a t u r a t i n g c o n d i t i o n s . The more obvious problems (such as p h y s i c a l d e n a t u r a t i o n during homogenisation) are mentioned by some authors i n v o l v e d i n s t u d i e s of t h i s type (Pette and Dolken, 1975; L i n z e n and G a l l o w i t z , 1975; Scrutton and O t t e r , 1968),but i t i s too c f t e n the case t h a t the p i t f a l l s are ignored and the c o n c l u s i o n s drawn without h e s i t a t i o n . C o n c e i v a b l y much of the problem could be overcome by measuring a c t i v i t i e s under " p h y s i o l o g i c a l " c o n d i t i o n s . Such c o n d i t i o n s are however almost i m p o s s i b l e to determine and do not take i n t o account the many d i f f e r e n t metabolic s t a t e s of the animal nor the d i f f e r e n c e i n metabolic a c t i v i t y between and a c t i v e and a sedentary animal. Thus maximum a c t i v i t i e s probably t u r n out to be the most meaningful of the two l i m i t e d methodologies. When a multi-enzyme pathway i s considered the problem becomes even more complex. The maximum f l u x through a l i n e a r pathway depends upon the maximum p o t e n t i a l of the r a t e l i m i t i n g s t e p i n t h a t pathway, which i s u s u a l l y a r e g u l a t o r y enzyme. I t i s o f t e n d i f f i c u l t t o measure the p o t e n t i a l of complex r e g u l a t o r y enzymes due to i n s t a b i l i t y and the l a r g e numbers of p o t e n t i a l enzyme e f f e c t o r s , and thus the a c t i v i t i e s of more a c t i v e enzymes at a n o n - r e g u l a t o r y s i t e , are u s u a l l y taken as a l t e r n a t i v e i n d i c e s . These l a t t e r enzymes are presumably present i n such high a c t i v i t i e s t o minimize or exclude the p o s s i b i l i t y t h a t the step they c a t a l y s e ever becomes rate l i m i t i n g . Therefore these a c t i v i t i e s may not be an a c c u r a t e i n d i c a t i o n o f the pathway's p o t e n t i a l , but may represent an 46 " o v e r k i l l " f a c t o r . T h i s i s not to say t h a t p r e c i s e r e l a t i o n s h i p s between enzyme l e v e l s and some aspect of metabolism have not been demonstrated. G l y c o l y t i c r a t e s have been c o r r e l a t e d with the a c t i v i t i e s of phosphorylase, hexokinase and PFK (Crabtree and Newsholme, 1972). Simon and Robin (1971 and 1972) have demonstrated a good c o r r e l a t i o n between b a s a l oxygen consumption and cytochrome oxidase a c t i v i t y and between PK a c t i v i t y and l a c t a t e p r o d u c t i o n under anaerobioses. Salminen e t . a l . , (1977) found t h a t i n mouse s k e l e t a l muscle, f a t t y a c i d o x i d a t i o n c a p a c i t y was h i g h l y c o r r e l a t e d with cytochrome oxidase a c t i v i t y , as was f a t t y a c i d o x i d a t i o n with MDH a c t i v i t y . Pande and Blanchaer,(1971) show a c o r r e l a t i o n between f a t t y a c i d o x i d i s i n g c a p a c i t y and the a c t i v i t i e s of the p a l m i t a t e - a c t i v a t i n g enzyme and the c a r n i t i n e p a l m i t o y l -t r a n s f e r a s e i n the mitochondria of r a b b i t red and white muscle. In a d d i t i o n many other c o r r e l a t i o n s e x i s t between enzyme a c t i v i t y , or simply enzyme e x i s t e n c e , and the known metabolic s t r a t e g y of the t i s s u e (See I n t r o d u c t i o n ) . Another type of c o r r e l a t i o n , one between the a c t i v i t i e s of •sets* of enzymes i n the same t i s s u e i s a l s o found to be widespread and i s a u s e f u l method of using more than one enzyme a c t i v i t y to determine the r e l a t i v e importance of metabolic pathways i n a t i s s u e . For example, Bass e t a l (1969) and Eette and Dolken (1975) f i n d t h a t groups of enzymes e x i s t whose a c t i v i t i e s are found i n comparable or even constant p r o p o r t i o n s i n a v a r i e t y of t i s s u e s or c e l l s . The a c t i v i t i e s of a v a r i e t y o f enzymes i n s k i p j a c k red and 47 white muscle are given i n Table 3-1. In terms of red and white muscle f u n c t i o n , the p i c t u r e i s a t y p i c a l one. A l l the g l y c o l y t i c enzymes, with the e x c e p t i o n of hexokinase are between 5 and 1 0 - f o l d more a c t i v e i n the white muscle, and the a c t i v i t y of CS, the onl y r e a l measure I have o f Krebs c y c l e a c t i v i t y , i s 7 - f o l d higher i n r e d muscle. In the s k i p j a c k , hexokinase i s e q u a l l y a c t i v e i n both t i s s u e s . Red muscle u s u a l l y has more hexokinase a c t i v i t y than white (Peter e t . a l . , 1968; c r a b t r e e and Newsholme, 1972; P e t t e , 1966; B u r l e i q h and Schimke, 1968), The r a t i o n a l behind t h i s hiqher a c t i v i t y i s t h a t the s u b s t r a t e of hexokinase i s g l u c o s e , a blood-born carbohydrate f u e l which i s burnt a e r o b i c a l l y (when blood supply i s adequate)by the more a e r o b i c muscle type. When compared to other f i s h red s k e l e t a l muscles (which on the whole do not d i f f e r s i q n i f i c a n t l y i n terras of enzyme a c t i v i t i e s from correspondinq mammalian s k e l e t a l muscles) the s k i p j a c k red muscle appears t y p i c a l with perhaps the e x c e p t i o n of LDH and GOT. The a c t i v i t i e s of these two enzymes are somewhat hiqher i n s k i p j a c k r e d muscle than i n other red muscles (Alp e t . a l . , 1976; Crabtree and Newsholme, 1972; Eeatty and Bocek, 1970). The white muscle on the other hand i s somewhat uniqiue., Phosphorylase values are hiqher than other f i s h white muscle values and rank with the hiqher values which are found i n seme mammalian white muscles.(Most mammalian and f i s h muscle values are about 50 0/q, but the r a t and the r a b b i t have hiqher values of around 100 D/g (Crabtree and Newsholme, 1972; Beatty and Bocek, 1970)). PFK values i n the s k i p j a c k white muscle are 48 r e l a t i v e l y low, but t h i s i s probably due to the extreme i n s t a b i l i t y of the enzyme which showed s i g n s of s i g n i f i c a n t d e t e r i o r a t i o n immediately a f t e r homogenisation, PK a c t i v i t i e s are about 3 - f o l d higher i n s k i p j a c k white muscle than i n other f i s h white muscles (Bochachka e t . a l * , 1978a; Beatty and Bocek, 1970) and the a c t i v i t y of LDH i s the highest so f a r found i n any t i s s u e . The LDH a c t i v i t y i n the s k i p j a c k white muscle i s at l e a s t 5 - f o l d h i g h e r , and u s u a l l y 1 0 - f o l d higher than any other value reported. CS a c t i v i t i e s i n s k i p j a c k white muscle are not unusual, but the HDH and GOT values are about 1.5 - 2 - f o l d higher than other white muscle values (Hochachka e t . a l . . 1978a; AlP §t«ai»# 1976). Srere (1969) has noted t h a t CS i s b e t t e r e x t r a c t e d a f t e r f r e e z i n g and thawing muscle t i s s u e . S k i p j a c k white muscle t r e a t e d t h i s way produces 7-8 D/g of CS, a value approaching t h a t i n some mammalian muscles. ( H o l l o s z y e t . a l . . 1970). The a c t i v i t y of GPDH i n the white muscle i s u n u s u a l l y high. T h i s enzyme, when i n high a c t i v i t i e s u s u a l l y f u n c t i o n s as t h e c y t o p l a s m i c arm of the GP c y c l e , which balances redox during a e r o b i c carbohydrate metabolism (Sacktor, 1976; Storey and Hochachka, 1975). There are u s u a l l y higher a c t i v i t i e s of t h i s enzyme i n white muscle than red (Crabtree and Newsholme, 1972; Blanchaer, 1964) but a t y p i c a l value i n v e r t e b r a t e white muscle i s 5 - 2 5 U/g, with the h i g h e s t values a t 50 U/g (Hochachka e t . a l . , 1978a; Beatty and Bocek, 1970; Crabtree and Newsholme, 1972). The enzyme p r o f i l e s both agree with and add to the c o n c l u s i o n s drawn from the h i s t c c h e m i c a l and u l t r a s t r u c t u r a l data presented i n Chapter 3. The red muscle i s the more a e r o b i c 49 of the two muscles as i s demonstrated by i t s g r e a t e r Krebs c y c l e a c t i v i t y and p o t e n t i a l f o r amino a c i d metabolism. The low a c t i v i t y of the g l y c o l y t i c enzymes i n red muscle suggests t h a t the m a j o r i t y of a c e t y l CoA f o r the Krebs c y c l e i n red muscle i s d e r i v e d from l i p i d , Thus the s k i p j a c k red muscle appears to be a t y p i c a l f i s h red muscle although the r e l a t i v e l y high a c t i v i t i e s of GOT and LDH suggest t h a t i t may have an a t y p i c a l l y high a b i l i t y to metabolise carbohydrate a e r o b i c a l l y (using a malate-aspartate s h u t t l e t o balance redox) and a n a e r o b i c a l l y , producing l a c t a t e . A l s o , the high a c t i v i t i e s o f LDH i n the red muscle (the red muscle LDH i s k i n e t i c a l l y H-type and thus has a higher a f f i n i t y f o r l a c t a t e than does the white muscle LDH (see F i g u r e 3-2 and Chapter 5)) c o u l d be f o r the o x i d a t i o n o f e x t r a c e l l u l a r l a c t a t e . Red muscle mitochondria o x i d i z e l a c t a t e at a f a s t e r r a t e than do white muscle mitochondria (Blanchaer, 1964), and Wittenberqer et.-aj,,-,- (1975) argues that carp red muscle i s the s i t e o f o x i d a t i o n f o r white muscle l a c t a t e . With the q u a n t i t i e s of l a c t a t e produced by the s k i p j a c k white muscle (Chapter 4 ) , white muscle l a c t a t e c o u l d w e l l be a major red muscle carbon source i n the skipjack.. In the white muscle the q l y c o l y t i c pathway i s more than adequately r e p r e s e n t e d and i s terminated by a hiqh a c t i v i t y of LDH. Thus t h e r e i s ample o p p o r t u n i t y f o r the anaerobic con v e r s i o n of the high c o n c e n t r a t i o n s of glycogen (Chapter 3 and 4) to l a c t a t e . The unusual a e r o b i c c a p a c i t y sugqested by the u l t r a s t r u c t u r e (Chapter 3) , i s supported by CS l e v e l s (in f r o z e n and thawed t i s s u e ) , l e v e l s of a-GPDH and hexokinase, and the hiqh GOT l e v e l s c ould r e p r e s e n t a c a p a c i t y f o r augmentinq 50 the Krebs c y c l e during a e r o b i c metabolism ( D r e i d z i c and Hochachka, 1976). The high l e v e l s of MDH may be c o r r e l a t e d to Krebs c y c l e a c t i v i t y , but as there i s a l s o a c y t o s o l i c form o f the enzyme ( S i e g a l and Englard, 1962), the added a c t i v i t y could j u s t as w e l l be due to the c y t o p l a s m i c form. The white muscle thus would appear to be a somewhat abnormal t i s s u e when compared to other v e r t e b r a t e white muscles. I t appears to have not only an e x t r a o r d i n a r y anaerobic p o t e n t i a l , but a s i g n i f i c a n t a e r o b i c one as w e l l . The c o n c l u s i o n s drawn i n t h i s chapter although c o n s i s t e n t with the data cannot be taken as f i n a l as was p o i n t e d out i n the i n t r o d u c t i o n t o t h i s c h a p ter. To begin with, there are no i n d i c a t o r s of l i p i d metabolism and many o f the values are a c t i v i t i e s of the n e a r - e g u i l i b r i u m enzymes, r a t h e r than those which are r a t e l i m i t i n g , as Atkinson (1977) p o i n t s out, the s o l v e n t c a p a c i t y of a c e l l i s not i n f i n i t e and thus the high a c t i v i t y of LDH and other enzymes could r e p r e s e n t an i n c r e a s e d turnover number r a t h e r than a higher c o n c e n t r a t i o n of enzyme. T h i s data does not have to stand alone however. I t i s supported by data which precede and f o l l o w i t and which are a l s o c o n s i s t e n t with the above metabolic arrangements. 51 Table 3-1. Enzyme p r o f i l e s i n r e d and white muscles. Enzyme a c t i v i t i e s (uM product/min/gram wet weight) at 25°C, and o p t i m a l s u b s t r a t e , c o f a c t o r and H* l e v e l s . , Enzyme Red Muscle White Muscle P h o s p h o r y l a s e Hexokinase Phosphoglucomutase Phosphoglucose isomerase Glucose-6-phosphate dehydrogenase P h o s p h o f r u c t o k l n a s e A l d o l a s e T r i o s e phosphate isomerase P h o s p h o g l y c e r a t e k i n a s e E n o l a s e P y r u v a t e k i n a s e L a c t a t e dehydrogenase a - G l y c e r o p h o s p h a t e dehydrogenase C i t r a t e s y n t h a s e Glutamate dehydrogenase M a l a t e dehydrogenase G l u t a m a t e - o x a l o a c e t a t e t r a n s a m i n a s e G l utamate-pyruvate t r a n s a m i n a s e C r e a t i n e phosphokinase Myokinase 22. 0( 0 . 6 4 ) * 106. 2(11.14) 1. 2(0.78) 0. 78(0.45) 31. 3(6.04) 152. 8(46.48) 84. 4(14.00) 426. 0(157.73) 0 0 10. 0(one v a l u e ) 25. 50(5.74) 35. 5(10.05) 269. 2(38.62) 1414. 6(2 v a l u e s ) 9886. 0(1763.23) 371. 1(34.8) 1982. 7(433.01) 77. 7(9.23) 522. 4(156.68) 195. 2(37.0) 1294. 9(249.5) 514. 4(74.25) 5492. 3(154.73) 21. 7(2.77) . 104. 5(9.24) 20. 6(0.00) 2. 15(0.87) 5. 9(0.58) 3. 0(1.15) 723. 4(30.95) 718. 0(160.85) 101. 9(6.18) 43. 0(3.41) 7. 7(2.25) 2. 0(2.31) 554. 2(268.7) 516. 4(101.21) 381. 8(23.21) 946. 7(102.02) *n = 4 (±1 S.D.) F i g u r e 3-1. A p l o t of f i b e r diameters f o r red and white muscle versus frequency. F i b e r s were sampled from deep red muscle, deep white muscle, and from the l a t e r a l , p o s t e r i o r wedqe, which i n the s k i p j a c k tuna appears t o be t y p i c a l white muscle. 55 F i g u r e 3-2. H i s t o c h e m i s t r y of the r e d and white muscle. In a l l cases except the lower r i g h t s e c t i o n , the r e d muscle i s on the l e f t and i s s t a i n e d more h e a v i l y ; i n the l a t t e r , the s e c t i o n i s composed e n t i r e l y of red muscle., Upper l e f t : s p e c i f i c s t a i n f o r s u c c i n a t e dehydrogenase {X 160). Upper r i g h t : s p e c i f i c s t a i n f o r l a c t a t e dehydrogenase (X 160). Lower l e f t : sudan black s t a i n i n g f o r l i p i d (X 160)., Lower r i g h t : sudan black s t a i n i n g f o r l i p i d (X 160). Note the sharp demarkation between red and white muscle i n the top two s e c t i o n s and the i n t e r d i g i t a t i o n s i n the lower l e f t s e c t i o n . A l s o note the d i f f e r e n t i a l u t i l i z a t i o n or d e p o s i t i o n of l i p i d i n the lower r i g h t s e c t i o n . 57 CHAPTER 4. METABOLITE LEVELS AND TEJillSAIUBIS IN THE £ED AND WHITE MUSCLES DURJNG JEST AND MSiMM 58 INTBOPPCTIOH Measurements of the non-protein components of metabolic pathways i s a technique which i s widely used t o c h a r a c t e r i z e metabolism i n a t i s s u e . The p r i n c i p l e of the technigue i n v o l v e s comparing m e t a b o l i t e l e v e l s before and a f t e r a s i t u a t i o n which has i n some way perturbed the t i s s u e ' s metabolism. The more obvious metabolite l e v e l changes occur i n g l y c o l y s i s , the Krebs c y c l e and a s s o c i a t e d amino a c i d pathways and i n the adenylate and c r e a t i n e phosphate systems. These changes, and the c o n c l u s i o n s t h a t can be drawn from them are now w e l l documented and are b r i e f l y summarized below. During the i n i t i a t i o n o f f l i g h t i n the b l o w f l y (an example of i n t e n s e a e r o b i c g l y c o l y s i s ) , t r e h a l o s e , glycogen, abdominal p o l y s a c c h a r i d e and p r o l i n e are depleted. During an hour of f l i g h t , ATP, ADP, arginine-phosphate and P i l e v e l s remain constant. These o b s e r v a t i o n s are c o n s i s t e n t with s u s t a i n e d a e r o b i c work based on non-blood born carbohydrate with Krebs c y c l e augmentation being d e r i v e d from p r o l i n e (Sacktor and Wormser-Shavit, 1966). When a e r o b i c g l y c o l y s i s i n the heart i s s t i m u l a t e d , t h e r e are no changes i n the l e v e l s of c r e a t i n e -phosphate, ATP, ADP and glycogen (Neely e t . a l . , 1972), and a s p a r t a t e l e v e l s f a l l (Safer and Williamson, 1973). These o b s e r v a t i o n s are c o n s i s t e n t with a e r o b i c work based on b l c c d -born glucose with a s p a r t a t e p r o v i d i n g the carbon f o r Krebs c y c l e augmentation. During a c e t a t e metabolism (Krebs c y c l e a c t i v a t i o n ) i n the heart, ATP l e v e l s remain constant and again a s p a r t a t e l e v e l s decrease (Williamson, 1965), and when the work load on the heart i s i n c r e a s e d mechanically, l o n g c h a i n a c y l 59 CoA l e v e l s decrease as f l u x through the B - o x i d a t i o n pathway i n c r e a s e s (Oram e t a l , 1973). With the onset of anaerobic work, the most obvious changes occur i n the l e v e l s o f end p r o d u c t ( s ) , which i n the m a j o r i t y of v e r t e b r a t e t i s s u e s i s l a c t a t e , a l s o d u r i n g a n a e r o b i o s i s the l e v e l s of pyruvate, ADP, AMP and c r e a t i n e i n c r e a s e , and l e v e l s of creatine-phosphate and AIP decrease (Edington, 1973; Bovetto gt. a l . , 1973; Lowry g t i j j , . , 1964; Neely e t . a l . , 1975; Ford and Candy, 1972; Sacktor, 1970). These k i n d s of changes which i n v o l v e m e t a b o l i t e s mainly a t the beginning and ends of pathways, and those which are a s s o c i a t e d with t h e energy s t a t u s of the c e l l * p rovide i n f o r m a t i o n on the f u e l used f o r energy g e n e r a t i o n , the pathway used, the end-products accumulated and the energy charge of the c e l l d u r i n g the work phase. L e v e l s of m e t a b o l i t e s throughout the pathway can o c c a s i o n a l l y i n c r e a s e or decrease, but u s u a l l y , as a group, show no c o n s i s t e n t c o r r e l a t i o n with f l u x . S p e c i f i c m e t a b o l i t e l e v e l s w i t h i n a pathway however, can be used very e f f e c t i v e l y f i r s t l y to l o c a t e r e g u l a t o r y enzymes and secondly to determine whether or not t h a t enzyme, and thus the pathway i n v o l v e d , i s a c t i v a t e d or i n h i b i t e d . Four enzymes i n g l y c o l y s i s have been found to be " f a r from e g u i l i b r i u m , , and thus p o t e n t i a l r e g u l a t o r y enzymes (see Newsholme and S t a r t , 1973 and B o l l e s t o n , 1972, f o r a d i s c u s s i o n of t h i s p o i n t ) . These are hexokinase, PFK, GAPDH, and pyruvate kinase, whose mass a c t i o n r a t i o s i n most t i s s u e s d i f f e r from t h e i r expected e q u i l i b r i u m by at l e a s t two orders of magnitude (Newshome and S t a r t , 1973), Thus d u r i n g the i n i t i a t i o n o f f l i g h t i n the b l o w f l y , the l e v e l s of FDP and pyruvate, products of two r e g u l a t o r y enzymes, r i s e 60 t r a n s i e n t l y as the pathway i s a c t i v a t e d (Sacktor and Wormser-Shavet, 1966 ). When a e r o b i c g l y c o l y s i s i n the heart i s s t i m u l a t e d , glucose and G-6-P l e v e l s f a l l as hexokinase and PFK are activated.,When g l y c o l y s i s i n the heart i s i n h i b i t e d , m e t a b o l i t e l e v e l changes are c o n s i s t e n t with i n h i b i t i o n a t PFK and GAPDH (Williamson, 1965), and when f a t t y a c i d o x i d a t i o n i s i n h i b i t e d , i n h i b i t i o n occurs before the Krebs c y c l e as evidenced by the b u i l d - u p of l o n g - c h a i n a c y l CoA d e r i v a t i v e s and the d e p l e t i o n of a c e t y l CoA (Neely e t . a l . , 1976). Metabol i t e s t u d i e s on the Krebs c y c l e have suggested c o n t r o l at a v a r i e t y of l o c i i n c l u d i n g c i t r a t e s y t h e t a s e (LaSoue e t . a l . . 1972), i s o c i t r a t e dehydrogenase (Hiltunen and Hassinen, 1977) and a - k e t o g l u t a r a t e dehydrogenase (Safer and Wi l l i a m s o n , 1973). Non-regulatory enzymes, c o n v e r s e l y , u s u a l l y have higher a c t i v i t i e s and t h e i r mass a c t i o n r a t i o s are c l o s e to t h e i r expected e q u i l i b r i u m (Newsholme and S t a r t , 1973; R o l l e s t o n , 1973; Williamson, 1965). 61 RESULTS -METABOLITES Le v e l s of v a r i o u s g l y c o l y t i c and Krebs c y c l e i n t e r m e d i a t e s , the a d e n y l a t e s , c r e a t i n e , creatine-phosphate, amino a c i d s and glycogen i n t i s s u e s and blood are given i n Tables 4-1 - 4 -4. Table 4-1 and 4-2 are r e s u l t s from 1976 when t i s s u e s were simply dropped i n t o l i q u i d n i t r o g e n . The data i n Tables 4-3 and 4- 4 are from 1977, when freeze-clamping with H o l l e n b e r g e r tongs was employed to q u i c k - f r e e z e the t i s s u e s i n case the s l i g h t l y l o nger f r e e z i n g time i n 1976 had l e d to a r t e f a c t s (see Chapter 2 ) . A comparison between Tables 4-1 and 4-3 demonstrates t h a t there are d i f f e r e n c e s which c o u l d be r e a l d i f f e r e n c e s , or p o s s i b l y technigue-based d i f f e r e n c e s . The m a j o r i t y of the values however, are very s i m i l a r , c o n s i d e r i n g the d i f f i c u l t y of s t a n d a r d i z i n g any work with s k i p j a c k . These d i f f e r e n c e s w i l l be mentioned i n the f o l l o w i n g d i s c u s s i o n . The glycogen data (Table 5- 4) can be r e l a t e d t o i n d i v i d u a l f i s h , which i s a v a l u a b l e as s e t and so has been presented i n t h i s form. Glycogen Glycogen l e v e l s i n the s k i p j a c k white muscle are high compared to other t e l e o s t white muscle values (1000 vs 40 0 mgSa) and are somewhat higher than s k i p j a c k red muscle values which i s c o n t r a r y to the s i t u a t i o n seen i n ether t e l e o s t s (Walker and Johansen, 1977; Johnston e t . a l . , 1977; P r i t c h a r d 62 e t . a l . , 1971; Johnston and Goldspink, 1973). The s k i p j a c k values are i n f a c t a k i n to mammalian values which tend t o be around 1000 mq% with s m a l l e r d i f f e r e n c e s between red and white f i b e r s (Baldwin e t . a l . , 1973; Ahlborg e t . a l . , 1967). B a r r e t and Conner (1964) found s i m i l a r glycogen l e v e l s i n white muscle of f r e s h l y caught s k i p j a c k . Glycogen l e v e l s i n the r e d muscle do not change between r e s t and s u s t a i n e d swimming, and the b u r s t swimming glycogen l e v e l s are too v a r i a b l e f o r an average to be meaningful (Table 4-4), Values f o r the t o t a l glycogen and l a c t a t e pool i n red muscle (Table 4-5), which are s i m i l a r f o r a l l three s t a t e s , simply demonstrate t h a t glycogen carbon i s e i t h e r i n glycogen or l a c t a t e and thus t h a t there i s no a e r o b i c metabolism of glycogen i . e . there i s no carbon l o s t as carbon d i o x i d e . / Glycogen l e v e l s i n the white muscle are s l i g h t l y lower during s u s t a i n e d swimming and drop markedly a f t e r burst swimming. F i g u r e s f o r t o t a l glycogen and l a c t a t e (Table 4-5) under burst c o n d i t i o n s are as high or higher than those under r e s t i n g c o n d i t i o n s i n d i c a t i n g t h at d u r i n g b u r s t swimming, glycogen i s q u a n t i t a t i v e l y converted t o l a c t a t e . T h i s r e l a t i o n s h i p can most e a s i l y be seen i n f i s h #3; glycogen v a l u e s i n the white muscle of f i s h #3 are the highest of the b u r s t swim white muscle values whereas l a c t a t e values f o r the white muscle of f i s h #3 are the lowest of the b u r s t swimming values (Table 4-4). However, the value f o r t o t a l glycogen and l a c t a t e under fee d i n g c o n d i t i o n s i s lower (Table 4-5) i n d i c a t i n g t h a t a e r o b i c carbohydrate metabolism i s t a k i n g place i n the white muscle durin g the f e e d i n g f r e n z y . 