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Patterns of anaerobic metabolism in molluscan muscle Fields, Jeremy Harold Austin 1976

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PATTERNS OF ANAEROBIC METABOLISM IN MOLLUSCAN MUSCLE by JEREMY HAROLD AUSTIN FIELDS B . S c , M c G i l l U n i v e r s i t y , Montreal, 1970 M.Sc, M c G i l l U n i v e r s i t y , Montreal, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1976 (c) Jeremy Harold Austin F i e l d s In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i ABSTRACT Anaerobic metabolism i n cephalopod muscle and i n b i v a l v e adductor muscle depends on the coupling of carbohydrate and amino acid metabolism. In cephal-opod muscle t h i s i s acheived by octopine dehydrogenase (E.C.1.5.1.11) , whereas i n the oyster adductor muscle i t i s acheived by transaminases and malate dehydrogenase (E.C.1.1.1.37). Therefore studies of the c a t a l y t i c properties (a) of octopine dehydrogenase from muscle of a group of cephalopods, and (b) of cytoplasmic aspartate aminotransferase (E.C.2.6.1.1) and malate dehydrogen-ase from adductor muscle of the oyster, Crassostrea gigas, were undertaken. Higher a c t i v i t i e s of octopine dehydrogenase were found i n the mantle of Octopus ornatus than i n the mantle of Symplectoteuthis oualaniensis, but the c a t a l y t i c properties of both enzymes were s i m i l a r . The a f f i n i t y f o r pyruvate was low (K approx. 1.7 mM), but increased with increasing concentrations of arginine; the a f f i n i t y for arginine s i m i l a r l y increased with increasing con-centrations of pyruvate. Octopine dehydrogenase from the spadix muscle of the chambered n a u t i l u s , Nautilus pompilius, had a higher a f f i n i t y f o r pyruvate (K^ approx. 0.3 mM), and t h i s was also increased by increasing arginine concentra-tio n s . I t ' i s suggested that octopine dehydrogenase maintains redox balance i n a manner analogous to l a c t a t e dehydrogenase (E.C.I.1.1.27), and c l o s e l y couples g l y c o l y s i s with arginine phosphate metabolism, such that an anaerobic reserve i s provided for high i n t e n s i t y "burst" work. The octopus mantle r e l i e s on t h i s mechanism more so than does the mantle of the oceanic squid, S_. oualanien- s i s , and the Nautilus spadix muscle appears to use t h i s anaerobic process for most of i t s energetic requirements. In contrast to cephalopod muscle, oyster adductor muscle maintains redox balance through coupling aspartate and alanine metabolism with carbohydrate fermentation. Adductor aspartate aminotransferase had a higher a f f i n i t y f o r i i a s p artate than f o r glutamate, and a higher a f f i n i t y f o r 2-ketoglutarate than f o r o x a l o a c e t a t e , suggesting that i t would f u n c t i o n more r e a d i l y i n the d i r e c -t i o n of a s p a r t a t e u t i l i z a t i o n . Adductor malate dehydrogenase had a higher a f f i n i t y f o r oxaloacetate than d i d aspartate aminotransferase, hence the major f a t e of oxaloacetate produced would be conversion to malate, and t h i s would d i r e c t the f l o w of a s p a r t a t e carbon towards s u c c i n a t e . Since adductor a l a n i n e aminotransferase (E.C.2.6.1.2) i s k i n e t i c a l l y adapted f o r a l a n i n e formation, these enzymes couple g l y c o l y s i s w i t h aspartate m o b i l i s a t i o n , such that a l a n i n e i s formed from glucose and succinate from asp a r t a t e . In a d d i t i o n , i t was found that pyruvate had another p o s s i b l e f a t e during anoxia i n the adductor, that i s conversion to an as yet u n i d e n t i f i e d compound that i s produced by a dehydrogenase r e q u i r i n g NADH, a l a n i n e and pyruvate as substrates. This enzyme has an extremely low a f f i n i t y f o r a l a n i n e , and i s p o t e n t l y i n h i b i t e d by succinate at low pH; hence during anoxia production of t h i s compound would be l i m i t e d , and the pathway l e a d i n g to succinate produc-t i o n favoured. I i i i TABLE OF CONTENTS Page Abstract i Table of Contents 1 1 1 L i s t of Tables i v L i s t of Figures v i Acknowledgements x Chapter I . I n t r o d u c t i o n 1 Chapter I I . M a t e r i a l s and Methods 24 Chapter I I I . Octopine Dehydrogenase i n Cephalopod Muscle 35 I n t r o d u c t i o n 35 Results and D i s c u s s i o n 36 Chapter IV. Oyster Adductor C i t r a t e Synthase 64 I n t r o d u c t i o n 64 Results and D i s c u s s i o n 65 Chapter V. Role of Oyster Adductor Cytoplasmic Aspartate Aminotransferase During Anoxia 84 I n t r o d u c t i o n 84 Results and D i s c u s s i o n 87 Chapter VI. Role of Oyster Adductor Cytoplasmic Malate Dehydrogenase During Anoxia '. 115 I n t r o d u c t i o n 115 Results and D i s c u s s i o n 115 Chapter V I I . Discovery of a Dehydrogenase Requiring A l a n i n e and Pyruvate as Substrates 158 I n t r o d u c t i o n 158 Methods 159 Results and D i s c u s s i o n 159 Chapter V I I I . Summary: Pathways of Anaerobic Metabolism i n Molluscs 202 L i t e r a t u r e C i t e d 209 Appendix: L i s t of Abbreviations 221 i v LIST OF TABLES Table Page I. A c t i v i t i e s of ODH and aGPDH i n cephalopod muscle 37 I I . Octopus mantle muscle ODH: apparent K values f o r s u b s t r a t e s . . 41 I I I . Squid mantle muscle ODH: apparent values f o r substrates ... 42 IV. N a u t i l u s spadix ODH: apparent K m values f o r substrates 43 V. Squid mantle ODH: K. values f o r various i n h i b i t o r s 49 VI. Octopus mantle ODH: K_ values f o r various i n h i b i t o r s 50 VI I . N a u t i l u s spadix ODH: values f o r various i n h i b i t o r s 51 V I I I . E f f e c t s of va r i o u s i n h i b i t o r s on c i t r a t e synthase 67 IX. M i c h a e l i s constants f o r adductor aspartate aminotransferase ... 101 X. K m values of aspartate aminotransferase from v a r i o u s sources.. 109 XI. I n h i b i t i o n constants (K. values) f o r v a r i o u s i n h i b i t o r s of adductor cytoplasmic aspartate aminotransferase 110 X I I . M i c h a e l i s constants f o r oyster adductor malate dehydrogenase .. 133 X I I I . . K values f o r cytoplasmic MDH from v a r i o u s sources 135 m XIV. I n h i b i t i o n constants of various i n h i b i t o r s of cytoplasmic MDH.. 147 XV. Summary of NCEADH p u r i f i c a t i o n 165 XVI. U t i l i z a t i o n of NADH by NCEADH i n the presence of excess a l a n i n e and l i m i t i n g pyruvate 167 XVII. U t i l i z a t i o n of pyruvate and a l a n i n e by NCEADH ... 168 XVII I . Keto a c i d s p e c i f i c i t y of N - ( l - c a r b o x y e t h y l ) - a l a n i n e dehydrogenase 172 XIX. Amino a c i d s p e c i f i c i t y of N-(1-carboxyethyl)-alanine dehydrogenase 173 V Table Page XX. M l c h a e l i s constants f o r NCEADH 179 XXI. I n h i b i t i o n constants f o r the adenylates w i t h respect to NADH.. 187 XXII. I n h i b i t i o n constants f o r NAD + at d i f f e r e n t pH values 188 XXII I . I n h i b i t i o n constants f o r succinate and 2-ketoglutarate at v a r y i n g pH values 194 XXIV. PEPCKrPK a c t i v i t y r a t i o s i n marine molluscs 204 v i LIST OF FIGURES Figure ' Page 1. The g l y c o l y t i c pathway as i t occurs i n v e r t e b r a t e muscles 4 2. The proposed pathway of glucose degradation i n the mantle of R. cuneata -8 3. Summary of metabolic c o n t r o l at the PEP branchpoint i n o y s t e r adductor muscle 11 4. Proposed pathway of anaerobic intermediary metabolism i n i n v e r t e b r a t e f a c u l t a t i v e anaerobes 14 5. Proposed p a t t e r n of anaerobic metabolism i n the anoxic o y s t e r heart 18 6. Intermediary metabolism i n squid mantle muscle 21 7. C a l i b r a t i o n of Sephadex G-200 f o r molecular weight determination .. 33 8. E f f e c t of pH on squid mantle ODH 38 9. Pyruvate and a r g i n i n e s a t u r a t i o n k i n e t i c s of squid mantle muscle ODH 44 10. Squid mantle ODH: e f f e c t of covarying a r g i n i n e and pyruvate concentrations at constant arginine:pyruvate 46 11. P a t t e r n of octopine i n h i b i t i o n w i t h respect to pyruvate and a r g i n i n e s a t u r a t i o n k i n e t i c s of octopus mantle ODH 52 12. P a t t e r n of pyruvate i n h i b i t i o n w i t h respect to octopine s a t u r a t i o n k i n e t i c s 54 13. P a t t e r n of a r g i n i n e i n h i b i t i o n w i t h respect to octopine s a t u r a t i o n k i n e t i c s of squid and octopus mantle ODH 56 14. P o s t u l a t e d summary scheme of metabolic o r g a n i z a t i o n i n cephalopod muscle 60 v i i Figure Page 15. E f f e c t of pH on adductor c i t r a t e synthase 68 16. E f f e c t s of MgSO^, ATP and MgATP on acetylCoA s a t u r a t i o n k i n e t i c s of adductor c i t r a t e synthase 70 17. E f f e c t of ATP on oxaloacetate s a t u r a t i o n k i n e t i c s of adductor c i t r a t e synthase 72 18. E f f e c t of c i t r a t e on oxaloacetate s a t u r a t i o n k i n e t i c s 76 19. E f f e c t of c i t r a t e on acetylCoA s a t u r a t i o n k i n e t i c s 78 20. E f f e c t of 2-ketoglutarate on oxaloacetate s a t u r a t i o n k i n e t i c s .... 80 21. E f f e c t of 2-ketoglutarate on acetylCoA s a t u r a t i o n k i n e t i c s 82 22. Malate-aspartate s h u t t l e 85 23. Starch g e l e l e c t r o p h o r e s i s of adductor a s p a r t a t e aminotransferase. 88 24. E l u t i o n of adductor aspartate aminotransferase from DEAE-Sephadex 90 25. E f f e c t of pH on a c t i v i t y of cytoplasmic aspartate aminotransferase 93 26. Aspartate and glutamate s a t u r a t i o n k i n e t i c s of cytoplasmic aspartate aminotransferase 95 27. Oxaloacetate and 2-ketoglutarate s a t u r a t i o n k i n e t i c s of cytoplasmic aspartate aminotransferase 98 28. E f f e c t of glutamate on asparta t e s a t u r a t i o n k i n e t i c s 103 29. E f f e c t of succinate on asparta t e s a t u r a t i o n k i n e t i c s 105 30. E f f e c t of succinate on 2-ketoglutarate s a t u r a t i o n k i n e t i c s 107 31. Proposed couple between glucose and aspartate metabolism during anoxia 113 32. Starch g e l e l e c t r o p h o r e s i s of adductor MDH 116 33. E f f e c t of pH on adductor cytoplasmic MDH 119 v i i i F igure Page 34. Malate s a t u r a t i o n of oy s t e r adductor malate dehydrogenase 121 35. NAD + s a t u r a t i o n of oyster adductor malate dehydrogenase 123 36. Oxaloacetate s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH .... 125 37. NADH s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH 127 38. Malate s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH 129 39. NAD + s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH 131 40. Malate i n h i b i t i o n of adductor cytoplasmic MDH 138 41. Oxaloacetate i n h i b i t i o n of adductor cytoplasmic MDH 140 42. E f f e c t of NADH on NAD + and malate s a t u r a t i o n k i n e t i c s 142 43. Dixon p l o t of NADH i n h i b i t i o n of adductor cytoplasmic malate dehydrogenase 144 44. E f f e c t of c i t r a t e on malate s a t u r a t i o n k i n e t i c s 148 45. E f f e c t of 2-ketoglutarate on malate s a t u r a t i o n k i n e t i c s 150 46. E f f e c t of c i t r a t e on oxaloacetate s a t u r a t i o n k i n e t i c s 152 47. E f f e c t of 2-ketoglutarate on oxaloacetate s a t u r a t i o n k i n e t i c s ... 154 48. E l u t i o n of NCEADH from a 95 x 5 cm column of Sephadex G-200 161 49. E l u t i o n of NCEADH from DEAE-Sephadex A-50 163 50. E l u t i o n of NCEA and a l a n i n e from a 40 x 2.5 cm column of Dowex 50W 170 51. E f f e c t of pH on NCEADH a c t i v i t y 175 52. NADH s a t u r a t i o n k i n e t i c s of adductor NCEADH 177 53. Pyruvate s a t u r a t i o n k i n e t i c s of adductor NCEADH at v a r y i n g a l a n i n e concentrations 181 54. Alanine s a t u r a t i o n k i n e t i c s at v a r y i n g concentrations of pyruvate 183 i x F igure Page 55. E f f e c t s of NAD + and ATP on NADH s a t u r a t i o n k i n e t i c s of NCEADH 185 56. P a t t e r n of succinate i n h i b i t i o n w i t h respect to pyruvate s a t u r a t i o n k i n e t i c s of NCEADH 189 57. P a t t e r n of succinate i n h i b i t i o n w i t h respect to al a n i n e s a t u r a t i o n k i n e t i c s of NCEADH 191 58. P a t t e r n of i n h i b i t i o n by 2-ketoglutarate of NCEADH wi t h respect to al a n i n e 195 59. I n h i b i t i o n of NCEADH by 2-ketoglutarate w i t h respect to pyruvate.. 197 60. P o s t u l a t e d metabolic map of anaerobic metabolism i n the cytoplasm of oyster adductor muscle 200 61. P o s t u l a t e d scheme f o r anaerobic metabolism of malate i n the mitochondria of b i v a l v e s 207 X ACKNOWLEDGEMENTS Thank you Peter f o r l e t t i n g me work on REAL ANIMALS while I was i n your lab. Many f a c u l t y and students have contributed to my education, research and s p i r i t u a l well-being over the past four years; i f your name does not appear among the following, then I apologize for my unthoughtfulness: W. S. Hoar, David Randall, John P h i l l i p s , John Gosline, Ray Reeves, David Jones, Ken Storey, Wiliam Driedzic, Terry Owen, Helga Guderley, John Himmelman, Michael Guppy, Janet C o l l i c u t t , Carol Norberg, Mark Anderson, Steve Haswell, Tony F a r r e l l , John Bailey, Barbara Moon, P o l l y Haswell, Bob Bryan. I was a r e c i p i e n t of a National Research Council Postgraduate Scholarship f o r part of my studies. Dr. Robert Kane and the s t a f f of the P a c i f i c Biomedical Research Center, Kewalo Basin, Hawaii, provided f a c i l i t i e s f o r part of th i s study, and t h i s was much appreciated. Thank you L e s l i e for typing the blasted manuscript, i t i s a chore. L a s t l y , without the incentive from the f a c u l t y members of the Depart-ment of Zoology, University of Washington, I doubt that I would ever have fi n i s h e d . 1 CHAPTER I INTRODUCTION The Scope of Anaerobiosls i n Molluscs A mammal was a f o r t u n a t e choice by L a v o i s i e r to study the r o l e of oxygen i n metabolism, because many animal species have s u c c e s s f u l l y invaded h a b i t a t s which have a very low or zero oxygen content, and others s u r v i v e i n areas . which are subject to p e r i o d i c anoxic s t r e s s . Several organisms which l i v e under permanently hypoxic or anoxic c o n d i t i o n s , such as A s c a r i s l u m b r i c o i d e s , possess a l t e r e d metabolic p a t t e r n s which preclude the u t i l i z a t i o n of oxygen to a s i g n i f i c a n t degree even i f present (Saz, 1971a). Many other species l i v i n g i n the i n t e r t i d a l area, f o r example b i v a l v e molluscs, use oxygen when present, but are a l s o capable of s u r v i v i n g v a r y i n g periods of anoxia. The term " f a c u l t a t i v e anaerobe" i s o f t e n used to describe these species (Hochachka and Somero, 1973). Although i n t e r t i d a l forms would only r a r e l y be exposed to anoxic s t r e s s f o r more than 24 hours, under l a b o r a t o r y c o n d i t i o n s s u r v i v a l f o r much longer periods i s p o s s i b l e . For example, L i t t o r i n a n e r i t o i d e s and L_. punctata can s u r v i v e s e v e r a l weeks i n pure n i t r o g e n (Patane, 1946a,b, 1955), Mya a r e n a r i a up to 8 days ( R i c k e t t s and C a l v i n , 1948), Rangia cuneata f o r 3 weeks (Chen and Awapara, 1969b), and Crassostrea gigas can l i v e f o r 22 days out of water w i t h no measurable oxygen uptake (Pedlow, 1974). Freshwater gastropods have more modest c a p a c i t i e s of 2-3 days (von Brand ejt a i l . , 1950), although Cole (1921) reported that P i s i d i u m idahoense survived the w i n t e r on the bottom of Lake Mendota (Wisconsin) where the oxygen content of the water was too low to be measured by techniques then a v a i l a b l e . More r e c e n t l y , Cepaea nemoralis (a t e r r e s t r i a l pulmonate) has been shown to s u r v i v e i n a n i t r o g e n atmosphere f o r 1 month (van der Horst, 1974). Cephalopods are g e n e r a l l y thought to be i n t o l e r a n t of hypoxic c o n d i t i o n s ( G h i r e t t i , 1966; Hochachka et a l . , 1975), 2 but the i s o l a t e d systemic heart of Octopus d o f l e i n i was found to withstand 48 hours of anoxic conditions (Pritchard et^ a l . , 1963). The oxygen storage capacity of bivalves (hemoglobin, hemocyanin and myoglobin) i s very l i m i t e d (ZsNagy, 1974) such that within a few minutes of s h e l l closure a l l oxygen stores are f u l l y depleted. In the oyster, C_. gigas, the pO^ of the p a l l i a l f l u i d decreased from 160 mmHg to 40-50 mmHg within a few minutes of s h e l l closure, and remained at t h i s l e v e l f o r several days (Pedlow, 1974), and a decrease from 130 to 0 mmHg within an hour of s h e l l closure has been recorded for the p a l l i a l f l u i d of Mytilus c a l i f o r n i a n u s (Moon and P r i t c h a r d , 1970). S h e l l closure i s also accompanied with a decrease i n pH from approximately 7.5 to approximately 6.5-6.7 within a few hours (Pedlow, 1974; Crenshaw and Neff, 1969; Wijsman, 1975). Thus, a f t e r an i n i t i a l short period, oxygen dependent metabolism can no longer function, so the b ivalve must modify i t s metabolism i n such a way that the energy require-ments of the tis s u e can s t i l l be met. An important adaptation i n t h i s regard i s a decrease i n the energy requirements of some tissues f o r example the heart rate i s decreased about 15-fold i n Mytilus edulis (Bayne, 1971) and about 20-fold i n Pecten maximus (Brand and Roberts, 1973), and also the c i l i a r y beat ceases i n M. edulis and Modiolus demissus (Malanga and A i e l l o , 1972). Energy Stores and End Products During Anoxia C e l l s which are capable of sustained anoxic metabolism have some pro-v i s i o n f o r (1) a storage form of energy which i s u t i l i z e d during anoxia, (2) the maintenance of redox balance i n the absence of the normal terminal electron acceptor, oxygen, and (3) the generation of high energy phosphate compounds such as ATP*. Several organisms use anaerobic g l y c o l y s i s to meet *See Abbreviations Used. 3 these requirements, where glycogen, the storage form of energy, i s metabolized to the l e v e l of l a c t a t e during anaerobiosis ( F i g . 1). Redox balance i s main-ta i n e d by c o u p l i n g LDH and GAPDH, thus maintaining l e v e l s of NAD + r e q u i r e d 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 . Substrate l e v e l phosphorylation at the PK and PGK l e v e l s s u p p l i e s ATP required f o r work f u n c t i o n s i n the c e l l . G l y c o l y s i s has been e x t e n s i v e l y s t u d i e d i n v e r t e b r a t e s k e l e t a l muscle (see Drummond, 1966) where i t s main f u n c t i o n i s to supply energy f o r short term, high l e v e l s of a c t i v i t y when the a e r o b i c c a p a c i t y of the system i s exceeded. However, t u r t l e s and d i v i n g mammals have accentuated the g l y c o l y t i c pathway as an adaptation to prolonged periods of anoxia (Hochachka and Storey, 1975). Several molluscs a l s o s t o r e glycogen i n t h e i r t i s s u e s , the highest l e v e l s being found i n the adductor muscles of some b i v a v l e s (Goddard and M a r t i n , 1966; Giese, 1969). Decrease i n glycogen s t o r e s during anoxia has been shown i n 16 species of freshwater gastropods (von Brand et_ a l . , 1950), the o y s t e r , C^. v i r g i n i c a ( G a l s t o f f , 1964 ), the b i v a l v e s M. e d u l i s (de Zwaan and Zandee, 1972), Anodonta cygnea (GSde ^ t a l . , 1975) and Cardium edule (GMde, 1975). Other carbohydrates, such as galactogen, are a l s o found i n some molluscs (Goudsmit, 1972) but no s t u d i e s have been performed to demonstrate u t i l i z a -t i o n of these during anoxia. Molluscs produce m u l t i p l e end products as a r e s u l t of g l y c o l y s i s under anaerobic c o n d i t i o n s . In some species of gastropods, f o r example Lymnaea s t a g n a l i s and Lynmaea n a t a l e n s i s , l a c t a t e i s a major end product (von Brand et^ a l . , 1950) but others such as A u s t r a l o r b i s glabratus and Helisoma d u r y i produce acetate and propionate as major end products (Mehlman and von Brand, 14 1951). Several b i v a l v e s have been shown to accumulate C-succinate when 14 incubated w i t h C-glucose, i n c l u d i n g R. cuneata, C_. v i r g i n i c a , V o l s e l l a 4 Figure 1. The g l y c o l y t i c pathway as i t occurs i n vertebrate muscles. 