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Enzyme mechanisms of facultative anaerobiosis in molluscs: regulation of the phosphoenolpyruvate crossroads… Mustafa, Tariq 1972

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ENZYME MECHANISMS OF FACULTATIVE ANAEROBIOSIS IN MOLLUSCS REGULATION OF THE PHOSPHOENOLPYRUVATE CROSSROADS IN THE OYSTER by TARIQ MUSTAFA B.Sc. (Hons.), University of Karachi, Pakistan, 1965 M.Sc. University of Karachi, Pakistan, 1966 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT of ZOOLOGY We accept this thesis as conforming to the required standard: THE UNIVERSITY OF BRITISH COLUMBIA May 1972 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 requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree 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 re ference and s tudy . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copy ing o f t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or 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 ga in s h a l l not be a l lowed without my w r i t t e n p e r m i s s i o n . ( TAEIQ MUSTAFA ) Department of Zoology The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date P<t3^ MU ABSTRACT C a t a l y t i c and regulatory properties of pyruvate kinase (EC 2.7.1.40) and phosphoenolpyruvate carboxykinase (EC 4.1.1.32) from oyster adductor muscle were studied. P a r t i c u l a r a t t e n t i o n was given to those properties of the enzymes which could help to explain the "switch over" from aerobic to anaero-b i c glucose degradation between a e r o ^ = ^ anaerobic conditions i n a f a c u l t a t i v e anaerobe, such as the oyster. A l l the a v a i l a b l e data can be summarized as follows: Situated s t r a t e g i c a l l y at the primary branching point between aerobic and anaerobic metabolism are the two enzymes, pyruvate kinase (favoured during aerobiosis) and P-enolpyruvate carboxykinase (favoured during anaerobiosis). H + ion plays a p i v o t a l r o l e i n the channelling of P-enolpyruvate through t h i s branching point. When 0 2 i s absent, the pH i s known to drop because of the build-up of various acid products. In the absence of any other f a c t o r s , t h i s would lead to an e f f e c t i v e a c t i v a t i o n of p-enolpyruvate carboxykinase due to (1) an increase i n absolute a c t i v i t i e s , and (2) an increase i n the a f f i n i t y f o r p-enolpyruvate. At the same time pyruvate kinase would be i n h i b i t e d by (1) a decrease i n the absolute a c t i v i t y , and (2) a decrease i n the a f f i n i t y f o r p-enolpyruvate. Alanine and ATP i n h i b i t i o n of pyruvate kinase potentiate these e f f e c t s p a r t i c u l a r l y at low pH. In contrast, alanine e f f e c t i v e l y a c t i -vates p-enolpyruvate carboxykinase by reversing ITP i n h i b i t i o n . The more a c i d i c the anaerobic system becomes, the more avid l y would p-enolpyruvate carboxykinase channel p-enolpyruvate towards oxaloacetate. Upon return to aerobic conditions, the pH would be expected to r i s e again and a l l of the above events would be reversed. The most important feature of such a regulatory system i s that i t i s a kind of a u t o c a t a l y t i c cascade. Once either pyruvate kinase a c t i v a t i o n or p-enolpyruvate carboxykinase activation is in i t ia ted , a l l the various regula-tory interactions potentiate one another. There is l i t t l e doubt that the specific control components at this point in the metabolism of molluscan facultative anaerobes are the outcome of selective tai loring of the 2 enzymes functioning at this point. What evolution seems to have done in molluscs was to arrange the control characteristics of pyruvate kinase and p-enolpyruvate carboxykinase in a reciprocal manner so that the two reactions cannot be ful ly active simultaneously. I l l TABLE OF CONTENTS Page Abstract i L i s t of Tables v i L i s t of Figures v i i Acknowledgements x Chapter I: Introduction 1 A l t e r a t i o n s i n the g l y c o l y t i c pathway f o r an 2 anaerobic way of l i f e The carboxylation reactions i n invertebrate 4 f a c u l t a t i v e anaerobes Oxidation of cytoplasmic NADH Phosphoenolpyruvate crossroads and the statement of the problem Chapter I I : Materials and Methods Animal c o l l e c t i o n and t h e i r handling Preparation of mantle and adductor muscle pyruvate kinases Preparation of mitochondria Preparation of cytoplasmic phosphoenolpyruvate carboxykinase, malate dehydrogenase and "malic enzyme" Assay of pyruvate kinase a c t i v i t y 14 Assay of phosphoenolpyruvate carboxykinase 15 (carboxylation reaction) a c t i v i t y Assay of phosphoenolpyruvate carboxykinase 15 (decarboxylation reaction) a c t i v i t y Assay of "malic enzyme" a c t i v i t y 16 6 8 12 12 12 13 13 i v Page Assay of malate and l a c t a t e dehydrogenases 17 Estimates of protein contents 17 Electrophoresis 17 I s o e l e c t r i c focusing of pyruvate kinases and 18 of phosphoenolpyruvate carboxykinase Chapter I I I : C a t a l y t i c and Regulatory Properties of Oyster 19 Pyruvate Kinases Introduction 19 Results 21 Electrophoresis and electrofocusing 21 Cation requirements 21 E f f e c t of f r u c t o s e - l , 6 - P 2 on pH optima 24 E f f e c t of f r u c t o s e - l , 6 - P „ on K of ADP 24 2 m E f f e c t of pH and f r u c t o s e - l , 6 - P „ on K of 24 z m p-enolpyruvate ATP i n h i b i t i o n 28 Interacting e f f e c t s of ATP and f r u c t o s e - 1 , 6 - P 2 on 32 the K of p-enolpyruvate m Search f o r other modulators 39 Nature of . alanine and phenylalanine i n h i b i t i o n 39 Discussion A3 Chapter IV: C a t a l y t i c and Regulatory Properties of Oyster 47 Phosphoenolpyruvate Carboxykinase Introduction 47 Results 49 Electrofocusing 49 Page Requirements f o r the p-enolpyruvate carboxykinase 49 catalyzed carboxylation reaction Tissue and s u b - c e l l u l a r d i s t r i b u t i o n of oyster 49 p-enolpyruvate carboxykinase a c t i v i t y R e v e r s i b i l i t y of pH e f f e c t 53 Inosine diphosphate saturation k i n e t i c s 58 P-enolpyruvate saturation k i n e t i c s 61 j | Cu i n h i b i t i o n k i n e t i c s 64 Discussion 67 Chapter V: C a t a l y t i c and Regulatory Properties of Oyster 71 Phosphoenolpyruvate Carboxykinase: I I . Regulation of the Enzyme A c t i v i t y and i t s Function i n Phosphoenolpyruvate Metabolism Introduction 71 Results 73 Nature of inosine triphosphate (ITP) i n h i b i t i o n 73 Search f o r other metabolic e f f e c t o r s 7 8 L-alanine effects 82 Discussion 85 Chapter VI: Summating Remarks 88 The enzymic control at p-enolpyruvate crossroads 89 Sources of alanine and reducing equivalents 92 Other metabolic sources of succinate 94 P o t e n t i a l metabolic sources of glutamate The y i e l d of high energy phosphate compounds 96 Chapter VII: L i t e r a t u r e Cited 98 VI LIST OF TABLES Table Page I I I , 1. E f f e c t of pH on K (p-enolpyruvate) i n absence and 29 presence of fructose-ljG-P^ I I I , 2. E f f e c t of ATP and fructose-1,6-P 2 on the K m (p-enolpyruvate) 33 of mantle pyruvate kinase I I I , 3. Comparison of the properties of mantle, adductor, rat 44 muscle and l i v e r pyruvate kinases IV, 1. Components of p-enolpyruvate carboxykinase catalyzed 51 carboxylation reaction IV, 2. Tissue and s u b - c e l l u l a r d i s t r i b u t i o n of oyster p-enol- 5 2 pyruvate carboxykinase IV, 3. R e v e r s i b i l i t y of pH e f f e c t on enzyme a c t i v i t y 56 LIST OF FIGURES Figure I I I , 1. Electrophoretic r e s o l u t i o n of oyster mantle, g i l l and adductor pyruvate kinase a c t i v i t y I I I , 2. Electrofocusing pattern of the mantle and adductor pyruvate kinases I I I , 3. E f f e c t of fructose-l }6 -P2° n t n e optima of mantle and adductor pyruvate kinase I I I , 4. E f f e c t of fructose-1,6-P„ on the K /.