6 3 Glucose Changes i n glucose l e v e l s are complex. In the 1976 experiments, g l u c o s e l e v e l s i n red muscle drop during f e e d i n g , but are unchanged d u r i n g b u r s t swimming; i n white muscle, i n c o n t r a s t , glucose c o n c e n t r a t i o n s drop s l i g h t l y during f e e d i n g , but r i s e d r a m a t i c a l l y d u r i n g b u r s t swimming. In the 1977 experiments, glucose c o n c e n t r a t i o n s i n r e s t i n g red muscle are lower than i n the 1976 samples and show l i t t l e change duri n g f e e d i n g f r e n z i e s ; however, on t r a n s i t i o n to b u r s t swimming, glucose l e v e l s r i s e s h a r p l y . Not enough i n f o r m a t i o n i s a v a i l a b l e on blood glucose l e v e l s t o be c e r t a i n of t r a n s i e n t p r o f i l e s ; however, i t does seem that red and white muscle glucose pools e q u i l i b r a t e with the blood during b u r s t swimming, but not d u r i n g steady s t a t e swimming. Glucose-6— pho sp ha t e G6P l e v e l s i n the r e d muscle do not change i n the 1976 experiments between r e s t and burst work and approximately double i n the 1977 experiment. There i s an i n c r e a s e i n white muscle G6P l e v e l s with e x e r c i s e i n both experiments, again the i n c r e a s e i s l a r g e r i n the 1977 experiments. The d i f f e r e n c e here between the two experiments i s the r e s t i n g l e v e l of G6P which d i f f e r s by a f a c t o r of 2. Fructose-6-phosphate Both red and white muscle l e v e l s of F6P i n c r e a s e with e x e r c i s e , the t r e n d being more pronounced i n the white muscle. 64 F r u c t o s e Diphosphate FDP l e v e l s i n the red muscle do not vary with e x e r c i s e i n the 1977 experiment although there was a s l i g h t i n c r e a s e during burst swimming i n the 1S76 f i s h . The same i s t r u e f o r the white muscle; there was a 2 - f o l d i n c r e a s e d u r i n g burst swimming i n the 1976 f i s h , but no changes with e x e r c i s e i n the 1977 f i s h . Pyruvate In both experiments pyruvate l e v e l s remain co n s t a n t i n the red muscle, but i n c r e a s e by a f a c t o r of 3 between r e s t and b u r s t swimming i n the white muscle. There i s no change i n pyruvate l e v e l s i n the white muscle between r e s t i n g and feeding f i s h i n the 1976 experiment, whereas there i s an i n c r e a s e (about 2-fold) i n pyruvate l e v e l s between r e s t i n g and f e e d i n g i n the 1977 f i s h . L a c t a t e I n i t i a l l a c t a t e l e v e l s are about 3 - f o l d lower i n both muscles i n the 1977 experiments. Apart from t h i s , the trend i s the same. There i s a s l i g h t i n c r e a s e i n l a c t a t e l e v e l s i n the red muscle during b u r s t swimming and i n the white.muscle l a c t a t e l e v e l s i n c r e a s e by 3-6 f o l d d u r i n g feeding and by a maximum of 2 0 - f o l d during b u r s t swimming. The 80-100 umol/g wet weight l a c t a t e c o n c e n t r a t i o n s produced i n the white muscle a f t e r 10 minutes of b u r s t work (Tables 4-1 and 4-3) are t o my knowledge, the f a s t e s t r a t e o f l a c t a t e p r o d u c t i o n , reaching the highest l e v e l s ever recorded f o r muscular work. The jack 65 mackerel can produce 70 umol/g wet weight i n 8 minutes, but c o n t r o l values were 40 umol/g wet weight ( P r i t c h a r d e t . a l . , 1971). In carp and c o a l f i s h e x e r c i s e d to f a t i g u e , the l a c t a t e c o n c e n t r a t i o n i n c r e a s e s to 12 and 20 umol/g wet weight r e s p e c t i v e l y ( D r i e d z i c unpub,; Johnston and G o l d s p i n k , 1973) and i t has been r e p e a t e d l y shown that the l e v e l of l a c t a t e i n salmonids can approach 30-50 umcl/g wet weight (Black e t . a l . , 1962; Stevens and Black, 1966; Hammond and Hickman, 1966; B i l i n s k y , 1974), & v a r i e t y of mammals, e x e r c i s e d to exhaustion were found to accumulate 10-30 mM l a c t a t e i n 10 minutes (Seeherman e t . a l . , 1976), Blood l a c t a t e i n the s k i p j a c k i s not i n e g u i l i b r i u m with the t i s s u e s under f e e d i n g or b u r s t c o n d i t i o n s , which a t l e a s t i n the l a t t e r s i t u a t i o n i s c h a r a c t e r i s i c of t e l e o s t s (Black e t . a l . , 1962). The l a c t a t e values i n the blood o f f e e d i n g f i s h however, as was the case f o r glucose, may not be comparable to the t i s s u e v a l u e s , although B a r r e t and Conner (1964) p u b l i s h e d s i m i l a r blood l a c t a t e l e v e l s with f r e s h l y caught s k i p j a c k tuna. GP GP l e v e l s approximately double dur i n g b u r s t swimming i n both t i s s u e s which i s i n accordance with other s t u d i e s on f i s h , i n s e c t s , and r a t s ( D r i e d z i c , Ph.D t h e s i s ; Edington e t . a l . , 1973; Ford and Candy, 1S72). C i t r a t e Arid p a l a t e C i t r a t e and malate l e v e l s r i s e with e x e r c i s e i n the red 66 muscle i n both 1976 and 1977 experiments. In the white muscle, c i t r a t e and malate l e v e l s do not change i n the 1976 experiment and r i s e d u r i n g b u r s t work i n the 1977 experiment. Adenylates And Creatine-phosphate There i s some f l u c t u a t i o n i n the l e v e l s of the adenylates i n both t i s s u e s under the d i f f e r e n t e x e r c i s e s t a t e s . The most dramatic change i s t h a t of ATP l e v e l s i n white muscle d u r i n g b u r s t swimming. The energy charge and adenylate pool s i z e (Table 4-6) are higher i n the white muscle. The energy charge i s highest during f e e d i n g and lowest during b u r s t swimming i n both t i s s u e s . The pool s i z e o f the adenylates i s h i g h e s t during r e s t i n both t i s s u e s and lowest d u r i n g f e e d i n g i n the red muscle, and during burst work i n the white muscle. C r e a t i n e -phosphate l e v e l s i n both experiments are a t l e a s t 6 - f o l d higher i n the white muscle and drop (most markedly i n the white muscle) i n both t i s s u e s with e x e r c i s e . Amino, JLsAds The amino a c i d values i n red and white muscle and blood d u r i n g the three e x e r c i s e s t a t e s are given i n Table 4-2. In the red muscle, a s p a r t a t e and glutamate l e v e l s f a l l d u r i n g b u r s t swimming, and a l a n i n e , h i s t i d i n e and p r o l i n e l e v e l s r i s e during f e e d i n g . In the white muscle, g l y c i n e and l y s i n e l e v e l s f a l l d u ring f e e d i n g and h i s t i d i n e l e v e l s , which are extremely high a l l the time, r i s e during f e e d i n g . H i s t i d i n e , which i s a l s o present i n high l e v e l s i n carp muscle (Creach, 1966) may be i n 67 the white muscle to b u f f e r t h e l a r g e l a c t i c a c i d f l u c t u a t i o n s . The i m i d a z o l e gioup on the h i s t i d i n e has a PK» of about 6.0, e n a b l i n g i t to act both as a proton donor and a proton acceptor at a pH near t h a t of b i o l o g i c a l f l u i d s . A l s o , h i s t i d i n e can be converted by h i s t i d i n e decarboxylase, to histamine, which i s a potent v a s o d i l a t o r and thus c o u l d i n c r e a s e blood flow i n the white muscle dur i n g burst work when h i s t i d i n e f a l l s (Table 4-2) . Blood amino a c i d l e v e l s are very low and the data o n l y r e p r e s e n t one sample. There may be a r i s e i n a s p a r t a t e and g l y c i n e l e v e l s i n the blood d u r i n g burst swimming., METABOLITE RATIOS Vario u s metabolite r a t i o s are presented i n Tables 4-7 and 4-8. As expected, hexokinase and PFK are f a r from e g u i l i b r i u m whereas PGI i s very c l o s e to e g u i l i b r i u m (Table 4-7). Although glucose measurements do r e p r e s e n t e x t r a - and i n t r a c e l l u l a r glucose pools, t h i s does not change the c o n c l u s i o n t h a t the hexokinase r e a c t i o n i s out of e g u i l i b r i u m . The mass a c t i o n r a t i o i s a t l e a s t 4 orders of magnitude removed from e g u i l i b r i u m implying t h a t glucose l e v e l s would have t o be i n e r r o r by at l e a s t 4 orders of magnitude i n order t h a t the hexokinase r e a c t i o n be i n e g u i l i b u i u m . The v a r i a t i o n i n the e g u i l i b r i u m of the hexokinase r e a c t i o n (mentioned below) could however be negated by a l t e r i n g glucose c o n c e n t r a t i o n s by only one crder of magnitude.. The degree to which the mass a c t i o n r a t i o of r e g u l a t o r y enzymes d i f f e r s from the expected e g u i l i b r i u m w i l l vary. 68 depending upon the a c t i v a t i o n / i n h i b i t i o n s t a t e of the enzyme. Thus i n the r a t heart, PFK i s f a r t h e r from e q u i l i b r i u m under a e r o b i c c o n d i t i o n s (when g l y c o l y s i s i s i n h i b i t e d ) than under anaerobic c o n d i t i o n s (when g l y c o l y s i s i s a c t i v a t e d ) (Williamson, 1965). In the s k i p j a c k muscles, t h e r e i s no change i n the mass a c t i o n r a t i o o f the PGI r e a c t i o n as the a c t i v i t y s t a t e v a r i e s , nor i n the mass a c t i o n r a t i o of the hexokinase and PFK r e a c t i o n s between r e s t and b u r s t swimming. However, upon the t r a n s i t i o n from r e s t t o f e e d i n q , the hexokinase r e a c t i o n mass a c t i o n r a t i o becomes c l o s e r t o e q u i l i b r i u m , i n d i c a t i n g an a c t i v a t i o n of hexokinase, c o n s i s t e n t with an a c t i v a t i o n of a e r o b i c g l y c o l y s i s . The mass a c t i o n r a t i o f o r the PFK s t e p however, moves f u r t h e r from e g u i l i b r i u m i n d i c a t i n g e i t h e r a PFK i n h i b i t i o n or simply t h a t PFK has become the rate l i m i t i n g step i n g l y c o l y s i s (Figure 4-1). I t i s obvious from the work of Williamson (1965) and Neely St.ajL., (1975) t h a t : 1. Meaningful changes i n mass a c t i o n r a t i o s can only r e s u l t from m e t a b o l i t e l e v e l changes which occur d u r i n g the t r a n s i t i o n from one s t a t e to another. What my measurments probably r e p r e s e n t , are s t e a d y - s t a t e m e t a b o l i t e l e v e l s a f t e r metabolism has a d j u s t e d to the new metabolic regime. 2. The locus i n a pathway at which r e g u l a t i o n i s o c c u r i n g v a r i e s as the t r a n s i t i o n i s underway,.Thus metabolite l e v e l -based evidence can e a s i l y present a p i c t u r e which l a b e l s f o r i n s t a n c e one s i t e as being r e g u l a t o r y , when i n f a c t t h i s i s only c o r r e c t f o r t h a t i n s t a n t i n time. I t h i n k what i s important t h e r e f o r e i n my data i s the f a c t t h a t 69 (a) the PFK and hexokinase r e a c t i o n s are out o f e q u i l i b r i u m and (b) t h a t the mass a c t i o n r a t i o s of these r e a c t i o n s chanqe by up to an order of magnitude. La c t a t e / p y r u v a t e r a t i o s (Table 4-8) are u n f o r t u n a t e l y not c o n s i s t e n t between the two experiments. There i s however, a d e f i n i t e i n c r e a s e i n the white muscle l a c t a t e / p y r u v a t e r a t i o upon b u r s t swimming i n the 1976 and 1977 f i s h . Assuming t h a t LDH i s a r e a c t i o n which i s c l o s e to e g u i l i b r i u m ( M e r r i l l and Guynn, 1976; T i s c h l e r e t . a l . , 1977), an i n c r e a s e i n t h i s r a t i o s i g n i f i e s e i t h e r a pH drop or t h a t the NAD/NADH couple i s beccming more reduced. Eoth occurences have been repo r t e d i n working and/or anaerobic t i s s u e (Macdonald and J o b s i s , 1976; Steenbergen e t . a l . , 1977)., CROSS OVER PLOTS Crossover p l o t s provide a method f o r l o c a l i z a t i o n of i n t e r a c t i o n or r e g u l a t o r y s i t e s i n complex enzyme systems (Williamson, 1970). The r e s t i n g m e t a b o l i t e l e v e l s are s e t a t 100% and the f e e d i n g , or b u r s t swimming me t a b o l i t e l e v e l s are p l o t t e d as r e l a t i v e v a l u e s on the v e r t i c a l a x i s ( F i g u r e s 4-1 and 4-2). A c r o s s - o v e r p o i n t i n d i c a t e s t h a t the changes i n f l u x , and the changes i n c o n c e n t r a t i o n of the r e a c t a n t s are ex p e r i m e n t a l l y i n c o n s i s t e n t ( i . e . some non-substrate agent must be a f f e c t i n g the r a t e of t h a t r e a c t i o n ; thus the r e a c t i o n i s being r e g u l a t e d (Williamson, 1970)). C r o s s o v e r s i n g l y c o l y s i s are t y p i c a l l y seen a t the hexokinase s t e p , the PFK s t e p , the GAPDH s t e p and at the PK step (Williamson, 1970; Rovetto e t . a l , , 1975; Williamson, 1965). My p l o t s show three 70 c r o s s o v e r p o i n t s ( F i g u r e s 4-1 and 4-2), a t hexokinase, PFK and LDH i n both red and white muscles under at l e a s t some c o n d i t i o n s . The c r o s s o v e r a t the PFK step probably r e p r e s e n t s t r u e s i t e s of l i m i t i n g f u n c t i o n (Williamson, 1970). The c r o s s o v e r at hexokinase may be i n v a l i d as the glucose measurement i n c l u d e s e x t r a c e l l u l a r as w e l l as i n t r a c e l l u l a r glucose and the one a t LDH i s probably due to the pH drop and the i n c r e a s e d r e d u c t i o n of the NAD/NADH couple which cause the i n c r e a s e i n the l a c t a t e / p y r u v a t e r a t i o seen i n Table 4-8. Since H + and NADH are s u b s t r a t e s of the r e a c t i o n t h i s c r o s s o v e r does not imply t h a t t h i s i s a r e g u l a t o r y s t e p . A s i a i l a r c r o s s o v e r at LDH i s a l s o shown i n the data of Minikami and Yoshikawa (1966). Again one must remember t h a t (a) my measurements do not r e p r e s e n t the a c t u a l t r a n s i t i o n and (b) r e g u l a t o r y s i t e s do not c o n s i s t e n t l y show up on c r o s s - o v e r p l o t s (Williamson, 1965; Rovetto e t . a l . , 1975). The f l u c u a t i o n s i n g l u c o s e , G6P, F6P and FDP l e v e l s , however, c l e a r l y show hexokinase a c t i v a t i o n and PFK r a t e l i m i t a t i o n during f e e d i n g and perhaps hexokinase i n h i b i t i o n d u r i n g b u r s t swimming. TEMPERATURES OF THE RED AND WHITE MUSCLE Ambient water temperatures, and the temperatures of the red and white muscle of i n d i v i d u a l f i s h under varying c o n d i t i o n s , are given i n Table 4-9. When the f i s h are r e s t i n g , both muscles are about 1°C above ambient, the deep white muscle tends to be about 0.5-1.0°C lower than the deep re d . The muscle temperatures are c o n s i s t e n t l y h i g h e s t d u r i n g f e e d i n g when they are about 10°C above ambient i n the red and 8-10°C above 71 ambient i n the white. Muscle temperatures are a l s o high a f t e r 10 minutes cf "on l i n e " swimming, but the maxima tend t o be lower and the ranges higher i n both muscles under these c o n d i t i o n s . 72 DISCUSSION T h i s chapter has pr o v i d e d informaton on f u e l s which are being burnt under the d i f f e r e n t c o n d i t i o n s , when end-products are accumulating and t o what l e v e l s over what period of time, a l s o , i t i s now p o s s i b l e t o say which enzymes have the p o t e n t i a l o f c o n t r o l l i n g f l u x through the g l y c o l y t i c pathway and when and to what degree, both muscles* temperatures are i n c r e a s i n g . During b u r s t swimming, the f o l l o w i n g m e t a b o l i t e c o n c e n t r a t i o n changes seem p a r t i c u l a r l y important: 1. Creatine-phosphate l e v e l s i n white muscle decrease by a f a c t o r of 10, i n red muscle by about a h a l f . 2. P a r t i c u l a r l y i n white muscle, the adenylate pool s i z e decreases. 3. Glycogen drops s l i g h t l y i n red muscle and markedly i n white muscle. 4. L a c t a t e accumulates i n white muscle to l e v e l s 7-20 times higher than i n the guiescent s t a t e . In red muscle, l a c t a t e l e v e l s i n c r e a s e , but modestly ( 1 . 5 - f o l d ) . These data, i n the absence o f any other, u n e g u i v o c a l l y i d e n t i f y anaerobic g l y c o l y s i s as the predominant c o n t r i b u t i o n to white muscle metabolism d u r i n g t h i s kind of swimming, a t the same time, they i n d i c a t e t h a t red muscle metabolism i s probably l a r g e l y a e r o b i c . That red muscle metabolism i s a c t i v a t e d under these c o n d i t i o n s i s i n d i c a t e d not only by the above data, p a r t i c u l a r l y by the f a l l i n ATP and creatine-phosphate l e v e l s , but a l s o by the f a c t t h a t t h e r e i s a l a r g e r i s e i n temperature, the hexose phosphates (G6P i n 1S77, and F6P) i n c r e a s e i n 73 c o n c e n t r a t i o n and the pool s i z e s of c i t r a t e and malate i n c r e a s e with a concommitant drop i n a s p a r t a t e l e v e l s . The 10-fold i n c r e a s e i n glucose c o n c e n t r a t i o n s i n both t i s s u e s i s c u r i o u s and i s confused by the f a c t t h a t t h e measurement i n c l u d e s i n t r a - and e x t r a c e l l u l a r g l u c o s e . Glycogen may be p r e f e r e n t i a l l y m o b i l i z e d d u r i n g burst swimming with the concommittant i n h i b i t i o n of hexokinase, which i s u s u a l l y i n h i b i t e d by the hexose-phosphates (Katzen and Soderman, 1975). T h i s would cause glucose ( e x t r a - and i n t r a c e l l u l a r ) l e v e l s t o r i s e as the i n t r a c e l l u l a r c o n t r i b u t i o n (an unknown guantity) would r i s e . However, why are blood and t i s s u e glucose l e v e l s i n e q u i l i b r i u m when blood and t i s s u e l a c t a t e l e v e l s are not (Table 4-1). A l t e r n a t i v e l y , perhaps glucose i s m o b i l i s e d from the l i v e r , f o r the red muscle d u r i n g b u r s t swimming. But i f blood flow t o the white muscle i s reduced d u r i n g b u r s t swimming (which i s the case i n other f i s h and which i s a l s o suggested by the n o n - e q u i l i b r i u m of the blood and muscle l a c t a t e pools) then glucose m o b i l i z a t i o n should not show up i n white muscle. U n f o r t u n a t e l y , i t i s c l e a r t h a t the qlucose numbers are d i f f i c u l t to i n t e r p r e t and should not be r e l i e d upon. In summary then, both red and white muscle metabolism c o n t r i b u t e s to b u r s t swimming; red muscle work i s s u s t a i n e d by an anaerobic and a e r o b i c metabolism while white muscle work depends upon an anaerobic g l y c o l y s i s more i n t e n s e than any other thus f a r found i n nature. T h i s c a p a b i l i t y of the white muscle f o r anaerobic f u n c t i o n i s almost c e r t a i n l y the d r i v i n g f o r c e behind the high burst speeds of which the s k i p j a c k i s capable. 74 Does a s i m i l a r d i v i s i o n of metabolic f u n c t i o n between red and white muscle occur during the steady s t a t e swimming a s s o c i a t e d with prey capture at sea? During such f e e d i n g f r e n z i e s at sea, when the h i g h e s t s u s t a i n e d a c t i v i t y l e v e l s of s k i p j a c k tuna are thought t o be reached, the r e l e v a n t m e t a b o l i t e changes i n red muscle can be summarized as f o l l o w s : 1. Creatine-phosphate l e v e l s drop more d r a m a t i c a l l y , but ATP c o n c e n t r a t i o n s are s u s t a i n e d w e l l w i t h i n t h e normal range, 2. Red muscle glucose c o n c e n t r a t i o n s are low while hexose phosphate l e v e l s r i s e (F6P i n the 1976 experiment, F6P and G6P i n 1977) i n d i c a t i n g a hexokinase a c t i v a t i o n F i g u r e ( 5-1) ; a t the same time, l a c t a t e l e v e l s remain i n the normal range. 3. C i t r a t e and malate l e v e l s r i s e . These data are taken to mean (a) that red muscle i s c o n t r i b u t i n g t o f r e n z y swimming, and (b) t h a t the red muscle c o n t r i b u t i o n i s t o t a l l y a e r o b i c , powered at l e a s t i n part by glucose o x i d a t i o n . By comparison, i n white muscle: 1. Creatine-phosphate l e v e l s drop (as during "on l i n e " swimming), but ATP c o n c e n t r a t i o n s , as i n red muscle, are maintained i n the normal range. 2. Glucose l e v e l s remain low, but G6P and F6P c o n c e n t r a t i o n s are h i g h , c o n s i s t e n t with a flow o f glucose carbon i n t o the g l y c o l y t i c path; some of t h i s carbon appears i n l a c t a t e , which accumulates to l e v e l s 1/5-1/4 those observed i n "on l i n e " swimming. These data are taken to mean t h a t white muscle i s c o n t r i b u t i n g t o f r e n z y swimming and t h a t t h i s c o n t r i b u t i o n , as 75 i n the case of red muscle, i s l a r g e l y a e r o b i c , with only a minor anaerobic component. Plasma glucose seems p r e f e r r e d to glycogen as a carbon and energy source f o r the white muscle c o n t r i b u t i o n t o a c t i v a t e d steady s t a t e swimming as glycogen l e v e l s remain high. Again, the unusually high c r u i s i n g speeds which the s k i p j a c k demonstrates, are a consequence of the c o n t r i b u t i o n by white muscle. What of the heat generated. Where i s i t produced, and how i s i t produced? And what does the temperature of these muscles t e l l us about the muscles* metabolic s t r a t e g i e s ? T h i s whole quest i o n i s dependent u n f o r t u n a t e l y upon an unknown q u a n t i t y , that i s the amount of heat t r a n s f e r between red and white muscle. .There are three p o t e n t i a l sources of heat a v a i 1 ab,1 e. 1. Anaerobic q l y c o l y s i s , y e i l d i n g 32 Kcal/mole of glucose. 