5 glycogen glyceraldehyde-3-P* • dihydroxyacetone-P "•NAD* NADH Pi 1,3-diphosphoglycerate -ADP "ATP 3-phosphoglycerate 2-phosphoglycerate ^ H 2 0 phosphoenolpyruvate -ADP r ATP pyruvate NADH-lactate 6 demissus (Simpson and Awapara, 1966) and M. e d u l i s (de Zwaan and van Marre-w i j k , 1973). In a d d i t i o n a l a n i n e i s accumulated during anoxia i n R. cuneata (Stokes and Awapara, 1968), M. e d u l i s (de Zwaan and Zandee, 1972), A. cygnea (Gade j i t a l . , 1975), Cardium edule (Gade, 1975) and C. gigas ( C o l l i c u t t , 1975). Prolonged anoxia leads to the production of short chain f a t t y a c i d s , acetate and propionate i n M. e d u l i s (Klutymans at a l . , 1975), A. cygnea (Gade et a l . , 1975) and Cardium edule (GMde, 1975). In t h i s regard i n t e r -t i d a l b i v a l v e s resemble i n t e s t i n a l p a r a s i t e s which produce su c c i n a t e and a v a r i e t y of short chain f a t t y a c i d s during anaerobiosis (von Brand, 1966). Metabolic O r g a n i z a t i o n A l l of the g l y c o l y t i c enzymes are present i n M. c a l i f o r n i a n u s and H a l i o t u s rufescens (Bennett and Nakada, 1968), and some have been described i n a number of other species (Goddard and M a r t i n , 1966; Goudsmit, 1972; G i l l e s , 1972). However i n order to account f o r succinate formation, carboxy-l a t i o n of a t r i o s e or pyruvate must occur. Simpson and Awapara (1964) demonstrated the presence of PEPCK i n 9 species of i n t e r t i d a l molluscs, and hypothesized that conversion of PEP to oxaloacetate was the major pathway f o r C O 2 i n c o r p o r a t i o n i n t o amino and d i c a r b o x y l i c a c i d s . Because LDH a c t i v i t i e s were found to be unusually low i n i n t e r t i d a l m o lluscs, i t was suggested that cytoplasmic MDH would provide an a l t e r n a t e means of ma i n t a i n i n g redox balance during anoxia (Simpson and Awapara, 1966), i n the f o l l o w i n g way: NAD + NADH NADH NAD+ Glucose-* —>•——>• —>—> PEP > oxaloacetate >malate->->succinate This scheme proposed f o r b i v a l v e t i s s u e s resembles the p a t t e r n found i n A s c a r i s lumbricoides (Saz and Lescure, 1969) and i t was presumed that s u c c i n -ate formation from malate occurred i n a s i m i l a r f a s h i o n i n both A s c a r i s and 7 i n t e r t i d a l b i v a l v e s . Subsequently i t was shown that s u c c i n a t e was not the only major end product, a l a n i n e was a l s o produced by mantle t i s s u e of R. cuneata (Stokes and Awapara, 1968) and a l s o M. e d u l i s (de Zwaan and van 14 Marrewijk, 1973). In R. cuneata approximately equal amounts of C were 1 4 incorporated i n t o a l a n i n e and succinate from C-glucose (Stokes and Awapara, 14 14 1968) whereas i n M. e d u l i s the r a t i o of C-alanine: C-succinate was 2:1 (de Zwaan and van Marrewijk, 1973), although succinate and a l a n i n e accumulate i n equimolar amounts (de Zwaan and Zandee, 1972). Stokes and Awapara (1968) proposed that glucose was metabolized normally to the l e v e l of PEP at which po i n t a dismutation occurred, one h a l f of the glucose carbon being converted to pyruvate and then to a l a n i n e , one h a l f being converted to oxaloacetate and then to succinate ( F i g . 2). Since 1 mole of NADH i s formed per mole PEP, reduc t i o n of oxaloacetate to malate would regenerate h a l f of the req u i r e d NAD+, there f o r e i t was proposed that the re d u c t i o n of fumarate to succinate would a l s o regenerate NAD + and hence maintain the system i n redox balance. S u b c e l l u l a r l o c a l i z a t i o n of the enzymes i n v o l v e d i n a l a n i n e and suc c i n a t e formation showed that a l l were present i n the cytoplasm w i t h the exception of fumarate reductase, which c a t a l y z e s the formation of succinate from fumarate (Chen and Awapara, 1969a). The enzyme "fumarate reductase" i s the same as succinate dehydrogenase (E.C. 1.3.99.1), which i s capable of f u n c t i o n i n g i n both d i r e c t i o n s (Singer, 1973). In A s c a r i s muscle, however, fumarate r e d u c t i o n i s favoured over succinate o x i d a t i o n (Kmectec and Beuding, 1961). Hammen and Lum (1966) i n v e s t i g a t e d the r e l a t i v e a c t i v i t i e s of fumarate r e d u c t i o n and succinate o x i d a t i o n i n s e v e r a l b i v a l v e s , and concluded that the species more t o l e r a n t to anoxia were more capable of producing s u c c i n a t e , having the higher fumar-8 Figure 2. The proposed pathway of glucose degradation i n the mantle of R. cuneata (adapted from Stokes and Awapara, 1968). glucose f r u c t o s e - l ^ - d i P ->NAD+ NADH phosphoenolpyruvate HCO; oxaloacetate NADH NAD malate fumarate NADH NAD +" succinate pyruvate •glutamate 2 - K G A alanine 10 ate reductase:succinate dehydrogenase r a t i o s . Succinate dehydrogenase nor-mally r e q u i r e s a f l a v o p r o t e i n as an e l e c t r o n acceptor (Singer, 1973), so when NADH i s the e l e c t r o n donor f o r fumarate r e d u c t i o n the p o t e n t i a l of using the f i r s t step i n o x i d a t i v e phosphorylation e x i s t s . This has been shown to occur i n F a s c i o l a h e p a t i c a (de Zoeten and Tipk e r , 1969), A s c a r i s lumbricoides (Saz, 1971b), and i n the anaerobic r a t heart (Wilson and Cascarano, 1970), hence the advantage of succinate production during anoxia. More r e c e n t l y Hammen (1975) has shown fumarate r e d u c t i o n to be st i m u l a t e d t h r e e - f o l d by ADP i n e x t r a c t s of the adductor muscle of M. e d u l i s , and a l s o succinate o x i d a t i o n was found to be g r e a t l y s t i m u l a t e d by ATP, suggesting that i n t h i s species a d d i t i o n a l ATP i s obtained by producing succinate during anoxia. 14 Although the f i x a t i o n of CO^ i n t o succinate had been e s t a b l i s h e d f o r oyster mantle (Hammen and Wilbur, 1959), the exact pathway was i n dis p u t e . Hammen (1966) claimed that the low a c t i v i t y of PEPCK precluded the formation of oxaloacetate being the major route, and i n s t e a d proposed that PEP was con-verted to pyruvate and that c a r b o x y l a t i o n of pyruvate to malate by m a l i c enzyme (E.C. 1.1.1.40) was the main pathway. This problem of PEP metabolism was r e i n v e s t i g a t e d by Mustafa (1972) i n a study of the c a t a l y t i c and r e g u l a -t o r y p r o p e r t i e s of PK and PEPCK from the adductor muscle of C_. gigas. The r e s u l t s of t h i s study suggested that PK and PEPCK were regulated i n such a way that they were not simultaneously a c t i v e ; PK would f u n c t i o n under aerobic c o n d i t i o n s and PEPCK under anaerobic c o n d i t i o n s . This e f f e c t would be pro-duced by i n c r e a s i n g concentrations of H + and a l a n i n e i n h i b i t i n g PK and simultaneously a c t i v a t i n g PEPCK ( F i g . 3) (Hochachka and Mustafa, 1972). Subsequently the p o t e n t i a l f o r a s i m i l a r e f f e c t was shown f o r M. e d u l i s through s t u d i e s on PK and PEPCK from the adductor muscle (de Zwaan and H o i -11 Figure 3. Summary of metabolic control at the PEP branchpoint i n oyster adductor muscle (from Hochachka and Mustafa, 1972). 12 FDP 5 i i 6 7 pH 8 13 werda, 1972; Holwerda and de Zwaan, 1973; de Zwaan and de Bont, 1975). In this proposal the role of MDH in maintaining cytoplasmic redox balance be-comes central to the channelling of a l l glucose carbon into oxaloacetate, rather than a dismutation at the level of PEP. The malate formed was consid-ered to have two possible fates: (1) conversion to fumarate and then to succinate, or (2) conversion to pyruvate by malic enzyme (E.C. 1.1.1.40), which possesses kinetic properties favouring malate decarboxylation rather than pyruvate carboxylation (Hochachka and Mustafa, 1973). The pyruvate thus formed would be converted to alanine by transamination with glutamate, yi e l d -ing 2-ketoglutarate which could then be converted to succinylCoA and subse-quently to succinate producing an additional high energy phosphate at the succinyl thiokinase step (Fig. 4) and also intramitochondrial NADH for fumar-ate reduction. Free amino acid levels in bivalves and other marine molluscs are high (Lynch and Wood, 1966; Schoffeneils and Gilles, 1972; Campbell and Bishop, 1970), where they play a role in adaptation to changing salinity through cellular volume regulation (Pierce, 1971; Pierce and Greenberg, 1972, 1973; Baginski and Pierce, 1975). DuPaul and Webb (1970) recognize a "fast" and "slow" component in adaptation to a higher salinity, alanine increase being the "fast" component occurring within a few hours of the salinity change. Further studies on the isolated g i l l of Mya arenaria showed that under anoxic conditions and also with an increase in salin i t y , the increase in alanine was correlated with a decrease in aspartate levels, with no net change in gluta-mate (Du Paul and Webb, 1971). Baginski and Pierce (1975) have also re-corded a decrease in aspartate that is correlated with an increase of alanine. The possibility exists, therefore, for the coupling of amino acid and 14 Figure 4. Proposed pathway of anaerobic intermediary metabolism i n i n v e r t e b r a t e f a c u l t a t i v e anaerobes (from Hochachka and Mustafa, 1972). 15 G6P T F6P FDP (~3M>- T R I O ? S E - P NADH 1,3-DPG ^ . TI SI v-—ADP y m-ATP 3-PG •DP fplpT >XXI W A \ y •nalate^^-|pyruvate| . NADP NADPH Itumaratel • Cytoplasm H*- pyruvate _ ^ _ q l u t a m a t e ESaEil -^Tkttoglutarate NAD—\T . . —NADH^Y succinylCoA N^NADH NAD { ^ G T P - fumnrateVs-^i>'<succinote| Mitochondrion 16 carbohydrate metabolism to respond e i t h e r to anoxic st r e s s or to s a l i n i t y s t r e s s . Few studies have been done on amino a c i d catabolism under anoxia i n bivalve s . In M. edu l i s concentrations of aspartate and glutamate were not found to change s i g n i f i c a n t l y during anoxia (de Zwaan and van Marrewijk, 1973), therefore another source of amino groups for alanine formation i s r e -quired. One poss i b l e source i s NH* f i x a t i o n v i a glutamate dehydrogenase (E.C. 1.4.1.2), and since glutamate dehydrogenase occurs i n M. e d u l i s t i s s u e s (de Zwaan and van Marrewijk, 1973), de Zwaan, van Marrewijk and Holwerda (1973) proposed that alanine aminotransferase and glutamate dehydrogenase were f u n c t i o n a l l y coupled i n alanine formation. Pyruvate + Glutamate < Alanine + 2-ketoglutarate 2-ketoglutarate + NH* + NADH^ ^ Glutamate + NAD + + H 20 Studies on the i s o l a t e d heart of C^. gigas showed that glutamate decreased s l i g h t l y during anoxia whereas aspartate l e v e l s decreased by about 5 to 10 mM 14 i n 1 hour ( C o l l i c u t t , 1975). Also i t was found that C-glucose was converted 14 mainly to alanine and C-aspartate mainly to succinate. Furthermore alanine and succinate were not formed i n an equimolar r a t i o as occurs i n M. e d u l i s , but i n a r a t i o of 2 a l a n i n e : ! succinate. In ad d i t i o n a t h i r d major end product i s formed which i s metabolically c l o s e l y l i n k e d to alanine ( C o l l i c u t t , 1975); therefore anoxic metabolism i n the oyster heart follows a d i f f e r e n t pattern from that established for mantle tis s u e of R. cuneata (Stokes and Awapara, 1968) and whole M. edulis (de Zwaan and Zandee, 1972; de Zwaan and van Marrewijk, 1973; Klutymans ^it a l . , 1975). A possible means of explaining the coupling between carbohydrate and aspartate metabolism i n the oyster heart i s by means of a transaminase couple 17 ( F i g . 5) w i t h a l a n i n e aminotransferase and aspartate aminotransferase ( C o l l i -c u t t , 1975) which permits maintenance of cytoplasmic redox balance by MDH. This i s s i m i l a r to the scheme proposed f o r aspartate m o b i l i z a t i o n i n d i v i n g mammals during anoxia (Owen and Hochachka, 1974). I t i m p l i e s that some means are a v a i l a b l e to the c e l l f o r maintaining high l e v e l s of a s p a r t a t e under aerobic c o n d i t i o n s , and then m o b i l i z i n g aspartate under anaerobic c o n d i t i o n s . The aspartate would be m o b i l i z e d to malate i n the cytoplasm, and t h i s would then be transported i n t o the mitochondria where the conversion to succinate occurs. I f t h i s i s the case, then the span of r e a c t i o n s i n the Krebs c y c l e 14 l e a d i n g from malate to 2-ketoglutarate must be stopped. Lack of CO2 pro-duction from glucose during anoxia ( C o l l i c u t t , personal communication) sug-gests that at l e a s t the dec a r b o x y l a t i o n r e a c t i o n s are not f u n c t i o n i n g to any s i g n i f i c a n t degree. The Anaerobic Reserve of Cephalopod Muscle Cephalopod molluscs are among the more a c t i v e i n v e r t e b r a t e s , a l l being predators of crustaceans or f i s h . Among the commoner species of octopods and decapods, the main method of swimming i s by j e t p r o p u l s i o n which i n v o l v e s vigorous use of the mantle musculature. The mantle muscle, however, i s a l s o i n v o l v e d i n normal r e s p i r a t o r y movements; hence i t s metabolism must be geared f o r continuous l o w - l e v e l work, and a l s o very h i g h - l e v e l work d u r i n g a c t i v e swimming whether i n hunting prey or escaping a predator. U n t i l r e c e n t l y very l i t t l e was known about cephalopod muscle metabolism, and i t was assumed to be s i m i l a r to that of v e r t e b r a t e s . However, u n l i k e v e r t e b r a t e s which u t i l i z e f a t f o r long-term, steady s t a t e work and glycogen f o r high i n t e n s i t y burst a c t i v i t y (Drummond, 1966, 1971), cephalopods use carbohydrate at a l l times (Hochachka et a l . , 1975). I t has been shown that 18 Figure 5. Proposed p a t t e r n of anaerobic metabolism i n the anoxic o y s t e r heart (from C o l l i c u t t , 1975). 19 g l u c o s e - 6 - P t r i o s e - P NAD -NADH + H V 1,3-DPG •ADP + P. ATP 3-PG V PEP ADP + P. ATP pyruvate a s p a r t a t e — - glutamate • • •., d - k e t o g l u t a r a t e • * K—> ot-ketoglutarate**' "••. glutamate 4 f I I Cytoplasm a l a n i n e o x a l o a c e t a t e --=7 NADH + H NAD+<= malate Mitochondrion + + NADH + H NAD FP s u c c i n a t e FP red fumarate ATP ADP + P. 20 the mantle muscle of the squid, Symplectoteuthis o u a l a n i e n s i s , contains very l i t t l e f a t , about 0.3 g carbohydrate/100 g dry wt, and possesses a l l of the g l y c o l y t i c enzymes i n high a c t i v i t y . Hence, mantle muscle presumably uses carbohydrates as i t s major energy source f o r work (Hochachka e^ t a l . , 1975). Few measurements of oxygen consumption of cephalopods have been made, but that of Sepia o f f i c i a n a l i s and Octopus d o f l e i n i i s q u i t e high (Goddard and M a r t i n , 1966; G h i r e t t i , 1966; Johansen and Lenfant, 1966), which suggests that the metabolism must be q u i t e a e r o b i c ; the very h i g h l y developed v a s c u l a r system, and the high m i t o c h o n d r i a l mass i n S_. o u a l a n i e n s i s (Moon and Hulbert, 1975), a l s o support t h i s contention. A very i n t e r e s t i n g f i n d i n g was the l a c k of s i g n i f i c a n t q u a n t i t i e s of LDH i n the mantle muscle. Maintenance of cytoplasmic redox balance i s necessary, and high a c t i v i t i e s of cxGPDH and aGP oxidase were found i n the muscle (Hochach-ka et_ _ a l . , 1975), i n d i c a t i n g the presence of an a c t i v e aGP s h u t t l e . The o r g a n i z a t i o n seemed to be s i m i l a r to that found i n i n s e c t f l i g h t muscle (Sacktor, 1970), and hence appeared to be t o t a l l y a erobic. Maintenance of cytoplasmic redox balance i s necessary, and the aGP c y c l e f u r n i s h e s one method of a c h i e v i n g t h i s under aerobic c o n d i t i o n s by t r a n s f e r r i n g c y t o p l a s m i c a l l y derived NADH equivalents across the m i t o c h o n d r i a l membrane (Sacktor and Dick, 1962) ( F i g . 6). Some molluscs, however, have evolved octopine dehydrogenase (ODH) (E.C. 1.5.1.11) which f u n c t i o n s i n an analogous manner to LDH (van Thoai and Robin, 1961; van Thoai et_ a l . , 1969; Regnouf and van Thoai, 1970), the end product of anaerobic g l y c o l y s i s being octopine ra t h e r than l a c t a t e . Octopine has been found i n the mantle of Sepia and Octopus (van Thoai and Robin, 1961), suggesting that cephalopods have r e t a i n e d some anaerobic c a p a c i -ty i n the muscle des p i t e some very impressive aerobic c a p a c i t i e s . 21 Figure 6. Intermediary metabolism i n squid mantle muscle (from Hochachka et a l . , 1975). 22 GLYCOGEN 23 Objectives of Study From the above c o n s i d e r a t i o n s , i t i s c l e a r that cephalopods and b i v a l v e s are two groups of organisms w i t h a considerable d i f f e r e n c e i n anaerobic capa-c i t y . There i s , however, a common feature between the two groups which i s expressed as a c l o s e connection between carbohydrate and amino a c i d metabolism. Nei t h e r the nature of t h i s connection nor i t s metabolic s i g n i f i c a n c e has been f u l l y described. Therefore, t h i s study was addressed to some of the outstanding questions of metabolic c o n t r o l i n these d i f f e r e n t groups of molluscs: ( 1 ) the r o l e of ODH i n redox r e g u l a t i o n of cephalopod muscle; (2) the r o l e of a l a n i n e and asparta t e aminotransferases (E.C. 2.6.1.2 and E.C. 2.6.1.1), and MDH i n coupling amino a c i d and glucose metabolism i n oyster adductor muscle; (3) the probable nature of the unknown compound produced dur-in g anoxia i n the oyste r heart; and (4) the r o l e of c i t r a t e synthase i n reduc-i n g Krebs c y c l e f u n c t i o n during anoxia i n the oyster adductor muscle. I t was found that i n cephalopods ODH fu n c t i o n s as an anaerobic reserve f o r " b u r s t " work c o n d i t i o n s , and the more quiescent species r e l y more on t h i s mechanism than the a c t i v e oceanic species. In the oyste r adductor muscle, a l a n i n e and aspartate aminotransferases are k i n e t i c a l l y s u i t e d to form a transaminase couple l e a d i n g to asparta t e d e p l e t i o n and a l a n i n e accumulation, and to aspar-t a t e carbon flow i n t o succinate. Furthermore, i t was found that pyruvate could have two f a t e s , conversion to a l a n i n e , or metabolism by an enzyme r e q u i r -i n g NADH, a l a n i n e and pyruvate as s u b s t r a t e s , which could account f o r the formation of the unknown compound by the anoxic o y s t e r heart. These systems i n v o l v e a simultaneous m o b i l i z a t i o n of amino a c i d s and carbohydrate, and pro-v i d e mechanisms f o r the maintenance of cytoplasmic redox balance. CHAPTER I I MATERIALS AND METHODS 24 Oysters were obtained from B i l l i n g s g a t e F i s h L t d . , Vancouver and sto r e d i n running, r e c i r c u l a t e d sea water at 15°C f o r at l e a s t one week before use. S_. o u a l a n i e n s i s were obtained from a commercial fisherman i n Hawaii, having been kept on i c e a f t e r capture. Octopus ornatus, Octopus cyanea and Euprymna  scolopes were c o l l e c t e d from shallow water r e e f s i n Hawaii. N a u t i l u s pompi- l i u s were c o l l e c t e d by Dr. Hochachka on the "Alpha H e l i x " e x p e d i t i o n to the P h i l i p p i n e s i n November 1975; spadix muscle was d i s s e c t e d from f r e s h l y k i l l e d animal, f r o z e n , and flown to Vancouver f o r the enzyme s t u d i e s . DEAE-Sephadex A-50 and Sephadex G-200 were purchased from Pharmacia Fine Chemicals, Montreal; h y d r o x y l a p a t i t e was a product of Bio Rad L a b o r a t o r i e s , Richmond, C a l i f o r n i a . NAD +-agarose and acetylCoA were purchased from P-L Biochemicals, Milwaukee, Wisconsin; a l l other biochemicals and commercial enzymes were obtained from Sigma Chemical Co., St. L o u i s , M i s s o u r i . Other chemicals used were reagent grade. A l l enzymes were assayed s p e c t r o p h o t o m e t r i c a l l y using a Unicam SP1800 UV Spectrophotometer equipped w i t h a Unicam AR25 l i n e a r recorder and a con-stant temperature c e l l holder. S p e c i f i c a c t i v i t y of an enzyme i s expressed as units/mg p r o t e i n . P r o t e i n Determination P r o t e i n concentrations 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, using the formula below (Layne, 1957). P r o t e i n (mg/ml) = 1.55 OD 2 g 0 - 0.76 OD 2 6 Q Assay of C i t r a t e Synthase C i t r a t e synthase was assayed by f o l l o w i n g the production of CoA w i t h 25 DTNB according to the method of Srere (1969). Routine assays were performed w i t h 0.05 mM acetylCoA, 0.05 mM oxaloacetate, 0.25 mM DTNB i n 50 mM T r i s - H C l pH 8.0 at 25°C. One u n i t of enzyme a c t i v i t y was defined as the amount c a t a -l y z i n g the formation of 1 umole CoA/min at 25°C, the e x t i n c t i o n c o e f f i c i e n t 3 of DTNB-CoA being taken as 13.6 x 10 (Srere, 1969). Assay of Aspartate Aminotransferase Aspartate + 2-ketoglutarate < ?