„.., of mantle 2 m(ADP) and adductor pyruvate kinases I I I , 5. E f f e c t of pH and fructose-1,6-P„ on the K of p-enol-2 m pyruvate of mantle pyruvate kinase I I I , 6. E f f e c t of pH and fructose-1,6-P„ on the K of p-enol-2 m r pyruvate of adductor pyruvate kinase I I I , 7. E f f e c t of ATP concentration on the reaction rate and ATP,.. . determination of mantle and adductor pyruvate i kinases at d i f f e r e n t pH and p-enolpyruvate concentrations I I I , 8. Double r e c i p r o c a l p l o t s of the reaction v e l o c i t y against p-enolpyruvate of mantle and adductor pyruvate kinases at d i f f e r e n t ATP concentrations at pH 8.5 I I I , 9. Interacting e f f e c t s of ATP and f r u c t o s e - l ^ - P ^ on the K . , , . f o r the mantle enzyme at pH 8.5 m(p-enolpyruvate) I I I , 10. Interacting e f f e c t s of ATP and fructose-1,6-P 2 on the K , , x for the mantle enzyme at pH 7.5 m(p-enolpyruvate) I I I , 11. E f f e c t of L-alanine concentration on the reaction rate and alanine (K\) determination f o r adductor and mantle pyruvate kinases at two d i f f e r e n t pH values V l l l Page I I I , 12. E f f e c t of phenylalanine concentration on the reaction 38 rate and phenylalanine (K^) determination for mantle and adductor pyruvate kinases at two d i f f e r e n t pH values I I I , 13. Double r e c i p r o c a l plots of the reaction v e l o c i t y against 40 p-enolpyruvate of mantle pyruvate kinase i n presence of alanine at pH 8.5 and 7.5 I I I , 14. Double r e c i p r o c a l plots of the reaction v e l o c i t y of 42 mantle and adductor pyruvate kinase i n presence of phenylalanine and f r u c t o s e - l ^ - P ^ IV, 1. Electrofocusing pattern of the adductor p-enolpyruvate 50 carboxykinase a c t i v i t y IV, 2. E f f e c t s of pH on p-enolpyruvate carboxykinase a c t i v i t y 54 I | | | i n presence of Zn and Mn IV, 3. E f f e c t s of metal ion a c t i v a t i o n at d i f f e r e n t pH values i n 57 I | | | presence of Mn or Zn I | IV, 4. E f f e c t s of Mn on the IDP saturation k i n e t i c s at pH 59 6.0 and 7.0 I | | | IV, 5. E f f e c t s of Zn and Mn on the IDP saturation k i n e t i c s 60 at pH 5.1 IV, 6. E f f e c t s of Mn on p-enolpyruvate saturation k i n e t i c s at 62 d i f f e r e n t pH values IV, 7. E f f e c t s of Zn on p-enolpyruvate saturation k i n e t i c s 63 at d i f f e r e n t pH values IV, 8. I n h i b i t i o n of p-enolpyruvate carboxykinase a c t i v i t y by 65 increasing concentration of Cu i n presence of Zn or Mn at pH 5.1 and 6.0 IV, 9. P-enolpyruvate carboxykinase a c t i v i t y as a function of 66 XX Page I | j | the concentration of Mn and Zn i n the presence or _| L. absence of Cu I | V, 1. Zn saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v 74 I | vs 1/Zn ) at varying ITP concentrations. I j V, 2. Mn saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v 75 I j vs 1/Mn ) at varying ITP concentrations. V, 3. IDP saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v 76 I | vs 1/IDP) at varying ITP concentrations i n presence of Zn V, 4. IDP saturation k i n e t i c s and i t s double r e c i p r o c a l plot (1/v 77 I | vs 1/IDP) at varying ITP concentrations i n presence of Mn V, 5. Double r e c i p r o c a l plots (1/v vs 1/p-enolpyruvate) at varying 79 ITP concentrations at pH 5.1 and 6.0. V, 6. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o c a l 80 plot (1/v vs 1/p-enolpyruvate) with varying concentrations I | of ITP and alanine with Mn as the divalent metal ion at pH 6.0. V, 7. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o c a l 81 pl o t (1/v vs 1/p-enolpyruvate) i n the presence and absence of 0.25 mM GTP. V, 8. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o c a l 84 I | plot with varying concentrations of ITP and alanine with Zn as the divalent metal ion at pH 6.0. X ACKNOWLEDGMENTS To my research supervisor Dr. Peter W. Hochachka who introduced me to the problems of environmental biochemistry, many thanks are extended f o r h i s wise, warm and i n t e l l i g e n t suggestions and c r i t i c i s m ; these kept my i n t e r e s t s a l i v e throughout t h i s study to reveal the mysteries of f a c u l t a t i v e anaerobio-s i s i n molluscs. I would l i k e to thank the members of my committee, Drs. W. S. Hoar, J . E. P h i l l i p s of Department of Zoology and Dr. D. H. Copp of Department of Physiology f o r t h e i r comments and c r i t i c i s m . Also, I would l i k e to thank my colleagues, Thomas Moon and Terry Owen for t h e i r invaluable help i n c o l l e c t i n g the research animals. I must thank my mother, s i s t e r s and brothers f o r t h e i r moral support from 12,000 miles away throughout my graduate work at U. B. C. And, f i n a l l y , the Canadian International Development Agency supported myself through the Canadian Commonwealth Scholarship program, and the National Research Council of Canada provided l o g i s t i c support throughout t h i s study. CHAPTER I: Introduction 1 There i s good geochemical and b i o l o g i c a l evidence suggesting that l i f e arose under reducing c o n d i t i o n s i n the t o t a l absence of molecular oxygen. Underlying c e l l u l a r metabolism presumably was l i n k e d to and " d r i v e n by" s u b s t r a t e - l e v e l phosphorylations viiere organic s u b s t r a t e , and not C^, acted as e l e c t r o n acceptors. Because of i t s high a f f i n i t y f o r e l e c t r o n s , molecular 0^ became the most important e l e c t r o n acceptor once i t appeared i n s i g n i f i -cant q u a n t i t i e s i n the hydrosphere and atmosphere. I t s h i g h e l e c t r o n a v i d i t y served to g r e a t l y i n c r e a s e the e f f i c i e n c y w i t h which the organism could capture bond energy of n u t r i e n t molecules i n "high chemical p o t e n t i a l " form, as n u c l e o t i d e t r i p h o s p h a t e , CoA d e r i v a t i v e s and so f o r t h . Yet to t h i s day, the b a s i c f a b r i c of intermediary metabolism i s fundamentally anaerobic (e.g. g l y c o l y s i s , the shunt pathway, amino a c i d metabolism, e t c . ) , w i t h r e a c t i o n s i n v o l v i n g molecular 0^ c l e a r l y r e p r e s e n t i n g e v o l u t i o n a r y embellishments added onto an already f u n c t i o n a l framework of anaerobic metabolism (Wald, 1964). Because of the use of c l a s s i c a l l a b o r a t o r y animals f o r most biochemical s t u d i e s , the degree to which h i g h l y s u c c e s s f u l metazoan organisms u t i l i z e anaerobic mechanisms to s u s t a i n temporary or i n d e f i n i t e periods of anoxia i s not widely appreciated. Nevertheless, i t i s now c l e a r that many i n v e r t e -brates are f a c u l t a t i v e