2. Glucose o x i d a t i o n , y e i l d i n g 423 Kcal/mole of glucose. 3. Fat o x i d a t i o n , y e i l d i n g 9.3 Kcal/gram of f a t . Durinq b u r s t swimming, the red muscle temperature i n c r e a s e s by 10 °C i n 10 minutes. B a s a l oxygen uptake measurements by N e i l l e t . a l . , (1976) of 2 mg oxygen/g/hr (assuming 1kg t i s s u e = 1L of water), would r a i s e red muscle temperature by 1.4°C/Kg/10 minutes i f carbohydrate only were o x i d i s e d . A 7 - f o l d i n c r e a s e i n heat production would t h e r e f o r e be necessary t o r a i s e the red muscle temperature 10°C i n 10 minutes. C o n s i d e r i n g t h a t the above f i g u r e f o r oxygen uptake i s f o r b a s a l metabolism, and that i t i s f o r the whole body, not j u s t the red muscle, and t h a t l i p i d o x i d a t i o n i s not c o n s i d e r e d , i t i s w e l l w i t h i n the c a p a b i l i t i e s of the red muscle to produce enough metabolic heat, i n 10 minutes, to r a i s e i t s temperature by 10°C. The 76 white muscle on the other hand, a l s o r a i s e s i t s temperature during b u r s t swimming by about 10°C i n some c a s e s , and i f t o t a l l y anaerobic, o x i d i z e s 50 mMoles/litre of glucose t o l a c t a t e i n 10 minutes. Again, on c o n d i t i o n of the assumptions used above, 50 mMoles of glucose being o x i d i s e d t o l a c t a t e i n 10 minutes w i l l only r a i s e the temperature of the white muscle by 2.3°C. The c o n c l u s i o n i s s e l f - e v i d e n t ; e i t h e r the white muscle i s r e c e i v i n g heat from the red muscle, or a e r o b i c metabolism i s c o n t r i b u t i n g to the temperature r i s e i n the white muscle as a r e s u l t o f burst swimming.^This l a t t e r e x p l a n a t i o n may e x p l a i n the i n c r e a s e i n c i t r a t e and malate l e v e l s i n b u r s t white muscle i n 1977. The s i t u a t i o n d u r i n g f e e d i n g i s a s i m i l a r one. The red muscle i s again the hot spot and g u i t e capable of producing i t s own heat. The white muscle under these c o n d i t i o n s heats up somewhat more than dur i n g b u r s t swimming; average excess temperatures f o r white muscle under these c o n d i t i o n s are 8-1G0C (Table 4-9), only 0.3<>C of which can be accounted f o r by l a c t a t e p r o duction (Table 4-4). I f we assume t h a t 60% of the glycogen d e p l e t e d , about 10 umol glucose/g, d u r i n g t h i s k i n d o f swimming i s f u l l y o x i d i s e d (Table 4-4), enough heat i s generated to r a i s e the white muscle temperature by 5-7<>C. Aerobic glycogen metabolism i n the white muscle d u r i n g f e e d i n g can o b v i o u s l y produce the m a j o r i t y of the temperature i n c r e a s e without even c o n s i d e r i n g glucose o x i d a t i o n or condu c t i o n from the red muscle. The q u e s t i o n of conduction (mentioned above) has not been r e s o l v e d . As 50% of the heat must be l o s t a cross the body from the red muscle ( N e i l l e t . a l . , 1976), conduction between r e d and white muscle seems l i k e l y , but there i s 77 evidence t h a t the m a j o r i t y of t h i s heat l o s s may occur over s p e c i f i c areas of the body (Dizon and B r i l l , i n p r e s s ) . 78 Table 4-1. 1976 m e t a b o l i t e c o n c e n t r a t i o n s . , M e t a b o l i t e c o n c e n t r a t i o n s (umol/g wet weight wet weight or /ml) i n red and white iruscle and blood during three d i f f e r e n t e x e r c i s e s t a t e s . Rest Feeding Burst Red White Red White Blood Red White Blood Glucose 1.83(0.39) 0.34(0.14) 0.18(0.09) 0.16(0.05) 1.25(0.57) 1.7(0.62) 2.35(0.51) 2.3 G6P 1.75(0.13) 1.57(0.26) 1.97(0.30) 2.7(1.02) 2.16(0.68) 3.87(1.06) F6P 0.25(0.14) 0.33(0.16) 0.58(0.12) 0.8(0.19) 0.46(0.07) 0.78(0.14) FDP 0.35(0.04) 0.35(0.1) 0.28(0.07) 0.35(0.14) 0.41(0.03) 0.69(0.11) DHAP 0.38(0.06) 0.32(0.05) 0.23(0.12) 0.12(0.05) 0.33(0.03) 0.3(0.08) G3P 0.22(0.04) 0.15(0.05) 0.11(0.03) 0.13(0.07) 0.18(0.05) 0.14(0.04) Pyruvate 0.14(0.03) 0.35(0.08) 0.18(0.10) 0.35(0.19) 0.19(0.04) 1.1(0.22) Lactate 12.3(2.1) 13.05(2.73) 10.47(1.82) 20.18(5.43) 4.9(1.82) 18.2(4.3) 84(10.4) 16.3 aGP 1.83(0.39). 2.0(0.54) 1.73(0.25) 1.65(0.24) 3.16(0.92) 0.37(0.34) Citrate 0.49(0.06) 0.25(0.09) 0.59(0.11) 0.25(0.03) 0.57(0.09) 0.20(0.04) aKG 0.14(0.02) 0.12(0.04) 0.14(0.06) 0.06(0.01) 0.12(0.04) 0.05(0.03) Malate 0.42(0.06) 0.19(0.22) 0.96(0.36) 0.25(0.045) 0.55(0.08) 0.31(0.06) Creatine 14.75(1.80) 17.35(2.80) 13.3(0.50) 23.5(1.76) vo Rest F e e d i n g B u r s t Red White Red White B l o o d Red White B l o o d C-P 3.4(0.56) 14.25(2.70) 0.73(0.12) 1.65(0.59) 1 .67(0.15) 1.35(0.48) ATP 4.2(0.39) 5.5(2.14) 3.55(0.44) 5.45(0.73) 3 .33(0.32) 2.9(1.51) ADP 1.17(0.29) 0.72(0.10) 0.49(0.07) 0.46(0.03) 1 .11(0.1) AMP 0.16(0.005) 0.09(0.03) 0.16(one v a l u e ) 0.11(0.32) n = 5 (± S.D.) 00 o 81 Table 4-2. Amino a c i d c o n c e n t r a t i o n s . C o n c e n t r a t i o n of amino a c i d s (umol/g wet weight or /ml) i n blood and red and white muscle during three d i f f e r e n t e x e r c i s e s t a t e s . Experiment done i n 1976. 82 White Muscle Rest Feeding On Line Aspartate 0.8* 0.4 0.55(0.5-0.6) Glutamate 0.7 0.65(0.4-0.9) 0.6(0.5-0.7) Glycine 1.8 0.6(0.4-0.8) 1.6(1.0-2.0) Alanine 5.3(3.1-7. 5) 1.3(1.2-1.4) 2.2(1.7-2.7) Histidine 34.5(20-49) 58(50-66) 17.5(13-22) Proline 0 0 0 Lysine 11.5 0 7.7 Red Muscle Rest Feeding On Line Aspartate 1.1 1.75(1.5-2.0) 0.4(0.3-0.5) Glutamate 2.8(2.4-3. 1) 3.9(3.7-4.2) 0.65(0.6-0.7) Glycine 3.1(2.0-4. 2) 4.4 (4.1-4.7) 2.8(1.9-3.6) Alanine 3.2(2.8-3. 6) 9.3(8.6-10.0) • 2.2(1.7-2.7) Histidine 0.9(0.3-1. 4) 5.1(4.2-6.0) 2.5(2.1-2.9) Proline 0 4.5(3.0-6.0) 0 Lysine Blood Rest Feeding On Line Aspartate - 0.13(0.1-0.17) 0.55(0.4-0.7) Glutamate - 0.3(0.2-0.4) 0.15(0.1-0.2) Glycine - 0.55(0.3-0.8) 1.1(1.0-1.2) Alanine - 1.4(0.7-2.0) 1.25(1.2-1.3) Histidine - 0.5(0.5-0.5) 0.35(0.3-0.4) Proline - 0 0 Lysine - 0.2(0.2-0.2) 0.3(0.2-0.4) * Values without accompanying ranges represent only one value. Other values are a mean of two. 83 Ta b l e 4-3. 1977 metabolite c o n c e n t r a t i o n s , m e t a b o l i t e c o n c e n t r a t i o n s (umol/g wet weight wet weight) i n r e d and white muscle duri n g three d i f f e r e n t e x e r c i s e s t a t e s . Rest Feeding Burst Red White Red White Red White Glucose 0.36(0.09)* 0.24(0.05) 0.52(0.20) 0.48(0.08) 2.03(0.43) 2.68(0.45) G6P 0.82(0.13) 0.71(0.26) 1.48(0.16) 3.34(0.54) 2.5(0.41) 5.0(2.21) F6P 0.14(0.05) 0.12(0.04) 0.27(0.09) 0.57(0.06) 0.38(0.09) 0.97(0.50) FDP 0.19(0.05) 0.18(0.08) 0.11(0.05) 0.28(0.06) 0.20*** 0.13(0.05) Pyruvate 0.22(0.20) 0.2(0.07) 0.14(0.007) 0.69(0.22) 0.23(0.05) 1.47(0.46) Lactate 3.1(1.17) 5.14(1.17) 5.82(2.26) 18.7(3.10) 7.2(1.86) 68.5(21.13) Citrate 0.96(0.46) 0.30(0.17) 0.62(0.25) 0.1** 1.51(1.2) 1.15(0.49) Malate 0.45(0.18) 0.12(0.05) 0.59(0.13) 0.18(0.06) 0.97(0.59) 0.47(0.31) Creatine-P 4.76(0.31) 30.7(13.2) 1.31(0.47) 6.3(1.34) 2.86(1.06) 3.45(2.06) *n = 5 (±S.D.) **A11 other values were 0.0 ***Average of two values 85 Ta b l e 4-4. Glycogen and l a c t a t e l e v e l s . Glycogen and l a c t a t e l e v e l s (umol/g wet weight wet weight) i n r e d and white muscle during three d i f f e r e n t e x e r c i s e s t a t e s . Means of glycogen v a l u e s are expressed as umcl/g (above) and as mq% (below). 86 Fish No. Red Muscle Lactate Glycogen White Muscle Lactate Glycogen Rest: 1 2 3 4 5 Mean ±S.D. Feeding: 1 2 3 4 5 Mean ±S.D. Burst: 1 2 3 4 5 Mean ±S.D. 4.7 2.1 2.0 3.9 2.8 3.1(1.17) 9.3 6.5 5.6 3.6 4.1 5.82(2.26) 9.6 8.1 6.0 7.5 4.8 7.2(1.86) 36.3 30.5 34.1 40.4 35.3(4.1) 635.0 42.4 40.1 46.0 25.9 44.2 39.7(8.0) 714.6 6.9 18.3 43.6 50.1 49 33.6(19.7) 604.8 4.0 3.8 5.9 5.6 6.4 41.8 44.5 41.5 58.0 60.5 5.14(1.17) 49.2(9.2) 885.6 19.2 15.4 23.7 17.4 17.9 18.7(3.10) 84.5 80.6 32.0 75.6 69.6 41.2 28.2 29.2 26.4 38.2 32.6(6.6) 586.8 21.6 19.1 59.05 9.6 22.6 68.5(21.13) 26.4(19.0) 475.2 87 Table 4-5. Glucose e q u i v a l e n t s . T o t a l glucose e g u i v a l e n t s (umol/g wet weight wet weight) from glycogen and l a c t a t e i n r e d and white muscle d u r i n g t h r e e e x e r c i s e s t a t e s . 88 Rest F e e d i n g B u r s t Red Muscle 36.3 (4.2) 41.9 (12.6) 37.6 (17.6) White Muscle 51.9 (9.5) 41.7 (7.0) 60.0 (11.7) *Values ± S.D. 89 Table 4-6. Energy charge and adenylate pool s i z e . Energy charge ({ATP «• 1/2ADP)/ATP +ADP • AMP) and adenylate pool s i z e {sum of the c o n c e n t r a t i o n s of ATP, ADP and AMP) i n the red and white muscle d u r i n g three e x e r c i s e s t a t e s using measured and c a l c u l a t e d AMP values. Experiment done i n 1976, 90 Red Muscle White Muscle Rest Feeding Burst Rest Feeding Burst Measured AMP level ymol/g 0.16 0.16 0.17 0.09 0.16 0.11 Calculated AMP level ymol/g 0.55 0.28 0.66 0.26 0.17 0.43 E. C. measured 0.86 0.9 0.84 0.93 0.94 0.88 E. C. calculated 0.81 0.88 0.76 0.9 0.93 0.8 Adenylate pool measured ymol/g 5.57 4.2 4.57 6.42 6.12 3.64 Adenylate pool calculated umol/g 5.92 4.32 5.09 6.48 6.08 3.96 91 Table 4-7. Metabolite r a t i o s f o r enzyme r e a c t i o n s . M e t a b o l i t e r a t i o s f o r the hexokinase, phoshoglucoseisomerase and phosphofructokinase r e a c t i o n s during three d i f f e r e n t e x e r c i s e s t a t e s . Expected values from Newsholme and S t a r t , 1973. 92 Hexokinase 76 Red 76 White Expected Equilibrium Constant 3.9-5.5 x 10" PGI 76 Red 77 Red 76 White 77 White 0.36-0.47 PFK 76 Red 76 White 0.9-1.2 x 10-Rest Feeding Burst 0.29 0.59 1.5 1.4 0.43 0.36 0.14 0.17 0.21 0.17 0.29 0.18 0.3 0.17 0.21 0.15 0.2 0.19 0.4 0.13 0.06 0.04 0.31 0.19 93 Table 4-8. L a c t a t e / p y r u v a t e r a t i o s . L a c t a t e / p y r u v a t e r a t i o d u r i n g three e x e r c i s e s t a t e s . 94 Rest Feeding Burst Lac/Pyr Red 76 87.9 58.3 95.8 Red 77 22.1(14.78) 36.85 36.5(11. 85) White 76 34.6 58 77.4 White 77 27.64(8.19) 28.76(7.00) 45.55(5. 76) n = 5 (± S.D.) 95 Table 4-9. Muscle temperatures. Temperatures of deep red and deep white muscle (QC) during t h r e e e x e r c i s e s t a t e s . Fish No. Temperature °C Red Muscle Ho0 White Muscle Rest: 1 25.0 23.0 25.0 2 26.8 23.0 25.0 3 25.0 23.0 24.0 4 . 25.0 23.0 24.0 5 25.0 23.0 24.0 Mean + S.D. 25.36(0.81)* 24 .04(0.55) Feeding: 1 34.3 24.4 31.9 2 33.7 24.4 31.4 3 33.6 24.4 32.5 4 34.7 24.4 31.5 5 35.2 24.4 32 Mean + S.D. 34.3(0.67) 31 .90(0.44) Burst: 1 34.6 23.0 31.0 2 34.6 23.0 31.0 3 32.0 23.0 27.6 4 33.8 23.0 31.0 5 32.0 23.0 27.0 Mean + S.D. 33.4(1.32) 29 .52(2.04) *±S.D. 97 F i g u r e 4-1. Cross over p l o t ; r e s t v e r s e s f e e d i n g . Cross over p l o t of the g l y c o l y t i c pathway i n red and white muscles. R e s t i n g values (100) v e r s e s feeding values. O, red muscle; x, white muscle. 98 Glucose G-6-P F-6-P FDP DHAP G'hde- Pyruvate Lactate 99 F i g u r e 4-2. Cross over p l o t ; r e s t verses b u r s t . Cross over p l o t of the g l y c o l y t i c pathway i n red and white muscle. R e s t i n g values (100) verses b u r s t values. O, red muscle; x, white muscle. Glucose G-6-P F-6-P FDP DHAP G'hde- Pyruvate Lactate 3-P 101 CHAPTER 5. BOLE OF DEHYDBOGBNASE COHPETITTON £R BEGOLATION: THE CASE OF LACTASE AND A-GIffCEBOPHOSPHATE DEHYDROGENASE 102 INTRODUCTION Evidence presented i n t h i s t h e s i s has s o - f a r shown the s k i p j a c k t o toe a very a c t i v e t e l e o s t (Chapter 1) with t y p i c a l t e l e o s t red muscle and a somewhat unusual white muscle. The white muscle d i s p l a y s a high g l y c o l y t i c p o t e n t i a l ( C h a p t e r s 3 and 4) which s u p p l i e s most of t h e energy f o r b u r s t swimming (Chapter 4 ) , and an a e r o b i c p o t e n t i a l based on carbohydrate f u e l (Chapter 3} , primed by a GP c y c l e (Appendix I) , which operates d u r i n g high speed , steady s t a t e swimming and p o s s i b l y during burst swimming (Chapter 4). According t o most c u r r e n t concepts, c o n t r o l of g l y c o l y s i s i n muscle and heart i s achieved through metabolite r e g u l a t i o n c f key r e g u l a t o r y enzymes such as glycogen phosphorylase (Hers, 1976), hexokinase (Katzen and Soderman, 1975), and phcsphofructokinase ( T s a i e t . a l . , 1975) . In lower v e r t e b r a t e s , muscle pyruvate kinase a l s o d i s p l a y s c h a r a c t e r i s t i c s c o n s i s t e n t with a r e g u l a t o r y r o l e i n g l y c o l y s i s (Johnston, 1975; Randall and Anderson, 1975). However, i n cases such as tuna white muscle, which d i s p l a y s an e x c e p t i o n a l c a p a c i t y f o r anaerobic as w e l l as a e r o b i c g l y c o l y s i s , c o n t r o l of the above enzyme steps cannot account f o r the r e l a t i v e l y e x c l u s i v e f u n c t i o n of e i t h e r LDH (in anaerobic g l y c o l y s i s ) or GPDH ( i n a e r o b i c g l y c o l y s i s ) . The metabolism of s k i p j a c k white muscle thus p r e s e n t s a problem with regards to r e g u l a t i o n . The s t r a t e g y i n most t i s s u e s i s to adopt carbohydrate metabolism as e i t h e r an a e r o b i c or an anaerobic mechanism of ATP production. Thus the renowned carbohydrate burners, the bl o w f l y and the bumblebee, are o b l i g a t e aerobes. Redox i s 103 balanced with a GP c y c l e and LDH has been e l i m i n a t e d from t h e i r metabolic machinery. Thus the problem of c o m p e t i t i o n between LDH and e i t h e r PDH or the hiqh l e v e l s of GPDH no l o n q e r e x i s t s (Sacktor, 1S76; Crabtree and Nensholme, 1972). At the other end of the s c a l e are the t i s s u e s with a hiqh g l y c o l y t i c p o t e n t i a l , which have hiqh a c t i v i t i e s of LDH and low a c t i v i t i e s of GPDH. Many v e r t e b r a t e white muscles f i t i n t o t h i s c a t e g o r y ; carbohydrate i s used almost e x c l u s i v e l y f o r anaerobic purposes and thus there i s never a need f o r r e g u l a t i o n of LDH and mainline g l y c o l y s i s i s r e g u l a t e d at the PFK, GAPDH, and PK s t e p s (Crabtree and Newsholme, 1972).Thus, r e q u l a t i o n of two p o t e n t i a l l y c o m p e t i t i v e dehydrogenases i s e s s e n t i a l l y bypassed i n most t i s s u e s at the qenome l e v e l . In the s k i p j a c k white muscle however, there i s an a c t i v e anaerobic metabolism terminated by hiqh a c t i v i t i e s of LDH, and hiqh a c t i v i t i e s of GPDH a s s o c i a t e d with a s i g n i f i c a n t a e r o b i c c a p a c i t y . These two enzymes (GPDH and LDH) e x i s t i n the same compartment of the c e l l (Crabtree and Newsholme, 1972) and c l e a r l y compete f o r the same cy t o p l a s m i c supply of NADH. The consequences of such c o m p e t i t i o n are p o t e n t i a l l y d e t r i m e n t a l . Under a e r o b i c c o n d i t i o n s , s i g n i f i c a n t LDH a c t i v i t y would reduce the flow of g l u c o s e - d e r i v e d carbon and hydrogen t o o x i d a t i v e metabolic pathways, while under anaerobic c o n d i t i o n s , s i q n i f i c a n t GPDH f u n c t i o n s i g h t d r a s t i c a l l y reduce the g l y c o l y t i c ATP y i e l d by c h a n n e l l i n q up to one-half of the glucose d e r i v e d carbon and hydrogen i n t o GP. Thus, when both enzymes occur i n the same c e l l , minimizing sumultaneous f u n c t i o n seems e s s e n t i a l . 104 Such c o n t r o l i s e v i d e n t l y achieved i n the s k i p j a c k white muscle, as during anaerobic g l y c o l y s i s , ooljr l a c t a t e accumulates. Although GP l e v e l s do i n c r e a s e under anaerobic c o n d i t i o n s , the o v e r a l l change i n c o n c e n t r a t i o n i s i n s i g n i f i c a n t compared to the t o t a l flow through g l y c o l y s i s . , During a e r o b i c carbohydrate metabolism over long p e r i o d s {as i n feeding) l a c t a t e only accumulates t o 1/10-1/5 of the anaerobic values (Chapter 4). S i m i l a r m e t a b o l i t e changes are a l s o seen during a e r o b i c and anaerobic carbohydrate metabolism i n the b r a i n (Lowry e t . a l . , 1964), h e a r t (Eovetto e t . a l . , 1975) and i n r a t s k e l e t a l muscle (Edington ej.a.1., 1973). To t r y to r e s o l v e how t h i s r e g u l a t i o n i s achieved, two types o f s t u d i e s were undertaken. 1. The GPDH and LDH from a t i s s u e with an a e r o b i c and an anaerobic carbohydrate based metabolism ( s k i p j a c k white muscle) were c h a r a c t e r i s e d i n order t o a s c e r t a i n what f a c t o r s a f f e c t e d t h e i r a c t i v i t i e s . 2. S t u d i e s i n v o l v i n g c o m p e t i t i o n f o r NADH, between v a r i o u s LDH's and GPDH's were undertaken i n order t o determine whether the a b i l i t y to compete f o r NADH was a f f e c t e d by (a) f a c t o r s which perturb the a c t i v i t i e s of these enzymes, and (b) the isozyme s e n s i t i v i t y to these f a c t o r s . Temperature, pH, GP l e v e l s and creatine-phosphate l e v e l s a f f e c t the a c t i v i t i e s of s k i p j a c k white muscle GPDH and LDH. In two-enzyme experiments, the outcome of competition f o r l i m i t i n g NADH depends upon both the kind of isozyme u t i l i z e d and the c o n c e n t r a t i o n of the two r e g u l a t o r y m e t a b o l i t e s , c r e a t i n e -phosphate and GP. 105 FAET 1. FACTORS AFFECTING TEE ACTIVITY OF PURIFIED LDH AND GPDH FROM SKIPJACK HJITE MUSCLE. RESULTS Gel E l e c t r o p h o r e s i s S t a r c h g e l e l e c t r o p h o r e s i s shows one band of LDH i n s k i p j a c k white muscle which migrates towards the cathode at pH 5.9, S k i p j a c k red muscle has four bands o f LDH; two migrate towards the cathode and two towards the anode a t pH 5.9. The white muscle band does not c o - e l e c t r o p h o r e s e with any of the red muscle bands. The s k i p j a c k isozyme patt e r n i s d e f i n i t e l y non-mammalian although the k i n e t i c comparison between the two t i s s u e s i s much l i k e an H-type (red muscle) and an M-type (white muscle) mammalian LDH comparison (Guppy, unpub.). LDH i s a four subunit enzyme. In a l l mammalian t i s s u e s except sperm c e l l s , t h e r e are two d i f f e r e n t s u b u n i t types which combine randomly to produce a maximum of f i v e p o s s i b l e LDH isozymes (Markert, 1963). There i s a unigue homotetramer i n mammalian sperm (Hawtrey e t . a l . , 1975). In f i s h e s , there i s a t h i r d subunit type, and combination i s not n e c e s s a r i l y random so the p a t t e r n i s more complicated (Horowitz and Whitt, 1972). There have been numerous LDH isozyme s t u d i e s done on f i s h "muscle"; the number of LDH types i n these muscles v a r i e s between 8 and 1 (Marguez, 1978; Wright e t . a l . , 1975; Toledo and R i b e i r o , 1978; 106 Horowitz and Whitt, 1972; M i l l e r and Whitt, 1975; Gesser and S u n d e l l , 1971). A more r e f i n e d study of muscle LDH isozymes has been done on the g o l d f i s h by Wilson e t . a l . (1975) ; the red muscle has 4 bands and the white, 1-2 bands. GPDH i s a dimer. Trout muscles can have 1,2 or 3 bands (Otter and H i d g i n s , 1972) and var i o u s organs i n b i r d s and mammals can have 0-4 bands (White and Kaplan, 1969; Tsao, 1960). S k i p j a c k white muscle has f o u r bands of GPDH; the p u r i f i e d enzyme corresponds t o the one major band t y p i c a l l y found (Figure 5-1)., PH P r o f i l e s The pH optima f o r the forward r e a c t i o n of LDH i s 6.2-6.5, f o r the forward r e a c t i o n of GPDH'is 7.0-7.4 (Figure 5-2).,There i s a sharp d r o p - o f f i n a c t i v i t y as the pH departs from the optimum i n both cases. At the pH optima o f GPDH (pH 7.4), the a c t i v i t y of LDH i s at about 1/3 Vmax, and vice--versa. The pfl optima of the r e v e r s e r e a c t i o n i s around 8.5-9.0 i n both cases. These are t y p i c a l pH responses of a dehydrogenase enzyme (Winer and Schwert, 1958). S u b s t r a t e A f f i n i t i e s Both enzymes obey Michaelis-Menten k i n e t i c s , the s u b s t r a t e s a t u r a t i o n curves being r e c t a n g u l a r hyperbolas and double-r e c i p r o c a l p l o t s being l i n e a r . , M i c h a e l i s c o n s t a n t s (Km) are given i n Table 5-1. The apparent a f f i n i t i e s of t h e enzymes f o r NADH are very s i m i l a r while the a f f i n i t y of LDH f o r pyruvate i s 107 5 - f o l d lower than t h a t of GPDH f o r DHAP. Due t o pH e f f e c t s , r e l a t i v e a f f i n i t i e s f o r NAD* and NADH, and i n h i b i t i o n of the back r e a c t i o n by NADH, a c t i v i t y i n the forward d i r e c t i o n i s s t r o n g l y favoured f o r both r e a c t i o n s . , A. E f f e c t s of c o - s u b s t r a t e Tuna white muscle LDH i s unusually r e f r a c t o r y t o high pyruvate l e v e l s , i t s a c t i v i t y at 20 mM pyruvate being 92% of t h a t a t optimal pyruvate c o n c e n t r a t i o n s . The a f f i n i t y of LDH f o r pyruvate i s s l i g h t l y dependent upon NADH l e v e l s and the Km drops from 0.33 to 0.25 mM as NADH l e v e l s drop from 0.1 to 0.02 mM (Figure 5-3a). NADH a f f i n i t i e s double as pyruvate drops 10-f o l d , from 1.0 to 0.1 mM (Fi g u r e 5-3b). The a f f i n i t y of GPDH f o r DHAP and NADH i s not a f f e c t e d by the c o - s u b s t r a t e , B. Temperature e f f e c t s The a f f i n i t y of LDH f o r pyruvate i s s t r o n g l y a f f e c t e d by temperature and r i s e s as temperature r i s e s , an e f f e c t p r e v i o u s l y observed f o r many LDH * s (Hazel and P r o s s e r , 1974). In c o n t r a s t , the a f f i n i t y f o r NADH i s v i r t u a l l y u n a f f e c t e d by temperature (Figure 5-4a and 6-4b), a r e s u l t t h a t i s r a t h e r unusual f o r e c t o t h e r m i c LDH's (Hazel and Pr o s s e r , 1975). High temperature decreases the a f f i n i t y of GPDH f o r DHAP and NADH, more so f o r the l a t t e r ; the Km values