• oxaloacetate + glutamate Aspartate ^ oxaloacetate d i r e c t i o n : The production of oxaloacetate was measured by coupling the r e a c t i o n w i t h an excess of MDH and NADH, the r a t e of oxaloacetate production being equal to the r a t e of o x i d a t i o n of NADH as determined by the decrease i n o p t i c a l d e n s i -ty at 340 nm. Each assay contained 0.2 mM NADH and approximately 12 u n i t s of commercial MDH i n a d d i t i o n to the s u b s t r a t e s . Oxaloacetate >aspartate d i r e c t i o n : The r a t e of production of 2-ketoglutarate was followed by co u p l i n g w i t h glutamate dehydrogenase. Twenty u n i t s of commercial glutamate dehydrogenase, 20 mM (NH^) 2S0^ and 0.2 mM NADH were added to each assay i n a d d i t i o n to the sub s t r a t e s . In both cases 0.1 M sodium phosphate b u f f e r of appropriate pH was used, and the r e a c t i o n was i n i t i a t e d by the a d d i t i o n of approximately 0.03 u n i t s of p a r t i a l l y p u r i f i e d aspartate aminotransferase. Routine assay c o n d i t i o n s were 40 mM a s p a r t a t e , 1 mM 2-ketoglutarate, 0.2 mM NADH, excess MDH i n 0.1 M sodium phosphate pH 7.4 at 25°C. One u n i t of enzyme a c t i v i t y was defined as the amount of enzyme producing 1 umole oxaloacetate per minute at 25°C, the 3 e x t i n c t i o n c o e f f i c i e n t of NADH being taken as 6.22 x 10 at 340 nm. 26 Assay of Malate Dehydrogenase NAD + + malate < > oxaloacetate + H + + NADH The r e a c t i o n was followed by measuring the r a t e of increase i n o p t i c a l d e n s i t y at 340 nm, which i s p r o p o r t i o n a l to the r a t e of NADH production, or by measuring the r a t e of decrease i n o p t i c a l d e n s i t y at 340 nm, which i s pro-p o r t i o n a l to the r a t e of NADH o x i d a t i o n . A l l assays were performed at 25°C i n 0.1 M T r i s - H C l pH 8.9 i n the malate —> oxaloacetate d i r e c t i o n ; 0.1 M T r i s -HC1 pH 7.5 and 0.1 M imidazole-HCl pH 6.5 were used i n the oxaloacetate > malate d i r e c t i o n . Routine assay c o n d i t i o n s were 0.1 mM NADH, 0.8 mM oxalo-acetate i n 0.1 M T r i s - H C l pH 7.5. One u n i t of malate dehydrogenase a c t i v i t y was defined as the amount of enzyme c a t a l y z i n g the o x i d a t i o n of 1 ymole NADH/min at 25°C. Assay of Octopine Dehydrogenase The f o l l o w i n g r e a c t i o n i s c a t a l y z e d by octopine dehydrogenase: NADH + a r g i n i n e + pyruvate < > octopine + NAD + + ^ 0 The enzyme a c t i v i t y was assayed by f o l l o w i n g the change i n o p t i c a l den-s i t y at 340 nm. A l l assays were performed at 25°C. In the d i r e c t i o n of octopine formation assays were performed i n 100 mM i m i d a z o l e - C l pH 7.0 b u f f e r , i n the reverse d i r e c t i o n , 100 mM T r i s - H C l pH 8.5 was used. Routine assay c o n d i t i o n s were 0.2 mM NADH, 30 mM a r g i n i n e , 6 mM pyruvate, i n 100 mM imida-zole-HCl pH 7.0 at 25°C. One u n i t of octopine dehydrogenase a c t i v i t y was defined as the amount of enzyme c a t a l y z i n g the o x i d a t i o n of 1 ymole NADH/min at 25°C. Pre p a r a t i o n of C i t r a t e Synthase A l l procedures were performed at 0-4°C. 10 g of adductor muscle were d i s s e c t e d f r e e of surrounding t i s s u e s , b l o t t e d dry w i t h f i l t e r paper, and homogenized i n 5 v o l of 0.1 M sodium phosphate pH 7.4 w i t h a S o r v a l l Omni-27 mixer set at the maximum speed. The suspension was c e n t r i f u g e d at 20,000 g f o r 20 min, the r e s u l t i n g supernatant set a s i d e , the p e l l e t rehomogenized i n a f u r t h e r 4 v o l of the same b u f f e r , and r e c e n t r i f u g e d as above. The super-natants were combined, t h i s s o l u t i o n being the crude homogenate. Ammonium sulphate 0.209 g/ml was s l o w l y added w i t h continuous s t i r r i n g to the crude homogenate, the suspension c e n t r i f u g e d at 20,000 g f o r 30 min and the p e l l e t discarded. The supernatant was then t r e a t e d w i t h a f u r t h e r 0.2 g/ml of ammonium sulphate, c e n t r i f u g e d and the supernatant discarded. The p e l l e t obtained was d i s s o l v e d i n a minimal volume of 5 mM sodium phosphate pH 7.4, d i a l y z e d against 2 changes of 1 l i t r e of the same b u f f e r f o r 2 hr each and a p p l i e d to a 10 x 1.6 cm column of DEAE Sephadex A-50 pre-v i o u s l y e q u i l i b r a t e d w i t h 5 mM sodium phosphate pH 7.4. A l i n e a r gradient of 5-100 mM sodium phosphate pH 7.4 was a p p l i e d to the column, the enzyme being e l u t e d as a s i n g l e peak. F r a c t i o n s c o n t a i n i n g enzyme a c t i v i t y were pooled and a p p l i e d to a 10 x 1.6 cm column of hydroxyapatite i n 20 mM sodium phos-phate pH 7.4, washed w i t h the same b u f f e r and then washed w i t h 80 mM sodium phosphate pH 7.4 u n t i l MDH a c t i v i t y had dropped to a low l e v e l . The column was then washed w i t h 100 mM sodium phosphate b u f f e r pH 7.4, f r a c t i o n s of 2 ml being c o l l e c t e d i n the f i n a l wash. F r a c t i o n s c o n t a i n i n g a c t i v i t y of 0.2 u n i t s /ml or greater were pooled and t h i s p a r t i a l l y p u r i f i e d enzyme was used f o r most of the k i n e t i c s t u d i e s . This procedure u s u a l l y y i e l d e d an enzyme w i t h a s p e c i f i c a c t i v i t y of 6.7 units/mg p r o t e i n and contained a s m a l l amount of MDH. In order to remove t h i s MDH, a 10 ml a l i q u o t was d i a l y z e d against 2 l i t r e s of 10 mM imidazole-HCl pH 7.2 b u f f e r and a p p l i e d to an agarose-NAD + a f f i n i t y column. F r a c t i o n s c o n t a i n i n g c i t r a t e synthase but no measurable MDH were c o l l e c t e d and used to study NADH i n h i b i t i o n of c i t r a t e synthase. 28 P r e p a r a t i o n of Aspartate Aminotransferase A l l procedures were c a r r i e d out between 0 and 4°C. Approximately 10 g of o y s t e r adductor muscle were d i s s e c t e d f r e e of surrounding t i s s u e , b l o t t e d dry w i t h f i l t e r paper, weighed and homogenized i n 5 v o l of 0.1 M sodium phos-phate pH 7.4 w i t h a S o r v a l l Omnimixer set at maximum speed. The homogenate was c e n t r i f u g e d at 20,000 g f o r 20 minutes, the supernatant set a s i d e and the p e l l e t rehomogenized i n a f u r t h e r 4 v o l of b u f f e r and r e c e n t r i f u g e d as above. The supernatants were combined, t h i s s o l u t i o n c o n s t i t u t i n g the crude homogen-ate. Ammonium sulphate, 0.209 g/ml, was s l o w l y added to the crude homogenate wi t h continuous s t i r r i n g , the r e s u l t i n g suspension c e n t r i f u g e d at 20,000 g f o r 30 minutes and the p e l l e t was discarded. The supernatant was then t r e a t -ed w i t h 0.2 g/ml ammonium sulphate, c e n t r i f u g e d as above and the supernatant discarded. The p e l l e t from the ammonium sulphate treatment was d i s s o l v e d i n 5 mM sodium phosphate pH 7.6 b u f f e r , d i a l y z e d against 2 changes of 1 l i t r e of the same b u f f e r f o r 2 hr each, and then a p p l i e d to a 10 x 1.6 cm column of DEAE Sephadex A-50, p r e v i o u s l y e q u i l i b r a t e d w i t h the same b u f f e r . The enzyme was e l u t e d w i t h a l i n e a r gradient between 5 and 100 mM sodium phosphate pH 7.6 i n a t o t a l volume of 400 ml, 2 ml f r a c t i o n s being c o l l e c t e d . Enzyme a c t i v i t y i s e l u t e d i n two peaks, f r a c t i o n s comprising the l a r g e r peak were pooled and a p p l i e d to a 1.6 x 5 cm column of h y d r o x l a p a t i t e e q u i l i b r a t e d w i t h 20 mM sodium phosphate pH 7.4. This column was then e l u t e d w i t h a l i n e a r gradient between 20 and 50 mM sodium phosphate pH 7.4, aspartate aminotransferase being e l u t e d i n a s i n g l e peak. A l l f r a c t i o n s which contained no malate de-hydrogenase a c t i v i t y were pooled, concentrated by u l t r a f i l t r a t i o n , and used f o r a l l assays without f u r t h e r p u r i f i c a t i o n . The s p e c i f i c a c t i v i t y of t h i s 29 p a r t i a l l y p u r i f i e d p r e p a r a t i o n was about 23 units/mg p r o t e i n . P r e p a r a t i o n of Malate Dehydrogenase A l l procedures were c a r r i e d out between 0 and 4°C. Approximately 5 g of oyster adductor muscle were d i s s e c t e d f r e e of surrounding t i s s u e , b l o t t e d dry w i t h f i l t e r paper, weighed and homogenized i n 5 v o l of 0.1 M sodium phos-phate pH 7.4 wi t h a S o r v a l l Omnimixer set at maximum. The homogenate was ce n t r i f u g e d at 20,000 g f o r 20 min, the supernatant set aside and the p e l l e t rehomogenized i n a f u r t h e r 4 v o l of b u f f e r and r e c e n t r i f u g e d . The supernat-ants were combined, t h i s s o l u t i o n c o n s t i t u t i n g the crude homogenate. Ammonium sulphate, 0.209 g/ml, was slowly added to the crude homogenate w i t h continuous s t i r r i n g , the r e s u l t i n g suspension c e n t r i f u g e d at 20,000 g fo r 30 min and the p e l l e t discarded. The supernatant was then t r e a t e d w i t h 0.2 g/ml ammonium sulphate, c e n t r i f u g e d as above, and the supernatant d i s -carded. The p e l l e t from the ammonium sulphate treatment was d i s s o l v e d i n 5 mM sodium phosphate pH 7.4, d i a l y z e d against 2 changes of 1 l i t r e of the same b u f f e r f o r 2 hr each, and then a p p l i e d to a 10 x 1.6 cm column of DEAE Sepha-dex A-50 p r e v i o u s l y e q u i l i b r a t e d w i t h 5 mM sodium phosphate pH 7.4. The malate dehydrogenase was e l u t e d w i t h a l i n e a r gradient from 5 mM to 100 mM sodium phosphate pH 7.4 i n a volume of 400 ml, 2 ml f r a c t i o n s being c o l l e c t e d . Malate dehydrogenase a c t i v i t y e l u t e s as a s i n g l e peak, the tubes c o n t a i n i n g maximal a c t i v i t y being pooled and used without f u r t h e r treatment. The s p e c i f -i c a c t i v i t y of t h i s p r e p a r a t i o n was u s u a l l y about 50 units/mg p r o t e i n . P r e p a r a t i o n of Octopine Dehydrogenase Mantle muscle from squid and octopus was d i s s e c t e d from the specimens, r i n s e d i n c o l d b u f f e r and homogenized i n 10 v o l of c o l d 100 mM sodium phos-30 phate pH 7.4 w i t h a commercial blendor at f u l l speed. The r e s u l t i n g homogen-ate was c e n t r i f u g e d at 20,000 g f o r 20 min. Ammonium sulphate was added to the supernatant w i t h continuous s t i r r i n g to 30% s a t u r a t i o n , the s o l u t i o n c e n t r i f u g e d and the p e l l e t discarded. This supernatant was f u r t h e r t r e a t e d w i t h ammonium sulphate to 55% s a t u r a t i o n , the suspension c e n t r i f u g e d and the supernatant discarded. E s s e n t i a l l y a l l of the ODH a c t i v i t y i s present i n t h i s p e l l e t . The p e l l e t was suspended i n a minimum volume of c o l d 100 mM im i d a z o l e -HC1 pH 7.0 b u f f e r , and t h i s p r e p a r a t i o n used f o r k i n e t i c s t u d i e s without f u r t h e r treatment. About 3 g spadix muscle was homogenized i n sodium phosphate pH 7.4 and c e n t r i f u g e d as above. The supernatant was t r e a t e d w i t h ammonium sulphate to 40% s a t u r a t i o n , c e n t r i f u g e d and the p e l l e t discarded. This supernatant was f u r t h e r t r e a t e d w i t h ammonium sulphate to 60% s a t u r a t i o n , c e n t r i f u g e d and the supernatant discarded. The p e l l e t was d i s s o l v e d i n a minimal volume of c o l d 50 mM imidazole-HCl pH 7.5, and t h i s p r e p a r a t i o n was used without f u r t h e r treatment f o r a l l k i n e t i c s t u d i e s . E l e c t r o p h o r e s i s Starch and polyacrylamide gels were used to separate the cytoplasmic and mi t o c h o n d r i a l isoenzymes of aspartate aminotransferase and malate dehydro-genase. The s t a r c h gels were 14% s t a r c h (Connaught L a b o r a t o r i e s , Toronto) i n 0.1 M T r i s c i t r a t e pH 8.5 or 5 mM phos p h a t e - c i t r a t e pH 7.7. The sample was a p p l i e d to the g e l by absor p t i o n on f i l t e r paper wicks which were i n s e r t e d i n t o s l o t s i n the g e l . E l e c t r o p h o r e s i s was c a r r i e d out at 400 v o l t s f o r 12 hr at 4°C, t h i s being the time p e r i o d r e q u i r e d to e f f e c t i v e l y separate the bands of enzyme a c t i v i t y . Polyacrylamide e l e c t r o p h o r e s i s was conducted by Davis' (1964) procedure 31 at 4°C using 7% (w/v) sep a r a t i n g gels and a Buchler apparatus. The s t a c k i n g ge l was omitted, the samples being a p p l i e d i n 10% sucrose. In order to i d e n t i f y the cytoplasmic and m i t o c h o n d r i a l isoenzymes on the g e l s , a supernatant f r a c t i o n was prepared from oys t e r adductor muscle by the method of de Zwaan and van Marrewijk (1973) and coelectrophoresed w i t h the samples. About 1 g of adductor muscle was homogenized i n a t e f l o n - g l a s s homogenizer i n 10 ml of an i c e - c o l d b u f f e r pH 7.5 c o n s i s t i n g of 10 mM potassium phosphate, 0.5 M KC1, 0.1% BSA, 10 mM EDTA, 2 mM c y s t e i n e . The homogenate was c e n t r i f u g e d at 1600 g f o r 10 min. The supernatant was then c e n t r i f u g e d at 29,000 g f o r 15 min g i v i n g a p e l l e t which contained the mitochondria. The supernatant was f r e e of any detectable c i t r a t e synthase a c t i v i t y ,which was used as a m i t o c h o n d r i a l marker enzyme. The supernatant contained the cyt o -plasmic isoenzymes of malate dehydrogenase and aspartate aminotransferase, and a l i q u o t s were coelectrophoresed w i t h the p a r t i a l l y p u r i f i e d enzymes that were used f o r k i n e t i c s t u d i e s . Aspartate aminotransferase was detected by s t a i n i n g w i t h a s o l u t i o n of aspartate (500 mg), 2-ketoglutarate (75 mg), pyridoxal-5-phosphate (50 mg), and f a s t v i o l e t B s a l t (200 mg) i n 100 ml of 50 mM T r i s - H C l b u f f e r adjusted to a f i n a l pH of 7.5 (Owen, 1974). Malate dehydrogenase was detected by s t a i n i n g w i t h a s o l u t i o n c o n t a i n i n g malate (1.34 g ) , NAD + (69 mg), n i t r o b l u e t e t r a z o l -ium (20 mg), phenazine methosulphate (4 mg) i n 100 ml of 100 mM T r i s - H C l adjusted to a f i n a l pH of 7.5. Molecular Weight Determination A column of Sephadex G-200 (35 x 1.5 cm) was used f o r determining the molecular weights of the enzymes. Samples were a p p l i e d at the bottom of the column and upward e l u t i o n c a r r i e d out w i t h 50 mM sodium phosphate pH 7.4, the 32 flow r a t e being 5 ml/hr. F r a c t i o n s of 1.08 ml were c o l l e c t e d and assayed f o r p r o t e i n at 280 nm, or f o r a s p e c i f i c enzyme. The column was c a l i b r a t e d w i t h Dextran blue 2000 (M.W. 2000000), a l d o l a s e (M.W. 158000), r a b b i t muscle l a c t a t e dehydrogenase (M.W. 130000), chymotrypsinogen A (M.W. 25000), o v a l -bumin (M.W. 45000) and ribonuclease A (M.W. 14000). The e l u t i o n volumes were determined by l o c a t i n g the peak of the sample. The parameter K was c a l c u -l a t e d from the r e l a t i o n s h i p V - V e o K - — — where V = e l u t i o n volume av V - V e t o V = t o t a l column volume V = v o i d volume o A l i n e a r p l o t of K vs. l o g M.W. was obtained f o r the standards. The molecu-r av & l a r weight of the enzymes was then obtained from t h i s c a l i b r a t i o n p l o t ( F i g . 7). This method was chosen because i t i s independent of flow r a t e and column packing (Winzor, 1969). Enzyme K i n e t i c s The s ubstrate s a t u r a t i o n k i n e t i c s of a l l enzymes were st u d i e d by means of double r e c i p r o c a l p l o t s . ( 1 / v vs. l / [ s ] ) . The K m values were obtained by use of the r e l a t i o n s h i p K = slope x V . A l l determinations were performed r m r max at l e a s t t w i c e , the values being r e p r o d u c i b l e w i t h i n 20%. On a l l graphs "v" i s i n a r b i t r a r y u n i t s that are p r o p o r t i o n a l to the r a t e of change i n o p t i c a l d e n s i t y . I n h i b i t i o n patterns were st u d i e d by means of double r e c i p r o c a l p l o t s w i t h at l e a s t two d i f f e r e n t concentrations of the i n h i b i t o r . I n h i b i t i o n con-st a n t s (K^ values) were c a l c u l a t e d from these p l o t s according to Webb (1963), and a l s o by means of Dixon p l o t s (1/v vs. [ l ] ) (Dixon, 1953). V a r i a t i o n s i n K^ values were between 10 and 20%. 33 Figure 7. C a l i b r a t i o n of Sephadex G-200 for molecular weight determinations. M.W. x 1 0 4 35 CHAPTER I I I OCTOPINE DEHYDROGENASE IN CEPHALOPOD MUSCLE  INTRODUCTION The presence of octopine i n muscles of various b i v a l v e s and cephalopods has long been known (Ackerman and Mohr, 1937; Moore and Wilson, 1937; van Thoai and Robin, 1959a). The suggestion that i t was produced b i o l o g i c a l l y from a r g i n i n e was made by Moore and Wilson (1937), and subsequently van Thoai and Robin (1959a,b) showed that i t was formed from pyruvate and a r g i n i n e , w i t h the u t i l i z a t i o n of NADH. They were a l s o quick to grasp the p o s s i b i l i t y that octopine would be an anaerobic end product of g l y c o l y s i s i n these t i s s u e s i n s t e a d of l a c t a t e (van Thoai and Robin, 1959b; Robin and van Thoai, 1961). S u r p r i s i n g l y there have been few attempts to measure octopine accumulation i n molluscan t i s s u e s i n i n t a c t animals. Gade and Greisbaher (1975) f a i l e d to detect any octopine accumulation i n Anodonta cygnea during a n a e r o b i o s i s , or i n Sepia muscle a f t e r e x e r c i s e (Gade, personal communication). Octopine dehydrogenase has been p u r i f i e d from the adductor muscle of Pecten maximus (van Thoai at a l . , 1969), Anodonta cygnea (Gade and Greisbaher, 1975), and the body w a l l of Sipunculus nudus (Haas et a l . , 1973). The k i n e t i c c h a r a c t e r i s t i c s of the enzyme from a l l three sources were found t o be s i m i l a r , and the molecular weight was a l s o s i m i l a r , being 38000-40000. The enzyme from Pecten maximus was shown to be a s i n g l e polypeptide by SDS polyacrylamide e l -e c t r o p h o r e s i s (Olomucki et a l . , 1972). Cephalopods are a d i v e r s e group of animals ranging from a c t i v e oceanic squid such as S_. o u a l a n i e n s i s , to more s l u g g i s h forms such as the octopus and N a u t i l u s . In order to assess the r o l e played by ODH i n these various types of cephalopods, a comparative k i n e t i c study was made on ODH from the mantle muscle of S_. o u a l a n i e n s i s , Octopus ornatus, and from the spadix muscle of N a u t i l u s pompilius. 36 RESULTS AND DISCUSSION  Molecular Weight Since a l l cephalopod ODH enzymes s a l t e d out d i f f e r e n t l y from the s c a l l o p homologue (van Thoai et a l . , 1969), i t was p o s s i b l e that the enzyme occurred i n d i f f e r e n t p o l y m e r i z a t i o n s t a t e s . However, the molecular weight of the 0_. ornatus enzyme as determined by g e l f i l t r a t i o n was 38,000±4,000, a value s i m i l a r to that reported f o r the enzyme from Pecten maximus (Olomucki ^ t a l . , 1972). I t i s t h e r e f o r e l i k e l y that the enzyme occurs as a s i n g l e chain s t r u c t u r e i n the cephalopods as w e l l as i n the l a m e l l i b r a n c h s , and that i t i s the s m allest dehydrogenase found i n animal t i s s u e s . 0DH:ctGPDH Ratios The r a t i o of ODH:aGPDH was measured i n the mantle muscle of two species of squid, two species of octopus and i n the spadix muscle of N a u t i l u s . Highest a c t i v i t i e s of ODH were found i n octopus mantle muscle, whereas the highest a c t i v i t i e s of aGPDH were found i n J>. o u a l a n i e n s i s mantle muscle (Table I ) . Assuming that ODH i s a measure of the anaerobic c a p a c i t y , the data suggest that the mantle muscle of C). ornatus, (). cyanea, and Euprymna, which are quiescent s p e c i e s , has a higher anaerobic c a p a c i t y than does the mantle muscle of the oceanic s q u i d , S_. o u a l a n i e n s i s , which appears to have a higher aerobic c a p a c i t y . ODH K i n e t i c s The pH p r o f i l e s of a l l enzymes were s i m i l a r ( F i g . 8) showing an optimum at about pH 6.5 i n the d i r e c t i o n of octopine formation, and about pH 8.0 i n the d i r e c t i o n of octopine o x i d a t i o n . The enzymes were c h a r a c t e r i z e d at pH 7.0 i n the d i r e c t i o n of octopine formation, and pH 8.5 i n the reverse d i r e c t i o n . 37 Table I . A c t i v i t i e s of ODH and ctGPDH i n cephalopod muscle. ODH aGPDH* ODH/ctGPDH Symplectoteuthis o u a l a n i e n s i s (mantle) 110 240 0. 45 Euprymna scolopes (mantle) 330 45 7. 3 Octopus cyanea (mantle) 500 32 15. 6 Octopus ornatus (mantle) 640 30 21. 3 N a u t i l u s pompilius (spadix) 42 1 42 A c t i v i t i e s expressed as ymole product/min/g wet wt. Assays performed at pH 7.0, 25°C. . *ctGPDH assay c o n d i t i o n s : 0.2 mM NADH, 2 mM DHAP, 100 mM imidazole-HCl pH 7.0, 25°C. 38 E f f e c t of pH on squid mantle muscle ODH i n both d i r e c t i o n s : (f) d i r e c t i o n of octopine formation, 0.2 mM NADH, 3 mM pyruvate, 30 mM a r g i n i n e ; (A) d i r e c t i o n of octopine o x i d a t i o n , 1 mM NAD +, 10 mM octopine. 39 P H 40 In a l l cases the enzymes followed Michaelis-Menten k i n e t i c s . The appar-ent K (K , .) for NADH decreased with lower concentrations of arginine or m N m(app) b pyruvate, although t h i s e f f e c t was less pronounced i n the spadix muscle than i n the mantle muscle (Table I I , I I I , IV). The ^ values f o r arginine and pyruvate were found to be s i m i l a r i n the squid and the octopus (Table II and I I I ) . The a f f i n i t y for pyruvate was low i n these species, as shown by the r e l a t i v e l y high K m values. The spadix muscle, however, had a higher a f f i n i t y f o r pyruvate and arginine (Table IV). No substrate i n h i b i t i o n by arginine was detected at concentrations as high as 60 mM, but pyruvate was found to be i n -h i b i t o r y at concentrations greater than 10 mM. In addition, the K , , for m(app) pyruvate or arginine was dependent on the concentration of the cosubstrate (arginine or pyruvate) (Fig. 9), r e s u l t i n g i n a decrease i n the K , , as the m\ aPP/ cosubstrate concentration was increased (Table I I , I I I , IV). Thus the enzyme a c t i v i t y would be ra p i d l y enhanced i f both substrates were increasing concomi-ta n t l y . This can be i l l u s t r a t e d by covarying arginine and pyruvate concentra-tions at a fi x e d r a t i o of arginine:pyruvate. The r e s u l t i n g saturation curve was sigmoidal (Fig. 10). The K , . for NAD+ and octopine decreased as the cosubstrate was increas-m(app) ed, i n a s i m i l a r manner to that of arginine and pyruvate. The ^ r o r NAD+ was markedly lower i n the Nautilus spadix than i n the octopus and squid mantle, and that for octopine was found to be much lower i n the octopus mantle and Nautilus spadix than i n the squid mantle. The higher a f f i n i t y f o r octopine i n the octopus mantle and Nautilus spadix suggests that these enzymes can function more r e a d i l y i n both d i r e c t i o n s under p h y s i o l o g i c a l conditions, or that octo-pine l e v e l s are higher i n the squid. So f a r data on octopine l e v e l s i n cephalopod muscle are a v a i l a b l e only for Nautilus spadix and re t r a c t o r muscle, values as high as 30 mM being found (Hochachka, H a r t l i n e and F i e l d s , unpublish-41 Table II. Octopus mantle muscle ODH: apparent K values f o r s u b s t r a t e s . Substrate Cosubstrate K , mM NADH 30 mM a r g i n i n e 3 mM pyruvate 0.031 6 mM a r g i n i n e 1.5 mM pyruvate 0.018 Ar g i n i n e 0.2 mM NADH 3 mM pyruvate 6.3 0.2 mM NADH 1.5 mM pyruvate 7.1 Pyruvate 0.2 mM NADH 30 mM a r g i n i n e 1.7 0.2 mM NADH 6 mM a r g i n i n e 2.9 NAD+ 5 mM octopine 0.07 0.7 mM octopine 0.13 Octopine 1 mM NAD + 0.8 0.1 mM NAD + 42 Table I I I . Squid mantle muscle ODH: apparent K values f o r s u b s t r a t e s . Substrate Cosubstrates NADH 30 mM a r g i n i n e 6 mM pyruvate 0.04 6 mM a r g i n i n e 1.2 mM pyruvate 0.032 A r g i n i n e 0.2 mM NADH 6 mM pyruvate 6.7 0.2 mM NADH 1.2 mM pyruvate 10.5 0.2 mM NADH 0.24 mM pyruvate 13.5 Pyruvate 0.2 mM NADH 30 mM a r g i n i n e 2.0 0.2 mM NADH 6 mM a r g i n i n e 2.9 0.2 mM NADH 3 mM a r g i n i n e 4.0 NAD + 10 mM octopine 0.15 Octopine 1.0 mM NAD + 4.4 0.1 mM NAD + 12 43 Table IV. N a u t i l u s spadix ODH: apparent K m values f o r s u b s t r a t e s . Substrate Cosubstrates K , mM m NADH 30 mM a r g i n i n e 6 mM pyruvate 6 mM a r g i n i n e 0.6 mM pyruvate 0.011 0.01 A r g i n i n e 0.2 mM NADH 0.2 mM NADH 0.2 mM NADH 6 mM pyruvate 0.9 mM pyruvate 0.45 mM pyruvate 3.7 5.3 7.7 Pyruvate NAD + Octopine 0.2 mM NADH 0.2 mM NADH 0.2 mM NADH 30 mM a r g i n i n e 6 mM a r g i n i n e 1.5 mM a r g i n i n e 5 mM octopine 0.5 mM octopine + 1 mM NAD 0.2 mM NAD + 0.33 0.67 1.3 0.02 0.06 0.44 0.59 44 Figure 9. Pyruvate and arginine saturation k i n e t i c s of squid mantle muscle ODH: a) double r e c i p r o c a l plot of pyruvate saturation at varying arginine concentrations: (A) 3 mM arginine; (•) 6 mM arginine; (0) 30 mM arginine. NADH concentration, 0.2 mM. b) double r e c i p r o c a l plot of arginine saturation at varying pyruvate concentrations: (A) 0.24 mM pyruvate; (t) 1.2 mM pyruvate; (0) 6 mM pyruvate. NADH concentration, 0.2 mM. 46 Figure 10. Squid mantle ODH: E f f e c t of covarying a r g i n i n e and pyruvate con-c e n t r a t i o n s at a constant arginine:pyruvate. Data are p l o t t e d w i t h respect to a r g i n i n e : (0) 0.5:1, (A) 1:1, (t) 5:1, (A) 10:1 a r g i n i n e pyruvate; 0.2 mM NADH. 47 48 ed), but no_data on squid or octopus muscle are a v a i l a b l e f o r comparison. Product I n h i b i t i o n NAD + and NADH were competitive i n h i b i t o r s w i t h respect to each other. A l l of the enzymes appear to be under strong redox c o n t r o l , as shown by the low K values f o r NAD + and NADH (Table V, VI and V I I ) . Octopine gave a mixed p a t t e r n of i n h i b i t i o n w i t h respect to both a r g i n i n e and pyruvate ( F i g . 