anaerobes, capable of s u r v i v i n g i n d e f i n i t e l y i n the absence of 0^ and capable of a c t i v e o x i d a t i v e metabolism i n i t s presence. Examples are p a r t i c u l a r l y common among wid e l y d i v e r s i f i e d helminths (Von Brand, 1966; Braynt, 1970), but during recent years i t has a l s o become evident that many molluscs have comparable metabolic c a p a c i t i e s (Hammen, 1969; Chen and Awapara, 1969). Newell (197o) concluded that many of the i n t e r t i d a l molluscs are adapted to a n a e r o b i o s i s , s u r v i v i n g long periods of time i n an oxygen f r e e 2 environment. Mya arenaria f o r example survives 8 days without oxygen (Ricketts and Calvins, 1948), while L i t t o r i n a n e r i t o i d e s and _L. punctata can survive a nitrogen atmosphere f o r several days (Patane, 1946, 1955). Rangia  cuneata have been kept i n deoxygenated water f o r 3 weeks without apparent harm to the animal (Chen and Awapara, 1969). Bivalves such as M. arenaria b u i l d up an oxygen debt during anaerobiosis to be paid by increasing the pumping rate vhen the ti d e returns. Others such as the R. cuneata simply close t h e i r valves and l i v e without oxygen f o r extended periods of time. Oyster i s l i k e Rangia i n t h i s regard. The term " f a c u l t a t i v e anaerobe" i s coined i n the l i t e r a t u r e f o r those organisms which can sustain anoxia i n -d e f i n i t e l y but which u t i l i z e 0^ when i t i s a v a i l a b l e . A l t e r a t i o n s i n the g l y c o l y t i c pathway for an anaerobic way of l i f e . It i s generally accepted that the degradation of the glucose to pyruvate i n many organisms i s by way of the Embeden-Meyerhof sequence of reactions, which i s , i n a sense, the basic way of l i f e . G l y c o l y t i c or fermentative processes underlie a l l other forms of metabolism, and v i r t u a l l y a l l types of c e l l s can survive f o r periods on g l y c o l y s i s i f deprived of oxygen (Wald, 1964). There i s evidence f o r b e l i e v i n g that most of the i n d i v i d u a l reactions of the Embeden-Meyerhof scheme are operative i n many f a c u l t a t i v e anaerobic invertebrates. Bueding (1962) has made i t clear that glucose i s degraded through the same reactions into pyruvate but the d i s -p o s i t i o n of pyruvate varies s u b s t a n t i a l l y from one organism to another. The p a r a s i t i c helminths have been the subject of much research into the nature of their metabolism. The breakdown of glycogen i n t o glucose and the subsequent g l y c o l y t i c reactions have been investigated i n Hymenolepis  dimunita (Read, 1951), Ascaris lumbricoides (Bueding and Saz, 1968), 3 D i r o f i l a r i a immltis (Hutchinson and McNeil, 1970), and others. In a l l of these helminths i t was apparent that the g l y c o l y t i c pathway i s i n operation, except that l i t t l e or no l a c t a t e could be measured when glycogen was broken down. In l i e u of l a c t a t e , a v a r i e t y of other products were formed, among which succinate and f a t t y acids were i d e n t i f i e d (Bueding, 1962). The anaerobic disappearance of glycogen without a concomitant l a c t a t e formation i s not l i m i t e d to helminths. This s i t u a t i o n i s known to occur i n oyster muscle, and the disappearance of glucose anaerobically i s not accounted f o r e n t i r e l y by the formation of pyruvate or l a c t a t e (Humphrey, 1944). The same observation has been made i n some fresh-water s n a i l s (von Brand, et^ a l . , 1950). At this juncture i t was evident that i n f a c u l t a t i v e anaerobes such as helminths and oysters, the formation of pyruvate could i n fac t proceed by the u n i v e r s a l l y occurring g l y c o l y t i c route but the f i n a l d i s p o s i t i o n of pyruvate was at variance with the reaction scheme. If l a c t a t e i s not formed then some other p r o v i s i o n must be made f o r the reoxidation of the NADH formed during the oxido-reduction step of g l y c o l y s i s . Bueding (1963) considered t h i s problem f o r the p a r a s i t i c worm, Ascaris lumbricoides and concluded that the formed pyruvate could react with CO^ to form malate and eventually fumarate. This conversion i s based on the observation that Ascarisi has a NAD-linked "malic enzyme" to catalyze the carboxylation reaction and also fumarase (Saz and Hubbard, 1957). In addition to these experimental f a c t s , Kemetec and Bueding (1963) discovered also i n Ascaris a fumarate reductase that functions with NADH. In other supporting experiments, Saz and V i r d i n e 14 14 (1959), demonstrated that C from lactate-3-C appears i n the methelyne carbon of succinate. Although mechanisms involved i n the d i s p o s i t i o n of f i n a l g l y c o l y t i c end products d i f f e r i n various f a c u l t a t i v e anaerobes, a carboxylation step and the e f f i c i e n t during g l y c o l y s i s as a r e s u l t of the i s mandatory f o r the continuation of 4 removal (or reoxidation) of NADH formed oxidation of glyceraldehyde-3-phosphate anaerobic g l y c o l y s i s . The carboxylation reactions i n invertebrate f a c u l t a t i v e anaerobes. CC^ f i x a t i o n reactions play important, although somewhat s p e c i a l i z e d , roles i n a v a r i e t y of metabolic areas and i t i s not too much to say that most, i f not a l l , hetrotrophic c e l l s are dependent on CO^ f i x a t i o n reactions. Carboxylation reactions have been shown to be quite common i n invertebrates (Hammen and Wilbur, 1959; Hammen and Osborne, 1959; Awapara and Campbell, 1963; Simpson and Awapara, 1964). One of the most common observations i s that succinate appears as the main product of carboxylation i n a v a r i e t y of marine invertebrates ( i n 14 species representing 12 phyla from P o r i f e r a to _Hemi-14 chordata) when given NaHC 0^ (Hammen and Osborne, 1959), At that time, i t was tempting to assume that the primary step was the carboxylation of propionate-CoA and Hammen and Wilbur (1959) f i r s t made th i s suggestion. They did i n f a c t obtain succinate upon incubation of propionate with oyster mantle. Later, the formation of succinate as the end product of a primary carboxyla-t i o n reaction became untenable, for organisms known to form succinate by CO^ f i x a t i o n were found to lack propionyl-CoA carboxylase a c t i v i t y (Simpson and Awapara, 1964). Hammen (1966), on the basis of the pattern of l a b e l l i n g of the acids 14 indicated that malic acid was the i n i t i a l major product of NaHC 0^ f i x a t i o n ; spectrophotometric evidence f o r the presence of a "malic enzyme" i n the supernatant of homogenate was demonstrated. The reduction of NADP i n propor-t i o n to homogenate concentration indicates that malate may be formed by the action of a malic enzyme of the type f i r s t described by 0choa,e£ al_. (1948). 