f o r both r i s e with temperature (F i g u r e s 5-4 c and d) . C. pH e f f e c t s Whereas the Km (NADH) i s har d l y i n f l u e n c e d by pH, the a f f i n i t y of LDH f o r pyruvate i s s t r o n g l y pH dependent (Figure 5-5 a and b). The Km(pyruvate) r i s e s from 0.33 mM a t pH 6.5 to 1.3 mM at pH 7.3. I n the g u i e s c e n t s t a t e , pyruvate occurs i n 108 s k i p j a c k white muscle at about 0.3 uraol/g wet weight (Chapter 4) and s i n c e the Km i s important i n s e t t i n g the r e a c t i o n v e l o c i t y 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 , the observed e f f e c t of pH could serve t o p o t e n t l y curb the LDH r e a c t i o n under c o n d i t i o n s of high pH. ATP I n h i b i t i o n Both enzymes are a f f e c t e d by ATP; i n h i b i t i o n i s 60% at 5 mM ATP (Figure 5-6). The i n h i b i t i o n i s c o m p e t i t i v e with NADH , as i s the us u a l case f o r dehydrogenases (Holbrock e t . ^ l , , 1975), but i s u n a f f e c t e d by temperature. GP And Creatine-phosphate E f f e c t s GP i s an e f f e c t i v e i n h i b i t o r of GPDH; the Ki i s about 0.5 mM and t h i s value i s independent of temperature. Thus, the enzyme i s 75% i n h i b i t e d a t 3 mM GP(Figure 5-7). GP has no e f f e c t on s k i p j a c k white muscle LDH. LDH i s i n h i b i t e d by creatine-phosphate (Figure 5-8); the i n h i b i t i o n i s mixed c o m p e t i t i v e , although the presence o f creatine-phosphate does lower the Km (pyruvate) somewhat a t a l l pH values t e s t e d ( F i g u r e 5-9). The Km{NADH) i s not a f f e c t e d by creatine-phosphate. The i n h i b i t i o n by creatine-phosphate i s s t r o n g l y a f f e c t e d by pH and temperature: i n h i b i t i o n drops as pH and temperature r i s e (Figure 5-8). A s i m i l a r c r e a t i n e - p h o s p h a t e i n h i b i t i o n of glyceraldehyde-3-phosphate dehydrogenase and of PK has been noted (Oguchi e t . a l , , 1973; Storey and Hochachka, 1974a). The e f f e c t of creatine-phosphate however, i s not a 109 general one f o r NADr-linked dehydrogenases s i n c e the compound does not a f f e c t GPDH a c t i v i t y a t e i t h e r low or high l e v e l s of s u b s t r a t e or coenzyme. DISCUSSION From our data, a complex modus operandi can be c o n s t r u c t e d of how, by an i n t e r a c t i o n of pH, cre a t i n e - p h o s p h a t e , and perhaps temperature, the o p e r a t i o n of the LDH r e a c t i o n can be c o n f i n e d to c e r t a i n s i t u a t i o n s , while under other circumstances, g l y c o l y s i s can f u n c t i o n a e r o b i c a l l y with GPDH s u p p l y i n g the r e q u i s i t e NAD, When the muscle i s not sh o r t of oxygen, the pH i s r e l a t i v e l y high (see Rahn, 1976 and A i c k i n and Thomas, 1977 f o r a d i s c u s s i o n o f i n t r a c e l l u l a r pH), and thus GPDH i s favoured because of the d i f f e r e n t pH optima of the two enzymes (Figure 5-2). Under these circumstances, pyruvate r e d u c t i o n i s at a minimum because of the low a f f i n i t y of LDH f o r pyruvate a t n e u t r a l pH's (Fi g u r e 5-5a). A l s o , c r e a t i n e - p h o s p h a t e , which i s at 15-30 mM i n r e s t i n g white muscle (Chapter 4 ) , i n h i b i t s the LDH r e a c t i o n (Figure 5-8). As soon as white muscle a c t i v a t i o n causes oxygen supply t o be r a t e l i m i t i n g , GP l e v e l s begin t o r i s e (Chapter 4 ) , and GPDH i s i n h i b i t e d (Figure 5-7). Meanwhile, creatine-phosphate l e v e l s drop d r a s t i c a l l y t o around 3 mfl (Chapter 4 ), and thus LDH i s somewhat d e - i n h i b i t e d . Further d e - i n h i b i t i o n i s brought about as the pH, and thus the Km (pyruvate), decrease (Figure 5-5a). The decrease i n pH could be due to the breakdown of creatine-phosphate (Macdonald and J o b s i s , 1976) or perhaps to an i n c r e a s e i n temperature i n the working white muscle (Rahn e t . a l . , 1975). An i n c r e a s e i n 110 temperature would a l s o decrease the a f f i n i t y of GPDH f o r NADH and DHAP. The a f f i n i t y of LDH f o r NADH i s not a f f e c t e d by temperature and although the Km(pyruvate) r i s e s with temperature, so under these c o n d i t i o n s does the pyruvate c o n c e n t r a t i o n . Hence, i t i s probable t h a t a temperature i n c r e a s e would not o f f s e t t he d e - i n h i b i t i o n of LDH.,When oxygen again becomes a v a i l a b l e as a c t i v i t y slows, GP i s again o x i d i z e d i n the mitochondria, GP c o n c e n t r a t i o n s r e t u r n t o normal, creatine-phosphate l e v e l s r i s e , pH r i s e s , and LDH a c t i v i t y f a l l s while GPDH a c t i v i t y r i s e s . Both enzymes are a f f e c t e d by ATE l e v e l s (Figure 5-6), but ATP l e v e l s do not drop below 2 mM even i n extreme b u r s t s of swimming (Chapter 4) and t h e r e f o r e ATE l e v e l s alone probably are not i n v o l v e d i n determining which of the two systems i s working at any given time. The energy charge however, which decreases with e x e r c i s e i n white muscle (Table 4-6) co u l d p o s s i b l y have a d i f f e r e n t i a l e f f e c t on the two dehydrogenases. The e f f e c t s of pH, ATP and c r e a t i n e -phosphate on a c t u a l r e l a t i v e r a t e s of the two enzymes are shown i n Figure 5-10. T h i s f i g u r e a l s c shows that the seemingly unsurmountable 10-20-fold d i f f e r e n c e i n the r e l a t i v e a c t i v i t i e s of the two enzymes can be reduced t o a 2- t o 3- f o l d d i f f e r e n c e by pH, ATP and creatine-phosphate. Thus, l o o k i n g at Fig u r e 5-10, as oxygen l e v e l s drop, pH drops and creatine-phosphate l e v e l s drop, the s i t u a t i o n on the l e f t s i d e of the graph i s obtained, with LDH a c t i v i t y d r a s t i c a l l y i n excess o f GPDH a c t i v i t y . On r e t u r n to steady-s t a t e swimming, oxygen r i s e s , pH r i s e s and creatine-phosphate r i s e s ; the s i t u a t i o n on the r i g h t s i d e of the graph i s now 111 o b t a i n e d , where the LDH excess i s g r e a t l y reduced. In t h i s way, a t i s s u e o b v i o u s l y geared to an impressive anaerobic metabolism can allow f o r a low, but s i g n i f i c a n t a e robic c o n t r i b u t i o n using the same f u e l source as i t does during anaerobic b u r s t s of swimming. The c o n t r i b u t i o n to t h i s r e g u l a t o r y scheme by temperature i s unknown. Obv i o u s l y temperature can have an e f f e c t , through pH changes (Rahn e t . a l . , 1975), and through s u b s t r a t e a f f i n i t i e s ; but s i g n i f i c a n t temperature changes only would occur between r e s t i n g and e i t h e r feeding and b u r s t swimming, not between fee d i n g and burst swimming (Chapter 4). T h i s i s a convenient p o i n t to s t r e s s t h a t I am using the words a e r o b i c and anaerobic i n r e l a t i v e terms. The white muscle i s almost c e r t a i n l y never f u l l y a e r o b i c or anaerobic and thus both r e a c t i o n s w i l l always be o c c u r i n g s i m u l t a n e o u s l y with a predominance of one over the other. These data appeared at f i r s t t o provide the s o l u t i o n t o the problem of p o t e n t i a l l y competing dehydrogenases.,However, the enzymes GPDH and LDH c a t a l y s e what are termed near-e g u i l i b r i u m r e a c t i o n s (Williamson e t . a l . , 1967; Hohorst et.§1.. 1959). Jn v i v o measurements of the s u b s t r a t e s and products of "equilibrium*' enzymes always show t h a t the mass a c t i o n r a t i o i s approximately equal to the thermodynamic e q u i l i b r i u m constant (Chapter 4). I t i s u s u a l l y assumed t h a t s i n c e e g u i l i b r i u m enzymes always tend toward e g u i l i b r i u m , and u s u a l l y d i s p l a y high a c t i v i t i e s , t h a t t h e i r f u n c t i o n i s t o simply t r a n s m i t along a pathway, f l u x changes being qenerated elsewhere, as these enzymes are by d e f i n i t i o n always near or at e q u i l i b r i u m , they are c o n s i d e r e d u n s u i t a b l e f o r r e g u l a t o r y l o c i and thus i t 112 has become dogma t h a t the " e g u i l i b r i u m " enzymes play secondary r o l e s , i f any, i n metabolic r e g u l a t i o n ( B o l l e s t o n , 1972). However, i f t h e r e i s e i t h e r c o m p e t i t i o n f o r the s u b s t r a t e of an e g u i l i b r i u m enzyme, or i f the s u b s t r a t e has an a l t e r n a t e way of being metabolised, a l t e r a t i o n s i n the the r a t e at which the r e a c t i o n comes to e g u i l i b u i u m c o u l d e f f e c t i v e l y r e - r o u t e carbon flow. So i f the v a r i o u s e f f e c t o r s d i s c u s s e d above a l t e r e d the r a t e at which GPDH and LDH come to e q u i l i b r i u m , the path of carbon flow should depend upon the c o n c e n t r a t i o n (in the case of GP and creatine-phosphate) of these e f f e c t o r s , and the s e n s i t i v i t y of the enzymes to these e f f e c t o r s . T h i s hypothesis was t e s t e d by f i r s t l y f i n d i n g isozymes of the same enzyme which d i f f e r i n s e n s i t i v i t y t o s p e c i f i c metabolite modulators and secondly, by doing c o m p e t i t i o n (competition f o r l i m i t i n g NADH) experiments between LDH and GPDH using d i f f e r e n t isozymes and d i f f e r e n t modulator c o n c e n t r a t i o n s . 113 PART 2. COMPETITION FOR NADH BETWEEN GPDH AND LDH: THE- EFFECTS OF IZOZYME FORM AND MODULATOR CONCENTRATION RESULTS LDH-creatine-phosphate I n t e r a c t i o n s The LDH enzymes used were prepared from t i s s u e s i n t e n t i o n a l l y chosen to r e p r e s e n t a wide spectrum, from h i g h l y anaerobic muscles t o the r e l a t i v e l y a e r o b i c metabolic o r g a n i z a t i o n of h e a r t and b r a i n . The kind of LDH present i n such t i s s u e s u s u a l l y c o r r e l a t e s with i t s oxygen dependence. In mammals LDH occurs as a tetramer formed from random combinations of H and M s u b u n i t s with the two homotetramers (H& and M) showing d i s t i n c t k i n e t i c c h a r a c t e r i s t i c s (Holbrook e t . a l , , 1975). U s u a l l y , M4 type LDH predominates i n h i g h l y g l y c o l y t i c t i s s u e s , while H type s u b u n i t s are more abundant i n a e r o b i c t i s s u e s . However, k i n e t i c f e a t u r e s a l s o can vary without c o r r e s p o n d i n g a l t e r a t i o n s i n 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 (Hcchachka and Storey, 1975). Moreover, i n f i s h e s , at l e a s t e i g h t subunit types are now known (Markert e t . a l . , 1975); k i n e t i c s p e c i a l i z a t i o n s , although probable, have not yet been f u l l y c l a r i f i e d . Despite these c o m p l e x i t i e s , creatine-phosphate was found to i n h i b i t a l l the LDH * s examined at l e a s t to some e x t e n t . Dixon p l o t s of 1 / v e l o c i t y versus creatine-phosphate 114 c o n c e n t r a t i o n f o r LDH o f tuna white muscle are c o n s i s t e n t with creatine-phosphate i n h i b i t i o n being mixed-competitive with r e s p e c t t o e i t h e r pyruvate or NADH (Figure 5-11 a and b ) . The c r e a t i n e phosphate s e n s i t i v i t y o f the 12 p r e p a r a t i o n s we s t u d i e d appears t o roughly c o r r e l a t e with the o x i d a t i v e c a p a c i t y of each t i s s u e . The sources of LDH l i s t e d i n Table 5-2, f o r example, are arranged approximately i n order of i n c r e a s i n g o x i d a t i v e c a p a c i t y (Randall and Hochachka, 1978; t h i s t h e s i s ) . T h i s c o r r e l a t i o n i s probably secondary and d e r i v e s from the f a c t that the LDH isozyme f u n c t i o n (Table 5-2) and content vary i n thes e t i s s u e s (French and Hochachka, 1978). That i s , creatine-phosphate s e n s i t i v i t y appears t o depend upon the r e l a t i v e abundance of LDH s u b u n i t s d i s p l a y i n g M type versus H type p r o p e r t i e s . Not s u r p r i s i n g l y , pure M4 LDH i s one of the l e a s t creatine-phosphate s e n s i t i v e p r e p a r a t i o n s s t u d i e s while pure H4 LDH i s one of the most s e n s i t i v e (Table 5-2). H4 And M4 LDH Vgrsus GPDH The above experiments e s t a b l i s h t h a t l a r g e d i f f e r e n c e s occur i n LDH s e n s i t i v i t y t o creatine-phosphate. T h e r e f o r e , i n the presence of creatine-phosphate, d i f f e r e n t LDH•s should show d i f f e r i n g c a p a c i t i e s to compete with GPDH f o r a common source of NADH. A c c o r d i n g l y , a p p r o p r i a t e competition experiments were s e t up to d i r e c t l y t e s t t h i s h y p o t h e s i s . ; Table 5-4a summarizes r e s u l t s of 2-enzyme co m p e t i t i o n experiments between r a b b i t muscle GPDH and e i t h e r of two (H4 and M4) kinds of LDH enzymes i n the presence and absence of creatine-phosphate, Egual i n i t i a l a c t i v i t i e s o f both 115 dehydrogenases lead to approximately equal c o n t r i b u t i o n s t o t o t a l NADH o x i d a t i o n i n both cases. In the case of the M4 LDH, a creatine-phosphate r e s i s t a n t enzyme, creatine-phosphate had no measurable e f f e c t on the f r a c t i o n of NADH o x i d i z e d by LDH. In c o n t r a s t , f u l l y t h r e e times more NADH was o x i d i z e d by GPDH compared t o H4 LDH when 20 mM creatine-phosphate was i n c l u d e d i n the medium. Thus under c o n d i t i o n s of l i m i t i n q NADH, creatine-phosphate i s an important modulator o f LDH c o n t r i b u t i o n t o redox r e g u l a t i o n . GPDH Product, I n h i b i t i o n My o r i g i n a l i n t e r e s t i n t h i s problem arose from the f i n d i n g t h a t tuna white muscle c o n t a i n s GPDH a t a c t i v i t y l e v e l s high enough to d r a i n up to 1/2 the g l u c o s e - d e r i v e d carbon i n t o GP and thus to s i g n i f i c a n t l y reduce the a l r e a d y low energy y i e l d of g l y c o l y s i s (Chapter 3; Hochachka and Guppy, 1977). During g l y c o l y t i c a c t i v a t i o n i n t h i s muscle, when LDH a c t i v i t y i s favoured, GPDH a c t i v i t y needs t o be and ap p a r e n t l y i s , dampened by GP product i n h i b i t i o n . Thus, i n tuna white muscle, GPDH i s unusually s e n s i t i v e t o product i n h i b i t i o n by GP, the K i determined from Dixon p l o t s being about 0.25 oM (Hochachka and Guppy, 1977). Table 5-3 i n d i c a t e s t h a t a s i m i l a r mechanism may operate i n s k e l e t a l muscles of ether v e r t e b r a t e s as well s i n c e a l l o f the GPDH's examined show r e l a t i v e l y low K i values f o r GP. In tuna muscle (Chapter 4) and mammalian muscle (Sovetto e t . a l . , 1975; Edington e t . a l . , 1973) GP accumulates to values above the Ki range of GPDH. ,Thus, t h e r e i s good c o r r e l a t i o n between enzyme data and t i s s u e metabolite measurements. 116 i m p l y i n g t h a t GP s e n s i t i v i t y may ne o f p h y s i o l o g i c a l s i g n i f i c a n c e . Although s u f f i c i e n t i n f o r m a t i o n i s not a v a i l a b l e on va r i o u s v e r t e b r a t e t i s s u e s to attempt to c l o s e l y c o r r e l a t e GPDH s e n s i t i v i t y t o GPDH isozyme type, two forms of the enzyme are known whose K i valu e s are f a r out o f l i n e with the t y p i c a l v e r t e b r a t e s i t u a t i o n . These are the GPDH's from bee f l i g h t muscle and s g u i d mantle muscle (Table 5-3). These two enzymes show the lowest s e n s i t i v i t y to product i n h i b i t i o n by GP of any GPDH's thus f a r known. I n t e r e s t i n g l y , they f u n c t i o n i n t i s s u e s t h at are extremely oxygen dependent and may never go anaerobic under normal p h y s i o l o g i c a l c o n d i t i o n s ; i f made anoxic e x p e r i m e n t a l l y , t h e s e muscles s u s t a i n an accumulation of GP t o much higher c o n c e n t r a t i o n s (up to 20 mH) than ever seen i n v e r t e b r a t e t i s s u e s (Hochachka e t . a l . , 1975; Sacktor, 1976). Taken t o g e t h e r , these data suggest t h a t the GP r e s i s t a n t GPDH's should be more c o m p e t i t i v e with LDH under c o n d i t i o n s of high GP l e v e l s than would be the t y p i c a l v e r t e b r a t e , GP s e n s i t i v e , GPDH. I f t h i s i s the case a p h y s i o l o g i c a l relevance of the low K i f o r GP seen i n v e r t e b r a t e t i s s u e s i s i n d i c a t e d . GP S e n s i t i v e And I n s e n s i t i v e GPDH's Versas H TypeLDH-The above hypothesis was d i r e c t l y t e s t e d by 2-enzyme competition experiments using one LDH form |H4) and two types of GPDH's (Table 5-4b), Babbit muscle GPDH d i s p l a y s a high s e n s i t i v i t y to GP i n h i b i t i o n w h i le the honey bee enzyme i s s t r o n g l y r e s i s t a n t to the r e a c t i o n product (Table 5-3). Hhen GP was absent from the i n c u b a t i o n medium, the two forms of GPDH 117 competed with s i m i l a r e f f e c t i v e n e s s f o r NADH. T h e i r c o n t r i b u t i o n s to t o t a l NADH o x i d a t i o n were not e x a c t l y equal {about 42% versus 50% o f t o t a l NADH o x i d a t i o n by the r a b b i t and bee GPDH*s, r e s p e c t i v e l y ) , p o s s i b l y because of d i f f e r e n c e s i n t h e i r r e s p e c t i v e Km va l u e s f o r s u b s t r a t e s and NADH {Storey and Hochachka, 1975). In sharp c o n t r a s t l a r g e d i f f e r e n c e s appeared i n the behaviour of the two enzymes i n the presence o f 2 mM GP. Under these c o n d i t i o n s , the f r a c t i o n a l LDH-dependent NADH o x i d a t i o n exceeded the o x i d a t i o n due to r a b b i t muscle GPDH by ne a r l y 10-fold while i t exceeded the o x i d a t i o n due to bee JJSsele GPDH by l e s s than 2 - f o l d (Table 5-4b). I t i s worth r e -emphasizing t h a t d u r i n g a naerobic work i n mammalian muscle, GP l e v e l s r i s e t o about 3 umol/g wet weight (Hovetto et.a.1., 1975; Edington e t . a l . , 1973), i . e . somewhat higher than the co n c e n t r a t i o n s used i n the above com p e t i t i o n experiments. Thus, with both LDH and GPDH competing f o r the same l i m i t i n g pool of NADH, a high GPDH s e n s i t i v i t y t o r e a c t i o n product s t r o n g l y d i m i n i s h e s the amount of carbon and hydrogen t h a t can be wa t e f u l l y c h a n n e l l e d from " m a i n l i n e " g l y c o l y s i s i n t o GP under c o n d i t i o n s of l i m i t i n g oxygen. D i s c u s s i o n So with f a c t o r s a f f e c t i n g the r a t e a t which GPDH and LDH come to e g u i l i b r i u m , and with a common l i m i t i n g s u b s t r a t e , one can see a r o l e f o r " e q u i l i b r i u m " enzymes i n the r e g u l a t i o n of the a e r o b i c t o anaerobic t r a n s i t i o n i n g l y c o l y s i s . Under a e r o b i c c o n d i t i o n s creatine-phosphate l e v e l s are high and GP l e v e l s are low; LDH cannot compete f o r NADH under these 118 c o n d i t i o n s , nor f o r pyruvate. The ma j o r i t y of carbohydrate carbon thus i s o x i d i z e d i n the mitochondrion and GPDH produces NAD* f o r continued g l y c o l y s i s using c a t a l y t i c amounts of DHAP. When oxygen becomes l i m i t i n g , t h e creatine-phosphate pool i s depleted and GP l e v e l s r i s e . The a c t i v i t y of GPDH conseguently drops and co m p e t i t i o n f o r NADH now favours the d e i n h i b i t e d LDH. Pyruvate dehydrogenase (which I could not succeed i n measuring i n the s k i p j a c k ) can no longer compete with the much higher a c t i v i t i e s of LDH and thus pyruvate i s c h a n e l l e d i n t o l a c t a t e with no wastage of carbon a t the t r i o s e phosphate l e v e l . Any othe r f a c t o r s which vary with the a n a e r o b i c - a e r o b i c t r a n s i t i o n , which i n f l u e n c e the r a t e of e i t h e r r e a c t i o n , such as pH, would a l s o be part of t h i s r e g u l a t o r y scheme. Competition i s the key element here. Even i f LDH and GPDH always tend towards thermodynamic e g u i l i b r i u m , i n slowing down t h i s tendency, the coenzyme of the r e a c t i o n becomes u n a v a i l a b l e as i t i s used by the competing dehydrogenase. The mass a c t i o n r a t i o does net de v i a t e from the e g u i l i b u i u m constant when the r e d u c t i o n r e a c t i o n i s slowed, s i n c e excess s u b s t r a t e can be c h a n e l l e d o f f i n t o glyceraldehyde-3-phosphate or a c e t y l CoA, r e s p e c t i v e l y . C e n t r a l t o t h i s i n t e r p r e t a t i o n i s the assumption that NADH at l e a s t under some c o n d i t i o n s occurs a t l i m i t i n g c o n c e n t r a t i o n s , which i s a p p a r e n t l y assumed by Jomain-Baum et . a l . , (1978) who mention the concept of dehydrogenase competition i n mitochondria. U n t i l r e c e n t l y , r e l i a b l e estimates of c y t o s o l i c NADH were not a v a i l a b l e . However, most previous measurements of NADH range between 0.03 and 0.15 umol/g wet weight; these values are f o r the whole c e l l and the NADH 119 c o n c e n t r a t i o n i n the c y t o s o l can only be l e s s (Williamson e t . a l . , 1971; Burch e t . a l . , 1963; K a l k h o f f e t . a l . , 1966). ¥ NAD+/NADH r a t i o s i n the c y t o s o l of l i v e r , b r a i n , and f i b r o b l a s t s vary from 7-2000 ( M e r r i l and Guynn, 1976; Swartz and Johnson, 1976; Stubbs e t . a l , , 1972) . Assuming about a 1 mM pool s i z e , NADH l e v e l s would be estmated at about 0.01 to 0,0005 mM. Such e a r l i e r e s t i m a t e s of c y t o s o l i c NADH c o n c e n t r a t i o n ranges have been c l o s e l y checked using the technigue o f t u r b u l e n t flow t o r a p i d l y l y s e i s o l a t e d hepatocytes ( T i s h l e r e t . a l . , 1977) . From these s t u d i e s , the c o n c e n t r a t i o n of f r e e NADH i n the c y t o s o l appears t o be i n the 0.06-1.5 uM range under d i f f e r i n g metabolic c o n d i t i o n s ( st a r v e d versus f ed n u t r i t i o n a l s t a t e s , with and without exogenous ammonia). Perhaps because the NADH b i n d i n g s i t e of dehydrogenases i s c o n s e r v a t i v e (Holbrook e t . a l . , 1975),the a f f i n i t y c o nstants f o r NADH a l s o are f a i r l y c o n s t a n t , u s u a l l y i n the 0.0 1 t o 0.02 mM range (Hochachka and Guppy, 1977; Holbrook e t . a l . , 1975; Storey and Hochachka, 1975; F i e l d s e t . a l . , 1976). Thus i t appears t h a t the a f f i n i t y c o n s t a n t s are s u b s t a n t i a l l y higher than the lower l i m i t s of c u r r e n t estimates of NADH c o n c e n t r a t i o n i n v i v o . So i t seems reasonable t o assume, at l e a s t t e n t a t i v e l y , t h a t NADH would o f t e n , i f not always, be l i m i t i n g i n the cytoplasm. At such times, c r e a t i n e -phosphate and GP e f f e c t s on LDH and GPDH r e s p e c t i v e l y , would profoundly i n f l u e n c e t r a n s i t i o n s between anaerobic and a e r o b i c g l y c o l y s i s i n a t i s s u e such as s k i p j a c k white muscle. 