11), however i t was more i n h i b i t o r y w i t h respect to a r g i n i n e (Table V, VI and V I I ) . The values were s i m i l a r i n the squid and octopus mantle ODH, but were con-s i d e r a b l y lower i n the N a u t i l u s spadix ODH. Pyruvate gave a mixed p a t t e r n w i t h respect to octopine i n a l l cases ( F i g . 12), but i n a l l cases the R\ values were high r e l a t i v e to the con c e n t r a t i o n of pyruvate i n the squid mantle muscle (Hochachka et_ a l . , 1975), and spadix muscle (Hochachka, H a r t l i n e and F i e l d s , i n p r e p a r a t i o n ) . A r g i n i n e i n h i b i t i o n was a l s o mixed i n the squid mantle muscle and spadix ODH, but i n the octopus mantle ODH the i n h i b i t i o n was complex, apparently t r e n d i n g towards an uncom-p e t i t i v e p a t t e r n ( F i g . 13). In the squid and octopus mantle muscle ODH the values were found to be about 5 mM, which i s i n the range of concentrations of a r g i n i n e (Hochachka et a l . , 1975). S u r p r i s i n g l y , the K., . . N was found r l ( a r g x n i n e ) to be 21.5 mM i n the spadix ODH. In the absence of any data on the ranges of octopine concentrations i n the squid and octopus mantle muscle, l i t t l e can be s a i d concerning the e f f e c t s of a r g i n i n e i n h i b i t i o n on the r a t e of octopine o x i d a t i o n . Octopine can accumu-l a t e i n the spadix muscle to values as high as 30 mM (Hochachka, H a r t l i n e and F i e l d s , i n p r e p a r a t i o n ) , and c e r t a i n l y at these concentrations octopine would have a strong e f f e c t on f u r t h e r octopine formation. I t i s of i n t e r e s t that the i n h i b i t i o n by a r g i n i n e was not n e a r l y as pronounced, the K. value being 49 Table V. Squid mantle ODH: K\ values f o r v a r i o u s . i n h i b i t o r s . Substrate I n h i b i t o r Type of I n h i b i t i o n K., mM x NADH NAD ATP ADP AMP Competitive Competitive Competitive Competitive 0.4 2.7 2.8 3.9 Pyruvate Octopine Mixed 12 Arginine" ,4 NAD + Octopine NADH Mixed Competitive 10 0.012 Octopine' Pyruvate A r g i n i n e Mixed Mixed 19.0 5.4 Conditions: ^30 mM a r g i n i n e , 6 mM pyruvate 230 mM a r g i n i n e , 0.2 mM NADH 3 6 mM pyruvate, 0.2 mM NADH 4 5 mM octopine 5 1 mM NAD + 50 Table VI Octopus mantle ODH: values for various i n h i b i t o r s . Substrate I n h i b i t o r Type of I n h i b i t i o n K., mM 1 NADH NAD ATP ADP AMP Competitive Competitive Competitive Competitive 0.16 1.9 0.8 4.2 Pyruvate Octopine Mixed 14.3 Arginine NAD + Octopine NADH ATP ADP' AMP Mixed Competitive Competitive Competitive Competitive 6.4 0.045 4.8 6.2 16 Octopine Pyruvate Arginine Mixed Uncompetitive 9 5 Conditions: as i n Table V. 51 Table VII. Nautilus spadix ODH: K. values f or various i n h i b i t o r s . r 1 Substrate NADH Pyruvate Arginine NAD+ Octopine I n h i b i t o r ATP ADP AMP NAD+ Octopine Octopine NADH Arginine Pyruvate Type of I n h i b i t i o n K\, mM Competitive Competitive Competitive Competitive Mixed Mixed Competitive Mixed Mixed 1.6 2.0 4.8 0.1 6.0 1.9 0.027 21.5 3.2 Conditions: as i n Table V. 52 Figure 11. P a t t e r n of octopine i n h i b i t i o n w i t h respect to pyruvate and a r g i n i n e s a t u r a t i o n k i n e t i c s of octopus mantle ODH: a) double r e c i p r o c a l p l o t showing p a t t e r n of octopine i n h i b i t i o n w i t h respect to a r g i n i n e . NADH c o n c e n t r a t i o n , 0.2 mM; pryuvate c o n c e n t r a t i o n , 3 mM. b) double r e c i p r o c a l p l o t showing p a t t e r n of octopine i n h i b i t i o n w i t h respect to pyruvate. NADH co n c e n t r a t i o n , 0.2 mM; a r g i n i n e c o n c e n t r a t i o n , 30 mM. 1/Pyruvate (mM - 1) 54 Figure 12. Pattern of pyruvate i n h i b i t i o n with respect to octopine satura-t i o n k i n e t i c s of squid mantle muscle ODH. NAD+ concentration, 1 mM. 55 -0.4 0 0.4 0.8 1.2 1.6 2.0 1/Octopine (mM - 1 ) 56 Figure 13. Pattern of arginine i n h i b i t i o n with respect to octopine saturation k i n e t i c s of squid and octopus mantle ODH. a) squid mantle ODH. b) octopus mantle ODH. NAD+ concentration, 1 mM. 58 r e l a t i v e l y h i g h , and i t i s p o s s i b l e that t h i s f a c i l i t a t e s r e v e r s a l of the spadix enzyme f o r octopine o x i d a t i o n under p h y s i o l o g i c a l c o n d i t i o n s . Other Met a b o l i t e s A s e r i e s of metabolites were t e s t e d f o r e f f e c t s on the enzyme a t s a t u r a t -i n g and approximate K m values of subs t r a t e s . Of these, only the adenylates had an e f f e c t , being competitive i n h i b i t o r s of NADH (Table V, VI, V I I ) , whereas a s p a r t a t e , glutamate, t a u r i n e , p r o l i n e , a l a n i n e , g l y c i n e , a r g i n i n e phosphate, a-glycerophosphate, f r u c t o s e diphosphate, phosphoenolpyruvate, dihydroxyacetone phosphate, 2-ketoglutarate, c i t r a t e , s u c c i n a t e and malate were found to have no e f f e c t . The i n h i b i t i o n by the adenylates i s s i m i l a r to that found f o r LDH (McPherson, 1970). The competitive p a t t e r n w i t h respect to NADH i s to be expected,since such b i n d i n g s i t e s i n dehydrogenases possess an adenine nucleo-t i d e domain (McPherson, 1970). P h y s i o l o g i c a l l y , there seems to be l i t t l e relevance s i n c e the K. values f o r ATP and ADP are s i m i l a r , hence ATP—>ADP conversions w i l l not a f f e c t enzyme a c t i v i t y . D e p l e t i o n of ATP and ADP to form AMP, however, may r e s u l t i n a s l i g h t i n c r e a s e i n a c t i v i t y . The Role of Octopine Dehydrogenase i n Cephalopod Muscle Metabolism As p r e v i o u s l y discussed, u t i l i z a t i o n of carbohydrates leads to the prob-lem of maintaining cytoplasmic redox balance, because of the production of NADH at the l e v e l of GAPDH. Vertebrate muscles u t i l i z e the malate-aspartate s h u t t l e (which t r a n s f e r s NADH across the m i t o c h o n d r i a l membrane under aerobic c o n d i t i o n s ) (LaNoue and Williamson, 1971), and LDH, which i s mainly anaerobic. In the mantle muscle of S_. ou a l a n i e n s i s there are high a c t i v i t i e s of aGPDH i n the cytoplasm (Storey and Hochachka, 1975) which couples w i t h a m i t o c h o n d r i a l aGP oxidase (Hochachka et a l . , 1975), thus p r o v i d i n g an aGP c y c l e f o r the 59 o x i d a t i o n of NADH v i a the mitochondrion as occurs i n i n s e c t s (Sacktor, 1970). The presence of ODH thus gives the squid and octopus mantle muscle another means of regenerating NAD + i n the cytoplasm ( F i g . 14), but u n l i k e LDH which re q u i r e s only pyruvate, ODH a l s o r e q u i r e s a r g i n i n e , and t h i s provides a l i n k w i t h a r g i n i n e phosphate metabolism. The mantle muscle of cephalopods must meet two demands, maintenance of r e s p i r a t o r y movements and swimming by j e t t i n g , and th e r e f o r e i t cannot remain i n a c t i v e f o r prolonged periods. Presumably during the lower order movements, such as normal r e s p i r a t i o n and slow swimming i n the squid, the aGP c y c l e i s s u f f i c i e n t f o r maintaining redox balance, energy i s obtained l a r g e l y by aero-b i c means, l i t t l e a r g i n i n e phosphate reserves are being u t i l i z e d and any pyru-vate produced i s being o x i d i z e d v i a the TCA c y c l e i n the mitochondrion. Under these c o n d i t i o n s , ODH i s probably i n a c t i v e because su b s t r a t e l e v e l s are w e l l below enzyme a f f i n i t i e s f o r both s u b s t r a t e s . During " b u r s t " work c o n d i t i o n s , the a r g i n i n e phosphate pool i s presumably used to generate a d d i t i o n a l ATP, thus b r i n g i n g about an increase i n the concentrations of a r g i n i n e . Simultaneously, decreasing a r g i n i n e phosphate w i l l serve to d e i n h i b i t the PK step i n g l y c o l y -s i s (Guderley et a l . , 1976), and p o s s i b l y a l s o the PFK step, s i n c e i n mammal-i a n muscle and t u r t l e h e a r t , c r e a t i n e phosphate i s an important r e g u l a t o r of PFK a c t i v i t y (Storey and Hochachka, 1974a,b). Therefore an increase i n the concentrations of pyruvate would be expected under these c o n d i t i o n s , and together w i t h the increase i n a r g i n i n e l e v e l s , would favour octopine formation. The above hypothesis r e q u i r e s that l a r g e f l u c t u a t i o n s i n a r g i n i n e concen-t r a t i o n occur w i t h muscle a c t i v i t y . In f a c t , p r e l i m i n a r y data on the N a u t i l u s spadix and r e t r a c t o r muscles (Hochachka, H a r t l i n e and F i e l d s , i n preparation) and from the mantle of L o l i g o (Storey, i n preparation) i n d i c a t e that a r g i n i n e 60 Figure 14. Postulated summary scheme of metabolic organization i n cephalopod muscle. 61 Arg in ine -P A D P A T P Carbohydrate \ F D P Arginine G-3P L N A D * f ^ N A D H . H * I I Pyruvate D H A P -, N A D H . H * ^ N A D * - G P • V i ^ - N A D H . H * • N A D * Octopine C Y T O P L A S M D H A P V r e d u c e d f lavin N flavin • - G P MITOCHONDRION 62 can vary from about 5 mM under r e s t i n g c o n d i t i o n s to about 30 mM during muscle a c t i v i t y . Therefore the observed decrease i n K , v w i t h i n c r e a s i n g m(pyruvate) a r g i n i n e may be p h y s i o l o g i c a l l y important i n r e g u l a t i n g octopine formation. This may w e l l provide one p o s s i b l e reason f o r e v o l v i n g ODH to replace LDH. In v e r t e b r a t e muscle u t i l i z i n g glucose under aerobic c o n d i t i o n s , LDH competes w i t h the malate-aspartate s h u t t l e f o r NADH, f o r example a r a t heart perfused w i t h glucose a e r o b i c a l l y produces s m a l l q u a n t i t i e s of l a c t a t e (Randle et_ a l . , 1970; Safer and Williamson, 1973). E n e r g e t i c a l l y i t i s advantageous to o x i -d i z e NADH v i a the e l e c t r o n t r a n s f e r system r a t h e r than by LDH, as the former y i e l d s 3 ATP/mole NADH, and the l a t t e r u t i l i z e s pyruvate which would otherwise be o x i d i z e d v i a the Krebs c y c l e y i e l d i n g 14 ATP/mole pyruvate. In cephalopod mantle muscle, the competition f o r NADH between ODH and ctGPDH would favour ctGPDH at low work l e v e l s because a r g i n i n e concentrations would be low. During " b u r s t " work c o n d i t i o n s , the increase of a r g i n i n e and the increase of the g l y c o l y t i c f l u x would a c t i v a t e an anaerobic reserve to meet the higher energy demand. A comparison of the l e v e l s of ODH i n the mantle muscle of va r i o u s cepha-lopods (Table I ) suggests that the more sedentary forms r e l y more on the anaerobic metabolism to supply the energy requirements during swimming than does the oceanic squid S_. o u a l a n i e n s i s . However, i n 0_. ornatus and S_. oualan- i e n s i s the a f f i n i t y of ODH f o r pyruvate i s at a l l times low, and i n these species the major f a t e of pyruvate i s thought to be o x i d a t i o n to CO2 and ^ 0 , octopine being formed only under extreme c o n d i t i o n s . The N a u t i l u s spadix i s a modified arm which i s i n v o l v e d w i t h the t r a n s -f e r of spermatophores during c o p u l a t i o n . The a c t i v i t i e s of c i t r a t e synthase and ctGPDH i n t h i s muscle are low (0.4 and 1.0 u n i t s / g wet wt, r e s p e c t i v e l y ) (Hochachka and F i e l d s , unpublished), suggesting the aerobic c a p a c i t y of t h i s 63 muscle i s low. Therefore the energy requirements of t h i s muscle would be met predominantly through anaerobic metabolism l e a d i n g to octopine accumulation. In t h i s context i t i s of i n t e r e s t that the N a u t i l u s spadix ODH has a higher a f f i n i t y f o r pyruvate than the octopus and squid mantle ODH, and the K m values (Table IV) were lower than the pyruvate c o n c e n t r a t i o n measured i n r e s t i n g and e x e r c i s e d muscle (approximately 0.5 and 1.0 mM, r e s p e c t i v e l y ) . Octopine accumulation has been observed i n the spadix a f t e r modest work c o n d i t i o n s (Hochachka, H a r t l i n e and F i e l d s , i n p r e p a r a t i o n ) , but, as p r e v i o u s l y mentioned, octopine accumulation was not observed i n Sepia muscle a f t e r e x e r c i s e (Gade, personal communication). The f a t e of the octopine accumulated during periods of burst muscle ac-t i v i t y i s unknown. I t i s p o s s i b l e that i t i s converted to a r g i n i n e and pyruvate i n the muscle when the a c t i v i t y i s low, thus a l l o w i n g the a r g i n i n e phosphate pools to be r e - e s t a b l i s h e d . Another p o s s i b i l i t y i s the r e l e a s e i n t o the bloodstream and transport to another t i s s u e where i t i s converted to a r g i n i n e and pyruvate, the a r g i n i n e then being transported back to the muscle. The l a t t e r hypothesis i s favoured by the f i n d i n g that the k i n e t i c s of ODH from the squid b r a i n seem to favour octopine u t i l i z a t i o n r a t h e r than produc-t i o n ( F i e l d s et a l . , 1976). Before these problems can be r e s o l v e d , a s u i t a b l e experimental animal has to be found, and techniques developed f o r simultaneous-l y monitoring a c t i v i t y , blood flow to and from the muscles, octopine and a r g i n i n e i n the blood, and a l s o octopine, a r g i n i n e and a r g i n i n e phosphate i n the muscle. 64 CHAPTER IV OYSTER ADDUCTOR CITRATE SYNTHASE: CONTROL OF CARBON ENTRY INTO THE  KREBS CYCLE OF A FACULTATIVE ANAEROBE INTRODUCTION Aerobic metabolism i n many organisms i s characterized by the use of the Krebs cycle to oxidize carbon compounds, which enter as two carbon units i n the form of acetylCoA. The oxidation reactions produce NADH and FAD^, which are oxidized v i a the.electron transport system, oxygen being the terminal e l e c -tron acceptor. The immediate response to anoxic conditions, therefore, w i l l be an increase i n the l e v e l s of NADH intra m i t o c h o n d r i a l l y , and t h i s has been well documented f o r i s o l a t e d mitochondria (Chance and Williams, 1955; J o b s i s , 1972; Johnson and Hasnford, 1975). The increased NADH/NAD+ r a t i o would e f f e c -t i v e l y i n h i b i t most of the NAD +-linked dehydrogenases i n the mitochondria by NADH i n h i b i t i o n or lack of NAD+ (Chen and Plaut, 1963; Garland, 1964; Raval and Wolfe, 1962a). In f a c u l t a t i v e anaerobes such as C_. gigas, the Krebs cycle i s f u n c t i o n a l under aerobic conditions (Hammen, 1969), but during anoxia the reactions of the Krebs cycle appear to be disconnected at the l e v e l of oxaloacetate forma-t i o n and u t i l i z a t i o n (Stokes and Awapara, 1968; Hammen, 1969; Mustafa and Hochachka, 1973a,b). Reversing the flow of carbon i n one arm of the cy c l e y i e l d s a sequence ( o x a l o a c e t a t e — > m a l a t e — > fumarate—> succinate) that con-t r i b u t e s to succinate accumulation during anoxia. The other arm of the cy c l e i n i t i a t e d by c i t r a t e synthase e i t h e r i s f u l l y turned o f f , as for example, i n the i s o l a t e d , anoxic oyster heart ( C o l l i c u t t , 1975), or i s held at a reduced a c t i v i t y supplying 2-ketoglutarate f o r a small but s i g n i f i c a n t synthesis of glutamate from glucose occurring during prolonged anoxia i s Mytilus e d u l i s (de Zwaan and van Marrewijk, 1973). C i t r a t e synthase c a t a l y z i n g the r e a c t i o n oxaloacetate + acetylCoA 4. > c i t r a t e + CoA 65 c l e a r l y i s p o s i t i o n e d at a p i v o t a l locus i n such a metabolic o r g a n i z a t i o n , and t h e r e f o r e the c a t a l y t i c and r e g u l a t o r y p r o p e r t i e s of o y s t e r adductor c i t r a t e synthase were s t u d i e d i n an attempt to e l u c i d a t e the c o n t r o l of the aerobic-anaerobic t r a n s i t i o n i n the Krebs c y c l e . RESULTS AND DISCUSSION Oyster adductor muscle has a very low a c t i v i t y of c i t r a t e synthase, there being approximately 1.5 u n i t s / g wet wt. Thus the t i s s u e has a very low poten-t i a l f o r aerobic metabolism, and t h i s i s r e f l e c t e d by the low numbers of mitochondria present (Hanson and Lowy, 1961). Molecular Weight The molecular weight as determined by g e l f i l t r a t i o n was found to be 67000±5000. Thus the enzyme appears to be somewhat smaller than the homologue from r a t heart, r a t l i v e r and p i g heart which have molecular weights ranging from 96000 to 10000O (Moriyama and Srere, 1971; Singh et a l . , 1970; Wu and Yang, 1970), and about the same s i z e as some b a c t e r i a l enzymes (Johnson and Hanson, 1974). E f f e c t of pH S h o r t l y a f t e r s h e l l c l o s u r e i n C_. gigas, the pH of the mantle f l u i d drops to about 6.5 and then continues to d e c l i n e slowly to values as low as 5.4 under prolonged anoxia. I n t r a c e l l u l a r pH presumably f o l l o w s a s i m i l a r p a t t e r n and has l e d to the suggestion that a decreasing pH i t s e l f i s an important c o n t r o l l i n g element i n the aerobic-anaerobic t r a n s i t i o n (Hochachka and Mustafa, 1972). For t h i s reason, i t was important at the outset to determine the pH dependence of the r e a c t i o n . U n l i k e the mammalian enzymes s t u d i e d thus f a r (Srere, 1974), o y s t e r adductor muscle c i t r a t e synthase i s e s s e n t i a l l y pH 66 independent between 7.5 and 9.0 ( F i g . 15), which are the p r a c t i c a l l i m i t s of the DTNB assay system (Srere, 1969), a r e s u l t which suggests that pH w i l l not have as s i g n i f i c a n t a r o l e i n r e g u l a t i n g t h i s branchpoint, as i s the case f o r the PEP branchpoint (Mustafa, 1972; de Zwaan and Holwerda, 1973; de Zwaan and de Bont, 1975). I t was decided to c h a r a c t e r i z e the enzyme at pH 8.0 to f a c i l i -t a t e d i r e c t comparison w i t h s t u d i e s on c i t r a t e synthase from other sources. Substrate A f f i n i t i e s L i k e most animal c i t r a t e synthases thus f a r s t u d i e d , the oyste r adductor enzyme f o l l o w s normal Michaelis-Menten k i n e t i c s f o r both substrates over the concen t r a t i o n ranges s t u d i e d (3-60 uM f o r acetylCoA; 3-50 pM f o r o x a l o a c e t a t e ) . The Kffi f o r acetylCoA i s 5.5 uM and f o r oxaloacetate i t i s 4.6 pM. The values f o r both acetylCoA and oxaloacetate are s i m i l a r to those reported f o r other c i t r a t e synthases (Srere, 1974). Regulation by Adenylates As w i t h other c i t r a t e synthases, the adenylates are p o t e n t i a l i n h i b i t o r s of adductor muscle c i t r a t e synthase (Table V I I I ) . ATP i s the most e f f e c t i v e ( K ± = 0.5 mM) while AMP i s the l e a s t e f f e c t i v e (K\ = 3.5 mM). The i n h i b i t i o n i n a l l cases i s competitive ( F i g . 16) w i t h respect to acetylCoA and noncom-p e t i t i v e w i t h respect to oxaloacetate ( F i g . 17). I n h i b i t i o n by ATP can be g r e a t l y reduced, but not abolished by the a d d i t i o n of MgSO^ ( F i g . 16), as p r e v i o u s l y shown f o r the p i g heart enzyme ( K o s i c k i and Lee, 1966). I t should be noted, however, that MgSO^ and Na2S0^ are a l s o i n h i b i t o r y , being competitive w i t h respect to acetylCoA as p r e v i o u s l y shown f o r mammalian enzymes. MgSO^ j | has a lower K\ than Na2S0^, presumably due to i n t e r a c t i o n s of the c a t i o n Mg wit h the polyphsophate group of acetylCoA. 