5 Hammen (1966) c o r r e c t l y asserts that the presence of "malic enzyme" does not ru l e out the presence of other carboxylating enzymes i n the same t i s s u e , and indeed there i s evidence for two others: priopionyl-CoA carboxylase (Hammen and Wilbur, 1959) as mentioned e a r l i e r , while phosphoenolpyruvate.carboxykinase occurs i n tissues of R. cuneata (Simpson and Awapara, 1964), and i n helminths (Saz and Lescure, 1969). Molluscs have a very a c t i v e phosphoenolpyruvate carboxykinase. In one bivalve mollusc, ]L cuneata, which i s one of the r i c h e s t sources of t h i s enzyme among invertebrates (Simpson and Awapara, 1964), the a c t i v i t y of the enzyme was found to be on the average ten times higher than i n chicken l i v e r . A rapid carboxylation of phosphoenolpyruvate could r e a d i l y account for the rapid formation of succinate from CO^ i n the marine organisms without invo l v -ing a carboxylation of pyruvate or the necessity of i t s formation. Further studies on R. cuneata revealed that i t produced from glucose large amounts of succinate and very l i t t l e l a c t a t e (Simpson and Awapara, 1966). Simpson and Awapara (1966), on the basis of balance studies and isotope d i s t r i b u t i o n , concluded that glucose i s degraded i n t h i s species and perhaps i n other molluscs to phosphoenolpyruvate with much of the phosphoenolpyruvate diverted ultimately to succinate by reacting with CO^ at a rapid rate. In contrast, only a small amount of succinate i s formed from the carboxylation of pyruvate as the malic enzyme i s only present i n small amounts and pyruvate carboxlase i s absent. In order to decide which pathway predominates, they 14 incubated mantle tis s u e from R. cuneata with glucose-6-C i n one experiment, 14 and with pyruvate-3-C i n another. The e f f i c i e n c y of succinate formation 14 14 i s about ten times greater with glucose-6-C as compared to pyruvate-3-C From these experiments i t became reasonable to assume that most of the p-enolpyruvate reacts with C0„ to form oxaloacetate and ult i m a t e l y succinate. 6 These workers have postulated the following reaction sequence f o r succinate formation: C0 2 NADH NADH p-enolpyruvate — — Oxaloacetate — ^ — Malate Fumarate — — — Succinate Chen and Awapara (1969) found that i n mantle of R. cuneata a l l but one of the enzymes-catalyzing the o v e r a l l flow of carbon from glucose to succinate are l o c a l i z e d i n the cyto s o l ; the s i n g l e exception, the enzyme catalyzing the reduction of fumarate, i s l o c a l i z e d i n the mitochondria. In some molluscs l a c t a t e dehydrogenase i s shown to be absent, and instead octopine dehydro-genase i s present (Regnouf and von Thoai, 1970). Octopine dehydrogenase catalyzes the production of octopine from pyruvate i n tissues of the cephalopod Sepia o f f i c i n a l i s and the bivalves Cardinal ed ule and Pecten maximus v i a the following reaction: NADH2 NAD L-arginine + pyruvate v >^ octopine At t h i s time i t i s not known whether molluscs that contain octopine dehydro-genase are f a c u l t a t i v e anaerobes. However, the regeneration of NAD f o r t r i o s e phosphate dehydrogenase i n most f a c u l t a t i v e anaerobes presents some unusual problems i n metabolic organization. Oxidation of cytoplasmic NADH. A feature of aerobic g l y c o l y s i s is that the NADH generated i n the glyceraldehyde phosphate dehydrogenase reaction i n the cytoplasm i s reoxidized at the expense of oxygen i n the mitochondria. E i t h e r the NADH i t s e l f must be able to penetrate across the mitochondrian or else reducing equivalents from the NADH must be transferred to the mitochondrial electron transport system by i n d i r e c t routes c a l l e d " s h u t t l e s " . Lehninger (1951, 1970) found that the respiratory chain of the i s o l a t e d l i v e r mitochondria i s almost i n a c c e s s i b l e to NADH from the external medium, and a s i m i l a r inacces-7 s i b i l i t y has been observed with kidney (Boxer and Devlin, 1961), tumor (Borst, 1962), and insect f l i g h t muscle(Sactor and Dick, 1962). From experiments i n 14 which (C ) n i c o t i n i c acid was in j e c t e d into r a t s , Purvis and Lowenstein (1961), deduced that the i n t r a - and extra-mitochondrial pyridine nucleotides of l i v e r do not undergo rapid e q u i l i b r a t i o n , and they calculated that NADH enters l i v e r mitochondria i n vivo at a speed quite inadequate to account for the res p i r a t o r y rate of the t i s s u e . I t i s now accepted that v i r t u a l l y a l l mitochondria are impermeable to NADH both i n vivo and i n v i t r o (Lehninger, 1964). I t seems, therefore, that reducing equivalents must be transferred from the cytoplasm to the res p i r a t o r y chain by means other than the transport of NADH. It i s evident that f o r every molecule of glucose metabolized a e r o b i c a l l y or anaerobically, two molecules of NADH are formed i n the cytoplasm by the action of the enzyme glyceraldehyde-3-phosphate dehydrogenase, and unless th i s co-enzyme i s immediately re-oxidized by the transfer of electrons to another acceptor, energy production must cease. The mechanism involved f o r the oxidation of cytoplasmic NADH seems to vary from t i s s u e to t i s s u e . In mammalian muscle and l i v e r , pyruvate acts as the electron acceptor, being reduced to l a c t a t e by the action of l a c t a t e dehydrogenase. Another known mechanism by which NADH can be oxidized i n l i v e r i s by the malate/oxaloacetate s h u t t l e . In yeast, alcohol dehydrogenase provides a t h i r d mechanism. In insect f l i g h t muscle, l a c t a t e dehydrogenase has a very low a c t i v i t y , and i t s function i s taken over by the soluble a-glycerphosphate dehydrogenase. Thus, dihydroxyacetone phosphate accepts electrons from NADH and i s reduced to a-glycerophosphate, while NAD i s restored f o r p a r t i c i p a t i o n i n the main Embeden-Meyerhof pathway. The a-glycerophosphate so formed d i f f u s e s r e a d i l y into the mitochondria, where i t is reoxidized by the mitochondrial a-glycero-8 phosphate dehydrogenase, t r a n s f e r r i n g electrons through the cytochromes to oxygen. The dihydroxacetone formed i n this reaction d i f f u s e s out of the mitochondria to complete the cycle, and i s then ready to accept electrons once more. Thus, the two a-glycerphosphate dehydrogenases and t h e i r substrate act as a powerful c a t a l y t i c cycle whereby reducing equivalents derived i n i t i a l l y from g l y c e r a l dehyde-3-phosphate are fed into the mitochondrial electron transport system, thus making possible the rapid and complete oxidation of glucose to carbon dioxide and water within the muscle c e l l . In the mantle ti s s u e of the R. cuneata, Stokes and Awapara (1969) have postulated that malic dehydrogenase and fumarate reductase are the enzymes responsible for the oxidation of cytoplasmic NADH. They postulate that the redox p a i r NAD/NADH i s kept i n the oxidized state by the reduction of oxaloacetate and of fumarate. The reduction of oxaloacetate and dehydration of malate i s catalyzed by the enzymes located i n the cytoplasmic f r a c t i o n , but fumarate i s reduced by NADH i n the mitochondria which i n R. cuneata mantle, unlike the mammalian mitochondria, appear to be permeable to the NADH. L i t t l e work has been done on this i n other invertebrates. Phosphoenopyruvate crossroads and the statement of the problem. From the foregoing d e s c r i p t i o n i t became clear that during anaerobiosis 14 i n a f a c u l t a t i v e anaerobe l i k e oyster, glucose-6-C