120 Table 5 - 1 . K i n e t i c c o n s t a n t s of LDH and GPDH from white muscle. LDH assays i n the forward d i r e c t i o n were done a t pH 6.5; i n the re v e r s e d i r e c t i o n a t pH 8.5. GPDH assays i n the forward d i r e c t i o n were done at pH 7.0; i n the r e v e r s e d i r e c t i o n a t pH 8.5. 50 mM im i d a z o l e was the b u f f e r used, and a l l assays were a t 25QC. LDH K m 0.01-0.02 (depends on pyruvate con-concentration) 0.33 (slightly dependent upon NADH concentra-tion) aGPDH K m 0.016 0.066 122 Table 5-2. I n h i b i t i o n of LDH by 20 mM creatine-phosphate. Assay c o n d i t i o n s : 0.1 mM pyruvate (0.3 mM pyruvate i n the case of the Amazon f i s h e s ) , 0.1 mM NADH, 25<>C, pH 7.0. 123 Type of LDH % I n h i b i t i o n by 20 mM Source of LDH funtion creatine phosphate Hoplias white muscle pyruvate reductase 25 Turtle white muscle pyruvate reductase 26 M. from rabbit muscle 4 pyruvate reductase 29 Hoplerythrinus white muscle pyruvate reductase 32 Skipjack white muscle pyruvate reductase 38 Hoplias heart b i f u n c t i o n a l 39 Arapaima heart b i f u n c t i o n a l 39 Hoplerythrinus heart b i f u n c t i o n a l 41 Osteoglossum heart b i f u n c t i o n a l 47 H^. from beef heart l a c t a t e oxidase 71 Rat brain l a c t a t e oxidase 71 Weddell seal heart l a c t a t e oxidase 77 124 Table 5-3. The a f f i n i t i e s f o r GP of GPDHs from v a r i o u s v e r t e b r a t e and i n v e r t e b r a t e muscles. K i v a l u e s were determined from Dixon p l o t s a t pH 7.0 (pH 7.4 f o r the t u r t l e and the trout) , 25°C. X Source o f GPDH K i ( G P ) ( m M ) Tuna w h i t e muscle 0.25 T r o u t w h i t e muscle 1.1 T u r t l e w h i t e muscle 0 .93 R a b b i t mixed muscle 0.5 Honey bee f l i g h t muscle 5.0 Squid mantle muscle 1 5 . 0 * * S t o r e y and Hochachka, 1975 126 Table 5-4. Competition f o r NADH o x i d a t i o n between LDH and GPDH. a. Competition between rabbit muscle GPDH and different LDH isozymes. 0.0 mM C-P . % Total % Total Oxidation Oxidation by LDH by GPDH 52.4 47.7 49.5 50.6 20 mM C-P % Total % Total Oxidation Oxidation by LDH by GPDH 25.6 74.3 48.3 51.7 b. Competition between H^ LDH and different GPDH isozymes. 0.0 mM GP % Total % Total Oxidation Oxidation by LDH by GPDH 2.0 mM GP % Total % Total Oxidation Oxidation by LDH by GPDH Rabbit muscle .'GPDH Honey bee 58.6 50.0 41.5 50.9 89.0 65.7 12.6 36.4 A l l values are a mean of 4 experiments. Variation is ± 2%. 128 F i g u r e 5-1. S t a r c h g e l e l e c t r o p h o r e s i s of s k i p j a c k red and white muscle LDH and GPDH. For c o n d i t i o n s of running and s t a i n i n g , see Chapter 2, 129 + O R I G I N LDH G P D H 130 F i g u r e 5-2. E f f e c t o f pH on s k i p j a c k white muscle LDH and GPDH, 50 mM i m i d a z o l e , 25°C. Assay c o n d i t i o n s f o r LDH: 0.3 mfl pyruvate, 0.1 mM NADH. Assay c o n d i t i o n s f o r GPDH: 0.14 mM DHAP, 0.1 mM NADH. 132 F i g u r e 5-3. E f f e c t of the c o - s u b s t r a t e on the Km of pyruvate and NADH of s k i p j a c k white muscle LDH. A. The e f f e c t s of NADH c o n c e n t r a t i o n on the K m of pyruvate o f s k i p j a c k white muscle LDH. 25°C, 50 mM i m i d a z o l e , pH 6.5. B. The e f f e c t s of pyruvate c o n c e n t r a t i o n on the Km of NADH of s k i p j a c k white muscle LDH. 25°C, 50 mM i m i d a z o l e , pH 6.5. U J 1 3 4 F i g u r e 5-4. The e f f e c t o f temperature on the s u b s t r a t e a f f i n i t y c o n s t a n t s o f s k i p j a c k white muscle LDH and GPDH. A. The e f f e c t of temperature on the Km of pyruvate o f s k i p j a c k white muscle LDH. 0.1 mM NADH, 50 mM i m i d a z o l e , pH 6. 5. 6. The e f f e c t of temperature on the Km of NADH of s k i p j a c k white muscle LDH. 0.5 mM pyruvate, 50 mM i m i d a z o l e , pH 6.5. C. The e f f e c t of temperature on the Km of DHAP of s k i p j a c k white muscle GPDH. 0 .1 mM NADH, 50 mM imidazole,pH 6.5. D. The e f f e c t of temperature on the Km of NADH of s k i p j a c k white muscle GPDH. 0.2 mM DHAP, 50 mM i m i d a z o l e , pH O 20 40 v -~ 1 / [DHAP] mM 1 /[NADH] mM 136 F i g u r e 5-5. The e f f e c t s o f pH on the Km of pyruvate and NADH of s k i p j a c k white muscle LDH. A. The e f f e c t s of pH on the Km of pyruvate of s k i p j a c k white muscle LDH. 0.1 mM N A CH, 25°C, 50 mM imi d a z o l e . B. The e f f e c t of pH on the Km of NADH of s k i p j a c k white muscle LDH. 1.0 mM pyruvate, 25°C, 50 mM i m i d a z o l e . IO / 20 r 1 / [Pyruvate] mM 20 0 40 80 1/[NADhfl mM 138 F i g u r e 5-6. The e f f e c t of ATP on the a c t i v i t i e s o f LDH and GPDH from s k i p j a c k white muscle. Assay c o n d i t i o n s f o r LDH: 0.3 mM pyruvate, 0.05 mM NADH, 50 mM im i d a z o l e , pH 6.5, 25°C. Assay c o n d i t i o n s f o r GPDH: 0.1 mM DHAP, 0.05 mM NADH, 50 mM i m i d a z o l e , pH 7.0, 250C. 139 [ A T P ] m M 140 F i g u r e 5-7. The e f f e c t o f GP on the a c t i v i t y of GPDH from s k i p j a c k s h i t e muscle. 0.1 mM DHAP, 0.05 mM NADH, 50 mM i m i d a z o l e , 25<>C, pH 7.0. 141 142 F i g u r e 5-8. The e f f e c t of creatine-phosphate on the a c t i v i t y of LDH from s k i p j a c k white muscle. 0.5 mM pyruvate, 0.05 mM NADH, 25°C, 50 mM i m i d a z o l e . 100 144 F i g u r e 5-9. The e f f e c t s o f pH and creatine-phosphate on the Km of pyruvate of s k i p j a c k white muscle LDH. 0.1 mM NADH, 20 mM creatine-phosphate, 25°C, 50 mM i m i d a z o l e . 0 5 10 I / [Pyruvate] mM 146 F i g u r e 5-10. R e l a t i v e a c t i v i t i e s o f LDH and GPDH i n a crude 1:9 supernatant. 25°C, 50 mM i m i d a z o l e . C l o s e d c i r c l e s : LDH, 0.1 mM pyruvate, 0.1 mM NADH; open squares: LDH, 0 . J mM pyruvate, 0.1 mM NADH, 30 mM creatine-phosphate, 6 mM ATP; c l o s e d t r i a n g l e s : GPDH, 0.1 mM DHAP, 0.1 mM NADH; open c i r c l e s : GPDH, 0.1 mM DHAP, 0.1 mM NADH, 30 mM c r e a t i n e -phosphate, 6 mM ATP. 147 148 F i g u r e 5-11. K i n e t i c s of creatine-phosphate i n h i b i t i o n o f s k i p j a c k white muscle LDH. A. Dixon p l o t of 1/OD/min versus creatine-phosphate c o n c e n t r a t i o n . 0.05 mM NADH, 25°C, 50 mM imidazole b u f f e r , pH 6,5. C, 0,5 mM pyruvate; X, 2.0 mM pyruvate., B. Dixon p l o t of 1/OD/min versus creatine-phosphate c o n c e n t r a t i o n . 0.5 mM pyruvate, 25<>C, 50 mM im i d a z o l e b u f f e r , pH 6.5. X, 0.05 mM NADH; 0, 0.02 mM NADH. 149 8 [ C r e a t i n e phosphate ] m M 150 CHAPTER 6. GJSJEAIt DISC0SSION 151 When my study of the s k i p j a c k began, background i n f o r m a t i o n , which c r e a t e d the impetus f o r the study, c o n s i s t e d almost e x c l u s i v e l y of b e h a v i o r a l , anatomical, and p h y s i o l o g i c a l data. The s k i p j a c k was known to swim at high speeds f o r l o n g p e r i o d s , i t s red muscle mass was r e l a t i v e l y l a r g e , warm and t o t a l l y d i s c r e t e from the white muscle mass which was a l s o warm; and i t s r e s p i r a t o r y c a p a b i l i t i e s approached those of mammals (Chapter 1). The aim of my study was t o add to the a l r e a d y a v a i l a b l e data i n f o r m a t i o n from the molecular l e v e l of o r g a n i z a t i o n which had been h i t h e r t o l e f t v i r t u a l l y untouched. C h r o n o l o g i c a l l y , my work began with a c o n s i d e r a b l e b i a s towards temperature r e l a t e d s t u d i e s . The bulk of t h i s work i s presented i n Appendix I I I , although s i m i l a r s t u d i e s were a l s o done on a c o l d water s p e c i e s , T.. a l u l u n q a . The r e s u l t s of t h i s work, although of i n t e r e s t to those working on pyruvate k i n a s e , l e f t l i t t l e doubt i n my mind t h a t : 1. Temperature and metabolism were not as o b v i o u s l y i n t e r c o n n e c t e d i n the s k i p j a c k as one might have thought. 2. More i n f o r m a t i o n on the s k i p j a c k , a t d i f f e r e n t o r g a n i z a t i o n a l l e v e l s , was needed be f o r e one c o u l d p o s t u l a t e on the r o l e of temperature i n the n a t u r a l h i s t o r y of t h i s f i s h . I t h e r e f o r e embarked upon the study which r e p r e s e n t s the bulk of my t h e s i s . Data from dead f i s h ( u l t r a s t r u c t u r e , h i s t o c h e m i s t r y and enzyme l e v e l s ) provide an unambiguous p i c t u r e of the d i f f e r e n t muscles 1 metabolic o r g a n i s a t i o n s , with the white muscle showing a s u r p r i s i n g p o t e n t i a l f o r d i v e r s e ( a e r o b i c and anaerobic ) f u n c t i o n . S t u d i e s with l i v e f i s h support the former r e s u l t s and show the s k i p j a c k white muscle 152 to have impressive anaerobic c a p a b i l i t i e s as w e l l as being a muscle capable of s i g n i f i c a n t a e r o b i c f u n c t i o n using carbohydrate as a f u e l source. These metabolite s t u d i e s a l s o c o n s i d e r a b l y expand the i n f o r m a t i o n concerning heat production i n the s k i p j a c k . When the heat i s produced (in terms of swimming speed and f u e l source) and i n which muscle the heat i s produced, are areas which are now c o n s i d e r a b l y c l e a r e r although the q u e s t i o n of heat conduction (mentioned i n Chapter 4) s t i l l makes the exact assessment of white muscle heat production i m p o s s i b l e , a l l of these r e s u l t s and t h e i r i m p l i c a t i o n s have alre a d y been d i s c u s s e d (Chapters 3 and 4 ) . From these r e s u l t s arose the q u e s t i o n of metabolic c o n t r o l i n the white muscle, a muscle capable of a e r o b i c and anaerobic carbohydrate metabolism. So my_interests descended to a lower l e v e l of o r g a n i z a t i o n with the end r e s u l t b e i n g a novel metabolic c o n t r o l s t r a t e g y whose i m p l i c a t i o n s are i n no way r e s t r i c t e d t c s k i p j a c k white muscle, again, t h i s l a t t e r p a r t of my t h e s i s , which i n c l u d e s appendix I b a s i c a l l y as r e s u l t s , and appendix I I as a r e l e v a n t ' a s i d e * , has already been d i s c u s s e d (Chapter 5). I i n i t i a l l y s e t out with 5 guestions to answer (Chapter 1). I have answered, or at l e a s t c l a r i f i e d , 4 out of 5 of these qu e s t i o n s . The question which s t i l l remains unanswered i s i n f a c t the one t o which I f i r s t addressed myself durinq my e a r l y s t u d i e s on the s k i p j a c k , i . e. what i s the advantaqe to the animal of e i t h e r a hiqh muscle temperature, or a c i r c u l a t o r y system which as an o f f s h o o t , produced hiqh temperatures i n the swimminq musculature? 153 The f u n c t i o n o f the r e t e i n the s k i p j a c k could be f o r the t r a n s f e r of gas, me t a b o l i t e s , or heat, or f o r t h a t matter, anything which d i f f e r s i n c o n c e n t r a t i o n between the venous and a r t e r i a l flow i n the r e t e . Heat exchange d e f i n i t e l y o c c u r s , metabolite exchange i s a d e f i n i t e p o s s i b i l i t y , but has never been i n v e s t i g a t e d , and S t e v e n s . e t . a l . , (1974) have r u l e d out the p o s s i b i l i t y of gas exchange. There i s l i t t l e one can say about metabolite* r a t h e r than heat exchange i n the r e t e , as t h e r e i s no evidence f o r or ag a i n s t t h i s p o s s i b i l i t y . The r e t e c o u l d be a method of dumping f o r i n s t a n c e , the end-products o f white muscle anaerobic metabolism i n t o the red muscle f o r continued o x i d a t i o n . But i s the advantage of such a mechanism s i g n i f i c a n t enpugh to j u s t i f y the f a c t t h a t s k i p j a c k tuna have a major heat l o s s problem. The magnitude of t h i s problem becomes obvious when one c o n s i d e r s that a s i g n i f i c a n t aspect o f s k i p j a c k behavior i s a migration to c o o l e r waters as they i n c r e a s e i n s i z e . T h i s m i g r a t i o n i s r e l a t e d to the problem of l a r g e r f i s h needing c o o l e r waters i n order t o thermoregulate s a t i s f a c t o r i l y (Dizon and B r i l l , 1978; N e i l l e t . a l . , 1976; Sharp pers. Comm.). I f the f u n c t i o n o f the r e t e i s t o heat the muscles (a p o i n t on which most workers agree), how i s i t s f u n c t i o n r e g u l a t e d and what i s the advantage of warm muscles. The f i r s t q u e s t i o n , d e s p i t e much work, remains unanswered. Nothing a s s o c i a t e d with the r e t e i t s e l f , nervous c o n n e c t i o n s , or shunts, have ever been found (I have used L.M. and scanning e l e c t r o n microscopy) which would o f f e r a reasonable mechanism f o r blocd passing from the d o r s a l a o r t a , through the red 154 muscle, and to the c a r d i n a l v e i n without going through the r e t e . The l a t e r a l c i r c u l a t i o n i n the s k i p j a c k , although much reduced (Kishinouye, 1923), c o u l d o f f e r an a l t e r n a t e route f o r the blood, but whether t h i s ever happens, and how much of the c i r c u l a t i o n c o u l d be accommodated t h i s way, i s completely unknown, as f o r the second guesion, t h e r e have been s e v e r a l s u g g e s t i o n s , the ma j o r i t y of which are untenable. 1. I f the P50 of s k i p j a c k haemoglobins e x h i b i t e d a a a m a l i a n - l i k e temperature s e n s i t i v i t y , the amount of oxygen unloaded at the muscle would be l a r g e compared to most t e l e o s t s . However, such a system would a l s o be v u l n e r a b l e t o water temperature a t the g i l l s , and i t would seem t h a t as the e f f e c t of temperature on most tuna haemoglobins i s s l i g h t {Sharp, 1974), the s k i p j a c k haemoglobins would be no d i f f e r e n t . 2. Walters (1962) suggests t h a t heat t r a n s f e r from the muscle to the immediate boundry l a y e r o f water may be s u f f i c i e n t t o a l t e r the kinematic v e l o c i t y of a very t h i n boundary l a y e r of water; t h i s would i n turn e n e r g i z e the boundary l a y e r and thus prolong laminar flow. However, the temperature near the s k i n i s low; a l s o one would t h i n k that i n t h i s case the white muscle should be the major t a r g e t f o r heat c o n s e r v a t i o n as i t i s contiguous with the s k i n almost a l l over the body. 3. Stevens (1974) suggests t h a t s i n c e the s p a c i a l g r a d i e n t between ambient and muscle temperature i s sharpest i n the re g i o n of the exchanger, red muscle temperature c o u l d be c o n t i n u o u s l y compared to water temperature ( a r t e r i a l blood) i n t h i s area and consequently sma l l changes i n water temperature 155 p e r c e i v e d . However, tunas as a group are no more responsive t o afcrubt temperature changes than are other f i s h e s (Dizon e t . a l . . 1974). Approaching the problem i n more general terms, N e i l l (1976) p o s t u l a t e t h a t t h e heat exchanger simply provides the muscles with thermal i n e r t i a and thus p r o t e c t s them from r a p i d changes i n temperature and from short-term exposure to extreme temperature. Graham (1973) and Carey e t . a l . , (1971) are both of the o p i n i o n that the high swimming speed of the tunas i s a consequence o f the higher muscle temperatures. Despite the wide range of p o s s i b i l i t i e s covered by these l a t t e r s u g g e s t i o n s , and the d i f f i c u l t y of t e s t i n g them, they do p o i n t out that a s i m i l a r i t y may e x i s t between tunas and endotherms i n g e n e r a l i . e. They both e x h i b i t r e l a t i v e l y c o nstant and hiqher than ambient body temperatures. In metabolic terms, the advantages of a c o n s t a n t body temperature are r e a d i l y e x p l a i n e d . Most b i o c h e m i c a l processses and s t r u c t u r e s u n d e r l y i n g metabolism are dependent f o r t h e i r i n t e g r i t y upon the s e g u e n t i a l formation (or breakage) of weak, non-covalent bonding (Low and Scmero, 1976; Hochachka and Somero, 1973; F e r s h t , 1978; Holbrook e j t . a l . , 1975). The important p o i n t t o emphasize, however, i s t h a t such weak bonds are d i f f e r e n t i a l l y a f f e c t e d by temperature, which i s why i t i s more d i f f i c u l t f o r organisms t o cope with thermal change than with,a given a b s o l u t e (high or low) temperature. B i o c h e m i c a l a d a p t a t i o n s t o low temperature are by d e f i n i t i o n maladaptive at high temperatures and v i c e versa (Hochachka. 1974). To be sure, many organisms can remain a c t i v e over l a r g e thermal ranges, but 156 t h i s i s u s u a l l y done at s i g n i f i c a n t c o s t i n terms of design of enzymes and probably metabolism. Thus, as pointed out by H e i n r i c h (1977), a good s t r a t e g y f o r highly a c t i v e organisms i s the t a i l o r i n g of enzymes f o r s p e c i f i c temperatures and the r e g u l a t i o n of body temperature.,But why choose temperatures higher than ambient? One p o s s i b i l i t y i s to allow higher a c t i v i t y r a t e s at high temperatures. T h i s i s an u n l i k e l y s e l e c t i v e f o r c e f o r s e v e r a l reasons. For example, s m a l l d i p t e r a n s weighing 1 mg or l e s s , can c o n t r a c t the t h o r a c i c muscles a t over 3 00 times/sec at 10°C while the s m a l l s i z e of these i n s e c t s makes endothermy of more than 10C above ambient, i m p o s s i b l e ( S o t a v a l t a , 1947). A l s c , Crcmpton e t . a l . , (1978) p o i n t out t h a t the speed of muscular c o n t r a c t i o n seems t o be l i m i t e d by f a c t o r s ether than temperature and i s p r o p o r t i o n a l t o (body weight)-°. * 2 S f o r s t r u c t u r a l reasons. The e x p l a n a t i o n f o r the high *set point* i n endotherms put forward by H e i n r i c h (1977) and Crompton e t . a l * , (1978) c e n t e r s on the need f o r c o n t r o l l e d heat l o s s . I f the body temperature i s above ambient, heat l o s s can r e a d i l y occur by mechanisms (such as conduction and r a d i a t i o n ) not i n v o l v i n g s i g n i f i c a n t water l o s s . But i f the body temperature i s not above ambient, e v a p o r a t i v e heat l o s s i s the only e f f e c t i v e way of h o l d i n g down the body temperature. P a r t i c u l a r l y f o r s m a l l t e r r e s t r i a l organisms, t h i s process would n e c e s s i t a t e d e t r i m e n t a l l y l a r g e l o s s e s of water. Thus the s e t p o i n t i n endotherms appears to be a compromise between the disadvantage o f the metabolic c o s t s of heating up to well above ambient and the advantage o f having a 157 high body temperature to f a c i l i t a t e heat l o s s to the environment. Set p o i n t s are thus r e l a t e d to the upper range of environmental temperatures, which i s why some p r i m i t i v e mammals, such as the hedgehog, are a c t i v e a t night ( a t lower environmental temperatures) and have lower set p o i n t s (Crcmpton e t . a l . , 1978). I propose by analogy t h a t the s k i p j a c k i s an endothermic t e l e o s t somewhat a k i n to the hedgehog i n terms of thermal r e g u l a t i o n . Since the tuna i s c o n t i n u o u s l y a c t i v e , muscle metabolism serves as an e x c e l l e n t heat source, while the sea serves as an e x c e l l e n t heat s i n k . Thus, the only b a r r i e r t o endothermy i s a way to dampen water temperature f l u c t u a t i o n s which would be t r a n s m i t t e d through the g i l l s t o the musculature i n 'normal* t e l e o s t s , and t h a t i s the r o l e of the r e t e . The r e t e m i r able i n the tuna c a n c e l s about 70% of the heat l o s s a t the g i l l s ( N e i l l e t . a l . , 1976), an admittedly d r a s t i c a d a p t a t i o n a l step, but without which there e v i d e n t l y i s no way of a v o i d i n g complete thermal e q u i l i b r a t i o n at the g i l l s (Stevens e t . a l . , 1974). As i n the hedhehog, a p r i m i t i v e mammalian homeotherm with about a 30°C body temperature (Crompton e t . a l . , 1 978), the balance between heat production and heat l o s s i s s e t to maintain a body temperature 5-10°C above ambient. I f body temperature were s e t at ambient, temperature r e g u l a t i o n by c o n t r o l l e d heat l o s s would be more d i f f i c u l t . I f body temperature were s e t much hi g h e r , the metabolic c o s t s would become p r o h i b i t i v e ; even with the c u r r e n t arrangment, the metabolic r a t e of the tuna i s many f o l d higher than t h a t of other t e l e o s t s , approaching t h a t of mammals of 158 comparable body s i z e (Dizon et.aJL,, 1978) . Because only a s h o r t b u r s t of swimming can warm up tuna muscles (Chapter 4) and because the tuna i s probably more a c t i v e at sea than i n c a p t i v i t y , I assume t h a t the r e s t i n g temperatures observed i n c a p t i v i t y are never found normally.,That i s , a t sea the muscle temperature of the s k i p j a c k i s probably as s t a b l e as i n p r i m i t i v e mammals (+ or - 3°C).,, Indeed, Carey and Lawson, (1973) found the temperature of f r e e swimming b l u e f i n tuna to be remarkably c o n s t a n t . Thus, the key combination of a constant and higher-than-ambient body temperature, t y p i c a l of other endotherms, seems a l s o to be w e l l expressed by the tuna. I f t h i s i n t e r p r e t a t i o n i s c o r r e c t , and the s e t p o i n t i n s k i p j a c k i s r e l a t e d to environmental temperature, i t f o l l o w s t h a t s k i p j a c k may not be a b l e t o l o s e enough heat t o t o l e r a t e water temperatures much above 26°C. T h i s p r e d i c t i o n seems to have been v e r i f i e d , as mentioned e a r l i e r i n the d i s c u s s i o n , by f i e l d o b s e r v a t i o n s (Dizon and B r i l l , 1978; N e i l l e t . . a l . , 1976; Sharp, pers. comm.)