67 Table VIII. E f f e c t s of various i n h i b i t o r s on c i t r a t e synthase I n h i b i t o r Type of I n h i b i t i o n vs. acetylCoA vs. oxaloacetate K ±, (mM) ATP ADP AMP MgATP NADPH NADH Na2SO^ MgS04 C i t r a t e 2-ketoglutarate competitive competitive competitive competitive competitive competitive competitive competitive mixed mixed noncompet i t ive noncomp e t i t i ve noncompetitive mixed mixed 0.5 1.6 3.5 1.1 0.9 2.0 9.7 1.7 3.0 6.0 68 Figure 15. E f f e c t of pH on adductor c i t r a t e synthase. Conditions: 0.05 mM acetylCoA, 0.05 mM oxaloacetate, 25°C. 69 i \ O CD O > O CM O og CCi CO 00 CO CM 00 a o •oo .co CD 70 Figure 16. E f f e c t s of MgSO^, ATP and MgATP on acetylCoA s a t u r a t i o n k i n e t i c s of adductor c i t r a t e synthase. (A) c o n t r o l ; ( • ) 2 mM MgSO^; (•) 1 mM ATP or 2 mM MgATP; (A) 2 mM ATP. Concentration of oxaloacetate 0.05 mM. 71 0.06 Y -160 -120 -80. -40 0 40 80 120 160 1/Acetyl C o A x 10 3 72 Figure 17. E f f e c t of ATP on oxaloacetate s a t u r a t i o n k i n e t i c s of adductor c i t r a t e synthase. ( • ) c o n t r o l ; ( A ) 2 mM ATP; (§) 4 mM ATP; 0.05 mM acetylCoA. 73 0.071 - 200 -150 -100 -50 0 50 100 150 200 • VOxaloacetate m M 74 Redox Regulation In some b a c t e r i a (Johnson and Hanson, 1974) c i t r a t e synthase i s s t r o n g l y i n h i b i t e d by NADH and t h i s has a l s o been found to be the case f o r the enzyme from squid muscle (Hochachka et a l . , 1975). During anoxia, one would a n t i c i -pate l a r g e a l t e r a t i o n s i n the NADH/NAD+ r a t i o i n the mitochondrion ( L a i and M i l l e r , 1973) which t h e o r e t i c a l l y could provide a s e n s i t i v e s i g n a l f o r reduc-i n g c i t r a t e synthase a c t i v i t y during anoxia. I t i s th e r e f o r e p a r t i c u l a r l y i n t e r e s t i n g that the adductor enzyme i s q u i t e r e f r a c t o r y to both NADH and NAD + at p h y s i o l o g i c a l concentrations. In f a c t , NAD + at a con c e n t r a t i o n of 2 mM had no e f f e c t , w h i l e the f o r NADH was found to be 2 mM, which i s c l e a r l y higher than the l e v e l s expected under the most severe c o n d i t i o n s . M e t a b o l i t e Regulation Of a l a r g e s e r i e s of amino a c i d s , d i c a r b o x y l i c and t r i c a r b o x y l i c a c i d s t e s t e d , only c i t r a t e and 2-ketoglutarate were found to have any s i g n i f i c a n t e f f e c t on c i t r a t e synthase a c t i v i t y . Both the l a t t e r compounds y i e l d a mixed i n h i b i t i o n p a t t e r n w i t h respect to oxaloacetate and acetylCoA ( F i g . 18, 19, 20, 21). I t i s presumed that these compounds are i n f a c t competitive w i t h oxaloacetate, but the presence of counter ions gives an o v e r a l l mixed p a t t e r n , as has been shown f o r mammalian enzymes (Srere, 1974). C i t r a t e and 2-ketoglu-t a r a t e are both metabolized f u r t h e r i n the Krebs c y c l e , or can be used f o r other b i o s y n t h e t i c purposes; hence, t h e i r e f f e c t s on c i t r a t e synthase can be viewed as c l a s s i c a l negative feedback c o n t r o l of an e a r l i e r step i n v o l v e d i n t h e i r formation. F u n c t i o n a l S i g n i f i c a n c e Perhaps the most s u r p r i s i n g outcome of t h i s study i s how "normal" the oyster adductor c i t r a t e synthase appears to be. L i k e other animal c i t r a t e 75 synthases, i t s a c t i v i t y would appear to be c o n t r o l l e d by the combination of three f a c t o r s , namely a v a i l a b i l i t y of s u b s t r a t e , "energy charge" of the c e l l , and the l e v e l s of c i t r a t e and 2-ketoglutarate (Srere, 1974). For an aerobic system t h i s arrangement would be e n t i r e l y s a t i s f a c t o r y , i n c r e a s i n g c i t r a t e synthase a c t i v i t y when the energy charge i s low or when c i t r a t e and 2-ketoglu-t a r a t e are being u t i l i z e d . However, i n a f a c u l t a t i v e anaerobe, these c o n d i -t i o n s a l s o e x i s t during anoxia ( C o l l i c u t t , 1975; Wij s man, 1976) and p o t e n t i a t -i n g c i t r a t e synthase a c t i v i t y under these c o n d i t i o n s would be extremely d i s -advantageous because i t would l e a d to a c t i v i t y of a b a s i c a l l y aerobic system during anaerobic c o n d i t i o n s . Furthermore, as pointed out e a r l i e r , oxaloace-t a t e i s channelled p r i m a r i l y towards s u c c i n a t e during anoxia, making i t even more necessary to c u r t a i l c i t r a t e synthase a c t i v i t y . The most probable point at which c o n t r o l i s achieved i s the a v a i l a b i l i t y of oxaloacetate, which i s known to be s i g n i f i c a n t i n r e g u l a t i n g mammalian c i t r a t e synthase jLn v i v o (Olson and Williamson, 1971). As the primary source of oxaloacetate i s malate, i t i s h i g h l y probable that a r e d u c t i o n i n m i t o c h o n d r i a l MDH a c t i v i t y i s the method used f o r "d i s c o n n e c t i n g " the oxaloacetate >2-ketoglutarate arm of the Krebs c y c l e . Indeed, i f o y s t e r adductor muscle m i t o c h o n d r i a l MDH i s as s e n s i t i v e to NADH i n h i b i t i o n as the cytoplasmic isoenzyme has been shown to be (Chapter 6 ) , then the l e v e l s of oxaloacetate would be very c l o s e l y regulated by the NADH/NAD+ r a t i o , and t h i s would preclude the n e c e s s i t y f o r c i t r a t e synthase to be regulated d i r e c t l y by the NADH/NAD+ r a t i o . 76 Figure 18. E f f e c t of c i t r a t e on oxaloacetate saturation k i n e t i c s . (0) c o n t r o l ; (t) 5 mM c i t r a t e ; (A) 10 mM c i t r a t e ; 0.05 mM acetylCoA. 77 0.051 1/Oxaloacetate mM 78 Figure 19. E f f e c t of c i t r a t e on acetylCoA saturation k i n e t i c s . (0) con t r o l ; ( • ) 5 mM c i t r a t e ; (A) 10 mM c i t r a t e ; 0.05 mM oxaloacetate. 80 Figure 20. E f f e c t of 2-ketoglutarate on oxaloacetate k i n e t i c s . (•) c o n t r o l ; ( • ) 5 mM 2-ketoglutarate; (A) 10 mM 2-ketoglutarate; 0.05 mM acetylCoA. 81 0.051 -150 -100 -50 0 50 100 150 200 250 VOxaloacetate mM" 1 82 Figure 21. E f f e c t of 2-ketoglutarate on acetylCoA k i n e t i c s . (•) c o n t r o l ; ( A ) 5 mM 2-ketoglutarate; ( • ) 10 mM 2-ketoglutarate; 0.05 mM oxaloacetate. 83 84 CHAPTER V THE ROLE OF OYSTER ADDUCTOR CYTOPLASMIC ASPARTATE AMINOTRANSFERASE  DURING ANOXIA  INTRODUCTION In the previous chapter the p o s s i b i l i t y of a l t e r i n g the metabolism of malate during anoxia was discussed. One p o s s i b l e source of malate would be aspartate. Aspartate l e v e l s i n b i v a l v e s are high under aerobic c o n d i t i o n s , being u t i l i z e d during anaerobiosis ( C o l l i c u t t , 1975; DuPaul and Webb, 1971). In the adductor, most of the aspartate would be found i n the cytoplasm of the c e l l s , because mitochondria account f o r a very small percentage of the t i s s u e (Hanson and Lowy, 1961). The cytoplasmic isoenzyme of aspartate aminotrans-ferase i s t h e r e f o r e i n v o l v e d i n converting aspartate to oxaloacetate under anaerobic c o n d i t i o n s , and hence i t must be adapted f o r f u n c t i o n at lower pH values (which occur during anoxia) than would be encountered by the mammalian homologue. The subsequent f a t e of the oxaloacetate i s conversion to malate i n the cytoplasm by malate dehydrogenase. Malate dehydrogenase i s an enzyme normally a s s o c i a t e d w i t h aerobic m i t o c h o n d r i a l f u n c t i o n , p a r t i c i p a t i n g i n the Krebs c y c l e . E u k aryotic c e l l s , however, possess a second isoenzyme form of malate dehydrogenase i n the cytoplasm which c a t a l y z e s the same r e a c t i o n but i s a s t r u c t u r a l l y d i s t i n c t p r o t e i n (Delbruck et a l . , 1959; S i e g e l and Englard, 1962). The cytoplasmic isoenzyme of malate dehydrogenase p a r t i c i p a t e s i n the malate-aspartate s h u t t l e ( F i g . 22) s e r v i n g to couple m i t o c h o n d r i a l and cyt o -plasmic metabolism (La Noue and Williamson, 1971; Safer and Williamson, 1973). The malate-aspartate c y c l e permits c y t o p l a s m i c a l l y derived NADH to be o x i d i z e d v i a the e l e c t r o n t r a n s p o r t chain i n the mitochondria and plays a major r o l e during aerobic catabolism of carbohydrate i n c a r d i a c muscle (La Noue and W i l l i a m s o n , 1971). Thus cytoplasmic aspartate aminotransferase and malate dehydrogenase have a s y n e r g i s t i c f u n c t i o n which generates malate from aspar-t a t e . B i v a l v e molluscs have apparently accentuated the cytoplasmic p o r t i o n 85 Figure 22. Malate-aspartate s h u t t l e (from La Noue and Williamson , 1971). 86 Aspartate f 2-Ketoglutarate • Glutamate Oxaloacetate NADH + H* r ^ NAD + Malate CYTOPLASM Aspartate 4 2- Ketoglutarate Glutamate J Oxaloacetate 4 NADH + H + NAD* ' Malate MITOCHONDRION 87 of the malate-aspartate c y c l e , adapting i t f o r an anaerobic f u n c t i o n i n order to regenerate NAD + and maintain redox balance. Because of t h i s , a spartate becomes another important energy source i n a d d i t i o n to glycogen, and has been shown to be depleted during anoxia (Du Paul and Webb, 1971), being converted mainly to succinate w h i l e glucose i s converted mainly to a l a n i n e ( C o l l i c u t t , 1975). The k i n e t i c p r o p e r t i e s of the cytoplasmic isoenzymes of aspartate amino-t r a n s f e r a s e and malate dehydrogenase would obviously play an important r o l e i n determining the method by which aspar t a t e l e v e l s are maintained at a high l e v e l during aerobic c o n d i t i o n s and then be m o b i l i z e d during anaerobic con-d i t i o n s . The p r o p e r t i e s of the cytoplasmic isoenzyme of aspartate aminotrans-ferase are reported here and those of cytoplasmic malate dehydrogenase i n a subsequent chapter. I t was found that aspartate aminotransferase had a higher a f f i n i t y f o r 2-ketoglutarate and a s l i g h t l y lower a f f i n i t y f o r oxaloacetate, a higher a f f i n i t y f o r aspart a t e than glutamate, thus favouring f u n c t i o n pre-dominantly i n the d i r e c t i o n of aspartate u t i l i z a t i o n . RESULTS AND DISCUSSION Aspartate aminotransferase occurs i n reasonably high a c t i v i t e s i n the oyster adductor muscle, ranging from 9 to 10 u n i t s / g wet weight. Starch g e l e l e c t r o p h o r e s i s at pH 8.5 revealed a major band, which i s the c y t o s o l i c i s o -enzyme, and a minor band, the m i t o c h o n d r i a l isoenzyme ( F i g . 23). Estimates from the s i z e s of the peaks from the DEAE Sephadex column ( F i g . 24) showed that the c y t o s o l i c isoenzyme accounts f o r about 90% of the t o t a l a c t i v i t y . The molecular weight was found to be 82,000±4,000 by g e l f i l t r a t i o n . This value corresponds to the values obtained f o r the cytoplasmic isoenzyme of 88 Figure 23. Starch g e l e l e c t r o p h o r e s i s of adductor aspartate aminotransferase. Conditions: 5 mM citrate-phosphate pH 7.7, 40 mAmp f o r 24 hr. a = crude homogenate; b = supernatant f r a c t i o n ; c = p a r t i a l l y p u r i f i e d cytoplasmic isoenzyme. 89 + Origin 90 Figure 24. E l u t i o n of adductor aspartate aminotransferase from DEAE Sephadex. The small peak i s the mitochondrial isoenzyme, the larger peak i s the cytoplasmic isoenzyme. Conditions as stated i n "Materials and Methods". 91 92 aspartate aminotransferase from beef heart, beef brain, pig heart, rat brain and porpoise s k e l e t a l muscle ( K r i s t a and Fonda, 1973; Magee and P h i l l i p s , 1971; Martinez-Carrion et a l . , 1967; Owen and Hochachka, 1974). Ef f e c t s of pH The enzyme shows a pH optimum i n the 7.4 to 8.4 range (Fig. 25), the a c t i v i t y decreasing below pH 7.0, the a c t i v i t y at pH 6.5 being approximately 75% of the a c t i v i t y at pH 7.4. Under the conditions of the assays, the a c t i v i t y was higher i n the d i r e c t i o n of aspartate formation than oxaloacetate formation. Enzyme Ki n e t i c s Under the conditions used the enzyme displayed normal Michaelis-Menten k i n e t i c s . Lineweaver-Burke plots of the data gave a series of p a r a l l e l l i n e s when the cosubstrate was varied (Fig. 26, 27), which i s consistent with a Ping Pong B i B i mechanism demonstrated for a v a r i e t y of other transaminases (Cleland, 1971). Kffi values were determined by p l o t t i n g 1/Km vs. 1/cosubstrate and extrapolating the r e s u l t i n g l i n e a r plot to 1/cosubstrate = 0 (Hopper and Segal, 1962); these are l i s t e d i n Table IX for both pH 7.4 and 6.5. TheK for aspartate was higher than that of a number of other cytoplas-m mic isoenzymes of aspartate aminotransferase i s o l a t e d from a v a r i e t y of d i f f e r e n t t i ssues, while the K m for 2-ketoglutarate was considerably lower than values reported for mammalian tissues (Henson and Cleland, 1964; Nisselbaum and Bodansky, 1966; Magee and P h i l l i p s , 1971; K r i s t a and Fonda, 1973; Owen and Hochachka, 1974) and s i m i l a r to the value reported for chicken heart (Bertland and Kaplan, 1970) (Table IX). The Kffi for glutamate was higher than values reported for other enzymes, whereas the K^ for oxaloace-tate was about the same as i n pig heart, but higher than that for porpoise 93 Figure 25. E f f e c t of pH on a c t i v i t y of cytoplasmic aspartate aminotransfer-ase. (A) aspartate >oxaloacetate: 40 mM a s p a r t a t e , 0.5 mM 2-ketoglutarate. (•) o x a l o a c e t a t e — > a s p a r t a t e : 40 mM glutamate, I I 1 mM oxaloacetate. Other c o n d i t i o n s as s t a t e d i n M a t e r i a l s and Methods". 94 95 Figure 26. Aspartate and glutamate s a t u r a t i o n k i n e t i c s of cytoplasmic aspartate aminotransferase. a) Aspartate s a t u r a t i o n at v a r y i n g 2-ketoglutarate: ( A ) 0.15 mM, (•) 0.1 mM, (•) 0.07 mM, (•.) 0.05 mM, and ( O ) 0.03 mM 2-ketoglutarate. Other c o n d i t i o n s : 100 mM soidum phosphate pH 7.4, 25°C. b) Glutamate s a t u r a t i o n at v a r y i n g oxaloacetate: (•) 0.5 mM, (•) 0.3 mM, ( A ) 0.2 mM, (•) 0.14 mM, ( O ) 0.1 mM oxaloacetate. Other c o n d i t i o n s : 100 mM sodium phsophate pH 7.4, 25°C. c) Replot of ! / K m ( a s p ) v s - 1/2-ketoglutarate. d) Replot of 1/K' , .. , vs. 1/oxaloacetate. m(glut) 96 Q01H 0.1 02 03 1/Aspartate mM" 1 26a Q O & i 001H 0.04 0.08 0.12 0.16 0.20 .1/Glutamate mM" 1 26b 97 98 Figure 27. Oxaloacetate and 2-ketoglutarate s a t u r a t i o n k i n e t i c s of c y t o p l a s -mic aspartate aminotransferase. a) 2-ketoglutarate s a t u r a t i o n at v a r y i n g a s p a r t a t e : ( O) 20 mM, (•) 15 mM, ( A ) 10 mM, ( #..) 7 mM, (•) 5 mM aspartate. Other c o n d i t i o n s : 100 mM sodium phosphate b u f f e r , pH 7.4, 25°C. b) Oxaloacetate s a t u r a t i o n at v a r y i n g glutamate: (•) 20 mM, ( •) 14 mM, ( A ) 10 mM, (O) 7 mM, (•) 5 mM glutamate. Other c o n d i t i o n s : 100 mM sodium phosphate pH 7.4, c) Replot of 1/K' m(2-kga) vs. 1/aspartate. d) Replot of 1/K' 'm(oxa) vs. 1/glutamate. 40n 0 Q04 Q08 0.12 0.16 02 1 /Glutamate mM 2 7 6 101 Table IX. M i c h a e l i s constants f o r adductor aspartate aminotransferase. Km, mM, at °° conc e n t r a t i o n of cosubstrate i n sodium phosphate b u f f e r at s t a t e d pH, and at 25°C. Substrate pH 7.4 pH 6.5 Aspartate 4.5 7.1 2-ketoglutarate 0.069 0.034 Glutamate 13.8 36.0 Oxaloacetate 0.09 0.065 102 muscle (Henson and C l e l a n d , 1964; Owen and Hochachka, 1974) (Table X). The K m values f o r 2-ketoglutarate and oxaloacetate decreased w i t h decreasing pH, whereas the values f o r aspartate and glutamate i n c r e a s e d , a phenomenon a l s o reported f o r porpoise muscle aspar t a t e aminotransferase (Owen and Hoch-achka, 1974). The decrease i n f o r 2-ketoglutarate at pH 6.5 probably a s s i s t s i n compensating f o r the decreased t o t a l a c t i v i t y at t h i s pH and main-t a i n i n g f u n c t i o n during anoxia. This i s s i g n i f i c a n t as the pH of the mantle f l u i d i n the oyster decreases to 6.7 i n the f i r s t twenty minutes a f t e r the s h e l l s have c l o s e d (Pedlow, 1974) and a decrease i n i n t r a c e l l u l a r pH would a l s o be expected. The higher a f f i n i t y f o r aspartate as compared w i t h g l u t a -mate, and the higher a f f i n i t y f o r 2-ketoglutarate as compared w i t h oxaloace-t a t e , coupled w i t h the high l e v e l s of aspartate under aerobic c o n d i t i o n s (12-15 mM) ( C o l l i c u t t , 1975) suggest that the enzyme would f u n c t i o n more r e a d i l y i n the d i r e c t i o n of aspartate u t i l i z a t i o n . I n h i b i t i o n Studies The enzyme was assayed i n both d i r e c t i o n s i n the presence of 10 mM a l a n i n e , a r g i n i n e , asparagine, a s p a r t a t e , g l y c i n e , glutamate, glutamine, h i s t i d i n e , i s o l e u c i n e , l e u c i n e , p r o l i n e , t a u r i n e , v a l i n e , malate, c i t r a t e and s u c c i n a t e ; 1 mM fumarate; 5 mM ATP, 1 mM ADP, FDP and PEP. In the a s p a r t a t e -oxaloacetate d i r e c t i o n only glutamate, malate, c i t r a t e and succinate were found to be i n h i b i t o r y ; i n the reverse d i r e c t i o n , a s p a r t a t e , s u c c i n a t e , malate and c i t r a t e were i n h i b i t o r y . Glutamate i s competitive w i t h a s p a r t a t e ( F i g . 28), and aspartate i s competitive w i t h glutamate. Succinate, malate and c i t r a t e a l l give a p a t t e r n of mixed i n h i b i t i o n w i t h both substrates ( F i g . 29, 30), the i n h i b i t i o n by malate being almost i d e n t i c a l w i t h that by c i t r a t e . The R\ values f o r s u c c i n a t e , malate and c i t r a t e (Table XI) at pH 7.4 were i n 103 Figure 28. E f f e c t of glutamate on aspartate s a t u r a t i o n k i n e t i c s . ( • ) con-t r o l , (•) 5 mM glutamate, (A) 10 mM glutamate. Other c o n d i t i o n s : 2-ketoglutarate c o n c e n t r a t i o n , 0.3 mM, 100 mM sodium phosphate pH 7.4, 25°C. -02 -C.l 5 -01 -0.05 0.05 Ol 0.15 0.2 025 Q3 0.35 1/Aspartate mM - 1 105 Figure 29. E f f e c t of succinate on aspartate s a t u r a t i o n k i n e t i c s . (0) c o n t r o l , (A) 5 mM s u c c i n a t e , ( • ) 10 mM succinate. Other c o n d i t i o n s : 0.3 mM 2-ketoglutarate, 100 mM sodium phosphate pH 7.4, 25°C. 106 107 E f f e c t of succinate on 2-ketoglutarate s a t u r a t i o n k i n e t i c s . ( • ) c o n t r o l , (•) 5 mM s u c c i n a t e , (A) 10 mM succinate. Other c o n d i t i o n s : 0.3 mM 2-ketoglutarate, 100 mM sodium phosphate pH 7.4, 25°C. 0.05 0.04 V 003 002 10 15 20 25 30 1/2-ketoglutarate mM" 109 Table X. K values f o r aspart a t e aminotransferase from v a r i o u s sources, m Source Conditions Km(ASP)' ™ Km(KGA)>m Beef b r a i n ( K r i s t a and Fonda, 1973) Beef heart and l i v e r (Wada and Morino, 1964) P i g heart (Nisselbaum and Bodansky, 1966) P i g heart (2 forms) (Martinez-Carrion e± a l . , 1967) Rat b r a i n (Magee and P h i l l i p s , 1971) Porpoise muscle (Owen and Hochachka, 1974) Chicken heart (3 forms) (Bertland and Kaplan, 1970) Oyster adductor muscle 67 mM T r i s - H C l pH 7.4 2.0 60 mM arsenate pH 7.4 2.0-2.5 67 mM sodium phosphate pH 7.4 100 mM T r i s - H C l pH 8.0 3.9 4.9 2.0 2.4 67 mM potassium phosphate 6.7 pH 7.4 100 mM sodium phosphate 2.4 pH 7.3 100 mM T r i s - H C l pH 7.4 3.67 4.5 3.77 100 mM sodium phosphate 4.6 pH 7.4 0.17 0.3-0.4 0.57 0.61 0.15 0.14 0.15 0.13 0.078 0.083 0.081 0.069 P i g heart (Henson and Cl e l a n d , 1964) Porpoise muscle (Owen and Hochachka, 1974) Oyster adductor muscle 100 mM sodium arsenate 8.9 pH 7.4 100 mM sodium phosphate 3.2 pH 7.3 100 mM sodium phosphate 13.8 pH 7.4 0.088 0.06 0.09 110 Table XI. I n h i b i t i o n constants (K_ values) (mM) f o r v a r i o u s i n h i b i t o r s of adductor cytoplasmic a s p a r t a t e aminotransferase. K_ values were obtained from Dixon p l o t s using three s u b s t r a t e concen-t r a t i o n s at a f i x e d c o ncentration of cosubstrate. Cosubstrate concentrations were: f o r glutamate i n h i b i t i o n , 0.3 mM 2-ketoglutarate; f o r aspart a t e i n -h i b i t i o n , 0.5 mM oxaloacetate; f o r s u c c i n a t e , malate and c i t r a t e i n h i b i t i o n , 20 mM aspartate or 20 mM glutamate. Conditions were 25°C and 0.1 M sodium phosphate at i n d i c a t e d pH. I n h i b i t o r Aspartate Oxaloacetate Oxaloacetate Aspartate pH 7.4 pH 6.5 pH 7.4 pH 6.5 Glutamate 8.0 15.0 Aspartate 4.5 11.4 Succinate 14.0 11.0 14.0 11.0 Malate 10.0 - 13.0 C i t r a t e 11.0 - 13.0 I l l excess of 10 mM, hence they are assumed to have very l i t t l e p h y s i o l o g i c a l sign i f i c a n c e , as the concentrations of malate and c i t r a t e do not exceed 1 mM ( C o l l i c u t t , 1975), and succinate l e v e l s are i n the 3 mM (de Zwaan and Zandee, 1972) to 5 mM range ( C o l l i c u t t , 1975). The competitive i n h i b i t i o n between the amino acids glutamate and aspartate i s expected on t h e o r e t i c a l grounds (Cleland, 1971), being found i n other transaminases as w e l l . The K^ f o r glutamate increased from 8 to 15 mM with a decrease i n pH from 7.4 to 6.5, a r e s u l t which also suggests that under anoxic conditions aspartate u t i l i z a -t i o n would be favoured. Considerations of Aspartate Aminotransferase Function Aspartate aminotransferase i s generally considered to catalyze an e q u i l i -brium r e a c t i o n i n vivo (Krebs and Veech, 1969), however, t h i s has never been conclusively proven, because of the compartmentation of the reactants between mitochondria and cytoplasm. In muscle i t i s thought to function s o l e l y i n aspartate u t i l i z a t i o n , as part of the malate-aspartate shuttle for t r a n s f e r of NADH across the mitochondrial membrane (Safer and Williamson, 1973; T i s c h l e r et_ s i l . , 1976; T i s c h l e r , personal communication). In mammalian muscle the aspartate concentration range i s about 2-5 mM (Safer and William-son, 1973; Randle et_ al., 1970), and t h i s corresponds c l o s e l y to the measured K m values for aspartate i n a v a r i e t y of tissues (Table X). However, i n the oyster adductor muscle, under aerobic conditions, the aspartate concentration would be s u f f i c i e n t to e f f e c t i v e l y saturate the enzyme, and the a v a i l a b i l i t y of 2-ketoglutarate would be a major factor i n determining the rate of aspartate u t i l i z a t i o n . As GDH a c t i v i t i e s are low, the most probable mode of producing 2-ketoglutarate from glutamate i s v i a alanine aminotransferase, which i s k i n e t -i c a l l y adapted for alanine and 2-ketoglutarate formation (Mustafa, 1974). 