i s a better source of 14 succinate accumulation than pyruvate-3-C , suggesting that the pathway to succinate branches o f f before pyruvate production. The brackish water clam R. cuneata has phosphoenolpyruvate carboxykinase a c t i v i t i e s that would favour carboxylation of p-enolpyruvate to oxaloacetate rather than i t s con-version to pyruvate. It was reported that i n the oyster, Crossostrea  v i r g i n i c a , however, the s p e c i f i c a c t i v i t y of phosphoenopyruvate carboxykinase 9 was only 5% of the pyruvate kinase a c t i v i t y , which would favour the continua-t i o n of g l y c o l y s i s to the stage of pyruvate (Hammen, 1969). This very observation i s indeed the crux of our problem here, and i t i s therefore necessary to consider b r i e f l y the p-enolpyruvate crossroads. In the case of mammalian tissues which are able to produce glucose from non-carbohydrate sources, the pyruvate kinase ( p h y s i o l o g i c a l l y i r -reversible) reaction i s bypassed by the successive action of two enzymes: pyruvate carboxylase and p-enolpyruvate carboxykinase. It i s generally accepted that this p a i r of enzymes i n rat l i v e r generates the p-enolpyruvate that w i l l be further metabolized to glucose. In tis s u e s , l i k e r a t l i v e r (gluconeogenic) a l l three enzymes, e.g., pyruvate kinase, p-enolpyruvate carboxykinase and pyruvate carboxylase occur i n cytosol (Henning, Stumpf, and Ohly, 1966; Nordlie, V a r r i c c h i o and Holten, 1965) and the a c t i v i t i e s of these enzymes i f not co n t r o l l e d could set up a " f u t i l e " cycle between pyruvate and p-enolpyruvate as follows: III Glucose-6-P (I) Pyruvate kinase, (II) Pyruvate carboxylase, and (III) P-enolpyruvate carboxykinase. In addition, such " f u t i l e " cycles often would function as net ATPases, i f t h e i r operation were not con t r o l l e d . Although this mode of operation may appear wasteful, i t o f f e r s a mechanism f o r regulation of both the ATP:ADP r a t i o and the r e l a t i o n s h i p between the g l y c o l y t i c and gluconeogenic f l u x . The mechanisms which may regulate the pyruvate/p-enolpyruvate cycle are 10 less well characterized. An acute requirement f o r the i n h i b i t i o n or compart-mentation of pyruvate kinase during gluconeogenesis exists because the maximal capacity of this enzyme i n the gluconeogenic t i s s u e exceeds those of pyruvate carboxylase and p-enolpyruvate carboxykinase by an order of magnitude. In r a t l i v e r , p-enolpyruvate formation occurs i n the cyto s o l ; also ATP and alanine i n h i b i t pyruvate kinase (Tanaka, Harano, Sue, and Morimura, 1967). However, i n chicken l i v e r , where p-enolpyruvate synthesis occurs within the mitochondria, GTP and ITP are i n h i b i t o r y , while alanine and ATP have no s i g n i f i c a n t e f f e c t (Scrutton and Utter, 1968). Although a l l these e f f e c t o r s s a t i s f y the c r i t e r i o n of a r e c i p r o c a l r e l a t i o n s h i p i n t h e i r e f f e c t s on the g l y c o l y t i c and gluconeogenic segments of th i s cycle, no evidence has been presented to prevent r e c y c l i n g at t h i s locus i n metabolism. In oyster t i s s u e s , the p-enolpyruvate crossroads poses a somewhat d i f f e r e n t s i t u a t i o n . Unlike the mammalian p-enolpyruvate carboxykinase, i n oyster, i t s net f l u x i s towards the synthesis of oxaloacetate, which i s further reduced to form succinate as the g l y c o l y t i c end product. Depending upon the environmental conditions, glycogen d i s s i m i l a t i o n i n oyster i s proposed as follows: I Pyruvate C0 2 + H 20 Glucose-6-P ,j==^  P-enolpyruvate II Oxaloacetate — S u c c i n a t e At the p-enolpyruvate branch-point, e s s e n t i a l l y two a l t e r n a t i v e routes are a v a i l a b l e . ( I ) , i f 0 2 i s present, the pyruvate formed i s "fed" d i r e c t l y into the Krebs cycle i n the usual manner. However, i f 0 2 i s absent, ( I I ) , p-enol-pyruvate i s converted to oxaloacetate v i a p-enolpyruvate carboxykinase. Evidently i n such a case the problem of pyruvate/p-enolpyruvate r e c y c l i n g i s 11 absent and p-enolpyruvate i s the common substrate f o r the pyruvate kinase and p-enolpyruvate carboxykinase. I f the above data are correct, what are the mechanisms a v a i l a b l e to the organism for holding pyruvate kinase and p-enolpyruvate carboxykinase i n "on and o f f " posi t i o n s i n a r e c i p r o c a l manner so that both are not f u l l y a c t i v e simultaneously? The studies reported i n this thesis deal i n d e t a i l with t h i s s i n g l e aspect of oyster metabolism: enzymic regulation at the p-enolpyruvate cross-roads. An understanding of this s p e c i f i c control point, however, has allowed a c r i t i c a l assessment of several a d d i t i o n a l problems which are evident i n the unusual metabolism of f a c u l t a t i v e anaerobes. These include (1) the redox (NADH/NAD) p o t e n t i a l , (2) pathways of succinate production, and (3) the high energy phosphate y i e l d . The approach adopted to inves t i g a t e t h i s problem i s outlined as follows: ( i ) Select the c r i t i c a l p h y s i o l o g i c a l branch point (p-enolpyruvate crossroads) through which channelling of the terminal g l y c o l y t i c end products i s c o n t r o l l e d during a e r o b i c ^ anaerobic t r a n s i t i o n s . ( i i ) I s olate and p a r t i a l l y p u r i f y key enzymes of th i s branch point (pyruvate kinase and p-enolpyruvate carboxykinase) and inves t i g a t e the e f f e c t of pH and other metabolites on the c a t a l y t i c and regulatory properties of the two enzymes. At the outset, pH was the only documented p h y s i o l o g i c a l f a c t o r known to fl u c t u a t e during aero- and anaerobiosis (Wilbur, 1964). ( i i i ) Construct a new metabolic map which accounts (1) for redox (NADH/ NAD) balance, (2) for the pathways of succinate production, and (3) f o r the high energy phosphate y i e l d . CHAPTER I I : Materials and Methods 12 Animal c o l l e c t i o n and t h e i r handling. Oysters (CrOssostrea gigas) were c o l l e c t e d from Chuckanut Bay, Bellingham, Washington, at low t i d e s , by the kind permission of Dr. Wallace Heath of the Aquaculture Training Programme, Lummi, Washington. Animals were brought to the laboratory i n an ice-box and opened quickly to excise the ti s s u e s . A l l tissues were washed thoroughly with i c e - c o l d homogenizing medium to remove exogenous algae and other microorganisms. A l l the work reported i n t h i s thesis was c a r r i e d out on mature oysters and no attempt was made to corr e l a t e enzyme a c t i v i t i e s with sexual differences or seasonal v a r i a t i o n s . Preparation of mantle and adductor pyruvate kinases. Mantle and adductor tissues were homogenized i n a S o r v a l l Omnimixer f o r 1 to 2 min with 3 to 4 volumes of i c e - c o l d 0.01 M Tris-HCl buffer, pH 7.5, containing 2 mM EDTA. The homogenates were s t i r r e d f o r 1 hr at 4° and then centrifuged at 12,000 X g for 15 min and the p e l l e t was discarded. The supernatants were f i l t e r e d through glass wool and then brought to 40% satura-t i o n with s o l i d ammonium sulphate and s t i r