., 159 APPENDIX I . THE ALPHA-GLYCEBOP HO SPHATE CXCLE IN SKIPJACK WHITE MUSCLE 160 INTRODUCTION During the a e r o b i c metabolism of glucose or glycogen i n a muscle c e l l , the co-enzyme NAD+ i s reduced t o NADH and pyruvate i s formed i n the c y t s o l i c compartment. The continued production, and o x i d a t i o n of pyruvate, r e g u i r e s an e g u i v a l e n t o x i d a t i o n of c y t o s o l i c NADH by the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t c h a i n , which regenerates NAD+. However, d i r e c t t r a n s f e r o f the reduced co-enzyme NADH to the r e s p i r a t o r y c h a i n i s prevented by a s e l e c t i v e p e r m e a b i l i t y b a r r i e r across the i n n e r m i t o c h o n d r i a l membrane to NADH as w e l l as other metabolic i n t e r m e d i a t e s (Lehninger, 1951; P u r v i s and Lowenstein, 1961; G r e v i l l e , 1969), There thus a r i s e s a reguirement f o r "hydrogen s h u t t l e s " f o r the t r a n s f e r of re d u c i n g e q u i l a v e n t s from the c y t o s o l i c to the m i t o c h o n d r i a l compartment. The two most commonly accepted hydrogen s h u t t l e s a re the malate-aspartate c y c l e and the GP c y c l e (Williamson e t . a l . , 1973)(Figure 1-1). In s k i p j a c k white muscle, the enzyme components of the malate-a s p a r t a t e c y c l e are prese n t . GOT a c t i v i t i e s however, although somewhat higher than those of other white muscles (Chapter 4) are lew compared t o GOT a c t i v i t i e s i n t i s s u e s such as the mammalian heart ( S c r u t t o n and U t t e r , 1968) i n which the malate-a s p a r t a t e c y c l e i s known to be the main reduced p y r i d i n e n u c l e o t i d e t r a n s p o r t system (Williamson e t . a l . , 1973; S a f e r , 1975). T h i s observaton, coupled with: 1. u l t r a s t r u c t u r a l evidence s u g g e s t i n g unusual a e r o b i c c a p a b i l i t i e s o f s k i p j a c k white muscle (Chapter 3 ) . 2. evidence from m e t a b o l i t e l e v e l s suggesting t h a t white muscle i s capable o f a e r o b i c work. , 161 3. unusually high a c t i v i t i e s of GPDH (the c y t o s o l i c arm of the GP c y c l e ) i n s k i p j a c k white muscle (Chapter 4 ) , r a i s e s the p o s s i b i l i t y t h a t s k i p j a c k white muscle r e s p i r a t i o n i s c l o s e l y l i n k e d to t h e GP c y c l e There i s evidence that suggests an important c o n t r i b u t i o n of the GP c y c l e i n white muscles of v e r t e b r a t e s ( C r a b t r e e and Newsholme, 1972; Blanchaer, 1964), but a t the time these experiments were i n i t i a t e d , d i r e c t demonstrations of f u n c t i o n i n g GP c y c l e s i n v e r t e b r a t e muscles were not a v a i l a b l e . MATERIALS AND METHODS Experimental animals and enzyme assays have a l r e a d y been mentioned i n Chapter 2. Pr e p a r a t i o n Of Mitochondria Samples of muscle were taken e i t h e r from guiescent or f a t i g u e d tuna. In the f i r s t i n s t a n c e , s l o w l y c r u i s i n g tuna were caught with a sharp dip of a net and q u i c k l y s a c r i f i c e d . Steaks about 3/4" i n t h i c k n e s s were cut from the mid-portion o f the body. About 8-10 gms of muscle were removed, b l o t t e d , weighed, then minced.To the mince was added C h a p p e l l - P e r r y medium (Cbappell and Pe r r y , 1954) or TES medium (Hansford and Johnson, 1975), i n a 1:1 r a t i o (w/v). The C h a p p e l l - P e r r y medium contained 0.1 M KCL, 0.05 M Tris-HCL b u f f e r , pH 7.4, 0.001 M ATP, 0.005 M MgS04, and 0.001 M EGTA. The TES medium contained 0.12 M KCL, 20 mM TES (N-Tr i s - (hydroxymethyl) *rmethtyl-2-amino-ethane s u l f o n i c a c i d ) , pH 7.2 and 10 mM KPi, pH 7.2. The mince 162 was ground i n a precooled mortar and p e s t l e . The r e s u l t a n t t i s s u e s l u r r y was d i l u t e d t o a f i n a l 1:3 or 1:4 d i l u t i o n using more homogenization medium; then homogenized i n a g l a s s homogenizer ( t e f l o n p l u n g e r ) . The homogenate was spun f o r 15 minutes at 600 x g, and the supernatant s o l u t i o n was then decanted and r e c e n t r i f u g e d at 12000 x g f o r 20 minutes. The 12 000g p e l l e t obtained was washed, r e c e n t r i f u g e d , then taken up i n running medium ( c o n t a i n i n g 0 . 1 2 M KCL, 20 mM TES, pH 7.2, and 10 mM KPi, pfi 7,2) f o r r e s p i r a t o r y s t u d i e s . The same procedure was used i n making p r e p a r a t i o n s from exhausted muscle, but i n t h i s case the animal was f i r s t run f o r 5-10 minutes on a hook and l i n e t o p a r t i a l l y d e p l e t e i t s glycogen r e s e r v e s i n white muscle (Chapters 3 and 4 ) , R e s p i r a t o r y Measurements Oxygen uptake r a t e by mitochondria was measured using a C l a r k ^ t y p e oxygen e l e c t r o d e a t t a c h e d to a Radiometer p02 meter, with the radiometer output being f o l l o w e d on a mV Bausch and Lomb c h a r t r e c o r d e r . The i n c u b a t i o n chamber was 2.5 ml i n volume; i t s temperature was r e g u l a t e d with a water-jacket connected to a Lauda co n s t a n t temperature bath and c i r c u l a t o r (Brinkman Instruments, N.Y.). Hamilton s y r i n g e s (10,50 and 100 uL) were used f o r the a d d i t i o n of m e t a b o l i t e s , i n h i b i t o r s , i o n s , and c o f a c t o r s to the i n c u b a t i o n medium without i n t e r r u p t i o n of continuous monitoring of oxygen consumption. 163 Spectrophotometrie S t u d i e s Of Mitochondria Mi t o c h o n d r i a , prepared as d e s c r i b e d above, were used i n attempts to r e c o n s t r u c t the GP c y c l e . GP c y c l i n g i n these experiments was monitered by f o l l o w i n q the chanqe i n OD (340) i n a LTnicam SP 1800 u l t r a v i o l e t r e c o r d i n q spectrophotometer a t 25°C using 1 ml c u v e t t e s and 1 cm l i g h t path. RESULTS AND DISCUSSION Isolated. White Muscle, MiJtp,c.h.ojid.r.i£ S k i p j a c k white muscle mitochondria are r a t h e r d i f f i c u l t to study because of two p o s s i b l y r e l a t e d problems. F i r s t l y , u s i n q standard technigues f o r v e r t e b r a t e muscle (Hansford and Johnson, 1975), i t was not p o s s i b l e t o get good c o u p l i n g i n s k i p j a c k muscle m i t o c h o n d r i a l p r e p a r a t i o n s . T h i s d i f f i c u l t y presumably stemmed from some unknown m e t a b o l i t e , i o n , or osmotic reguirement. The second problem arose from the presence of r a t h e r massive glycogen r e s e r v e s i n s k i p j a c k white muscle (Chapters 3 and 4). In white muscle e x c i s e d from g u i e s c e n t animals, a l a r g e amount of glycoqen was spun down d u r i n g c e n t r i f u g a t i o n and contaminated the m i t o c h o n d r i a l p e l l e t . In white muscle e x c i s e d from animals t h a t had swum t o f a t i g u e , much of the glycogen was d e p l e t e d and hence i t s contamination of subseguent m i t o c h o n d r i a l p r e p a r a t i o n s was reduced. Of p a r t i c u l a r importance however, i s t h a t i n such i n s t a n c e s , the glycogen g r a n u l e s c a r r y with them a number of enzymes 164 (Heilmeyer e t . a l . , 1970), including, i n the case of the skipjack white muscle, c y t o s o l i c GPDH and LDH. A close q u a n t i f i c a t i o n of t h i s e f f e c t was not attempted, but i n a t y p i c a l mitochondrial preparation from quiescent skipjack, a 1:10 d i l u t i o n contained about 20 0/ral of GPDH and about 4C0 0/ml of LDH. In mitochondrial preparations made from muscle whose qlycogen reserves had been at lea s t p a r t i a l l y depleted by strenuous swimming, the amounts of GPDH and LDH p r e c i p i t a t i n q out with the glycogen were reduced to about 1/3 of the above values, due to the decreased amount of pr e c i p i t a t i n g glycogen. As i s noted i n Table 1-1, GP by i t s e l f could serve as an acceptable substrate for skipjack white muscle mitochondria, increasing r e s p i r a t i o n over endogenous rates by about 6-fold. NADH alone causes a two-fold increase i n oxygen uptake (demonstrating leakyness of the mitochondria), pyruvate and malate added together cause an oxygen uptake which i s 2/3 that with GP as the substrate and the addition of succinate, which l i k e GP donates i t s electrons to FAD, causes a 6-fold r i s e i n oxygen uptake. These r e s u l t s demonstrate that at le a s t one half of the GP cycle i s operative i n skipjack white muscle i . e . GP oxidase must be present i n skipjack white muscle mitochondria and thus GP i s oxidised, FADH i s produced and r e s p i r a t i o n i s stimulated. The r e l a t i v e levels of oxygen uptake with GP vs malate-pyruvate as a substrate are s i m i l a r to those i n mammalian white muscle mitochondria (Blanchaer, 1964) and insect f l i g h t muscle mitochondria (Sacktor, 1976). 165 R e c o n s t r u c t i o n Of The GP Cycle S k i p j a c k white muscle mitochondria were i s o l a t e d as before and the p e l l e t was taken up i n a minimal volume of runninq medium, pH 7.2. A s m a l l volume (200 uL) of the m i t o c h o n d r i a l p r e p a r a t i o n was added to 1 ml of runninq medium c o n t a i n i n q 0. 1 mM NADH. In such p r e p a r a t i o n s made from muscle of q u i e s c e n t tuna, each cuvette would c o n t a i n about 20 u n i t s o f endoqenous GPDH; i n p r e p a r a t i o n s from f a t i q u e d muscle, there would be about 7-10 u n i t s i n each c u v e t t e . A d d i t i o n of GP to these mitochondria would then l e a d to NADH o x i d a t i o n , which was followed at 340 nm. T h i s technique was s e n s i t i v e enouqh t o allow a demonstration of the dependence o f NADH o x i d a t i o n on GP a v a i l a b i l i t y , t o allow i n e f f e c t the c o n s t r u c t i o n of GP s a t u r a t i o n curves (Fiqure 1-2) and the c a l c u l a t i o n of an apparent M i c h a e l i x constant f o r GP(about 0.4 mM). There i s only cne i n t e r p r e t a t i o n that can e x p l a i n these d a t a ; i t assumes t h a t the added GP i s o x i d i z e d by the mitochondria t o DHAP, which then serves as the s u b s t r a t e f o r c y t o p l a s m i c GPDH, NADH beinq o x i d i z e d as a consequence. T h i s experiment i s thus a r e c o n s t r u c t i o n of the GP c y c l e (Fiqure 1-1) . I f t h i s i n t e r p r e t a t i o n i s c o r r e c t , i t leads t o another important p r e d i c t i o n : namely, t h a t under such c o n d i t i o n s , s i n c e the c y c l e only uses c a t a l y t i c amounts of s u b s t r a t e s , DHAP and GP should be a t , or c l o s e to, some s o r t of steady s t a t e . On the other hand, NADH, should be, and was, dem£ust£ably_ d e c l e t e d : once the change i n E 340 was reduced t o zero, a d d i t i o n of more NADH l e d to f u r t h e r o x i d a t i o n and thus to r e a c t i v a t e d GP c y c l i n g . In one experiment, 3-4 such rounds o f NADH o x i d a t i o n could be r e a d i l y 166 demonstrated, with no f u r t h e r a d d i t i o n of GP to the pr e p a r a t i o n . The c o n t r o l p r o p e r t i e s o f t h i s r e c o n s t r u c t e d GP c y c l e were not f u l l y c h a r a c t e r i z e d , but s e v e r a l s i q n i f i c a n t o b s e r v a t i o n s were noted. 1. A d d i t i o n of NAD* to the pr e p a r a t i o n caused a s t r c n q i n h i b i t i o n of r e s p i r a t i o n , presumably by a c o m p e t i t i v e block at the GPDH step. 2. ADP d e f i n i t e l y a c t i v a t e d the GP c y c l e at a l l l e v e l s of GP t e s t e d , s a t u r a t i n g and no n - s a t u r a t i n q {Figure 1-2) , but as ex p l a n a t i o n s of the r e s u l t s , these experiments c o u l d not d i s t i n g u i s h between {a) a d i r e c t a c t i v a t i o n of GP ox i d a s e , or (b)a simple c o u p l i n g e f f e c t of ADP on p h o s p h o r y l a t i o n and r e s p i r a t i o n . 3. The e f f e c t s of GP c o n c e n t r a t i o n on the r e c o n s t r u c t e d GP c y c l e should be mentioned. Skipjack white muscle GPDH i s unusually s e n s i t i v e to GP {Chapter 6), thus hiqh c o n c e n t r a t i o n s of GP should i n h i b i t a t l e a s t the c y t o s o l i c arm of the GP c y c l e . However, GP at up to 25 mM l e v e l s , d i d not i n h i b i t NADH o x i d a t i o n (Fiqure 1-2). T h i s suqqests t h a t the GP oxidase s u p p l i e s the r a t e - l i m i t i n g step i n the aer o b i c process, althouqh I am aware t h a t t h i s c o n c l u s i o n i s not supported by (1.) above. A s i m i l a r c o n c l u s i o n a r i s e s from measures of the r e l a t i v e a c t i v i t i e s of c y t o s o l i c GPDH and m i t o c h o n d r i a l GP oxidase i n v a r i o u s animals; the former a c t i v i t y i s a t l e a s t 3 times higher than the l a t t e r {Crabtree and Newsholme, 1972). The f u n c t i o n of GP i n h i b i t i o n of c y o s o l i c GPDH, t h e r e f o r e appears to take on importance only under anaerobic c o n d i t i o n s , 167 when GP oxidase and e l e c t r o n t r a n s f e r are blocked by an oxygen l a c k , and GPDH i s i n h i b i t e d so as t o avoid a d r a i n o f carbc n frcffi mainline g l y c o l y s i s to GP (Chapter 6). From the above data, i t i s t e n t a t i v e l y concluded that redox balance d u r i n q white muscle r e s p i r a t i o n i n s k i p j a c k i s conserved by a GP c y c l e . T h i s c o n c l u s i o n r a i s e s two i a p c r f a n t p o i n t s : 1, Why would v e r t e b r a t e white muscle, i n s e c t f l i q h t nuscle and squid mantle muscle depend on a GP c y c l e i n s t e a d of a malate-aspartate c y c l e as i n mammalian heart (Williamson e t . a l . , 1973) and v i c e versa? 2. Why are a l l the known NAD* r e g e n e r a t i n g systems { which i n c l u d e the malate-aspartate c y c l e , the GP c y c l e , the f a t t y a c i d c y c l e and the l a c t a t e pyruvate c y c l e ) based on the same desiqn i . e . a s h u t t l e o f hydrogen a c r o s s the m i t o c h o n d r i a l mesbrane with a r e s u l t a n t production of NAD* i n the c y t o s o l ? The second q u e s t i o n i s somewhat out of pl a c e here and i s considered i n d e t a i l i n Appendix I I . The f i r s t q u e s t i o n needs c l a r i f i c a t i o n b efore i t i s co n s i d e r e d . I s t h e r e a mutually e x c l u s i v e s e p a r a t i o n of the malate-aspartate c y c l e and the GP c y c l e between the mammalian heart ( f o r example) on one hand, and some v e r t e b r a t e white muscles, i n s e c t f l i g h t muscle and sgui d mantle muscle on the other? V e r t e b r a t e white muscle d i s p l a y s higher a c t i v i t i e s o f GPDH than GOT, whereas i n mammalian h e a r t , a c t i v i t i e s show the opposite trend (Opie and Newsholme, 1967; Crabtree and Newsholme, 1972; Scr u t t o n and U t t e r , 1968). In S i raphes tote.u t h i s mantle, GPDH a c t i v i t i e s are 10-f o l d higher than GOT a c t i v i t i e s and i n the b l o w f l y f l i g h t 168 muscle and s k i p j a c k white muscle, oxyqen uptake by mitochondria i s g r e a t e s t when GP i s the s u b s t r a t e (Table 1-1; Hochachka e t . a l , , 1975; Sacktor, 1976). The evidence thus suggests that there i s simply a predominance c f the GP c y c l e system over the other i n t i s s u e s which u t i l i z e carbohydrate as the primary carbon and energy source. However, t i s s u e s u s u a l l y have the components of both systems and i t has been shown that more than one c y c l e can be o p e r a t i v e i n one t i s s u e (Williamson e t . a l . , 1973; Cederbaum e t . a l . , 1973). What are the c o n s i d e r a t i o n s when choosing one system over any other? In terms o f energy y i e l d , f o r each s p i n of the GP c y c l e (or, per mcle of G6P converted to p y r u v a t e ) , 7 moles of ATP are netted, 4 moles through the GP mediated t r a n s f e r c f r e d u c i n g e q u i v a l e n t s to oxyqen, 3 moles through s u b s t r a t e phosphorylations i n g l y c o l y s i s . However, the o v e r a l l a e r o b i c y i e l d i s 36 moles ATP/mole G6P, a v a l u e s l i g h t l y lower than the 38 moles obtained (by the mammalian heart f o r example) where the malate-aspartate c y c l e i s used. Of these 36 moles of ATP, the formation of only 4 moles ( i . e . Only 1/9th the energy y i e l d ) i s o b l i g a t o r i l y l i n k e d to the GP c y c l e . In t h e s e terms, t h e r e f o r e , the GP c y c l e does not seem t o r e p r e s e n t an unusually advantageous, or f o r that matter, disadvantaqeous, system. Another d i f f e r e n c e i s t h a t the m i t o c h o n d r i a l component of the GP c y c l e uses PAD as i t s co-enzyme, r a t h e r than NAD as i n the malate-aspartate c y c l e (fcigure 1-1; Appendix I I ) . Thus i f NAD were ever l i m i t i n g i n the mitochondria, s e l e c t i o n f o r an GP c y c l e , r a t h e r than a malate-aspartate c y c l e , might ensue. Other c o n s i d e r a t i o n s are that the malate-aspartate c y c l e may be more 169 r e a d i l y r e v e r s a b l e than the GP c y c l e simply because of the p o s i t i v e standard f r e e enerqy change i n v o l v e d with r e v e r s i n q both GPDH and GP oxidase (Appendix I I ) . Thus the malate-a s p a r t a t e c y c l e may be u s e f u l i n s u p p l y i n q c y t o p l a s m i c NADH f o r qluccneogenesis. The t i s s u e l o c a t i o n of the l a t t e r however, c e r a i n l y does not sugqest t h i s (the heart uses almost e x c l u s i v e l y blood qlucose as i t s carbohydrate enerqy source (Safer, 1975) and a l s o Safer (1975) p o i n t s out that the aspartate-glutamate exchanger i n the malate-aspartate c y c l e i s e s s e n t i a l l y u n i d i r e c t i o n a l . The f a c t t h a t the presence of an GP c y c l e c o r r e l a t e s with a r e s p i r a t o r y system based on carbohydrate, suqqests that a f u n c t i o n a l l i n k e x i s t s between the two, but i t s exact nature i s as yet undefined. 170 f a b l e 1-1. R e l a t i v e r e s p i r a t i o n r a t e o f i s o l a t e d white muscle mitochondra. 0.12 M KCL, TES runninq medium, pH 7.2, 30°C. Mitochondria i s o l a t e d as de s c r i b e d i n M a t e r i a l s and Methods. A slow endoqenous r a t e o f 02 uptake i s a r b i t r a r i l y s e t at 100. C o n d i t i o n s R e l a t i v e R e s p i r a t i o n Rate Endogenous 100 1 mM NADH 200 2 mM GP 610 1 mM p y r u v a t e + 0.05 m a l a t e 400 10 mM s u c c i n a t e 600 2 mM GP + 1 mM NADH + 1 mM NAD+ 530 172 F i g u r e 1-1. T r a n s p o r t of reducing e g u i v a l e n t s between c y t o s o l and mitochondria: the GP c y c l e and the malate-a s p a r t a t e c y c l e . 173 O A H 2 0 17a F i g u r e 1-2. Reconstructed GP c y c l e i n i s o l a t e d mitochondria from s k i p j a c k s h i t e muscle. In each experiment, 200 uL of f r e s h l y i s o l a t e d s h i t e muscle mitochondria were added to a t o t a l volume of 1 ml of the running medium (see M a t e r i a l s and Methods), pH 7.2, 25°C, c o n t a i n i n g 0.1 mM NADH. The e f f e c t of i n c r e a s i n g a v a i l a b i l i t y of GP was assessed i n each experiment a f t e r s u b t r a c t i n g a lew " c o n t r o l " r a t e of endogenous NADH o x i d a t i o n by the m i t o c h o n d r i a l p r e p a r a t i o n s . C o n t r o l curve (open c i r c l e s ) r e f e r s t o the e f f e c t of GP alon e ; the e f f e c t of i n c l u d i n g 1 mM MgADP i s shown i n the marked curve (dark c i r c l e s ) . 0.10 P l u s A D P h-GP],mM 176 APPENDIX I I . THE IMPORTANCE OF WATER AND OXYGEN INTHE MQLUIIQM. Ql HYDROGEN SHUTTLE MECHANISMS 177 INTBODOCTIOH Primary metabolic pathways show l i t t l e v a r i a t i o n through the v a r i o u s b i o l o g i c a l kingdoms. Anaerobic energy production i s accomplished by anaerobic g l y c o l y s i s with l a c t a t e or e t h a n o l as accumulating end-products (Bovetto e t . a l . , ,1973; Edington e t . a l . , 1973). In some cases, anaerobic energy production i n v o l v e s p o r t i o n s of the Krebs c y c l e and metabolites such as s u c c i n a t e accumulate (de zwaan and Zandee, 1972). A e r o b i c a l l y , ATP can be produced i n the mitochondria by the o x i d a t i o n of f a t t y a c i d s , glucose or glycogen, but l i p i d o x i d a t i o n i s the p r e f e r r e d a e r o b i c pathway i n most muscles which are c a l l e d upon to provide a long term s t e a d y - s t a t e power output ( B i l i n s k i , 1974; Baldwin e t . a l . , 1973). Whatever the pathway i n v o l v e d , energy production i n the c e l l i n v o l v e s a p a r t i a l or complete o x i d a t i o n of s u b s t r a t e . T h e r e f o r e , the design of an energy producing pathway must take i n t o account a hydrogen acceptor which must (1) accumulate as long as s u b s t r a t e i s being o x i d i z e d , and (2) always be a v a i l a b l e t o accept e l e c t r o n s i f s u b s t r a t e o x i d a t i o n i s to continue. T h i s b r i n g s us t o the r o l e of hydrogen s h u t t l e s i n the a e r o b i c metabolism of carbohydrate. Carbohydrate metabolism can assume a major r o l e i n a e r o b i c ATP production. I t i s the only source o f energy i n some i n s e c t f l i g h t muscles (Sacktor, 1976), and i t assumes prime importance i n a e r o b i c metabolism i n s g u i d mantle muscle (Storey and Hochachka, 1975). Mammalian b r a i n , an o b l i g a t e l y a e r o b i c t i s s u e which can account f o r up to 20% of the t o t a l oxygen used i n the body, i s s t r i c t l y a glucose-burner (Dunn and Bondy, 1974), and 178 glucose i s metabolised a e r o b i c a l l y i n the r a t heart (Safer and Williamson, 1973). In the g l y c o l y t i c sequence, q l y c e r a l d e h y d e -3-phosphate dehydrogenase reduces NAD. Under anaerobic c o n d i t i o n s , NAD i s regenerated by the LDH r e a c t i o n , redox balance i s maintained and l a c t a t e accumulates. Under a e r o b i c c o n d i t i o n s , pyruvate i s no longer reduced t o l a c t a t e s i n c e i t e n t e r s the mitochondrion f o r f u r t h e r o x i d a t i o n . Regeneration of NAD, however, i s s t i l l necessary f o r the c o n t i n u a t i o n of g l y c o l y s i s . S i n c e the inne r m i t o c h o n d r i a l membrane i s g e n e r a l l y considered t o be impermeable t o p y r i d i n e n u c l e o t i d e s ( G r e v i l l e , 1969, Lehninger, 1951; Lowenstein, 1961) cyt o p l a s m i c NADH cannot be o x i d i z e d d i r e c t l y by the m i t o c h o n d r i a l e l e c t r o n t r a n s p o r t system. Thus, with r e s p e c t t o redox balance, a e r o b i c carbohydrate metabolism would seem to present somewhat of a problem. , THE SHUTTLES Four mechanisms have been demonstrated by which the r e q u i r e d NAD can be produced under a e r o b i c c o n d i t i o n s ; these are d e s c r i b e d i n d e t a i l below. The four mechanisms a r e the GP c y c l e , the malate a s p a r t a t e c y c l e , the f a t t y a c i d c y c l e and the l a c t a t e pyruvate c y c l e (Williamson e t . a l . , ,1973; Sack t o r , 1976; Klingenberg, 1970; Grunnet, 1970; Storey and Kayne, 1977). These f o u r c y c l e s are s i m i l a r i n t h a t i n a l l cases r e d u c i n g e q u i v a l e n t s from the NADH produced by GAPDH are donated t o a hydroqen c a r r i e r which then passes these e q u i v a l e n t s t o an i n t r a - m i t o c h o n d r i a l c a r r i e r which i s f i n a l l y o x i d i s e d by the e l e c t r o n t r a n s p o r t system ( F i q u r e s 11-1,2,3). 