112 During aerobic c o n d i t i o n s the major f a t e of pyruvate i s conversion to CC>2 and B^O ( C o l l i c u t t , personal communication). On the t r a n s i t i o n to anaerobic c o n d i t i o n s , the g l y c o l y t i c a c t i v i t y w i l l be enhanced and pyruvate production w i l l be increased. This w i l l r e s u l t i n an accumulation of a l a n i n e and an increase i n the l e v e l of 2-ketoglutarate, the l a t t e r then i n i t i a t i n g the m o b i l i z a t i o n of aspartate. The net e f f e c t i s a s t a b i l i z a t i o n of g l u t a -mate l e v e l s , which i s observed to be the case (DuPaul and Webb, 1971; C o l l i c u t t , 1975), production of oxaloacetate from a s p a r t a t e , and maintenance of cytoplasmic redox balance by the conversion of oxaloacetate to malate by cytoplasmic malate dehydrogenase ( F i g . 31). I t should be noted that the cou p l i n g of al a n i n e aminotransferase and aspartate aminotransferase i s a l s o found i n mammalian heart muscle, but the f u n c t i o n i n t h i s t i s s u e i s the generation of malate, which i s r e q u i r e d to augment the pool of Krebs c y c l e intermediates during increased work (Safer and Williamson, 1973). In the oyster adductor and heart muscle t h i s t r a n s -aminase couple permits maintenance of redox balance under aerobic c o n d i t i o n s i n the absence of LDH. 113 Proposed couple between glucose and aspartate metabolism during anoxia. 114 aspar ta te oxa loaceta te NADH •--NAD malate g lycogen FDP i G 3 P NAD + -NADH-1,3DPG P E P • pyruvate 2 - kga - ^—. g lu tamate • • g l u t a m a t e ^ V — 2 - k g a alanine succinate 115 CHAPTER V I THE ROLE OF CYTOPLASMIC MALATE DEHYDROGENASE DURING ANOXIA  INTRODUCTION As p r e v i o u s l y d i s c u s s e d , t he o y s t e r a d d u c t o r m u s c l e p o s s e s s e s a h i g h l y a c c e n t u a t e d c y t o p l a s m i c a rm o f t h e m a l a t e - a s p a r t a t e s h u t t l e . The k i n e t i c p r o p e r t i e s o f c y t o p l a s m i c a s p a r t a t e a m i n o t r a n s f e r a s e were f o u n d t o be a d a p t e d f o r a s p a r t a t e u t i l i z a t i o n u n d e r a n a e r o b i c c o n d i t i o n s , t h u s p r o d u c i n g o x a l o a c e -t a t e f o r t h e c y t o p l a s m i c MDH. C o n v e r s i o n o f o x a l o a c e t a t e t o m a l a t e p e r m i t s t h e r e g e n e r a t i o n o f c y t o p l a s m i c N A D + f o r t h e c o n t i n u e d f u n c t i o n o f g l y c o l y s i s , i n t h i s r e g a r d MDH f u n c t i o n s a s a s u b s t i t u t e f o r L D H . Thus MDH h a s a c e n t r a l r o l e i n m a i n t a i n i n g c y t o p l a s m i c r e d o x b a l a n c e i n t h i s scheme o f c o u p l e d amino a c i d and c a r b o h y d r a t e m e t a b o l i s m , and a l s o i n t h e o t h e r schemes p r o p o s e d where PEP i s c o n v e r t e d d i r e c t l y t o o x a l o a c e t a t e . The k i n e t i c p r o p e r t i e s o f a d d u c t o r c y t o p l a s m i c MDH were s t u d i e d i n a n a t t e m p t t o u n d e r s t a n d how t h i s enzyme i s a d a p t e d f o r m e t a b o l i c f u n c t i o n i n a f a c u l -t a t i v e a n a e r o b e . I t was f o u n d t h a t t h e K f o r o x a l o a c e t a t e was l o w , and m d e c r e a s e d w i t h d e c r e a s i n g p H . F u r t h e r m o r e t h e enzyme i s i n s e n s i t i v e t o i n -h i b i t i o n by m a l a t e and N A D + a t p h y s i o l o g i c a l l e v e l s , w h e r e a s i n t h e r e v e r s e d i r e c t i o n i t i s e x t r e m e l y s e n s i t i v e t o NADH and o x a l o a c e t a t e i n h i b i t i o n . RESULTS AND DISCUSSION I n t h e o y s t e r a d d u c t o r m u s c l e , m a l a t e d e h y d r o g e n a s e o c c u r s i n h i g h a c t i v i t y , a b o u t 120 u n i t s / g wet w t . On s t a r c h g e l s and a l s o on p o l y a c r y l a -mide g e l s two e l e c t r o p h o r e t i c f o rms were d i s t i n g u i s h e d , c y t o p l a s m i c and m i t o c h o n d r i a l ( F i g . 3 2 ) , t h e c y t o p l a s m i c f o r m p r e d o m i n a t i n g . D e s p i t e s e v e r a l a t t e m p t s u n d e r v a r i o u s e x p e r i m e n t a l c o n d i t i o n s , t h e m i t o c h o n d r i a l i s o e n z y m e 116 Figure 32. Starch g e l e l e c t r o p h o r e s i s of adductor MDH. Con d i t i o n s : 14% st a r c h i n 5 mM phosphate-citrate pH 7.7, 40 mAmp f o r 24 hr. a, crude homogenate; b, supernatant f r a c t i o n ; c, p a r t i a l l y p u r i f i e d cytoplasmic isoenzyme. 1 1 7 118 was never s u c c e s s f u l l y i s o l a t e d from the adductor muscle. The molecular weight was found to be 6700015000 by gel f i l t r a t i o n on Sephadex G-200. This i s s i m i l a r to the values reported for MDH from a va r i e t y of sources (Grimm and Doherty, 1961; K i t t o and Lewis, 1967; Dowda and Betterton, 1974; Leskovac et a l . , 1975). C a t a l y t i c Properties In the oxaloacetate —>malate d i r e c t i o n , adductor MDH had a pH optimum between 7.2 and 7.6, although there was not much decrease i n a c t i v i t y at pH 6.5 (80% of maximum). In the reverse d i r e c t i o n the pH optimum was between 8.5 and 9.0 (Fig. 33). For oxaloacetate and NADH normal Michaelis-Menten k i n e t i c s were followed over the concentration ranges 0.03 to 0.2 mM NADH, and 0.03 to 0.8 mM oxaloacetate. At concentrations of oxaloacetate i n excess of 2 mM, some s l i g h t substrate i n h i b i t i o n was observed. Malate saturation k i n e t i c s were normal between 0.1 and 4 mM, however at concentrations i n excess of 5 mM, substrate a c t i v a t i o n was observed (Fig. 34). In addition NAD+ also showed anomalous saturation k i n e t i c s at concentrations higher than 1 mM (Fig. 35), whereas i n the 0.05 to 1 mM range normal satura-ti o n k i n e t i c s were obtained. Dehydrogenases have been shown to possess an ordered k i n e t i c mechanism i n which NAD+ or NADH binds to the enzyme f i r s t ( D a l z i e l , 1975). In order to ca l c u l a t e the absolute K^, substrate saturation data at varying cosubstrate concentrations were pl o t t e d on double r e c i p r o c a l p l o t s , and then the ordinate intercepts (1/V') were rep l o t t e d vs. 1/cosubstrate and extrapolated to 1/V* = 0, which gives the value of 1/K , , ' - (Fig. 36, 37, 38, 39). m(cosubstrate) In a l l cases the concentration range used was that over which normal M i c h a e l i s -Menten k i n e t i c s were obtained. The K values are l i s t e d i n Table XII. 119 Figure 33. E f f e c t of pH on adductor cytoplasmic MDH. (0) oxaloacetate malate 0.1 mM NADH, 0.5 mM oxaloacetate; ( • ) malate oxaloacetate 1 mM NAD+, 4 mM malate. 120 121 Figure 34. Malate s a t u r a t i o n of oys t e r adductor malate dehydrogenase; i n s e t double r e c i p r o c a l p l o t showing anomalous a c t i v a t i o n . Conditions: 1 mM NAD+, 0.1 M T r i s - H C l pH 8.9, 25°C. 1 2 2 Malate, mM 123 Figure 35. NAD s a t u r a t i o n of oyster adductor malate dehydrogenase; i n s e t double r e c i p r o c a l p l o t showing anomalous a c t i v a t i o n . C onditions: 4 mM malate, 0.1 M T r i s - H C l pH 8.9, 25°C. 124 80h NADt m M 125 Figure 36. Oxaloacetate s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH. a) double r e c i p r o c a l p l o t s of oxaloacetate s a t u r a t i o n at var y i n g NADH (0) 0.05 mM, (A) 0.03 mM, ( • ) 0.02 mM, (•) 0.014 mM, (a) 0.01 mM NADH. Other c o n d i t i o n s : 0.1 T r i s - H C l pH 7.5, 25°C. b) r e p l o t of 1/V' from Figure 37 vs. 1/oxaloacetate. 126 0.0 5i -28 -24 -20 -16 -12 -8 -4 0 4 1/Oxaloacetate mM 36b 8 12 ,-1 16 20 1 2 7 Figure 37. NADH s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH. a) double r e c i p r o c a l p l o t s of NADH s a t u r a t i o n at v a r y i n g oxaloacetate (0) 0.2 mM, (A) 0.13 mM, ( • ) 0.1 mM, (•) 0.07 mM, ( A ) 0.05 mM oxaloacetate. Other c o n d i t i o n s : 0.1 M T r i s - H C l pH 7.5, 25°C. b) r e p l o t of 1 / V from Figure 36 vs. 1/NADH. 128 0.051 -60 -40 -20 20 40 6 0 80 100 1/ N A D H m M 37a Q03n -60 -40 -20 0 20 40 60 80 100 1/ N A D H m M " 1 37b 129 Figure 38. Malate s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH. a) double r e c i p r o c a l p l o t of malate s a t u r a t i o n at v a r y i n g NAD ( A ) 1 mM, (0) 0.3 mM, ( • ) 0.2 mM, (A) 0.13 mM NAD+. Other c o n d i t i o n s : 0.1 M T r i s - H C l pH 8.9, 25°C. b) r e p l o t of 1 / V from Figure 39 vs. 1/malate. 130 0.041 -1.6 -12 -08 -0.4 0 0.4 08 1.2 1.6 20 1/ Malate mM" 1 38a -8 -7 -6 -5 -4 - 3 - 2 - 1 1 2 1/Malate mM' 38b 131 Figure 39. NAD s a t u r a t i o n k i n e t i c s of adductor cytoplasmic MDH. a) double r e c i p r o c a l p l o t of NAD + s a t u r a t i o n at v a r y i n g malate (•) 4 mM, ( Q ) 2 mM, (A) 1 mM, (0) 0.5 mM malate. Other c o n d i t i o n s : 0.1 M T r i s - H C l pH 8.9, 25°C. b) r e p l o t of 1/v' from Figure 38 vs. 1/NAD+. 132 -25 -20 -15 -10 -5 5 10 1 / N A D + m M " 1 39b 133 Table X I I . M i c h a e l i s constants f o r oyste r adductor malate dehydrogenase. A l l values are at °° conce n t r a t i o n of coenzymes or cosubstrate, 25 C and st a t e d pH. NAD+, oxaloacetate and malate con c e n t r a t i o n ranges were those i n which normal Michaelis-Menten k i n e t i c s were obtained. Substrate K (mM) m NADH pH 7.5 0.019 pH 6.5 0.022 oxaloacetate pH 7.5 0.041 pH 6.5 0.01-0.02 NAD + pH 8.9 0.043 malate pH 8.9 0.13 134 The K m values f o r oxaloacetate and NADH were found to be s i m i l a r to thos obtained f o r cytoplasmic MDH from a v a r i e t y of other sources, whereas the values f o r NAD + and malate were found to be lower (Table X I I I ) (Grimm and Doherty, 1961; Englard and Breigher, 1962; K i t t o and Kaplan, 1966; Zee and Zinkham, 1968; Leskovac et a l . , 1975). S i g n i f i c a n t s u b s t r a t e i n h i b i t i o n by oxaloacetate has been observed f o r MDH's, but normally i t occurs at higher concentrations of oxaloacetate (1-2 mM) f o r the cytoplasmic isoenzyme than f o r the m i t o c h o n d r i a l isoenzyme (0.1-0.2 mM) (Englard and Breigher, 1962; S i e g e l and Englard, 1962; K i t t o and Kaplan, 1966; K i t t o and Lewis, 1967). With regard to oxaloacetate i n h i b i t i o n , adductor MDH resembles other cytoplas mic MDH's from v e r t e b r a t e s . Anomalous malate a c t i v a t i o n has been observed fo the m i t o c h o n d r i a l isoenzyme from beef heart and p i g heart (Davies and Kun, 1957; T e l e g d i ej; a l . , 1973), but NAD+ a c t i v a t i o n has not been observed. In t h i s regard adductor MDH resembles the m i t o c h o n d r i a l isoenzyme of p i g heart and beef heart. The mechanism of malate a c t i v a t i o n has been i n v e s t i g a t e d f o r the p i g heart m i t o c h o n d r i a l isoenzyme, and shown to be produced by malate b i n d i n g to a s i t e d i s t i n c t from the a c t i v e s i t e , thereby reducing the Michael i s constant f o r NAD + 1 0 - f o l d (Telegdi j i t a l , 1973). The concentrations of malate r e q u i r e d to produce t h i s e f f e c t are much higher than those normally found jLn vivo, being 30 mM f o r porcine heart MDH (Telegdi et_ a l , 1973), and 5 mM f o r adductor MDH, i n v i v o concentrations being approximately 0.2 mM i n heart (Randle et a l . , 1970; Safer and Williamson, 1973) and approximately 0.3 mM i n oyste r heart ( C o l l i c u t t , 1975). Therefore i t appears that t h i s phenomenon i s not p h y s i o l o g i c a l l y s i g n i f i c a n t as a r e g u l a t o r of MDH a c t i v i t y i n v i v o , although i t i s d i f f i c u l t to understand why such a mechanism should have evolved i n widely d i f f e r i n g species. On the other hand, the anomalous a c t i v a t i o n by NAD + may be p h y s i o l o g i c a l l y s i g n i f i c a n t , because i t occurs at 135 Table X I I I . values (mM) f o r cytoplasmic MDH from v a r i o u s sources. Source Conditions K m(OXA) K m(NADH) Beef heart (Grimm and Doherty, 1961) (Englard and Breigher, 1962) 100 mM EDTA pH 9.0 500 mM potassium phosphate pH 6.7 0.051 0.042 0.038 0.027 Chicken heart ( K i t t o and Kaplan, 1966) 100 mM sodium pyro-phosphate pH 9.0 0.05 Human erythr o c y t e s (Shrago and Falcone, 1963) 33 mM T r i s - H C l pH 7.4 0.0095 0.035 -Pig e r y t h r o c y t e s (Leskovac et a l . , 1975) 100 mM sodium phos-phate pH 7.4 0.062 0.056 A s c a r i s suum (2 forms) (Zee and Zinkham,1968) 90 mM potassium phos- 0.024 phate pH 7.6 0.029 Drosophila v i r i l i s (McReynolds and K i t t o , 1970) 0.04 Penaeus s e t i f e r o u s , muscle (Hodnett et a l . , 1976) 100 mM potassium phosphate pH 7.5 0.103 Penaeus aztecus, muscle (Hodnett et a l . , 1976) 100 mM potassium phosphate pH 7.5 0.0577 Oyster adductor muscle 100 mM T r i s - H C l pH 7.5 0.041 0.019 136 Table X I I I (Continued) K values f o r cytoplasmic MDH from v a r i o u s sources, m Source Conditions K K "I-m(malate) m(NAD ) Beef heart (Grimm and Doherty, 1961) (Englard and Breigher, 1962) Chicken heart ( K i t t o and Kaplan, 1966) Human erythrocytes (Shrago and Falcone, 1963) P i g e r y t h r o c y t e s (Leskovac et a l . , 1975) A s c a r i s suum (2 forms) (Zee and Zinkham, 1968) Drosophila v i r i l i s (McReynolds and K i t t o , 1970) Penaeus s e t i f e r o u s (Hodnett et a l . , 1976) Penaeus aztecus (Hodnett et a l . , 1976) Oyster adductor muscle 100 mM EDTA pH 9.0 0.054 100 mM T r i s - H C l 0.47 pH 8.4 100 mM sodium pyro-phosphate pH 9.0 90 mM g l y c i n e , 10 mM sodium pyrophos-phate pH 10.5 0.8 100 mM g l y c i n e KOH 0.38 pH 10.0 g l y c i n e T r i s - H C l 1.2 pH 9.0 1.9 1.7 8.0 100 mM potassium 0.398 phosphate pH 7.5 100 mM potassium 0.22 phosphate pH 7.5 100 mM T r i s - H C l 0.13 pH 8.9 0.2 0.099 0.093 0.135 0.043 137 q u i t e low concentrations of NAD + (1 mM) which approximates the NAD+ pool s i z e i n c e l l s (Chance et _ a l . , 1965; Leroy and Bachand, 1975). Product I n h i b i t i o n Malate was found to be a potent i n h i b i t o r , being uncompetitive w i t h respect to NADH at an oxaloacetate concentration of 0.5 mM, and noncompetitive w i t h respect to oxaloacetate at .0.1 mM •NADH (Fig.40). The K\ f o r malate was found to be 2.4 mM w i t h respect to oxaloacetate at pH 7.5, t h i s value i n -c r e a s i n g to 5 mM at pH 6.5. NAD + was a competitive i n h i b i t o r w i t h respect to NADH, R\ values being 2.4 and 1.3 mM at pH 7.5 and 6.5 r e s p e c t i v e l y . This decrease i s s u r p r i s i n g , because the ^NAIDH.) ^ O E S N O T C N A N S E very much between these pH values. Furthermore, the r e l a t i v e l y high (NAD-*") a t ^ ^"^ s u g g e s t s that redox con-t r o l i s not very important i n r e g u l a t i n g the enzyme a c t i v i t y at t h i s pH. Oxaloacetate gave mixed k i n e t i c s w i t h respect to NAD"*" and malate ( F i g . 41), the being 0.03 mM w i t h respect to malate at 1 mM NAD+. NADH a l s o gave a mixed p a t t e r n w i t h respect to both NAD + and malate ( F i g . 42), but s u r p r i s i n g l y the i n h i b i t i o n was found to be non l i n e a r w i t h respect to NADH when p l o t t e d on a Dixon p l o t ( F i g . 43). The i n h i b i t i o n p a t t e r n i s suggestive of there being more than one NADH bi n d i n g s i t e per a c t i v e u n i t , and the + + anomalous NAD k i n e t i c s support t h i s contention.. Since NAD i n h i b i t i o n and NADH s a t u r a t i o n k i n e t i c s were normal, however, extensive s t u d i e s of the k i n e t i c p r o p e r t i e s would be r e q u i r e d to c l a r i f y t h i s . Other I n h i b i t o r s A search was made f o r other metabolic e f f e c t o r s of adductor MDH. Only c i t r a t e , 2-ketoglutarate and the adenylates were found to have any e f f e c t , and a l l were i n h i b i t o r y . Taurine, glutamate, a s p a r t a t e , a l a n i n e , g l y c i n e , 138 Figure 40. Malate i n h i b i t i o n of adductor cytoplasmic MDH. a) Double r e c i p r o c a l p l o t of NADH s a t u r a t i o n at v a r y i n g malate, (0) c o n t r o l , (A) 3.5 mM malate, ( • ) 5 mM malate. Other c o n d i t i o n s : 0.5 mM oxal o a c e t a t e , 0 . 1 M T r i s - H C l pH 7.5, 25°C. b) Double r e c i p r o c a l p l o t of oxaloacetate s a t u r a t i o n at v a r y i n g malate, (A) c o n t r o l , ( • ) 5 mM malate, (0) 10 mM malate. Other c o n d i t i o n s : 0.1 mM NADH, 100 mM T r i s - H C l pH 7.5, 25°C. 140 Figure 41. Oxaloacetate i n h i b i t i o n of adductor cytoplasmic MDH. a) Double r e c i p r o c a l p l o t s of NAD + s a t u r a t i o n at v a r y i n g oxaloacetate, ( A ) c o n t r o l , (# ) 0.02 mM oxaloacetate, (•) 0.05 mM oxaloacetate. Other c o n d i t i o n s : 4 mM malate, 0.1 M T r i s - H C l pH 8.9, 25°C. b) Double r e c i p r o c a l p l o t s of malate s a t u r a t i o n at v a r y i n g oxaloacetate, ( •) c o n t r o l , (•) 0.02 mM oxaloacetate, ( A ) 0.05 mM oxaloacetate. Other c o n d i t i o n s : 1 mM NAD+,. 0.1 M T r i s - H C l pH 8.9, 25°C. 141 1/ Malate mM 41b 142 Figure 42. E f f e c t of NADH on NAD and malate s a t u r a t i o n k i n e t i c s . a) Double r e c i p r o c a l p l o t of NAD + s a t u r a t i o n at v a r y i n g NADH, (0) c o n t r o l , (•) 0.02 mM NADH, (A) 0.04 mM NADH. Other co n d i -t i o n s : 4 mM malate, 0.1 M T r i s - H C l pH 8.9, 25°C. b) Double r e c i p r o c a l p l o t of malate s a t u r a t i o n at v a r y i n g NADH, (A) c o n t r o l , (0) 0.02 mM NADH, (•) 0.1 mM NADH. Other c o n d i t i o n s : 1 mM NAD+, 0.1 M T r i s - H C l pH 8.9, 25°C. 1 4 3 ' — e — i r-* ' , , , , , -3 -2 -1 0 1 2 3 4 5 1/Malate mM"1 4 2 b 144 Figure 43. Dixon p l o t of NADH i n h i b i t i o n of adductor malate dehydrogenase; (0) 0.3 mM NAD+; (•) 0.5 mM NAD+; (A) 1.0 mM NAD+; other condi-t i o n s : 4 mM malate, 0.1 M T r i s - H C l pH 8.9, 25°C. 145 0.02 0.06 Q10 0.14 N A D H , m M 146 p r o l i n e , pyruvate, succinate, PEP and FDP had no e f f e c t . The adenylates were found to be competitive with NADH and NAD+, an e f f e c t s i m i l a r to that reported for porcine heart MDH (Kuramitsu, 1966; Oza and Shore, 1973). The binding of adenylates to NADH and NAD+ binding s i t e s would be expected as such s i t e s possess an adenine nucleotide binding domain (McPherson, 1970). Furthermore the R\ values for ATP and ADP were s i m i l a r (Table XIV),- hence any change i n the r e l a t i v e amounts of ATP and ADP would not have any s i g n i f i c a n t e f f e c t on enzyme a c t i v i t y . C i t r a t e and 2-ketoglutarate were competitive i n h i b i t o r s with respect to malate (Fig. 44, 45). C i t r a t e gave a mixed pattern with respect to oxaloace-tate (Fig. 46), whereas 2-ketoglutarate gave a competitive pattern (Fig. 47). The K., . x was 6 mM with respect to malate, and 8 mM with respect to l ( c x t r a t e ) oxaloacetate, both values being i n considerable excess of jLn vivo c i t r a t e con-centrations of 2 mM ( C o l l i c u t t , 1975). values f o r 2-ketoglutarate were lower, 1.7 mM with respect to both substrates, but these values are higher than i n vivo l e v e l s ( C o l l i c u t t , 1975). Therefore c i t r a t e and 2-ketoglutarate would have l i t t l e s i g n i f i c a n t e f f e c t on the function of MDH i n the adductor muscle. Considerations on Malate Dehydrogenase Function As discussed e a r l i e r , the cytoplasmic arm of the malate-aspartate s h u t t l e has been accentuated i n the oyster adductor muscle, and g l y c o l y s i s i s li n k e d to aspartate fermentation through a transaminase couple, thus permitting cytoplasmic MDH to function i n maintaining redox balance instead of LDH. The k i n e t i c c h a r a c t e r i s t i c s of adductor cytoplasmic MDH appear to be adaptive for function i n the d i r e c t i o n of malate formation under anaerobic conditions. The a f f i n i t y f o r NADH and oxaloacetate was high, " . • . 