r e d f o r 1 hr at 4°. The suspension were then centrifuged as above, the p e l l e t s were discarded, and the super-natant were brought to 75% saturation with s o l i d ammonium sulphate. A f t e r 1 hr with s t i r r i n g , the solutions were centrifuged at 37,000 X g f o r 20 min. The p e l l e t s were dissolved i n a minimal volume of 0.01 M Tris-HCl b u f f e r , pH 7.5. The dissolved p e l l e t s were further centrifuged at 84,000 X g for 90 min i n r e f r i g e r a t e d Beckman model L preparative u l t r a c e n t r i f u g e to remove glycogen and the high speed supernatant were used as the sources of pyruvate kinases. Portions of enzymes were dialyzed before use against 0.05 M T r i s -HCl buffer, pH 7.5. The enzyme was stable at 0-4° f o r a few days and i f frozen, was stable f o r several weeks without bringing any change i n the K 13 of the substrate p-enolpyruvate. Mantle enzyme was somewhat unstable to d i a l y s i s , showing a loss of a c t i v i t y of approximately 10% within 2 hours of d i a l y z i n g . Preparation of mitochondria. In the study of the c e l l u l a r d i s t r i b u t i o n of the enzymes, mitochondria were prepared by an adaptation of the method of Hogboom (1965). Mantle, g i l l and adductor tissues were obtained and washed i n a cold buffer medium pH 7.2 at 4° containing 0.02 M T r i s - H C l , 1 mM MgCl 2, 0.025 M sucrose. A l l subsequent operations were conducted at 0° to 4°. A f t e r grinding i n the S o r v a l l Omnimixer fo r 1 to 1.5 min, the t i s s u e was f i l t e r e d through cheese c l o t h . The crude homogenate was centrifuged i n a S o r v a l l superspeed RC 2-B centrifuge f o r 10 min at 650 X g. Following c e n t r i f u g a t i o n , the ti s s u e pulp was discarded and the supernatant was centrifuged at 7,000 X g. The p e l l e t from the preceding step was resuspended and washed i n the homogenizing medium. Aft e r several cycles of washing and c e n t r i f u g i n g , the mitochondrial p e l l e t was obtained by c e n t r i f u g a t i o n i n the homogenizing medium at 24,000 X g. The presence and pu r i t y of mitochondria i n the p e l l e t was confirmed with phase contrast micro-scopy. The suspension of mitochondria so obtained was frozen and thawed three times, then centrifuged and the supernatant was used as the source of the mitochondrial enzyme. Preparation of cytoplasmic phosphoenolpyruvate carboxykinase. As no a c t i v i t y was found i n the mitochondrial f r a c t i o n , tissues were homogenized i n a Sorval Omnimixer f o r 1 to 2 min with 4 to 5 volumes of i c e -cold 0.01 M Tris-HCl buffer, pH 7.2 containing 2 mM EDTA. The homogenate was centrifuged at 12,000 X g f o r 15 min and the p e l l e t was discarded. The 14 supernatant was adjusted to pH 5.5 with 0.1 M a c e t i c a c i d ; a f t e r centrifuga-t i o n at 37,000 X g f o r 7 min, the p r e c i p i t a t e was discarded and the super-natant was neut r a l i z e d with 0.1 M KHCO^. The neut r a l i z e d supernatant was then brought to 40% saturation with s o l i d ammonium sulphate and s t i r r e d f o r 1 hr at 4°. The suspension was then centrifuged as above, the p e l l e t was discarded, and the supernatant was brought to 75% saturation with s o l i d ammonium sulphate. Aft e r 1 hr with s t i r r i n g , the suspension was centrifuged at 37,000 X g for 15 min. The p e l l e t was dissolved i n a minimal volume of 0.01 M Tris-HCl buffer, pH 7.2. The dissolved p e l l e t was further centrifuged at 41,300 X g for 15 min and the high speed supernatant was used as the source for phosphoenol-pyruvate carboxykinase (PEPCK). Portions of enzyme were dialyzed before use against cold 0.05 M Tris-HCl buffer, pH 7.2. The enzyme was stable at 0° to 4° for a few days and i f frozen was stable f o r several weeks. Above prepara-t i o n was also used as the source of malate dehydrogenase and "malic enzyme". Assay of pyruvate kinase a c t i v i t y . Pyruvate kinase was assayed by the method of BUcher and P f l e i d e r (1959). Pyruvate formation was coupled to l a c t a t e dehydrogenase and the rate of pyruvate kinase a c t i v i t y was measured as the decrease i n E ^ Q due to NADH. Tris-HCl buffers were used i n a l l assay reactions. Standard assay mixtures contained the following i n a f i n a l volume of 2 ml: 50 mM Tris-HCl buffer, Mg , K , ADP, p-enolpyruvate, NADH and excess of dialyzed Sigma l a c t a t e dehydrogenase at concentrations s p e c i f i e d i n the f i g u r e legends. Saturating concentrations f o r each of the reactants for both mantle and adductor enzymes were 6 mM Mg"1-1", 50 mM K +, 0.2 mM ADP, 1.4 mM p-enolpyruvate, 5 X 1 0 _ 5 M fructose-1,6-P 2, 5 mM ATP, 3 mM L-alanine (adductor pyruvate kinase), 8 mM L-alanine (mantle pyruvate kinase), and 10 mM phenylalanine. A l l reactions 15 were started by the addition of pyruvate kinase preparation. A l l experiments were performed at 20° since K , , , was found temperature independ-c m(p-enolpyruvate) ent over a temperature range of 5-30°. Assay of the phosphoenolpyruvate carboxykinase (carboxylation reaction) a c t i v i t y . The enzyme was assayed i n the d i r e c t i o n of oxaloactate synthesis by the method of Utter and Kurahashi (1954), Oxaloacetate formation was coupled to excess quantities of dialyzed malate dehydrogenase, and the rate of p-enolpyru-vate carboxykinase a c t i v i t y was followed as the decrease i n E^Q due to NADH. Sodium acetate or Tris-Maleate buffers were used i n a l l assay reactions to cover a wide pH range. Standard assay mixture contained the following i n a I | | | f i n a l volume of 1 ml: 50 mM Tris-Maleate b u f f e r , Mn , or Zn , Inosine diphosphate (IDP), p-enolpyruvate, NaHCO^, NADH and an excess of dialyzed Sigma pig heart malate dehydrogenase at concentrations s p e c i f i e d i n the fi g u r e legends. Saturating concentrations for each of the reactants f o r the p-enolpyruvate carboxykinase reaction were: 10 mM NaHCO^, 1 mM Mn , or Zn , 1 mM IDP, and 1.4 mM p-enolpyruvate. A l l reactions were i n i t i a t e d by the addition of the p-enolpyruvate carboxykinase preparation. A l l experiments were performed at 25°. Assay of phosphoenolpyruvate carboxykinase (decarboxylation reaction) a c t i v i t y . Decarboxylation assay (synthesis of p-enolpyruvate) was measured by the method described by Nordlie and Lardy (1963) with s l i g h t modifications. P-enolpyru-vate formed as a r e s u l t of the decarboxylation reaction was measured enzymically by the method of BUcher and P f l e i d e r e r (1959). The assay medium contained i n a f i n a l volume of 1 ml: 0.05 mM Tris-Maleate buffer, 120 uM oxaloacetate, 1 mM Mn ,20 uM Inosine triphosphate (ITP), 16 KF. This mixture was preincubated f o r 5 min at 25°, a f t e r which p-enolpyruvate carboxykinase was added. The reaction was stopped by adding approximately 1 mg of potassium borohydrate (KBH^). The reaction tubes were kept i n an i c e bath and 0.2 ml of i c e - c o l d 6% HCIO^ was added. Denatured protein was removed by ce n t r i f u g a t i o n and supernatant was ne u t r a l i z e d by the addition of 3 M T r i s . The volume of the neut r a l i z e d supernatant was brought to 2 ml by adding of 0.05 mM Tris-Maleate buffer pH 7.6, 50 mM K +, 1 mM ADP, 1 mM Mn"1-*", 0.15 mM NADH