179 The c y t o s o l i c hydrogen c a r r i e r s of the malate-aspartate, f a t t y a c i d and l a c t a t e pyruvate s h u t t l e s a c t u a l l y c r o s s the i n n e r m i t o c h o n d r i a l membrane (Safe r , 1975; Whereat e.t.a.1. , ,1969; Cederbaum e t . a l . , ,1973; Storey and Kayne, 1977). GP however, the c y t o s o l i c hydrogen c a r r i e r i n the GP c y c l e , cannot c r o s s the i n n e r m i t o c h o n d r i a l membrane and GP oxidase i s l o c a t e d on the o u t s i d e of the i n n e r membrane and t r a n s f e r s reducing e q u i v a l e n t s from GP, a c r o s s the i n n e r membrane, t o FAD (Klingenberg, 1970). The NAD/NADH system i s more reduced i n the mitochondria (Williamson e t . a l . , 1971) n e c e s s i t a t i n g the NAD-linked s h u t t l e s to work a g a i n s t a g r a d i e n t . So f a r only i n one of the s h u t t l e s , has an energy d r i v e n s t e p been found which could account f o r the " u p h i l l " nature of these s h u t t l e s . T h i s i s the glutamate -a s p a r t a t e exchanger i n the malate-aspartate c y c l e , which i s l o c a t e d i n the i n n e r membrane. T h i s exchanger app a r e n t l y takes advantage of the inward proton g r a d i e n t (Safer, 1975) and thus c o u l d be the energy i n p u t i n t o the malate a s p a r t a t e s h u t t l e . In the other s h u t t l e mechanisms, no such energy d r i v e n s t e p has been d e s c r i b e d , although the gradient w i l l not be as i n h i b i t o r y i n the case of the GP c y c l e , as a f l a v i n i s the i n t r a m i t o c h o n d r i a l hydrogen c a r r i e r r a t h e r than a p y r i d i n e n u c l e o t i d e . The malate a s p a r t a t e c y c l e ( c o n s i d e r i n g the c y c l e as a r e a c t i o n ) e x h i b i t s i d e n t i c a l standard f r e e energy changes f o r the forward and r e v e r s e d i r e c t i o n s , as the c y t o s o l i c and m i t o c h o n d r i a l enzyme components are i d e n t i c a l . Thus t h i s system has the p o t e n t i a l o f running backwards and p o s s i b l y s u p p l y i n g 180 reducing e q u i v a l e n t s f o r c y t o p l a s m i c gluconeogenesis, although the glutamate-aspartate exchange mechanism i s s a i d to be d i f f i c u l t t o r e v e r s e (Safer, 1975). The GP and f a t t y a c i d c y c l e s are more e n e r g e t i c a l l y f a v o u r a b l e i n the forward d i r e c t i o n because of the m i t o c h o n d r i a l f l a v i n involvement (the GP oxidase r e a c t i o n has a s t a n d a r d f r e e energy change of -8 Kcal/mole i n the o x i d i z i n g d i r e c t i o n ) and thus would need i m p o s s i b l y l a r g e c o n c e n t r a t i o n s o f s u b s t r a t e s i n order to f o r c e the c y c l e backwards.,The l a c t a t e - p y r u v a t e s h u t t l e i s not r e a l l y a c y c l e at a l l as the pyruvate formed i n the mitochondria i s probably o x i d i z e d i n the mitochondria i n s t e a d of being r e c y c l e d . T h i s s h u t t l e , however, i f disconnected from the Krebs c y c l e , could p o s s i b l y run backwards and supply reducing e g u i v a l e n t s to the cytoplasm (Figure I I - 3 ) . The o p e r a t i o n of the GP c y c l e i t s e l f , produces 4 moles of ATP/mole G6P, the l a c t a t e pyruvate and malate a s p a r t a t e c y c l e s produce 6 and the f a t t y a c i d c y c l e produces 10. DISCUSSION Having d e s c r i b e d these s h u t t l e mechanisms i n d e t a i l , i t i s c l e a r t h a t they accomplish the second of the two design f e a t u r e s mentioned i n the i n t r o d u c t i o n ( i . e. they provide a continued supply of e l e c t r o n a c c e p t o r ) . However, f a r s i m p l e r mechanisms, which do not i n v o l v e the mitochondria, but which accomplish the same end, come to mind. 1. Cytoplasmic NADH oxidase (Figure I I - 4 ) . T h i s system, although e n e r g e t i c a l l y unfavourable, c o u l d produce NAD f o r g l y c o l y s i s and would have no m i t o c h o n d r i a l components. 181 Membrane-bound NADH oxidase has been found i n 'procaryotes {Junks and Matz, 1976) and N&DH o x i d a t i o n o u t s i d e the pe r m e a b i l i t y b a r r i e r of the mitochondria has been demonstrated i n pidgeon heart (Rassmussen, 1969). There i s no evidence that these o x i d a t i o n s i n v o l v e cytochromes, oxygen o r water, and the guesti o n of p h y s i o l o g i c a l s i g n i f i c a n c e i s as y e t unc l e a r . 2. A c y t o p l a s m i c dehydrogenase system (Figure II-5 a and b). such a system would a l s o accomplish the g o a l of a complicated hydrogen s h u t t l e system i . e . s u p p l y i n g NAD f o r g l y c o l y s i s . However, again, such a system has never been d e s c r i b e d f o r an a e r o b i c system and presumably does not e x i s t . Why i s i t that such complicated systems have been s e l e c t e d over r e l a t i v e l y simple ones which c o u l d accomplish the same end? T h i s i s where the i n c o r p o r a t i o n of the f i r s t design f e a t u r e (see i n t r o d u c t i o n ) i s r e a l i z e d . Aerobic work (carbohydrate or f a t based) i s a s t e a d y - s t a t e , long term process, which completely o x i d i z e s t he s u b s t r a t e {in t h i s case, carbohydrate), to C02 and H20. I t i s terminated by a r e a c t i o n which disposes of hydrogen, the l a s t remaining element of the su b s t r a t e . The s u b s t r a t e pool f o r t h i s t e r m i n a l r e a c t i o n must be at l e a s t as l a r g e as the i n i t i a l s u b s t r a t e pool ( i . e . glucose or glycogen) i f i t i s not to l i m i t the whole process. C o r r e s p o n d i n g l y , the f i n a l c o n c e n t r a t i o n of the end-product o f the t e r m i n a l r e a c t i o n w i l l at l e a s t equal the decrease i n the c o n c e n t r a t i o n of the i n i t i a l carbohydrate pool which occurs durinq the work phase. I t i s only because of cytochrome oxidase t h a t a e r o b i c metabolism i s not c o n s i d e r a b l y burdened by the s u b s t r a t e s and products o f i t s own t e r m i n a l r e a c t i o n . The co-182 s u b s t r a t e ( e l e c t r o n s and protons are the other) f o r cytochrome oxidase i s oxygen, an i d e a l e l e c t r o n acceptor, which i s a v a i l a b l e d u r i n g s t e a d y - s t a t e a e r o b i c metabolism i n q u a n t i t i e s which are i n e x h a u s t i b l e . Oxygen i s d i s s o l v e d i n the c e l l f l u i d s , but the r e a l oxygen pool i s the atmosphere, an i n f i n i t e l y l a r g e pool, which i s i n e q u i l i b r i u m with the c e l l f l u i d s during a e r o b i c metabolism.,So the problem of s u b s t r a t e pool s i z e does not e x i s t , The end-product of the r e a c t i o n i s water, the only " m e t a b o l i t e " which can accumulate without s i g n i f i c a n t l y a f f e c t i n g the already 40 M c o n c e n t r a t i o n i n the c e l l . The q u a l i t i e s of the a e r o b i c t e r m i n a l r e a c t i o n are perhaps more obvious i f one examines the correspondinq anaerobic r e a c t i o n , LDH. T h i s enzyme r e a c t i o n (somewhat a k i n to the system proposed i n Figure II-4) accumulates l a c t a t e , a p o t e n t i a l l y noxious m e t a b o l i t e , and anaerobic g l y c o l y s i s i s c h a r a c t e r i s t i c a l l y a short-term process which i s probably l i m i t e d by l a c t a t e b u i l d - u p (Hulten e t . a l . , 1975). With these s t r a t i g i e s i n mind, the disadvantages of the c y t o p l a s m i c a e r o b i c , NAD r e g e n e r a t i o n mechanisms are very c l e a r . Both processes would r e s u l t i n d e l e t e r i o u s end-product accumulation; hydrogen gas i n the case of NADH oxidase (Figure II-4) and m e t a b o l i t e "b" or "d" i n the case of the c y t p l a s m i c dehydrogenase system. Also i n the case of the l a t t e r , pool q u a n t i t i e s of s u b s t r a t e "a" or " c " would be neccessary (Figure II-5 a and b). Hydrogen gas and the s u b s t r a t e s and products of the c y t o p l a m i c dehydrogenase system could not be as innocuous as oxygen and water. So, these c y t o p l a s m i c mechanisms would balance redox, but would l i m i t and d e t r a c t from the long-term 183 nature of mainline a e r o b i c metabolism. T h e i r absence i n nature i s t h e r e f o r e h a r d l y s u r p r i s i n g . The c y t o p l a s m i c redox b a l a n c i n g mechanisms have been d i s c a r d e d f o r what are now apparent reasons, and the nature of the a e r o b i c metabolic process has been conserved. Nature has c h a r a c t e r i s t i c a l l y taken advantage of the s u i t a b i l i t y of oxygen and water as s u b s t r a t e and product of the t e r m i n a l step o f a process i n v o l v e d i n long-term, steady s t a t e a e r o b i c work. The r e s u l t i s that NAD i s regenerated d u r i n g a e r o b i c carbohydrate metabolism by a mechanism with a design which ensures redox balance and yet p l a c e s no burden on the e f f i c i e n c y of the a e r o b i c process. Since a r e g e n e r a t i o n system i n v o l v i n g a m i t o c h o n d r i a l component produces e x t r a reduced coenzyme i n the mitochondria (Figures 1 1-1 , 2 and 3 ) , another "advantage" of these processes i s the i n c r e a s e d amount of ATP produced per mole of carbohydrate. T h i s added ATP amounts t o between 11 and 20% per mole of glycogen depending on the s h u t t l e mechanism i n v o l v e d . The r e l a t i v e importance o f t h i s a d d i t i o n a l ATP i s i m p o s s i b l e t o a s s e s s ; i t c o u l d d e f i n i t e l y be an advantage, but a t e r m i n a l r e a c t i o n with an innocuous end-product and a l i m i t l e s s supply of s u b s t r a t e i s a n e c e s s i t y . 184 F i g u r e I I - 1 . T r a n s p o r t of r e d u c i n g e q u i v a l e n t s between c y t o s o l and mitochondria: the GP c y c l e and malate-aspartate c y c l e . 185 186 F i g u r e 11-2* T r a n s p o r t of reducing e g u i v a l e n t s between c y t o s o l and mitochondria: the f a t t y a c i d s h u t t l e C Y T O P L A S M 2NADH + H + 2 N AD + outer m e m b r a ^ C H 3 " ( C H 2 ) n " f - S C o A inner Co C H 3 - C V S C 0 A » C H 3 - ( C H 2) n +2 - C ' ^ S C o A membrane CH 3 -C^9 SCoA .O C H 3 - ( C H 2 ) n - C ^ S C NADH+H FADH MATRIX P CH 3 - (CH 2 ) n+2- C <>SCoA NAD FAD 2 H 2 0 1 8 8 F i g u r e I I - 3 . T r a n s p o r t of r e d u c i n g e q u i v a l e n t s between c y t o s o l and mitochondria: the l a c t a t e - p y r u v a t e c y c l e , 189 CYTOPLASM G L Y C E R A L D E H Y D E -3- P H O S P H A T E NAD • N A D -NADH 3- PHOSPHOGLYCERATE PYRUVATE N A D H LACTATE .(accumulates under anaerobic condit ions) MITOCHONDRIA LACTATE N A D NADH PYRUVATE KREB'S C Y C L E • C O , I/ ETS 0 2 H 2 0 190 F i g u r e Il-H,,Mechanisms f o r the generation of NAD* from NADH i n the cytoplasm: c y t o p l a s m i c NADH oxidase. 191 H (accumulates) GLYCERALDEHYDE -3 - PHOSPHATE NAD NADH OXIDASE NADH 3- PHOSPHOGLYCERATE 192 F i g u r e I I - 5 , a and b. Mechanisms f o r the gen e r a t i o n of NAD+ from NADH i n the cytoplasm; a cy t o p l a s m i c dehydrogenase. G L Y C E R A L D E H Y D E - 3 - P H O S P H A T E NAD • NADH 3 - PHOSPHOGLYCERATE B (accumulates) B D E H Y D R O G E N A S E A (pool quanti t ies needed ) G L Y C E R A L D E H Y D E - 3 - P H O S P H A T E - N A D -N A D H <N > L I A nu E N Z Y M E 1 3 - P H O S P H O G L Y C E R A T E • B D (accumulates) J | ^ ^ - N A D , E N Z Y M E 2 E N Z Y M E 3 ^ N A D H (A and B needed in cata ly t ic quanti t ies only) C (pool quanti t ies of C needed) mitochondr ia l m e m b r a n e if this w e r e t h e o c - G P cyc le 194 APPENDIX I I I PYBOVATE KINASE FONCTIQNS IN HOT AND COLD ORGANS OF TONA 195 INTRODUCTION Because d i f f e r e n t major t i s s u e s f u n c t i o n a t s i g n i f i c a n t l y d i f f e r e n t temperatures i n the s k i p j a c k (Chapter 1), t h i s animal o f f e r s the unigue o p p o r t u n i t y f o r s t u d y i n g w i t h i n a s i n g l e organism, the i n f l u e n c e of d i f f e r e n t i a l temperatures on c o n t r o l l e d enzyme f u n c t i o n . I chose to examine i n t h i s context, the c a t a l y t i c and r e g u l a t o r y p r o p e r t i e s o f pyruvate kinase i n red muscle (highest c e l l temperatures) and i n the heart (ambient c e l l temperatures). Pyruvate kinase ( I . C. 2. 7. 1. 40) i s an enzyme whose p h y s i o l o g i c a l f u n c t i o n s are well understood. I t occurs i n mammals i n two primary (L and M) forms. The L-type, or r e g u l a t o r y pyruvate kinase, has a low a f f i n i t y f o r PEP, i s s t r o n g l y a c t i v a t e d by-'FDP, and i s under i n h i b i t o r y c o n t r o l by ATP, a l a n i n e , and p h e n y l a l a n i n e ; PEP s a t u r a t i o n curves are s t r o n g l y s i g m o i d a l . The M-type, or non-regulatory enzyme, has a high a f f i n i t y f o r PEP and i s l a r g e l y r e f r a c t o r y to modulation by ATP, a l a n i n e , p h e n y l a l a n i n e , and FDP.,Moreover, s u b s t r a t e s a t u r a t i o n curves are Michaelis-Menten hyperbolas (Tanaka e t . a l . , 1967; L e v e i l l e , 1968; Van B e r k e l , 1974; Seubert and Schonen, 1971). In f i s h e s , the s i t u a t i o n seems more complicated. In the ma j o r i t y o f cases, evidence suggests t h a t f i s h muscle pyruvate k i n a s e , although L-type i n some r e s p e c t s , d i s p l a y s h y p e r b o l i c PEP s a t u r a t i o n curves. These pyruvate kinases are s e n s i t i v e t o modulation by ATP and FDP, but the degree of s e n s i t i v i t y i s low. Eat l i v e r pyruvate kinase can be a c t i v a t e d 1 0-fold by FDP and i n h i b i t e d 90% by 5 mM ATP (Van B e r k e l e.t.aJL., 1972; Tanaka 196 e t . a l , * 1967) whereas the r e s p e c t i v e f i g u r e s f o r f i s h pyruvate k i n a s e s are 1.5-2.5-fold and 20-50% {Somero and Hochachka, 1968; Mustafa e t . a l . , 1971; Johnston, 1975). The Km (PEP) of the f i s h enzyme i s i n the range o f 0.1-0.4 mfl which i s i n t e r m e d i a t e between the PEP a f f i n i t i e s shown by the L- and M-type mammalian pyruvate kinases, and the a f f i n i t i e s f o r ADP appear to be s l i g h t l y lower i n f i s h pyruvate k i n a s e s than i n the mammalian forms (Van B e r k e l , 1974; Johnston, 1975; Hustafa e t . a J L . , 1971). Temperature s t u d i e s on pyruvate kinase have been almost t o t a l l y concerned with enzyme-PEP i n t e r a c t i o n s i n f i s h and i n v e r t e b r a t e enzymes although t h e r e are some data on a c t i v a t i o n e n e r g i e s f o r the r e a c t i o n (Low and Somero, 1976). In the organisms thus f a r s t u d i e d , the Km(PEP) responds i n one of three ways to temperature. F i r s t l y , and most u s u a l l y , i t r i s e s with temperature, but i t a l s o can be temperature independent and i n one case a c t u a l l y f a l l s with temperature (Somero, 1975; Hoffman, 1976). For the pyruvate kinase r e a c t i o n , s t r u c t u r a l volumes of a c t i v a t i o n appear to be c o r r e l a t e d with the a d a p t a t i o n temperature of the p r o t e i n (Low and Somero, 1975) and c a t a l y t i c e f f i c i e n c i e s are r e l a t e d t o c e l l temperatures (Low and Somero, 1976). There i s no r e l a t i o i n s h i p , however, between the a c t i v a t i o n enthalpy and the p h y s i o l o g i c a l temperature range f o r a v a r i e t y of pyruvate k i n a s e s from v e r t e b r a t e s and i n v e r t e b r a t e s (Low and Somero, 1976; Hoffman, 1976). D i f f e r e n t pyruvate k i n a s e s are s e n s i t i v e t o metabolite e f f e c t s at a v a r i e t y o f p h y s i o l o g i c a l temperatures, but no s t u d i e s have been done on the e f f e c t of d i f f e r e n t temperatures 197 on the metabolite s e n s i t i v i t y of a s i n g l e pyruvate k i n a s e . In the s k i p j a c k tuna, the heart and muscle pyruvate kinases occur i n t i s s u e - s p e c i f i c i s ozymic forms. The two enzymes d i f f e r i n terms of the response of the Km (PEP) to temperature, but are s i m i l a r i n t h a t both are more s e n s i t i v e t o metabolite r e g u l a t i o n as temperature i n c r e a s e s . Both enzymes are r e l a t i v e l y i n s e n s i t i v e t o modulators which i s a c h a r a c t e r i s t i c t y p i c a l of f i s h muscle pyruvate kinases. 198 MATERIALS AND METjHODS Fi s h Samples Fresh and frozen f i s h samples were obtained as i s d e s c r i b e d i n the main t e x t (Chapter 2). P r e p a r a t i o n Of Pyruvate Kinase P r e p a r a t i o n of pyruvate kinase f o r s p e c i f i c a c t i v i t i e s has alr e a d y been d e s c r i b e d (Chapter 2). For e l e c t r o p h o r e t i c purposes, f r o z e n t i s s u e samples were (a) homogenized i n a minimal amount o f 10 mM sodium phosphate b u f f e r , 2 mM EDTA, pH 7.0, c e n t r i f u g e d at 12,000g f o r 20 minutes, (b) then concentrated by p r e c i p i t a t i o n a t 80% ammonium su l p h a t e , and (c) resuspended i n b u f f e r . Before use, the muscle ammonium su l p h a t e p r e p a r a t i o n s were d i a l y s e d f o r one hour, with one change, a g a i n s t 1L of 50 mM sodium phosphate b u f f e r , pH 5.7. The heart ammonium sulphate p r e p a r a t i o n s were d i a l y s e d as above a g a i n s t T r i s b u f f e r (5. 8g NaCl/L) 0.5 M, pH 9.0. For k i n e t i c s t u d i e s the t i s s u e s were homogenized i n 5 volumes of 10 mM sodium phosphate b u f f e r , 2 mM EDTA, pH 7.0 (pH 6.5 f o r the heart enzyme), and then concentrated with 80% ammonium sulphate. The muscle f r a c t i o n was d i a l y s e d twice a g a i n s t 1L of 50 mM sodium phosphate b u f f e r , 50 mM K C l , pH 6.5, and then a p p l i e d t o a 2.5 x 40 cm c e l l u l o s e phosphate column e g u i l i b r a t e d with the d i a l y s i s b u f f e r . A 50-400 mM KCl g r a d i e n t 199 was then run through the column and the peak f r a c t i o n s c o l l e c t e d and again c o n c e n t r a t e d with 80% ammonium s u l p h a t e . The p r e c i p i t a t e was resuspended, then d i a l y s e d twice a g a i n s t 1L of 50 mM sodium phosphate b u f f e r , 50 mM K C l , pH 6.0, and a p p l i e d t o a 2.4 x 40 cm SE Sephadex column e q u i l i b r a t e d to the d i a l y s i s b u f f e r . A 50-400 mM KCl g r a d i e n t was then run through the column, the peak f r a c t i o n s were c o l l e c t e d , c o n c e n t r a t e d with ammonium sulphate and s t o r e d a t 0°C. P u r i f i c a t i o n was between 40- and 5 0 - f o l d . The procedure f o r the heart enzyme was e s s e n t i a l l y t he same. The f i r s t column was a SE Sephadex e g u i l i b r a t e d with 20 mM sodium phosphate b u f f e r , pH 6.5, and the g r a d i e n t was 20-600 mM potassium phosphate. The second column was c e l l u l o s e phosphate e g u i l i b r a t e d with 50 mM sodium phosphate b u f f e r , 50 mM KCl, pH 6.0, and the g r a d i e n t was 50-400 mM KCl. The p u r i f i c a t i o n f a c t o r f o r the heart enzyme was about 9 0 - f o l d . By two c r i t e r i a ( t o t a l a c t i v i t y and Km (PEP)), the p u r i f i e d enzymes were s t a b l e a t 0°C f o r at l e a s t one month. Before use, a l i g u o t s were d i a l y s e d , with one change, f o r one hour, a g a i n s t one l i t r e of the assay b u f f e r . Assays Pyruvate k i n a s e a c t i v i t y was monitored i n 1 ml c u v e t t e s (1 cm l i g h t path) using a Unlearn SP 1800 r e c o r d i n g spectrophotometer. The r e a c t i o n c u v e t t e s were h e l d i n c e l l h o l d e r s t h e r m a l l y e q u i l i b r a t e d with a constant temperature bath and c i r c u l a t o r . The pyruvate kinase r e a c t i o n was l i n k e d to the LDH r e a c t i o n and the r a t e was determined by the decrease i n absorbance of NADH at 340 nm. 200 The assays f o r s p e c i f i c a c t i v i t e s have already been de s c r i b e d i n Chapter 2. The assays f o r the k i n e t i c data were done i n 50 mM i m i d a z o l e b u f f e r using 0.15 mM NADH, 15 mM MgS04, 100 mM K C l . A l l other c o n c e n t r a t i o n s are given i n the t e x t . Values f o r the Michaelis-Menten c o n s t a n t s (Km) were determined by d o u b l e - r e c i p r o c a l p l o t s of 1 / v e l o c i t y versus 1/substrate c o n c e n t r a t i o n . I n h i b i t i o n c o n s t a n t s (Ki) f o r ATP were obtained from Dixon p l o t s o f 1 / v e l o c i t y versus i n h i b i t o r c o n c e n t r a t i o n at v a r y 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 . Values obtained were r e p r o d u c i b l e w i t h i n 15%. E l e c t r o p h o r e s i s E l e c t r o p h o r e s i s was done on 13% s t a r c h gels..The e l e c t r o d e b u f f e r used was the d i a l y s i s b u f f e r (see p r e p a r a t i o n of pyruvate k i n a s e ) ; the g e l b u f f e r was a 1:20 d i l u t i o n of the e l e c t r o d e b u f f e r . The g e l s were run f o r three hours at 50 mA and 400 V. To d e t e c t a c t i v i t y , the g e l s were o v e r l a i d with f i l t e r paper s t u r a t e d with 0.15 mM NADH, 20 mM MgS04, 100 mM KCl, 0.2 mM PEP, 0.5 mM ADP, 0.5 I•U. LDH, and 50 mM im i d a z o l e b u f f e r , pH 6,5. Glucose(20 mM) and 5 I,0, Hexokinase were added to c o n t r o l ATP l e v e l s and to regenerate ADP. The presence of a c t i v i t y was determined under 0.V. l i g h t ; NADH absorbs at 340 nm and appears yellow, NAD+ tr a n s m i t s the l i g h t and appears purple (Susor and B u t t e r , 1971). 