147 Table XIV. I n h i b i t i o n constants (K\ values) of va r i o u s i n h i b i t o r s . Values were obtained from Dixon p l o t s . For i n h i b i t o r s w i t h respect to NADH, a constant concentration of 0.5 mM oxaloacetate was used; w i t h respect to oxaloacetate, NADH was 0.1 mM; w i t h respect to NAD+, malate was 4 mM; w i t h respect to malate, NAD + was 1 mM. Substrate I n h i b i t o r i NADH NAD + pH 7.5 2.4 pH 6.5 1.3 NADH ATP pH 7.5 2.8 NADH ADP pH 7.5 2.8 NADH AMP pH 7.5 3.5 oxaloacetate malate pH 7.5 3.0 pH 6.5 5.0 oxaloacetate 2-ketoglutarate pH 7.5 1.6 pH 6.5 1.3 oxaloacetate c i t r a t e pH 7.5 8.0 NAD + ATP pH 8.9 1.9 NAD + ADP pH 8.9 1.7 NAD + AMP pH 8.9 4.1 malate oxaloacetate pH 8.9 0.03 malate 2-ketoglutarate pH 8.9 1.7 malate c i t r a t e pH 8.9 6.0 148 Figure 44. E f f e c t of c i t r a t e on malate s a t u r a t i o n k i n e t i c s . Double r e c i p r o c a l p l o t of malate s a t u r a t i o n at v a r y i n g c i t r a t e . (A) c o n t r o l , (t) 5 mM c i t r a t e , ( • ) 10 mM c i t r a t e . Other c o n d i t i o n s : 1 mM NAD+, 0.1 M T r i s - H C l pH 8.9, 25°C. 149 0.07n 150 Figure 45. E f f e c t of 2-ketoglutarate on malate s a t u r a t i o n k i n e t i c s . Double r e c i p r o c a l p l o t of malate s a t u r a t i o n at v a r y i n g 2-keto-g l u t a r a t e . ( t ) c o n t r o l , ( • ) 3 mM 2- k e t o g l u t a r a t e , (A) 5 mM 2-ketoglutarate. Other c o n d i t i o n s : 1 mM NAD+, 0.1 M T r i s - H C l pH 8.9, 25°C. 151 0.1 -1 Q08-V 0.06-0.04-1 -3 i -2 -1 0 1 i 2 •1 r-" r 3 4 5 1/ Malate mM 152 Figure 46. E f f e c t of c i t r a t e on oxaloacetate saturation k i n e t i c s . Double r e c i p r o c a l p l o t s of oxaloacetate saturation at varying c i t r a t e . (•) c o n t r o l , ( A ) 4 mM c i t r a t e , (•) 10 mM c i t r a t e . - Other conditions: 0.1 mM NADH, 0.1 M Tris-HCl pH 7.5, 25°C. 153 0.041 0 5 10 15 20 V Oxaloacetate m M " 1 154 Figure 47. E f f e c t of 2-ketoglutarate on oxaloacetate s a t u r a t i o n k i n e t i c s . Double r e c i p r o c a l p l o t s of oxaloacetate s a t u r a t i o n at v a r y i n g 2-ketoglutarate. ( O ) c o n t r o l , ( A ) 2.5 mM 2- k e t o g l u t a r a t e , (•) 5 mM 2-ketoglutarate. Other c o n d i t i o n s : 0.1 mM NADH, 0.1 M T r i s - H C l pH 7.5, 25°C. 155 156 and decreasing the pH to 6.5 increased the a f f i n i t y f o r oxaloacetate which would a s s i s t i n maintaining a c t i v i t y during anoxia. Also, adductor MDH had a higher a f f i n i t y for oxaloacetate ( ^ m ( O x a ) = ^ " ^ than did adductor aspartate aminotransferase (K N=0.09 mM), further suggesting that the major fate of m(Oxa) oxaloacetate would be conversion to malate. S u r p r i s i n g l y , there are no exceptional regulatory properties of cyto-plasmic MDH or aspartate aminotransferase, such as metabolite a c t i v a t o r s or i n h i b i t o r s which would provide a mechanism f or maintaining high l e v e l s of aspartate under aerobic conditions and then m o b i l i z i n g aspartate during anaerobiosis. Instead i t appears that the concentrations of the substrates are the l i m i t i n g factor for the function of these enzymes. As previously discussed, i t appears that 2-ketoglutarate would be the l i m i t i n g f a c t o r i n determining aspartate u t i l i z a t i o n . Since t h i s i s coupled with g l y c o l y s i s , the re s u l t would be an increase i n oxaloacetate and NADH (from the GAPDH step i n g l y c o l y s i s ) and t h i s would serve to " a c t i v a t e " cytoplasmic MDH. The r e l a t i v e -l y high values f o r NAD+ and malate at pH 7.5 further suggest that i n h i b i -t i o n by the products w i l l not have any s i g n i f i c a n t e f f e c t on the rate of f l u x through the reaction . Malate l e v e l s do not increase during anoxia to any s i g n i f i c a n t degree (de Zwaan and Zandee, 1972; Klutymans ejt a l . , 1975; C o l l i c u t t , 1975), instead the malate i s converted to succinate which was found to have no e f f e c t on cytoplasmic MDH at concentrations as high as 20 mM. Other considerations of MDH function must centre around r e p l e n i s h i n g the aspartate pool at some stage a f t e r the anoxic period. Presumably the succi n -ate formed during anoxia would be the major source, but other metabolic sources could also be used. In ei t h e r event, aspartate aminotransferase and MDH would be involved, but i n the reverse d i r e c t i o n to the one used during 157 anoxia. Although the K values f o r malate and NAD + do not appear to be a • m r r r e s t r i c t i o n on the f u n c t i o n of MDH i n the malate >oxaloacetate d i r e c t i o n , the marked NADH and oxaloacetate i n h i b i t i o n observed would c e r t a i n l y be a l i m i t a t i o n on the r a t e at which t h i s would occur. Furthermore, continued production of oxaloacetate would increase the l e v e l s of NADH i n the cytoplasm, and unless t h i s process i s coupled w i t h another dehydrogenase which maintains NADH at low l e v e l s , the r a t e of oxaloacetate production, and hence aspartate production, would be very low. On the other hand, these o b j e c t i o n s do not apply to replenishment of asparta t e by a m i t o c h o n d r i a l route. In t h i s com-partment NADH l e v e l s can be maintained at a low l e v e l by the e l e c t r o n t r a n s -f e r system, the pH i s higher than that i n the cytoplasm ( T i s c h l e r et_ a l . , 1976) which would make the e q u i l i b r i u m of the r e a c t i o n more favourable f o r oxaloacetate production (Raval and Wolfe, 1962b), and f i n a l l y , there are two e l e c t r o p h o r e t i c forms of al a n i n e aminotransferase i n adductor muscle (Mustafa, personal communication) and i t i s f e a s i b l e that one of these i s m i t o c h o n d r i a l , which would provide a source of amino groups from the a l a n i n e accumulated during anoxia, f o r the m i t o c h o n d r i a l isoenzyme of aspartate aminotransferase. 158 CHAPTER V I I DISCOVERY OF A DEHYDROGENASE RQUIRING ALANINE AND PYRUVATE AS  SUBSTRATES In the preceding chapters the metabolic source of succinate during anaero-b i o s i s , and the r o l e of cytoplasmic aspartate aminotransferase and malate dehydrogenase i n maintaining cytoplasmic redox balance has been discussed. The k i n e t i c p r o p e r t i e s of these enzymes suggest that aspartate would be the major source of s u c c i n a t e , a l a n i n e being formed mainly from glucose during the e a r l y stages of anae r o b i o s i s . This hypothesis i s f u l l y supported by the 14 14 experiments of C o l l i c u t t (1975) on u t i l i z a t i o n of C-glucose and C-aspartate by the oyster heart during short term anoxia. In a d d i t i o n to succinate and a l a n i n e , a t h i r d major end product was a l s o found, which accounted f o r as much as 30% of the glucose carbon metabolized. The unknown compound was always found i n the amino a c i d f r a c t i o n , and si n c e t h i s was obtained by chromatography on Amberlite IR120 r e s i n , i t i n d i -cates the presence of a p o s i t i v e l y charged N-containing group. The compound was not n i n h y d r i n p o s i t i v e , i n d i c a t i n g the absence of a 1° amino group, and i t was s t a b l e to b o i l i n g 6N HC1 f o r 12 hours, thus i n d i c a t i n g i t was not a peptide. 14 Incubating o y s t e r hearts w i t h C-alanine under anoxia r e s u l t e d i n the l a b e l -l i n g of pyruvate and the unknown compound o n l y , i n d i c a t i n g i t was m e t a b o l i c a l -l y c l o s e l y l i n k e d to e i t h e r a l a n i n e or pyruvate ( C o l l i c u t t , 1975). Although separable from octopine, the unknown cochromatographed w i t h octopine (which contains a 2° amino group, and th e r e f o r e i s not n i n h y d r i n p o s i t i v e ) i n three solvent systems used ( C o l l i c u t t , personal communication) suggesting that the s t r u c t u r e was s i m i l a r to that of octopine. The p o s s i b i l i t y that an enzyme s i m i l a r to octopine dehydrogenase, but r e q u i r i n g another keto a c i d or amino a c i d , was considered. Such an enzyme would account f o r the production of the unknown metabolite formed during 159 anoxia i n the oyster heart. Subsequent experiments l e d to the discovery of a new enzyme which o x i d i z e d NADH i n the presence of pyruvate and, s u r p r i s i n g l y , a l a n i n e . C l e a r l y such an enzyme could account f o r the formation of the un-known, and would a l s o a s s i s t i n m a i n t a i n i n g redox balance during a n a e r o b i o s i s i n the oyst e r . This chapter discusses the r e g u l a t o r y and c a t a l y t i c p r o p e r t i e s of t h i s enzyme, and how i t f i t s i n t o the o v e r a l l p a t t e r n of metabolism during anoxia. METHODS Enzyme a c t i v i t y was determined by f o l l o w i n g the decrease i n o p t i c a l den-s i t y at 340 nm, r o u t i n e assay c o n d i t i o n s were 0.2 mM NADH, 200 mM a l a n i n e , 3 mM pyruvate and 100 mM imidazole HC1 pH 7.0, at 25°C. Pyruvate was assayed s p e c t r o p h o t o m e t r i c a l l y using beef heart l a c t a t e dehydrogenase and 0.16 mM NADH i n T r i s - H C l pH 7.5, the decrease i n o p t i c a l d e n s i t y at 340 nm being measured (Lowry and Passonneau, 1972). Alanine was determined c o l o r o m e t r i c a l l y w i t h n i n h y d r i n according to the method of Lee and Takahashi (1966). The sample c o n t a i n i n g 0.05 to 0.2 umole of a l a n i n e i n 0.1 ml was added to 1.9 ml of a n i n h y d r i n - c i t r a t e - g l y c e r o l mixture that c o n s i s t s of 0.5 ml of 1% n i n h y d r i n i n 0.5 M sodium c i t r a t e b u f f e r (pH 5.5), 1.2 ml g l y c e r o l , and 0.2 ml of 0.5 M sodium c i t r a t e b u f f e r (pH 5.5). The r e a c t i o n mixture was heated i n a b o i l i n g water bath f o r 12 min, cooled to room tempera-ture i n tap water, and the o p t i c a l d ensity determined at 570 nm. RESULTS AND DISCUSSION Pr e p a r a t i o n of Enzyme A l l procedures were c a r r i e d out between 0 and 4°C. Approximately 35 g of oys t e r adductor muscle were d i s s e c t e d f r e e of surrounding t i s s u e , b l o t t e d dry, 160 weighed and homogenized i n 5 v o l 0.1 M imidazole HC1 pH 7.4 w i t h a S o r v a l l Omnimixer set at the maximum speed. The suspension was c e n t r i f u g e d at 20000 g f o r 20 min, and the supernatant set aside. The p e l l e t was rehomogenized i n a f u r t h e r 4 v o l of b u f f e r and r e c e n t r i f u g e d as above. The supernatants were combined, t h i s being the crude homogenate. Ammonium sulphate, 0.243 g/ml, was slowly added w i t h continuous s t i r r i n g to the crude homogenate, the suspension c e n t r i f u g e d at 20000 g f o r 30 min, and the p e l l e t discarded. The supernatant was then t r e a t e d w i t h 0.168 g/ml ammonium sulphate, c e n t r i f u g e d , and the supernatant discarded. The p e l l e t obtained was d i s s o l v e d i n a minimum volume of 50 mM sodium phosphate pH 7.4 and chromatographed on a 95 x 5 cm column of Sephadex G-200, e l u t i o n being achieved w i t h the same b u f f e r . The enzyme e l u t e s as a s i n g l e peak ( F i g . 48). F r a c t i o n s c o n t a i n i n g more than h a l f the maximum peak a c t i v i t y were pooled and t r e a t e d w i t h 0.472 g/ml ammonium sulphate to p r e c i p i t a t e the enzyme, which was c o l l e c t e d by c e n t r i f u g a t i o n at 20000 g f o r 20 min. The p e l l e t obtained was d i s s o l v e d i n a minimal volume of 5 mM sodium phosphate pH 7.4 b u f f e r , d i a l y z e d against two 1 - l i t r e changes of the same b u f f e r , and a p p l i e d to a 10 x 1.6 cm column of DEAE-Sephadex A-50 p r e v i o u s l y e q u i l i b r a t e d w i t h 5 mM sodium phosphate pH 7.4. E l u t i o n was achieved w i t h a 5 to 100 mM gradient of sodium phosphate pH 7.4, the enzyme e l u t i n g as a s i n g l e peak ( F i g . 49). A summary of the p u r i f i c a t i o n i s given i n Table XV. The s p e c i f i c a c t i v i t y averaged about 250 units/mg p r o t e i n . This p a r t i a l l y p u r i f i e d p r e p a r a t i o n was used f o r proving the nature of the r e a c t i o n , and a l s o f o r s t u d i e s on the sub-s t r a t e s p e c i f i c i t y . 161 Figure 48. E l u t i o n of NCEADH from a 95 x 5 cm column of Sephadex G-200. Flow r a t e was 50 ml/hr. F r a c t i o n s of 7 ml were c o l l e c t e d . ( O ) O p t i c a l density at 280 nm, (A) NCEADH a c t i v i t y micromoles/ min/ml. 391 163 Figure 49. E l u t i o n of NCEADH from DEAE Sephadex A-50. ( •) O p t i c a l d e n s i t y at 280 nm, ( A ) NCEADH a c t i v i t y . 2 ml f r a c t i o n s were c o l l e c t e d , a gradient of 5-100 mM sodium phosphate, i n a t o t a l volume of 400 ml, was a p p l i e d to the column. 164 12-j 10-E 8-6" c » 4-2-0 0 20 40 60 80 Tube # Table XV. Summary of NCEADH p u r i f i c a t i o n . S p e c i f i c a c t i v i t y Units/ml P r o t e i n , mg/ml T o t a l u n i t s units/mg p r o t e i n P u r i f i c a t i o n ( f o l d ) Y i e l d Crude homogenate Ammonium sulphate 40-65% 8.1 51.0 5.2 21.2 2100 1780 1.6 2.4 1.5 x 100% 85% ON G-200 chromatography DEAE-Sephadex A-50 pH 7.4, 5-100 mM sodium phosphate 7.0 11.0 0.25 0.04 830 510 28.4 275.0 17.8 x 170 x 39.5% 24.7% 166 Stoichiometry of the Reaction In order to prove that the observed NADH o x i d a t i o n was dependent on the u t i l i z a t i o n of al a n i n e and pyruvate, d e p l e t i o n of the three compounds was followed independently. In the presence of excess a l a n i n e (200 mM), 0.2 mM NADH and var i o u s l i m i t i n g concentrations of pyruvate, i t was shown that the NADH u t i l i z e d was equal to the i n i t i a l pyruvate concentration (Table XVI). That i s , NADH and pyruvate were depleted i n a 1:1 sto i c h i o m e t r y . The r a t i o between a l a n i n e and pyruvate d e p l e t i o n was determined i n a r e a c t i o n mixture of 2 mM NADH, 3 mM a l a n i n e , 3 mM pyruvate i n 100 mM imidazole HC1 pH 7.0. The f i n a l volume was 15 ml; a l i q u o t s of 1 ml were removed at various i n t e r v a l s a f t e r a d d i t i o n of enzyme, d e p r o t e i n i z e d w i t h 0.5 ml 7% p e r c h l o r i c a c i d , n e u t r a l i z e d w i t h 0.1 ml 3 M K 2 C°3 a n c* a s s a Y e d f ° r a l a n i n e and pyruvate. The r e s u l t s (Table XVII) i n d i c a t e that pyruvate and al a n i n e were a l s o consumed i n a 1:1 sto i c h i o m e t r y . A l i q u o t s taken 120 min a f t e r the r e a c t i o n had been s t a r t e d were used as a source of the product being formed. This product i n the presence of 1 mM NAD+, 100 mM T r i s - H C l pH 8.5 reacted w i t h the enzyme l e a d -ing to an increase i n o p t i c a l d e n s i t y at 340 nm. The r e a c t i o n t h e r e f o r e appears to be of the general type R-C=0 + R'-C-NH2 + NADH + H + < » R-CH-NH-C-R' + ^ 0 + NAD + Other enzymes that f a l l i n t o t h i s category are glutamate dehydrogenase, a l a -nine dehydrogenase (E.C. 1.4.1.1), octopine dehyrogenase and saccharopine de-hydrogenase (E.C. 1.5.1.7), ther e f o r e i t would seem reasonable that the r e a c t i o n c a t a l y z e d by the enzyme i s NADR -f H + + pyruvate + al a n i n e <—> NAD + + ^ 0 + N-(1-carboxyethyl)-alanine In order to s i m p l i f y f u r t h e r d i s c u s s i o n , the enzyme w i l l be r e f e r r e d to as N-(1-carboxyethyl)-alanine dehydrogenase (NCEADH). 167 Table XVI U t i l i z a t i o n of NADH by NCEADH i n presence of excess a l a n i n e and l i m i t i n g pyruvate. Conditions were 200 mM a l a n i n e , 0.2 mM NADH, pyruvate as s t a t e d , 100 mM imidazole HC1 pH 7.0, 25°C. Change i n o p t i c a l Pyruvate, mM den s i t y at 340 nm* NADH u t i l i z e d , mM** 0.03 0.22 ± 0.03 0.035 ± 0.004 0.06 0.43 ± 0.02 0.069 ± 0.003 0.09 0.58 ± 0.02 0.093 ± 0.003 0.12 0.76 ± 0.02 0.122 ± 0.003 0.15 0.94 ± 0.04 0.151 ± 0.006 *Average of 4 experiments * * C a l c u l a t e d on ba s i s of molar e x t i n c t i o n c o e f f i c i e n t f o r NADH of 6.22 x 1 0 3 168 Table XVII U t i l i z a t i o n of pyruvate and alanin e by NCEADH. Conditions were 2.0 mM NADH, 3.0 mM a l a n i n e , 3.0 mM pyruvate i n 100 mM imidazole HC1 pH 7.0, 25°C. Experiment performed i n t r i p l i c a t e , average values being reported. Time a f t e r a d d i t i o n Decrease i n Decrease i n of enzyme, min pyruvate, mM a l a n i n e , mM 0 0 0 5 0.14 0.03 10 0.25 0.12 20 0.44 0.23 30 0.47 0.41 45 0.59 0.6 60 0.78 0.76 75 0.89 0.87 90 1.09 1.03 105 1.17 1.14 120 1.32 1.26 I 169 14 C-alanine has been used to l a b e l the product. The r e a c t i o n mixture contained NADH (226 mg), a l a n i n e (40 mg), pyruvate (80 mg), about 1.5 x 10 dpm 14 of C(U)-alanine (13 mCi/mmole; New England Nuclear) i n 100 ml of 5 mM imidazole HC1 pH 7.2. The i n c u b a t i o n was s t a r t e d w i t h 10 ml of p a r t i a l l y p u r i f i e d enzyme, and l e f t at room temperature (23-25°C) f o r 6-8 hours. At the end of t h i s p e r i o d , the r e a c t i o n was stopped by the a d d i t i o n of 5 ml 10 N HC1, and the mixture concentrated by r o t a r y evaporation at 50°C. The concentrated s o l u t i o n was then chromatographed on a 40 x 2.5 cm column of Dowex 50W e q u i l i -brated w i t h 200 mM sodium phosphate pH 3.0. E l u t i o n was achieved w i t h the same b u f f e r , f r a c t i o n s of 3.8 ml being c o l l e c t e d . Two r a d i o a c t i v e peaks were obtained from the column ( F i g . 50), the f i r s t l a r g e r peak corresponding to the product, the second smaller peak being unconsumed a l a n i n e . F r a c t i o n s c o n t a i n -i n g the product were c o l l e c t e d and experiments are c u r r e n t l y i n progress to p o s i t i v e l y i d e n t i f y i t . S p e c i f i c i t y of NCEADH The enzyme was t e s t e d f o r a c t i v i t y w i t h a v a r i e t y of keto a c i d s , amino acids and NADPH. I t was found to be s p e c i f i c f o r NADH, a c t i v i t y w i t h NADPH being 0.1% of the a c t i v i t y w i t h NADH. The s p e c i f i c i t y f o r pyruvate was a l s o high (Table X V I I I ) , a c t i v i t y w i t h 2-oxobutyrate was about 38% of c o n t r o l , and low a c t i v i t i e s were obtained w i t h g l y o x y l a t e and hydroxypyruvate. The enzyme i s l e s s s p e c i f i c f o r the amino a c i d f o r g l y c i n e was found to be as good a su b s t r a t e as a l a n i n e ; s e r i n e and c y s t e i n e were about one-half as e f f e c t i v e , and g-alanine, s u r p r i s i n g l y , could a l s o be used to some extent (Table XIX). Tissue D i s t r i b u t i o n The enzyme was found i n high a c t i v i t y i n the adductor muscle (about 80 u n i t s / g wet wt) and i n the heart (about 40 u n i t s / g wet wt). The a c t i v i t y i n 170 Figure 50. E l u t i o n of NCEA and a l a n i n e from a 40 x 2.5 cm column of Dowex 50W. The e l u t i n g b u f f e r was 200 mM sodium phosphate pH 3.0, 3.8 ml f r a c t i o n s were c o l l e c t e d . i product alanine A ? 3l - O L O / P / 5 0 1 0 0 Tube no. 1 5 0 2 0 0 172 Table XVIII Keto a c i d s p e c i f i c i t y of N - ( l - c a r b o x y e t h y l ) - a l a n i n e dehydrogenase. Keto a c i d , 3 mM R e l a t i v e a c t i v i t y Pyruvate 100 2-oxobutyrate 38.4 Gly o x y l a t e 4.7 Hydroxypyruvate 4.0 Oxaloacetate 1.3 2-oxoglutarate 1.3 Conditions: 0.2 mM NADH, 200 mM a l a n i n e , 100 mM imidazole HC1 pH 7.0, 25°C. 173 Table XIX Amino a c i d s p e c i f i c i t y of N-(1-carboxyethyl)-alanine dehydrogenase. Amino a c i d , 200 mM R e l a t i v e a c t i v i t y A l a n i n e 100 Glycine 98 Cysteine 50.5 Serine 50.0 3-alanine 16.0 Taurine 0 Glutamate 0 Conditions: 0.2 mM NADH, 3 mM pyruvate, 100 mM imidazole HC1 pH 7.0, 25°C. 174 the mantle, g i l l s and d i g e s t i v e gland was barel y d e t e c t a b l e , and was l e s s than 0.1 u n i t s / g wet wt. Molecular Weight The molecular weight of the enzyme was found to be 43000±4000, which i s s i m i l a r to the molecular weight of octopine dehydrogenase. Octopine dehydro-genase has been shown to be a s i n g l e subunit enzyme (Olomucki et a l . , 1972), and i t semms l i k e l y that t h i s enzyme i s a l s o a s i n g l e chain p r o t e i n molecule. K i n e t i c s During p u r i f i c a t i o n the enzyme underwent a change i n k i n e t i c p r o p e r t i e s a f t e r d i a l y s i s against 5 mM sodium phosphate. The r e s u l t i n g preparations had e x c e p t i o n a l l y high K^ values f o r pyruvate and a l a n i n e , and sometimes showed non-Michaelis k i n e t i c s w i t h respect to a l a n i n e . These phenomena suggested the l o s s of a c o f a c t o r during d i a l y s i s , but t h i s has not yet been c l a r i f i e d . Therefore a l l of the f o l l o w i n g k i n e t i c s t u d i e s were performed on the p a r t i a l l y p u r i f i e d enzyme obtained from the g e l f i l t r a t i o n step ( s p e c i f i c a c t i v i t y about 25 units/mg p r o t e i n ) . 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 the pH optimum i s about 6.0, but i f lower concentrations of al a n i n e are used the pH optimum i s 6.7-7.0 ( F i g . 51). The enzyme was c h a r a c t e r i z e d at pH 7.5, 7.0, and 6.5 i n order to f u l l y understand i t s r o l e during anoxia. In a l l cases the enzyme f o l l o w s normal Michaelis-Menten k i n e t i c s . The apparent Kffi f o r NADH i s dependent on the co n c e n t r a t i o n of the substrates a l a n i n e or pyruvate (Table XX, F i g . 52). In general decreasing e i t h e r a l a n i n e or pyruvate leads to a s l i g h t decrease i n K m ( a p p ) a t 7.0 or 6.5, but at pH 7.5 there i s only a small change. Decreasing pH from 7.5 a l s o increases the K m f o r NADH, but the range of values observed (0.01-0.02 mM) i s s i m i l a r 175 Figure 51. E f f e c t of pH on adductor NCEADH a c t i v i t y . Conditions: 3 mM pyruvate, 0.2 mM NADH, (•) 40 mM, (A) 200 mM, (,•) 400 mM a l a n i n e . 176 12Ch 65 7 0 75 BO P H 177 Figure 52. NADH s a t u r a t i o n k i n e t i c s of adductor NCEADH. Conditions: (•) 200 mM a l a n i n e , 3 mM pyruvate, (O ) 70 mM a l a n i n e , 3 mM pyruvate, (A) 200 mM a l a n i n e , 0.8 mM pyruvate. 178 V 0.03-1 002H -100 -80 -60 -40 -20 0 20 1/ NADH mM -1 40 179 Table XX M i c h a e l l s constants f o r adductor NCEADH. Substrate Cosubstrates K m pH 7.5 / x , mM (app) pH 7.0 PH 6.. NADH 200 mM a l a n i n e , 3.0 mM pyruvate 0.01 0.016 0.019 200 mM a l a n i n e , 0.9 mM pyruvate 0.01 0.013 0.016 70 mM a l a n i n e , 3.0 mM pyruvate 0.008 0.012 0.008 Alanine 0.04 mM NADH, 3.0 mM pyruvate 95 77 87 0.2 mM NADH, 3.0 mM pyruvate 120 110 170 0.2 mM NADH, 1.2 mM pyruvate 200 140 190 0.2 mM NADH, 0.6 mM pyruvate 285 190 235 Pyruvate 0.04 mM NADH, 200 mM al a n i n e 0.67 0.48 0.38 0.2 mM NADH, 200 mM a l a n i n e 1.0 0.62 0.56 0.2 mM NADH, 70 mM al a n i n e 1.6 1.1 0.8 0.2 mM NADH, 50 mM a l a n i n e 1.8 1.3 0.91 180 to that of other dehydrogenases from a v a r i e t y of organisms, and t h i s i s not considered to be a s i g n i f i c a n t change. Pyruvate s a t u r a t i o n k i n e t i c s are a l s o normal up to concentrations of 3 to 6 mM, at higher concentrations s u b s t r a t e i n h i b i t i o n i s observed. The K f o r m pyruvate i s dependent on the co n c e n t r a t i o n of a l a n i n e and NADH (Table XX), i n c r e a s i n g w i t h decreasing concentrations of a l a n i n e ( F i g . 