and excess l a c t a t e dehydrogenase. The reaction was i n i t i a t e d by the addition of constant amount of Sigma rabbit pyruvate kinase. The p-enolpyru-vate concentration can be calculated from the decrease i n E„ / r. a f t e r the 340 addition of pyruvate kinase. I | | [ Decarboxylation reaction was assayed between pH 5.1 and 8.2, Mn , Zn I j or Mg . A l l the components of the decarboxylation assay were tested by deleting or adding, by increasing or decreasing the concentrations of the reactants at a l l pH values. The e f f e c t of acetylCoA was also tested. In no instance, could s i g n i f i c a n t p-enolpyruvate synthesis be detected with the assay system. Low and v a r i a b l e a c t i v i t y was detected by measuring P^ released from p-enolpyruvate by the method of Seubert and Huth (1965). Further studies are required to assess the properties of the decarboxylation reacti o n . Assay of the "malic enzyme" a c t i v i t y . "Malic enzyme" a c t i v i t y was measured by the method of Ochoa- (1965). Rate of enzyme a c t i v i t y was measured by the increase or decrease i n absorbance at due to the reduction of NADP or oxidation of NADPH. Standard assay mixture contained the following i n a f i n a l volume of 1 ml: 50 mM Tris-Maleate I | buffer, pH 8.2, 1 mM Mn , 1 mM malic acid and 0.15 mM NADP. A l l reactions were i n i t i a t e d by the addition of enzyme preparation. CHAPTER I I I : C a t a l y t i c and Regulatory Properties of Oyster Pyruvate Kinases 17 Assay of malate and l a c t a t e dehydrogenases . Malate and l a c t a t e dehydrogenase a c t i v i t i e s were measured by the methods described by Ochoa ( 1 9 6 5 ) and Romberg ( 1 9 6 5 ) . Reduction of oxaloacetate and pyruvate was measured i n a medium containing 50 mM Tris-HCl buffer, pH 7.5, 1 mM of eith e r substrate, 0.15 mM NADH and enzyme preparation. The a c t i v i t y was determined from the decrease i n e x t i n c t i o n at E ^ Q (*see page 18) . Estimates of pro t e i n content. The p r o t e i n content of a l l enzyme preparations wherever required were determined by the method of Lowry, Rosebrough, Farr and Randall ( 1 9 5 1 ) . A l l samples were compared with a standard curve determined from 0 to 100 ugm/ml bovine serum albumen prot e i n . Electrophoresis. Mantle, g i l l , and adductor muscle pyruvate kinases were prepared and studied e l e c t r o p h o r e t i c a l l y to determine whether t i s s u e - s p e c i f i c forms are present i n the oyster. Electrophoresis was c a r r i e d out on c e l l u l o s e polyace-tate s t r i p s at a p o t e n t i a l gradient of 17 v/cm f o r 2 hr at 4 ° i n buffer containing 0.5 M sucrose 0.01 M Tr i s - H C l , pH 7.5 and 0.001 M f r u c t o s e - l , 6 - P 2 . Samples were applied to the center of the s t r i p . Pyruvate kinase a c t i v i t y was detected by coupling with the l a c t i c dehydrogenase system. The region producing NADH oxidation was v i s u a l l y observed by the loss of fluorescence. I t was recorded by exposing the s t r i p s with 340 mu l i g h t . The c e l l u l o s e acetate s t r i p s to be assayed were placed on an agar f i l m containing 8 mg/ml Nobel agar, 50 mM Tr i s - H C l , pH 7.5, 2 mM EDTA, 20 mM Mg44", 60 mM X +, 2 mM p-enolpyruvate, 1 mM ADP, 1 mM NADH, 0.2 mM fructose-l,6-P„ and excess sigma dialyzed LDH. Development was allowed to 18 proceed f o r 1 to 3 min at room temperature. Pyruvate kinase a c t i v i t y was recorded by contact p r i n t i n g on Kodak photographic enlarging paper using a 340 mu l i g h t source. Areas of enzymatic a c t i v i t y (low NADH) transmit the incident l i g h t and expose the photographic paper while other areas (high NADH) do not. These experiments were performed i n co l l a b o r a t i o n with Dr. Walter Susor of C a l i f o r n i a Medical Center, San Francisco, C a l i f o r n i a . Electrofocusing. Technique of electrofocusing was used to determine whether isozymes having d i f f e r e n t p i values were present. Electrofocusing experiments were performed by the method of Haglund (1967). For pyruvate kinase both mantle and adductor enzymes were run at pH 5 to 8 gradient (LKB-8133) at 900 v o l t s for 53 hours. For phosphoenolpyruvate carboxykinase, a pH 3 to 10 gradient (LKB-8141) was used at a p o t e n t i a l of 300 v o l t s . The temperature of the apparatus was maintained at 4° for the e n t i r e period of the experiments. Small f r a c t i o n s of 15 drops each were c o l l e c t e d . For pyruvate kinase a c t i v i t y a l l f r a c t i o n s were assayed i n presence of 2.5 X 10 6 fructose-1,6-P 2 at pH 8.5. Phosphoenolpyruvate carboxykinase a c t i v i t y was measured i n the presence of I | Zn at pH value 5.1. *A11 the spectrophotometric assays were repeated 2 to 3 times; change i n O.D. was reproducible between 90-100% i n a l l cases. K measurements of d i f f e r e n t r m metabolites f o r one enzyme preparation i f repeated were reproducible but varied between 3-5% when compared with d i f f e r e n t enzyme preparations. 19 INTRODUCTION In theory pyruvate occupies a ce n t r a l crossroads p o s i t i o n i n energy metabolism since i t may be metabolized by a number of d i f f e r e n t pathways. At lea s t f i v e such pathways are present i n oyster tissues (Hammen, 1969). In oyster adductor muscle the major source of pyruvate i s storage glycogen. According to Hammen (1969), the pyruvate, which i s produced i n high quantities during periods of anaerobiosis, can have several metabolic f a t e s , the most important one being conversion to malic acid. The l a t t e r i s r e a d i l y metabol-ized to succinate, which i s a major end product of anaerobic metabolism i n oyster. Lactate i n contrast does not accumulate (Hammen, 1969). Under aerobic conditions pyruvate can be oxidized by the usual Krebs cycle reactions. In mantle, on the other hand, the pyruvate branching point i s more complex and the degree to which these pathways operate d i f f e r s from that i n the adductor 14 muscle. During anaerobiosis glucose-6-C i s a better source of succinate 14 than i s pyruvate-3-C , suggesting that the pathway to succinate branches o f f before pyruvate production. Simpson and Awapara (1966) suggest that carboxy-l a t i o n of p-enolpyruvate by phosphoenolpyruvate carboxykinase i s the major mechanism leading to succinate accumulation. Also, i n contrast to adductor muscle, mantle i s a major gluconeogenic t i s s u e . Under gluconeogenic conditions, pyruvate and p-enolpyruvate may be produced from non-carbohydrate sources as wel l as from Krebs cycle intermediates. The pyruvate kinase step, which i n most organisms i s p h y s i o l o g i c a l l y i r r e v e r s i b l e (Scrutton and Utter, 1968) may be bypassed during glucose (or glycogen) synthesis by the p-enolpyruvate carboxykinase which generate p-enolpyruvate f o r further metabolism to glucose. In mantle of most molluscs examined, however, pyruvate kinase a c t i v i t y apparently exceeds p-enolpyruvate carboxykinase by a fac t o r of 10 (Hammen, 1969). Hence i f the data are correct there are s p e c i a l requirements f o r holding pyruvate kinase i n a "shut o f f " conformation i n t h i s t i s s u e . From the above consideration i t i s evident that the requirements f o r the regulation of pyruvate kinase reaction i n adductor and mantle tissues are quite d i s t i n c t from each other. One obvious way of meeting these d i f f e r e n t requirements i s the elaboration of ti s s u e s p e c i f i c variants of the enzyme. Therefore t h i s study was i n i t i a t e d by examining mantle and adductor tissues for d i f f e r e n t e l e ctrophoretic forms of pyruvate kinase. 