201 RESULTS E l e c t r o p h o r e s i s S t a r c h g e l e l e c t r o p h o r e s i s of muscle e x t r a c t s d i s p l a y s one band of pyruvate kinase which migrates toward the cathode at pH 5.7. There are two bands, one major and one minor, i n the heart of the s k i p j a c k ; both migrate toward the anode at pH 9.0 and are c l e a r l y s e p a r a b l e from the muscle form of pyruvate k i n a s e . I t was net p o s s i b l e to separate the two heart forms by column chromatography. The a c t i v i t i e s of pyruvate kinase (uJ3 s u b s t r a t e converted/min/gm wet weight) i n red muscle and heart are 195.2 (160.-8-241. 1; three values) and 126.6 (107. 1-150.0; four v a l u e s ) , r e s p e c t i v e l y . Both enzymes showed a broad pH optima around pH 6.5 at 20°C. The peak becomes sharper and s l i g h t l y more a c i d i c at 40°C ( f i g u r e I I I - 1 ) . a f f i n i t i e s f o r PEP find ADP Both forms e x h i b i t e d h y p e r b o l i c s a t u r a t i o n curves with r e s p e c t to PEP and ADP, at 10*C through 40<>C. The Km (PEP) of i o A c t i v i t i e s E f f e c t Of PH 202 the heart enzyme r i s e s very s l i g h t l y as temperature i n c r e a s e s from 10°C to 30°C and then takes a f o u r - f o l d jump to 0.55 mM at 40QC ( f i g u r e III-2) . The Km (ADP) of the heart enzyme r i s e s very g r a d u a l l y from 10°C to 40°C. The Km(PEP)of the muscle enzyme i s always between 1.5- to 2 - f o l d higher than that of the heart enzyme; i t begins t o r i s e at a s i g n i f i c a n t r a t e a t 20°C and r i s e s c o n s t a n t l y between 20°C and 35°C. The Km (ADP) of the muscle enzyme i s between 2 and 3 f o l d higher than t h a t of the heart enzyme and i s u n a f f e c t e d by temperature (Figure III-2).-E f f e c t Of ATP At Km c o n c e n t r a t i o n s of PEP and ADP, both enzymes are weakly i n h i b i t e d by ATP (Figure I I I - 3 ) . I n h i b i t i o n of the two enzymes i s s i m i l a r {U0% at 5 mM ATP) at 20°C. At 40°C the i n h i b i t i o n of the muscle enzyme i s as i t was a t 20°C, but the e f f e c t on the he a r t enzyme i s more pronounced, being 62% at 5 mM ATP (Figure I I I - 3 ) . , ATP i s co m p e t i t i v e with PEP and non-c o m p e t i t i v e or uncompetitive with ADP as determined from Dixon p l o t s a t 10, 20, 30 and 40°C. The K i values f o r ATP are high, r e f l e c t i n g the lack o f i n h i b i t i o n and do not show any c o n s i s t e n t v a r i a t i o n with temperature. The K i v a l u e s f o r the heart enzyme are s l i g h t l y lower than f o r the muscle enzyme (Table I I I - 1 ) . E f f e c t Of FDP FDP a c t i v a t e s the heart enzyme a t 20°C and at 40°C by i n c r e a s i n g the a f f i n i t y of the enzyme f o r PEP; i n c o n t r a s t , the 203 Km f o r ADP i s not a f f e c t e d Jby FDP. FDP a c t i v a t e s the muscle enzyme only at 40°C and the a c t i v a t i o n i s gr e a t e r than t h a t seen i n the heart enzyme at 40°C. .Again, the mechanism of a c t i v a t i o n i n v o l v e s a decrease i n the Km (PEP), but no change i n the Km (ADP) (Table I I I - 2 ) . E f f e c t Of Alanine Alanine has a s l i g h t i n h i b i t o r y e f f e c t at 20°C on both enzymes (25% i n h i b i t i o n at 5 mM a l a n i n e ) . At <40°C the i n h i b i t i o n i s more potent and the muscle enzyme i s a f f e c t e d t o a g r e a t e r extent, 50 and 75% i n h i b i t i o n of the heart and muscle enzymes, r e s p e c t i v e l y , o c c u r r i n g at 5 mM a l a n i n e (Figure I I I -4) . T h i s i n c r e a s i n g i n h i b i t i o n i s graded over the temperature range. The mechanism of i n h i b i t i o n appears to be r a t h e r complex. Both the Vmax and Km (PEP) are a f f e c t e d by a l a n i n e at a l l temperatures. Combined E f f e c t s Of FDP, M P And i i a n j s e I n both t i s s u e s , 0.1 mM FDP p a r t i a l l y overcomes the e f f e c t s of 5 mM ATP at 20<>C; at 40°C the i n h i b i t i o n i s t o t a l l y r e v e r s e d . I n h i b i t i o n by 2 mM a l a n i n e i s f u l l y overcome by FDP at 20«C and 40<>C i n both t i s s u e s (Table III-3) . Q1Q Values Q10 v a l u e s , when determined at Km values of s u b s t r a t e do not d i f f e r s i g n i f i c a n t l y between the two enzymes. The values are s l i g h t l y lower than expected f o r a non-catalyzed r e a c t i o n between 10 and 20°C and s u b s t a n t i a l l y lower between 20°C a 30°C. The value between 30°C and 40°C i s negative f o r both enzymes due to the l a r g e r i s e i n tbe Km (PEP) a t these temperatures (Table III-4) . 205 DIS CJUS ION There are two major e l e c t r o p h o r e t i c types of pyruvate kinase found i n a d u l t mammals. . S k e l e t a l muscles tend t o have a s i n g l e form whereas the l i v e r , kidney, b r a i n and heart can have two or more forms. Some of the forms are t e t r a m e r i c h y b r i d s of the major p a r e n t a l forms (Cardenas e t . a l . , 1973 a and b; Osterman and F r i t z , 1973).,The pyruvate kinase isozymes i n Rana Ei-PiSSS appear to be d e r i v e d from the same p a r e n t a l types as i n the mammals. M u l t i p l e forms e x i s t i n the l i v e r and there i s one t i s s u e s p e c i f i c form i n the c a r d i a c and s k e l e t a l muscle (Schloen e t . a l . , 1974) . The onl y e l e c t r o p h o r e t i c work on f i s h i n v o l v e s salmon which have f i v e bands i n the he a r t (probably tetramers formed from two subunit t y p e s ) , and one i n the muscle (Guderley, pers. Comm.). The s k i p j a c k conforms to the trend i n having only one form i n the muscle and m u l t i p l e forms i n the heart . In mammals and salmon, however, one form i s common to the heart and s k e l e t a l muscle, whereas t h i s i s not the case i n the s k i p j a c k . The s p e c i f i c a c t i v i t i e s o f pyruvate kinase i n the red muscle and heart of the s k i p j a c k are s i m i l a r t o t h a t found i n f o u r s p e c i e s of Amazon f i s h e s (Hochachka e t . a l . , 1978 a and b). Pyruvate k i n a s e a c t i v i t y i n carp r e d muscle i s about h a l f t h at found i n s k i p j a c k red muscle (Van den T h i l l a r t , 1977). Most values f o r mammalian muscles are somewhat higher and vary from 200 to 700 units/gram wet weight (Osterman and F r i t z , 1973). These values however, are f o r mixed (red and white) muscles and are t h e r e f o r e not r e p r e s e n t a t i v e . The only value f o r a " r e d " mammalian muscle i s 280 units/gram wet weight (Prewitt and 206 S a l a f s k y , 1967). The muscle used f o r t h i s d e t e r m i n a t i o n was the s o l e u s muscle of the c a t , which may be composed of mammalian in t e r m e d i a t e f i b e r s (Edgerton and Simpson, 196 9) and t h e r e f o r e again i s not r e p r e s e n t a t i v e . I f e e l t h a t s p e c i f i c a c t i v i t i e s are probably not s i g n i f i c a n t l y d i f f e r e n t between comparable muscles of mammals and t e l e o s t s . A d i s t i n c t i o n between f i s h and mammals however, becomes obvious when the k i n e t i c s of pyruvate kinase and the e f f e c t s of pH are c o n s i d e r e d . Pyruvate kinase from mammals, g e n e r a l l y has an a l k a l i n e pH optimum, somewhere between 7.5 and 8,5 (Randall and Anderson, 1975; Borgmann and Moon, 1976), whereas the pH optima f o r most t e l e o s t pyruvate k i n a s e s are a c i d i c (Johnston, 1975; Mustafa e t . a l . , 1971). The pH p r o f i l e s of the pyruvate k i n a s e s from s k i p j a c k red muscle and heart are thus t y p i c a l l y t e l e o s t . The k i n e t i c s of the two enzymes over t h e i r p h y s i o l o g i c a l temperature ranges (18-25°C f o r the heart (Sharp, pers. Comm.); 20-34°C f o r the red muscle (Chapter 5) are t y p i c a l of n e i t h e r the M nor L form found i n mammalian t i s s u e s . At these temperatures the Km (PEP) of the heart enzyme i s 0.1 mM and t h a t of the muscle enzyme v a r i e s between 0.14 and 0.5 mM. The Km (ADP) of the heart enzyme i s 0.3 mM and the Km (ADP) of the muscle enzyme i s 0.7 mM, which i s unusually high. ATP i n h i b i t i o n and FDP a c t i v a t i o n are r e l a t i v e l y modest, and a l a n i n e i n h i b i t i o n i s n e g l i g a b l e a t temperatures below 30^0. These two enzymes a r e thus t y p i c a l l y t e l e o s t ; they are p a r t i a l l y r e g u l a t e d enzymes e x h i b i t i n g c h a r a c t e r i s t i c s i n between those of the two major forms found i n a d u l t mammals. The e f f e c t of temperature on enzyme r e a c t i o n s has been the 207 o b j e c t of prolonged i n v e s t i g a t i o n . I t i s now w e l l e s t a b l i s h e d what should be the e f f e c t s of temperature on enzyme r e a c t i o n s and how a p r o t e i n can evolve to e i t h e r d i m i n i s h o r heighten t h i s e f f e c t {Somero, 1S75). Much of t h i s work has centered around the pyruvate k i n a s e s of f i s h e s , r e s u l t i n g i n a l a r g e data base f c r comparison. The b i n d i n g of the h i g h l y charged PEP molecule t o the enzyme must be h i g h l y dependent upon i o n i c i n t e r a c t i o n s and thus should be adversely a f f e c t e d by temperature change (Somero, 1975). Conversely, the b i n d i n g of the ADP molecule probably i n v o l v e s hydrophobic as w e l l as i c n i c i n t e r a c t i o n s and should not be as temperature dependent (Hochachka, 1974). Information to date shows t h a t i n the m a j o r i t y of pyruvate k i n a s e s , the Km(PEP) i n c r e a s e s as temperature r i s e s through the p h y s i o l o g i c a l range (Somero, 1975; Hoffman, 1976). In the one study on the i n f l u e n c e of temperature on the Km(ADP), i t was found that the a f f i n i t y of t r o u t pyruvate kinase f o r ADP was temperature independent (Somero and Hochachka, 1968).The s k i p j a c k muscle enzyme shows a t y p i c a l two-fold r i s e i n the Km(PEP) over 10°C, whereas the Km (ADP) i s u n a f f e c t e d by temperature. Both of the a f f i n i t y c o n stants of the heart enzyme however, are v i r t u a l l y temperature independent over the p h y s i o l o g i c a l temperature range. The muscle enzyme i s thus a t y p i c a l f i s h muscle enzyme (Somero, 1975) whereas the heart enzyme shares c h a r a c t e r i s t i c s i n common with some i n v e r t e b r a t e enzymes whose a f f i n i t y c onstants do not vary with temperature (Hoffman, 1976). An important f e a t u r e o f s k i p j a c k red muscle i s t h a t i t s temperature i s dependent upon muscle a c t i v i t y . Thus, a d i r e c t 208 Km-temperature r e l a t i o n s h i p c o u l d prevent s a t u r a t i o n of red muscle pyruvate k i n a s e i f PEP l e v e l s were to r i s e with a c t i v i t y . However, although PEP l e v e l s i n s k i p j a c k red muscle are too low to be measured a c c u r a t e l y , they d e f i n i t e l y do not r i s e t o l e v e l s which c o u l d present s a t u r a t i o n problems. The Km (PEP)-temperature r e l a t i o n s h i p t h e r e f o r e probably serves to render the pyruvate kinase r e a c t i o n temperature independent i n an a c t i v e t i s s u e s u b j e c t to sudden, s i g n i f i c a n t temperature v a r i a t i o n s . T h i s l a t t e r c o n c l u s i o n i s supported by the low Q10 values f o r the red muscle pyruvate kinase r e a c t i o n (Table I I I -U). ..The s i g n i f i c a n c e of the f l a t Km(PEP)-temperature r e l a t i o n s h i p of the heart enzyme i s not c l e a r . , T h i s shape of curve appears to be c h a r a c t e r i s t i c of eurythermic organisms which "opt" f o r the maintenance of enzyme-substrate a f f i n i t i e s w i t h i n c e r t a i n optimal values, r a t h e r than temperature independence (Hoffman, 1S76). I n t e r e s t i n g l y , however, i n c o n t r a s t to the enzymes of eurytherms, the heart pyruvate kinase shows r e l a t i v e temperature independence. How the low Q10 f o r the heart enzyme (Table I I l - u ) i s achieved i s s t i l l unknown. ATP l e v e l s i n the red muscle do not vary s i g n i f i c a n t l y under the t h r e e e x e r c i s e c o n d i t i o n s (Chapter 5) and ATP e f f e c t s are not temperature s e n s i t i v e ; the r o l e of ATP i n r e g u l a t i o n at t h i s s i t e would thus seem t o be minor. This i s not to say t h a t the energy charge, which may f l u c u a t e s i g n i f i c a n t l y (Table 5-6) does not play a s i g n i f i c a n t r o l e i n r e g u l a t i o n of red muscle pyruvate k i n a s e . The e f f e c t of a l a n i n e , however i s s t r o n g l y temperature 209 dependent (Figure I I I - 4 ) . The temperature of the red muscle i s 1°C above ambient when the f i s h i s a t " r e s t " , i t i s c o n s i s t e n t l y high d u r i n g f e e d i n g f r e n z i e s ( i . e . d u r i n g very f a s t s u s t a i n e d swimming), and may a l s o be r a i s e d during b u r s t swimming (Chapter 5 ) . Alanine c o n c e n t r a t i o n s i n the red muscle r i s e from 4 umol/g wet weight wet weight under r e s t i n g c o n d i t i o n s to 9 umol/g wet weight wet weight d u r i n g f e e d i n g c o n d i t i o n s (Chapter 4). Thus, at t h e high f e e d i n g temperatures, a l a n i n e would be i n h i b i t o r y (Figure . 1 1 1-4). FDP l e v e l s i n red muscle f l u c t u a t e s l i g h t l y between the v a r i o u s a c t i v i t y s t a t e s , but never drop below 0.25 umol/g wet weight wet weight which i s almost c e r t a i n l y enough to modulate the e f f e c t s of both ATP and a l a n i n e a t any temperature. T h i s i s a common paradox a s s o c i a t e d with pyruvate kinase r e s e a r c h and t h e r e are many o b s e r v a t i o n s showing minimal FDP c o n c e n t r a t i o n s to completely overcome the r e g u l a t o r y e f f e c t s of other m e t a b o l i t e s (Koster e t . a l . , 1972; Mustafa e t . a l . , , 1971; Guderley e t . a l . , 1976; Storey and Hochachka, 1974b). PEP a v a i l a b i l i t y i s o b v i o u s l y important, but u n f o r t u n a t e l y PEP l e v e l s are too low to be measured acc u r a t e l y . , P E P may become a key element i n c o n t r o l when l e v e l s drop due to i n h i b i t i o n of r e g u l a t o r y s i t e s i n g l y c o l y s i s above pyruvate k i n a s e . M e t a b o l i t e l e v e l s i n the heart o f the s k i p j a c k are not known, but blood l e v e l s o f glucose and a l a n i n e h a r d l y change between r e s t e d and a c t i v e f i s h (Chapter 4). In the heart, temperature bears no r e l a t i o n t o a c t i v i t y and thus the s i g n i f i c a n c e of the temperature induced pyruvate kinase s e n s i t i v i t y to m e t a b o l i t e s i s as yet obscure. 211 T a b l e I I I - 1 . ATP i n h i b i t i o n c onstants f o r the red muscle and heart pyruvate kinases from the s k i p j a c k . Dixon p l o t s sere done a t three c o n c e n t r a t i o n s o f PEP and at 0.2 mM ADP , pH 6.5. 2 1 2 K 1 ( A T P ) M Temperature °C M u s c l e H e a r t 1 0 0 . 5 1 .7 20 3 . 5 2 . 5 30 2 . 0 2 . 4 40 4 . 0 1 . 5 213 Table I I I - 2 . The e f f e c t o f FDP on pyruvate kinase from the heart and red muscle o f s k i p j a c k . Heart assays done a t 0.5 mM ADP and 0.1 mM PEP; muscle assays done a t 0.2 mM ADP and 0.1 mM PEP. A l l assays done at pH 6.5. % Activation by 0.5 mM FDP 20°C 40°C Muscle 0 50 Heart 14 30 215 T a b l e I I I - 3 . The e f f e c t of 0.1 mM FDP O H the a c t i v i t y of the heart and red muscle pyruvate k i n a s e s i n the presence of 5 mM ATP or 2 mM a l a n i n e . 2.0 mM PEP, 2.0 mM ADP, pH 6.5. 216 Activity (% of control) FDP + ATP FDP + Alanine 20°C 40°C 20°C 40°C Red muscle 73 100 100 180 Heart 73 113 130 244 217 Table I I I - 4 . Q10 v a l u e s of s k i p j a c k red muscle and heart pyruvate k i n a s e s . 0.04 mB PEP and 0.5 mH ADP. ,218 Temperature range C Red muscle H e a r t 10-20 20-30 30-40 2.3 1.57 .66 1.9 1.3 .66 219 Fig u r e I I I - 1 . R e l a t i v e a c t i v i t y of pyruvate k i n a s e versus pH at 200C and 40<>C. a. R e l a t i v e a c t i v i t y versus pH at 20°C. ., muscle; x, heart; 0.2 mM PEP; 0.5 mM ADP. B. R e l a t i v e a c t i v i t y versus pH at 40°C. muscle; x, heart. 0.2 mM PEP; 0.5 mM ADP. Relative Activi ty 221 Fig u r e I I I - 2 . Km(mM) versus T«>C: s k i p j a c k red muscle and he a r t pyruvate kinases. muscle ADP; sguares, muscle PEP; o, heart ADP; x # h e a r t PEP. 0.2 mM PEP; 0.5 mM ADP; pH 6.5., 2 2 2 T e m p e r a t u r e °C 223 F i g u r e I I I - 3 . % of c o n t r o l a c t i v i t y versus ATP c o n c e n t r a t i o n . Assays done at pH 6.5, 0.2 mM PEP and 0.2 mM ADP. ., muscle, 20°C; squares, muscle (H0°C); o, h e a r t (20°C); t r i a n g l e s , heart (IQOC). , 224 M g ATP C o n c e n t r a t i o n ( m M ) 225 3 f i g u r e % o f c o n t r o l a c t i v i t y versus a l a n i n e c o n c e n t r a t i o n . Muscle assays at 0.2 J»M PEP and 0.2 mM ADP; heart assays a t 0.1 mM PEP and 0.2 mM ADP. A l l assays a t pH 6.5. ., muscle (20°C); sguares, muscle (40°C); o, heart (20°C) ; t r i a n g l e s , heart (40<>C). 226 Alan ine Concen t ra t i on ( m M ) 227 REFERENCES CITED Ahlborg, B., Bergstrom, J . , Ekulund, L.G. and Hultman, E. 1967. 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P h y s i o l . 43B: 47-54. , 256 ABBREVIATIONS OSED ADP: adenosine diphosphate AMP: adenosine monophosphate ATP: adenosine t r i p h o s p h a t e CS: c i t r a t e s y nthetase DHAP: dihydroxyacetone phosphate DTNB: 5, 5' d i t h i o b i s - ( 2 - n i t r o b e n z o i c acid) EDTA: ethylene diamine t e t r a a c e t i c a c i d EGTA: e t h y l e n e g l y c o l - b i s - ( -amino-ethyl ether) N, N * - t e t r a a c e t i c a c i d FAD: f l a v i n adenine d i n u c l e o t i d e (oxidized) FADH: f l a v i n adenine d i n u c l e o t i d e (reduced) FDP: f r u c t o s e diphosphate F6P: fructose-6-phosphate GAFDH; g l y c e r a l d e h y d e 3-phosphate dehydrogenase GOT: glutamate o x a l o a c e t a t e transaminase GP: c< -glycerophosphate GPDH: o<-glycerophosphate dehydrogenase G6PDH: glucose 6-phosphate dehydrogenase G6P: glucose-6-phosphate LDH: l a c t a t e dehydrogenase MDH: malate dehydrogenase NAE+; ni c o t i n a m i d e adenine d i n u c l e o t i d e (oxidized) NADH: nico t i n a m i d e adenine d i n u c l e o t i d e (reduced) NADP; n i c o t i n a m i d e adenine d i n u c l e o t i d e phosphate (oxidized) PDH: pyruvate dehydrogenase PEP: phosphoenol pyruvate PFK: phosphofructokinase EK: pyruvate k i n a s e SDH: s u c c i n a t e dehydrogenase SR: s a r c o p l a s m i c r e t i c u l u m T-: t r a n v e r s e tubule rj: u n i t s of enzyme a c t i v i t y tuM product produced/minute) PUBLICATIONS Guppy, M. (1974) B i r d s o f the Murrumbidgee and Murray R i v e r s . B i r d s 8: 85-88. Hochachka, P. W. and M. Guppy. (1976) V a r i a t i o n s on a theme by Embden, Meyerhof and Parrias. I n Oxygen and the Organism (Ed. F. J B b s i s ) P r o f e s s i o n a l I n f o r m a t i o n L i b r a r y . D a l l a s , Texas. 292-310. Hochachka, P. W., C. F r e n c h and M. Guppy. (1977) When and how the a - g l y c e r o p h o s p h a t e c y c l e works. P r o c . T h i r d I n t l . Congress on the B i o c h e m i s t r y o f E x e r c i s e . In p r e s s . Hochachka, P. W., W. H u l b e r t and M. Guppy. (1977) The tuna power p l a n t and f u r n a c e . In P h y s i o l o g i c a l E c o l o g y o f Tuna. (Ed. G. Sharp and A. D i z o n ) . Academic P r e s s , i n p r e s s . Guppy, M. and P. W. Hochachka. (1978) C o n t r o l l i n g t h e h i g h e s t ^ l a c t a t e dehydrogenase v a l u e s known i n n a t u r e . Am. J . P h y s i o l . 234: R136-140. Hochachka, P. W,, M. Guppy, H. E. G u d e r l e y , K. B. S t o r e y and W. C. H u l b e r t . (1978) M e t a b o l i c b i o c h e m i s t r y o f water vs a i r b r e a t h i n g f i s h e s : m u s c l e enzymes and u l t r a s t r u c t u r e . Can. J . Z o o l . 5 6 ( P t . 2 ) : 736-750. Guppy, M. and P. W. Hochachka. (1977) Tuna w h i t e muscle: a b l u e p r i n t f o r the i n t e g r a t i o n o f a n a e r o b i c and a e r o b i c c a r b o h y d r a t e m e t a b o l i s m . Ln P h y s i o l o g i c a l E c o l o g y o f Tuna (Ed. G. Sharp and A. D i z o n ) Academic P r e s s , i n p r e s s . Hochachka, P. W. , M. Guppy, H. E. G u d e r l e y , K. B. .'Storey and W. C. H u l b e r t . (1977) M e t a b o l i c b i o c h e m i s t r y o f water vs a i r b r e a t h i n g o s t e o g l o s s i d s : h e a r t enzymes and u l t r a s t r u c t u r e . Can. J . Z o o l . 5 6 ( P t . 2 ) : 759-768. S t o r e y , K. B., H. E. G u d e r l e y , M. Guppy and P. W. Hochachka. (1977) C o n t r o l o f ammoniagenesis i n t h e k i d n e y o f water and a i r b r e a t h i n g o s t e o g l o s s i d s : c h a r a c t e r i z a t i o n o f g l u t a m a t e dehydrogenase . Can. J . Z o o l . 5 6 ( P t . 2 ) : 845-851. Guppy, M. and P. W. Hochachka. (1978) P y r u v a t e k i n a s e i n the m u s c l e s o f warm-bodied f i s h . J . Exp. B i o l , s u b m i t t e d . Guppy. M. and P. W. Hochachka. (1978) C o m p e t i t i o n between dehydrogenases i n m e t a b o l i c r e g u l a t i o n : the c a s e o f l a c t a t e and a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e s . J . B i o l . Chem., i n p r e s s . H u l b e r t , W. C , M. Guppy and P. W. Hochachka. (1979) M e t a b o l i c s o u r c e o f h e a t and power i n tuna m u s c l e s . I . Rete and m u s t l e f i n e s t r u c t u r e . J . Exp. B i o l , ( s u b m i t t e d ) . Guppy, M., W. C. H u l b e r t and P. W. Hochachka. (1979) M e t a b o l i c s o u r c o f h e a t and power i n tuna m u s c l e s . I I . Enzyme and m e t a b o l i t e p r o f i l e s . J . Exp. B i o l , ( s u b m i t t e d ) . 

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