53), and decreasing w i t h decreasing concentrations of NADH. Likewise the K values f o r a l a n i n e & m increase w i t h decreasing concentrations of pyruvate ( F i g . 54), and decrease w i t h lower concentrations of NADH (Table XX). I t should be noted that the K m values f o r a l a n i n e are e x c e p t i o n a l l y h i g h , o f t e n considerably higher than the highest l e v e l s of al a n i n e found i n the oyste r heart of adductor muscle ( C o l l i -c u t t , 1975, personal communication). The k i n e t i c s of the reverse d i r e c t i o n have not been s t u d i e d , and are awai t i n g p u r i f i c a t i o n of la r g e q u a n t i t i e s of the product, N-(1-carboxyethly)-a l a n i n e (NCEA). Regulation by Me t a b o l i t e s A s e r i e s of metabolites were t e s t e d f o r e f f e c t s on the enzyme at s a t u r a t -i n g and approximate values of su b s t r a t e s . NAD + and the adenylates (ATP, ADP, AMP) were found to be competitive i n h i b i t o r s w i t h respect to NADH ( F i g . 55). The K. values are shown i n Tables XXI and XXII. Succinate and 2-keto-x g l u t a r a t e were a l s o found to be i n h i b i t o r s , whereas a s p a r t a t e , glutamate, p r o l i n e , f r u c t o s e diphosphate, t a u r i n e , v a l i n e , l e u c i n e , phosphoenolpyruvate, malate, c i t r a t e , fumarate, glucose-6-P, fructose-6-P, a r g i n i n e , and a r g i n i n e phosphate had no e f f e c t . The i n h i b i t i o n by succinate was a mixed p a t t e r n w i t h respect to a l a n i n e and pyruvate ( F i g . 56, 57). The K\ values f o r succinate were the same w i t h respect to both a l a n i n e and pyruvate, and showed a s u r p r i s i n g l y l a r g e v a r i a -181 Figure 53. Pyruvate saturation k i n e t i c s of adductor NCEADH at varying a l a -nine concentrations. (A) 200 mM, (O) 70 mM, ( •) 50 mM alanine; NADH concentrations, 0.2 mM, 100 mM imidazole pH 7.0. 183 Figure 54. Alanine s a t u r a t i o n k i n e t i c s at v a r y i n g concentrations of pyruvate. (A) 3.0 mM pyruvate, ( O) 1.2 mM, (•) 0.6 mM pyruvate; other c o n d i t i o n s , 0.2 mM NADH, 100 mM imidazole HC1 pH 7.0. 1 8 4 Q05i -0.01 0 001 0.02 1/Alanine m M " 1 185 Figure 55. E f f e c t s of NAD and ATP on NADH s a t u r a t i o n k i n e t i c s of NCEADH. a) P a t t e r n of i n h i b i t i o n of NAD + w i t h respect to NADH. ( A ) con-t r o l , (•) 1.0 mM NAD+, ( •) 2.0 mM NAD+; other c o n d i t i o n s , 200 mM al a n i n e , 3 mM pyruvate, 100 mM imidazole HC1 pH 7.0. b) P a t t e r n of i n h i b i t i o n of ATP with respect to NADH. ( O ) con-t r o l , (•) 3.0 mM ATP, ( A ) 5.0 mM ATP; other c o n d i t i o n s , 200 mM a l a n i n e , 3 mM pyruvate, 100 mM imidazole HC1 pH 7.0. 186 187 Table XXI I n h i b i t i o n constants f o r the adenylates w i t h respect to NADH. I n h i b i t o r ATP ADP AMP Conditions: 200 mM a l a n i n e , 3 mM pyruvate, 100 mM imidazole HC1, pH 7.0. K., mM 0.74 1.5 4.0 188 Table XXII I n h i b i t i o n constants f o r NAD + at d i f f e r e n t pH values. K., mM 0.13 0.22 0.53 7.5 7.0 6.5 Conditions: 200 mM a l a n i n e , 3 mM pyruvate, 100 mM imidazole HC1, 25° C. 189 F i g u r e 56. P a t t e r n of succinate i n h i b i t i o n w i t h respect to pyruvate s a t u r a -t i o n k i n e t i c s i n NCEADH. ( A ) c o n t r o l , ( O ) 10 mM s u c c i n a t e , (•) 20 mM suc c i n a t e ; other c o n d i t i o n s , 200 mM a l a n i n e , 0.2 mM NADH, 100 mM imidazole HC1 pH 7.0. 190 1 9 1 Figure 57. P a t t e r n of succinate i n h i b i t i o n w i t h respect to a l a n i n e s a t u r a t i o n k i n e t i c s of NCEADH. ( A ) c o n t r o l , (O) 10 mM su c c i n a t e , (•) 20 mM suc c i n a t e ; other c o n d i t i o n s , 0.2 mM NADH, 3 mM pyruvate, 100 mM imidazole HC1 pH 7.0. 192 193 t l o n over the pH range 7.5-6.5 (Table XXIII). The i n h i b i t i o n by succinate i s f a r more pronounced at pH 6.5, and values i n t h i s range have been recorded f o r the p a l l i a l f l u i d ; therefore succinate modulation may be of extreme p h y s i o l o g i -c a l importance. I n h i b i t i o n by 2-ketoglutarate was found to be complex, being uncompetitive with respect to alanine and pyruvate (Fig. 58, 59), but the R\ values of 0.9-0.5 mM were higher than the concentrations of 2-ketoglutarate measured i n oyster heart or adductor muscle ( C o l l i c u t t , 1975, personal communication). The i n h i b i t i o n with respect to NADH was also uncompetitive, the K\ value was 1.3 mM at pH 7.0. Considerations on Metabolic Function C l e a r l y the r o l e t h i s enzyme plays i n anaerobic metabolism i s one s i m i l a r to l a c t a t e dehydrogenase i n mammals or octopine dehydrogenase i n cephalopods. Therefore during anoxia one would expect accumulation of NCEA, the alanine required for the synthesis being derived from the free amino acid pool. In t h i s regard the a b i l i t y to use glycine as an alternate substrate i s important, alanine l e v e l s being low at the beginning of an anaerobic period and glycine l e v e l s are often as high as alanine i n bivalve tissues (Lynch and Wood, 1966; Baginsky and Pierce, 1975; DuPaul and Webb, 1970). This would suggest that the unknown found by C o l l i c u t t (1975) i s i n fact two compounds, NCEA and N-(l-carboxyethyl)-glycine (NCEG). So far only one radioactive peak has been obtained by paper chromatography, possibly the question could be resolved by gas chromatography, t h i s method being more s e n s i t i v e . In t h i s regard the f a i l u r e to detect any s i g n i f i c a n t decrease i n glycine concentrations during anoxia would suggest that very l i t t l e NCEG i s formed. An o v e r a l l view of oyster adductor anaerobic metabolism can now be postu-194 Table XXIII I n h i b i t i o n constants f o r succinate and 2-ketoglutarate at v a r y i n g pH values . K ./ . \»mM K. ,„ , .. \>mM 1 ( s u c c i n a t e ) 1 ( 2 - k e t o g l u t a r a t e )  7.5 22 0.95 7.0 6.5 0.7 6.5 1.8 0.51 C o n d i t i o n s : 0.2 mM NADH, 100 mM imidazole HC1, 25°C. 195 Figure 58. P a t t e r n of i n h i b i t i o n by 2-ketoglutarate of NCEADH w i t h respect to a l a n i n e . ( A ) c o n t r o l , ( O ) 1 mM, ( •) 2 mM 2-keto g l u t a r a t e ; other c o n d i t i o n s , 0.2 mM NADH, 3 mM pyruvate, 100 mM imidazole HC1 pH 7.0, 25°C. 196 0 0 1 0 0 2 1/Alanine mM"1 197 Figure 59. Inhibition of NCEADH by 2-ketoglutarate with respect to pyruvate. (•) control, (O) 1 mM, (A) 2 mM 2-ketoglutarate; other conditions, 200 mM alanine, 0.2 mM NADH, 100 mM imidazole HC1 pH 7.0, 25°C. 198 199 la t e d (Fig. 60), that encompasses both aspartate m o b i l i z a t i o n and formation of NCEA, or NCEG. There i s competition for pyruvate between alanine aminotrans-ferase and NCEADH. On the basis of t h e i r respective K values for pyruvate, which are about equal (Mustafa, 1974), l i t t l e can be s a i d concerning the major fate of pyruvate. However, NCEADH has a low a f f i n i t y for alanine and glycine, thus suggesting that more alanine would be formed than NCEA or NCEG. That t h i s 14 i s indeed the case i s demonstrated by C o l l i c u t t ' s (1975) data on C-glucose 14 u t i l i z a t i o n i n the oyster heart. The r a t i o of C i n alanine to that i n the unknown was 1.8:1. Therefore the formation of alanine, which leads to the production of oxaloacetate from aspartate, and then to succinate formation, i s favoured under i n v i t r o anoxic conditions. In t h i s context, the s i g n i f i c a n c e of succinate i n h i b i t i o n becomes rather obvious, for production of succinate from aspartate w i l l lead to a decrease i n pH, and these two factors w i l l i n h i b -i t any further production of s i g n i f i c a n t amounts of NCEA. This contention i s also supported by C o l l i c u t t ' s (1975) data, which show that by decreasing the 14 14 pH of the s a l i n e medium from 7.8 to 7.0, the % of C incorporated from C-glucose into the unknown decreased from 30% to 20%, as opposed to 50% incor-porated into alanine. 200 Figure 60. P o s t u l a t e d metabolic map of anaerobic metabolism i n the cytoplasm of oyster adductor muscle. 201 glycogen • I F D P N A D * N A D H H C O ; aspartate G3P N A D H pyruvate •2-kga^_. . .g lutamate glutamate oxaloacetate N A D H -* — - N A D * alanine (glycine) N A D H N A D * — malate N C E A ( N C E G ) succinate 202 CHAPTER V I I I SUMMARY: PATHWAYS OF ANAEROBIC METABOLISM IN MOLLUSCS Molluscs produce a v a r i e t y of anaerobic end products, but a common energy source i s a form of carbohydrate. In some freshwater gastropods the end product i s l a c t a t e (von Brand et a l , 1950), and the g l y c o l y t i c pathway i s unmodified from that of v e r t e b r a t e s k e l e t a l muscle. In marine molluscs, amino a c i d metabolism i s o f t e n coupled to carbohydrate fermentation, and one f u n c t i o n of t h i s c o u p l i n g i s to maintain cytoplasmic redox balance. The sim p l e s t form of t h i s c o u p l i n g i s shown by ODH i n cephalopods and some b i v a l v e s (Gade and Zebe, 1973; van Thoai and Robin, 1959b), and by NCEADH i n oyst e r adductor and heart muscle. These enzymes are analogous to LDH i n f u n c t i o n , producing octopine or NCEA as a l t e r n a t e end products of g l y c o l y s i s . In the cephalopods ODH appears to be used f o r meeting short term, high energy demands on the muscle, and t h i s i s i l l u s t r a t e d by the v a r y i n g amounts of ODH and ctGPDH i n the mantle muscle of squids and o c t o p i where the more a c t i v e forms have high l e v e l s of ctGPDH and high l e v e l s of ODH. The s i t u a t i o n s that ODH has been evolved to compensate f o r are d i f f e r e n t from those encountered by i n t e r t i d a l m olluscs, which have to withstand periods of anoxia l a s t i n g f o r s e v e r a l hours, but both groups do show the property of coupled amino a c i d and carbohydrate metabolism. In t h i s case the c o u p l i n g i s achieved by the a l a n i n e and aspar t a t e aminotrans-ferase couple which tumbles glutamate and 2-ketoglutarate between them. These enzymes are present i n a l a r g e number of marine i n v e r t e b r a t e s (Read, 1962; Hammen, 1968), and hence the p o t e n t i a l f o r such a couple i s widespread. There may be s e v e r a l advantages of t h i s couple over the NCEADH or ODH type of couple, an obvious one being the formation of oxaloacetate from a s p a r t a t e , which permits redox balance to be maintained by cytoplasmic MDH l e a d i n g to malate production, and t h i s malate can then be f u r t h e r metabolized i n the mitochondri-on to s u c c i n a t e , y i e l d i n g 1 mole ATP/mole succinate (de Zoeten and T i p k e r , 203 1969; Wilson and Cascarano, 1970; Saz, 1971b). That t h i s pathway i s preferred is consistent with the regulatory properties of NCEADH i n oyster adductor muscle, since decreasing pH and increasing succinate would c u r t a i l the a c t i v i t y of t h i s enzyme as anoxia progresses. In t h i s connection i t i s i n t e r e s t i n g that the more anoxia-tolerant species, such as R. cuneata and M. e d u l i s , have a higher PEPCK:PK a c t i v i t y r a t i o (Table XXIV). I t should also be pointed out that the aminotransferase couple and the PEPCK pathway are not mutually exclusive, as both lead to the produc-t i o n of oxaloacetate (the aminotransferase couple i n d i r e c t l y from aspartate), and from t h i s succinate i s ulti m a t e l y produced. Over a prolonged period of anoxia the PEPCK pathway would be favoured as glycogen can be stored i n much higher q u a n t i t i e s than free aspartate (about 15 mM). During the i n i t i a l period of anoxia PK a c t i v i t y would be higher than PEPCK a c t i v i t y , and alanine would be accumulated. Simultaneously aspartate would be depleted and succinate accumulated leading to a decrease i n pH which i n conjunction with the accumu-l a t i o n of alanine would begin the t r a n s i t i o n from PK to PEPCK (Hochachka and Mustafa, 1972; de Zwaan and Holwerda, 1973; Holwerda and de Zwaan, 1972; de Zwaan and de Bont, 1975). Moreover, recently i t was found that i n M. e d u l i s alanine accumulation peaks as anoxia i s prolonged, and succinate, propionate and acetate become the predominant anaerobic end products (Klutymans et a l . , 1975), thus providing further evidence that PK a c t i v i t y i s c u r t a i l e d and PEPCK is accentuated with progressive anoxia. The production of succinate, acetate and propionate can a l l be accounted for by metabolism of malate i n the mito-chondrion. Malate i s one of several d i c a r b o x y l i c acids that can penetrate the inner mitochondrial membrane (Rongstad and Katz, 1973), but during anoxia i n bival v e s 204 Table XXIV. PEPCK:PK r a t i o s i n marine molluscs, Species PEPCK:PK Rangia cuneata* 1.1:1 v i r g i n i c a * 0.05:1 V o l s e l l a demissus* 0.16:1 L i t t o r i n a i r r o r a t a * 0.16:1 Thais hemostoma* 0.05:1 M. e d u l i s * * 1.3:1 Cardium edule** 0.08:1 Mya arenaria** 0.77:1 Dreissena polymorpha** 1.67:1 Unio sp.** 0.67:1 'C_. gigas (adductor) 0.05:1 *Data for acetone powders of whole animals (Simpson and Awapara, 1966) **Data f o r adductor muscle (Gade and Zebe, 1973) 205 normal metabolism through MDH and c i t r a t e synthase appears to be blocked at the MDH step by NADH accumulation (perhaps one reason why adductor c i t r a t e synthase shows no unusual r e g u l a t o r y f e a t u r e s , as these would be unnecessary). For t h i s reason, one major f a t e of malate i s conversion to suc c i n a t e . For t h i s conversion reducing equivalents (NADH or reduced f l a v i n ) are re q u i r e d a t the l e v e l of fumarate r e d u c t i o n , and these must be obtained i n t r a m i t o c h o n d r i a l -l y . In A s c a r i s muscle t h i s i s achieved by a dismutation at the l e v e l of malate, one-half being converted to s u c c i n a t e , one-half being converted to pyruvate and CO2 by a NAD +-linked malic enzyme (E.C. 1.1.1.39) (Saz, 1971b; Saz and Lescure, 1969) which s u p p l i e s the NADH req u i r e d f o r the r e d u c t i o n of fumarate. The p o s s i b i l i t y that a s i m i l a r pathway e x i s t s i n b i v a l v e s i s one that merits f u r t h e r c o n s i d e r a t i o n . NADP +-linked malic enzyme (E.C. 1.1.1.40) occurs i n the cytoplasm of oyster adductor muscle (Hochachka and Mustafa, 1973), and at l e a s t 30% of "^C-aspartate flows to pyruvate and a l a n i n e ( C o l l i -c u t t , 1975) by t h i s or a s i m i l a r route i n v o l v i n g d ecarboxylation at the l e v e l of oxaloacetate or malate. Function of NADP +-malic enzyme during anoxia would lead to accumulation of NADPH i n the cytoplasm and increase the problem of redox r e g u l a t i o n , unless there were some other NADPH r e q u i r i n g dehydrogenase present i n the cytoplasm. On the other hand, i f an NAD +-malic enzyme occurs i n the mitochondria, as i s the case f o r r a b b i t heart, pigeon breast muscle and pigeon heart mitochondria ( L i n and Davis, 1974), then a ready e x p l a n a t i o n of the f i n d i n g s i s a v a i l a b l e . This enzyme i s e s s e n t i a l l y i r r e v e r s i b l e , has a pH optimum of 6.8-7.0, i s i n h i b i t e d by ATP, a c t i v a t e d by fumarate and suc c i n a t e -9 ( L i n and Davis, 1974; Sauer, 1973) and the K £ q i s 3.44 x 10 (Veech et a l . , 1969), whereas the K e q f o r MDH i s 2.78 x 1 0 ~ 1 2 (Williamson et a l . , 1967), t h e r e f o r e i f an enzyme w i t h s i m i l a r k i n e t i c p r o p e r t i e s occurred i n the mito-chondria of b i v a l v e s , i t would be r e a d i l y a c t i v a t e d under anoxic c o n d i t i o n s 206 and would a l s o have a more favourable e q u i l i b r i u m f o r m e t a b o l i z i n g malate than does the MDH r e a c t i o n . The proposed pathway f o r the m i t o c h o n d r i a l metabolism of malate i s shown i n Figure 61. Conversion to pyruvate leads to a v a r i e t y of other end products, i n c l u d i n g a l a n i n e , by transamination w i t h glutamate, as occurs i n the oyster heart ( C o l l i c u t t , 1975), and NCEA which a r i s e s from a l a n i n e . Acetate a l s o can be formed from pyruvate by d e c a r b o x y l a t i o n , as occurs i n M. edulis,. A. cygnea, Helisoma d u r y i , A u s t r a l o r b i s glabratus and Cardium edule (Gade, 1975; Gade j i t a l . , 1975; Klutymans j i t al. , 1975; Mehlman and von Brand, 1951), but whether t h i s d e carboxylation i s by a d i r e c t mechanism, or by con-v e r s i o n of pyruvate to acetylCoA by pyruvate dehdyrogenase, and subsequent conversion of acetylCoA to acetate by acetylCoA synthase, a p o s s i b i l i t y suggested by Hochachka et a l . (1973), remains to be demonstrated experimentally. The other major product of malate metabolism, s u c c i n a t e , i s excreted i n t o the p a l l i a l f l u i d (Crenshaw and Neff, 1969), or converted to propionate as occurs i n M. e d u l i s , A. cygnea and Cardium edule (Gade, 1975; Gade et a l . , 1975; Klutymans et a l . , 1975). Research on b i v a l v e anaerobic metabolism has been d i r e c t e d towards an understanding of the metabolic processes o c c u r r i n g i n the cytoplasm, but i t i s obvious that the mitochondria a l s o have a s i g n i f i c a n t r o l e i n anaerobic pro-cesses and the aerobic-anaerobic t r a n s i t i o n . At the present time l i t t l e i s known about mitochondrial-cytoplasmic i n t e r a c t i o n s i n b i v a l v e s , but c l e a r l y these w i l l be important i n changes i n metabolic pathways s i n c e the e f f e c t s of anoxia w i l l be f i r s t manifested at the l e v e l of the e l e c t r o n t r a n s f e r chain i n mitochondria. 207 Figure 61. 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P h y s i o l . 43B: 47-54. 221 APPENDIX: LIST.OF ABBREVIATIONS AcetylCoA - acetyl-S-coenzyme A Asp - aspartate AMP, ADP, ATP - adenosine 5'-mono-, d i - , triphosphate BSA - bovine serum albumin CoA - coenzyme A DEAE - di e t h y l a m i n o e t h y l DHAP - dihydroxyacetonephosphate 1,3DPG - 1,3-diphosphoglycerate 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 - ethylenediamine t e t r a a c e t i c a c i d F6P - fructose-6-phosphate FDP - fructose-1,6-diphosphate G1P - glucose-1-phosphate G6P - glucose-6-phosphate G3P - glyceraldehyde-3-phosphate GAPDH - glyceraldehyde-3-phosphate dehydrogenase (E.C. 1.2.1.12) GDH - glutamate dehydrogenase (E.C. 1.4.1.2) Glut - glutamate aGP - glycerol-1-phosphate aGPDH - gly c e r o l - l - p h o s p h a t e dehydrogenase IDP, ITP - i n o s i n e 5 ' - d i - , triphosphate 2-kga - 2-ketoglutarate LDH - l a c t a t e dehydrogenase (E.C. 1.1.1.27) MDH - malate dehydrogenase (E.C. 1.1.1.37) NAD + - nicotinamide adenine d i n u c l e o t i d e 222 NADH - reduced NAD NADP + - nicotinamide adenine d i n u c l e o t i d e phosphate NADPH - reduced NADP + NCEA - N - ( l - c a r b o x y e t h y l ) - a l a n i n e NCEG - N-(1- c a r b o x y e t h y l ) - g l y c i n e NCEADH - N - ( l - c a r b o x y e t h y l ) - a l a n i n e dehydrogenase ODH - octopine dehydrogenase (E.C. 1.5.1.11) O.D. - o p t i c a l d ensity Oxa - oxaloacetate P^ - inorganic- phosphate PEP - phosphoenolpyruvate PDPCK - phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) PFK - phosphofructokinase (E.C. 2.7.1.11) PGK - phosphoglycerate kinase PK - pyruvate kinase (E.C. 2.7.1.40) SuccinylCoA - succinyl-S-coenzyme A PUBLICATIONS F i e l d s , J . H . A . 1 9 7 2 . P a r t i c u l a t e e x c r e t i o n i n H e r m o d i c e  c a r u n c u l a t a P a l l a s . M . S c . T h e s i s , M c G i l l U n i v e r s i t y . H o c h a c h k a , P . W . , F i e l d s , J . H . A . and M u s t a f a , T . 1973 . A n i m a l l i f e w i t h o u t o x y g e n : b a s i c b i o c h e m i c a l m e c h a n i s m s . Am. Z o o l . 13 5 4 3 - 5 5 5 . H o c h a c h k a , P . W . , N o r b e r g , C . , B a l d w i n , J . and F i e l d s , J . H . A . 1 9 7 6 . E n t h a l p y e n t r o p y c o m p e n s a t i o n o f oxamate b i n d i n g b y homo logous l a c t a t e d e h y d r o g e n a s e s . N a t u r e 260 6 4 8 - 6 5 0 . F i e l d s , J . H . A . 1976 . A d e h y d r o g e n a s e r e q u i r i n g a l a n i n e and p y r u v a t e as s u b s t r a t e s f r o m o y s t e r a d d u c t o r m u s c l e . F e d . P r o c . 35 1687 . G u d e r l e y , H . E . , S t o r e y , K . B . , F i e l d s , J . H . A . and H o c h a c h k a , P .W. 1 9 7 6 . C a t a l y t i c and r e g u l a t o r y p r o -p e r t i e s o f p y r u v a t e k i n a s e i s o z y m e s f r o m o c t o p u s m a n t l e and l i v e r . C a n . J . Z o o l . 54 8 6 3 - 8 7 0 . F i e l d s , J . H . A . , B a l d w i n , J . and H o c h a c h k a , P .W. 1976 . On t h e r o l e o f o c t o p i n e d e h y d r o g e n a s e i n c e p h a l o p o d m a n t l e m u s c l e m e t a b o l s i m . C a n . J . Z o o l . 54 8 7 1 - 8 7 8 . F i e l d s , J . H . A . , G u d e r l e y , H . E . , S t o r e y , K . B . and H o c h a c h k a , P .W. 1 9 7 6 . The p y r u v a t e b r a n c h p o i n t i n s q u i d b r a i n : c o m p e t i t i o n b e t w e e n o c t o p i n e d e h y d r o g e n a s e and l a c t a t e d e h y d r o g e n a s e . C a n . J . Z o o l . 54 8 7 9 - 8 8 5 . F i e l d s , J . H . A . , G u d e r l e y , H . E . , S t o r e y , K . B . and H o c h a c h k a , P .W. 1 9 7 6 . O c t o p u s m a n t l e c i t r a t e s y n t h a s e . C a n . J . Z o o l . 54 8 8 6 - 8 9 1 . F i e l d s , J . H . A . and H o c h a c h k a , P .W. 1 9 7 6 . O y s t e r c i t r a t e s y n t h a s e : c o n t r o l o f c a r b o n e n t r y i n t o t he K r e b s c y c l e o f a f a c u l t a t i v e a n a e r o b e . C a n . J . Z o o l . 54 8 9 2 - 8 9 5 . 

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