21 RESULTS Electrophoresis and electrofocusing. Electrophoretic r e s o l u t i o n of pyruvate kinase a c t i v i t y i n three d i f f e r e n t tissues i s shown i n F i g . 1. Mantle pyruvate kinase a c t i v i t y appears as a s i n g l e band, moving towards the cathode, pyruvate kinase i n the g i l l t i s s u e displays a s i m i l a r electrophoretic m o b i l i t y . Adductor pyruvate kinase, showing a d i f f e r e n t pattern, moves as a "doublet" towards the cathode. The ele c t r o p h o r e t i c differences were confirmed by electrofocusing experiments (F i g . 2). Mantle pyruvate kinase appears as a s i n g l e major a c t i v i t y peak with a p i value of 6.35. In contrast, the adductor pyruvate kinase appears as two d i s t i n c t peaks having p i values of 5.6 and 6.5. Further i t was noted that mantle pyruvate kinase i s more unstable than the adductor pyruvate kinase. It should be mentioned here that i n a l l of the k i n e t i c experiments performed on adductor pyruvate kinase the two components were not separated because the tissue i s small and i t i s d i f f i c u l t to obtain large quantities of material. Cation requirements. In common with pyruvate kinases from other species, oyster pyruvate j | kinases show absolute requirements f o r divalent and monovalent cations. Mg can s a t i s f y the former requirement (Ka values at pH 8.5 are 1.25 and 2.27 mM for mantle and adductor pyruvate kinases respectively) and K + or NH^+ can s a t i s f y the l a t t e r requirement (Ka f o r K + at pH 8.5 i s 7.7 mM f o r both enzyme systems). High K + concentrations i n h i b i t e d the oyster enzymes, i n contrast to pyruvate kinases from other sources (Carminatti et a l . , 1968). Cu at concentration of up to 10 mM do not a f f e c t these enzymes. 22 F i g . I l l , 1. Electrophoretic r e s o l u t i o n of oyster mantle, g i l l , and adductor pyruvate kinase a c t i v i t y . Procedure f o r electrophoresis and a c t i v i t y s t a i n i n g are according to Susor and Rutter (1968). Electrophoresis was c a r r i e d out on c e l l u l o s e polyacetate s t r i p s at a p o t e n t i a l gradient of 17 v o l t s per cm for 3 hours at 4° i n a buffer containing 0.5 M sucrose, 0.01 M Tris-HCl (pH 7.5 at 4°), and 0.001 M f r u c t o s e - l ^ - P ^ . Samples are applied to the center of the s t r i p s . MANTLE GILL ADDUCTOR 23 F i g . I l l , 2. Electrofocusing pattern of the mantle and adductor pyruvate kinases. For experimental procedures see "Materials and Methods". A c t i v i t y i s p l o t t e d against f r a c t i o n numbers; the numerical values plotted on the abscissa i n d i c a t e the pH of selected f r a c t i o n s . Reactant concentrations are the same as described under F i g . 3. Activity ( A E 3 4 0 /unit time) 24 E f f e c t of f r u c t o s e - 1 , o n the pH optima. As shown i n F i g . 3 i n the absence of fructose-1,6-P 2 both forms show a pH optima at pH 8.5 but the shape of the a c t i v i t y curves d i f f e r f o r both, with adductor pyruvate kinase being more s e n s i t i v e to pH changes. In the presence of fructose-1,6-P 2, the a c t i v i t y of the mantle enzyme i s approximately constant between pH 7.5 and 9, while the pH optimum f o r the adductor enzyme i s displaced towards pH 7. Ef f e c t of fructose-1,6-P 2 on ADP saturation curves. The e f f e c t of increasing ADP concentration at a f i x e d p-enolpyruvate concentration on the a c t i v i t y of 2 enzymes i n the absence and presence of fructose-1,6-P 2 at pH 8.5 are given i n F i g . 4 (upper panel) along with double -4 r e c i p r o c a l p l o t s (lower panel). Both enzymes require 2 X 10 M ADP for -4 maximal a c t i v i t y at 1 X 10 M p-enolpyruvate. For both enzymes the curves f o r the reaction v e l o c i t y against ADP i n the presence and absence of fructose-1, 6-P„ have the Michaelis-Menton form. For mantle pyruvate kinase the K / . ^ v 2 m(ADP) -4 i n the absence of f r u c t o s e - l , 6 - P 2 i s 6.1 X 10 M, while fructose-1,6-P 2 increases the K / A T , „ s at l e a s t by 6 f o l d . In contrast to t h i s , fructose-1, m(ADP) 6-P2 has no e f f e c t on the K m ^ ^ r j p ) ° f adductor pyruvate kinase. E f f e c t of pH and fructose-1,6»P'2 on the of_ p-enolpyruvate. P-enolpyruvate saturation curves at d i f f e r e n t pH values i n the presence and absence of fructose-1,6-P 2 are shown i n F i g . 5, 6 ( l e f t panel), double r e c i p r o c a l p l o t s (right panel). For mantle enzyme ( F i g . 5, middle panel), the minimum K i n the absence of fructose-1,6-P 0 appears at pH 8.5 and the K m 2 ^ r m increases markedly below pH 8.5 being maximum at pH 6.5. In the presence of fructose-1,6-P 2 the for the mantle enzyme i s markedly lowered and remains 25 F i g . I l l , 3. E f f e c t of fructose-l,6-P2 on the pH optima no fru c t o s e - l , 6 - P 2 ; • , A , 2.5 X 10~ 6 M fructose-1,6-P 2). Reaction contents were: 50 mM Tris-HCl buffer at d i f f e r e n t pH values, 6 mM Mg**, 50 mM K +, 0.2 mM ADP, 0.5 mM p-enopyruvate, 0.15 mM NADH and excess of la c t a t e dehydrogenase. PyK, pyruvate kinase. 26 F i g . I l l , 4. E f f e c t of fructose-1,6-P„ on the K of ADP of mantle & ' 2 m and adductor pyruvate kinases. Reactant contents were: 50 mM Tris-HCl buffer, pH 8.5, 6 mM Mg"1"*", 50 mM K +, 0.1 mM p-enolpyruvate, increasing concentrations of ADP, 0.15 mM NADH and excess l a c t a t e dehydrogenase; • , mantle pyruvate kinase; A, adductor pyruvate — 6 kinase; • , A , with 2.5 X 10 M fructose-1,6-P,. 27 F i g . I l l , 5. E f f e c t of pH and fructose-1,6-?^ on the K m of p-enolpyru-vate of mantle pyruvate kinase. Reactant contents were: 50 mM Tris-HCl, 6 mM Mg**, 50 mM K +, 0.2 mM ADP, 0.15 mM NADH, increasing concentrations of p-enolpyruvate and excess l a c t a t e dehydrogenase. 2 7 <*-28 constant over a pH range of 6.5 to 8.5. F i g . 6 shows the same kind of response for the adductor pyruvate kinase. These r e s u l t s summarized i n Table 1, are i n contrast to those reported by Rozengurt eit al_. (1969) f o r mouse l i v e r pyruvate kinase. Rozengurt demonstrated that as the pH of the assay medium i s lowered, s u s c e p t i b i l i t y to fructose-1,6-P 2 a c t i v a t i o n correspondingly decreases. In case of oyster pyruvate kinases both enzymes are much more susceptible to fructose-1,6-P^ a c t i v a t i o n at a c i d i c pH than at a l k a l i n e pH. Adenosine-triphosphate (ATP) i n h i b i t i o n . Like pyruvate kinases from other sources (Rozengurt et a l . , 1969; and Tanaka et al., 1967) ATP also i n h i b i t s both forms of the oyster enzymes. In F i g . 7 ( l e f t panel) the e f f e c t of ATP at d i f f e r e n t pH values and p-enolpyruvate concentrations i s shown f o r both enzymes, along with Dixon p l o t of the data (r i g h t panel). While ATP i n h i b i t s enzymes at both pH values examined, several pH differences can be noted. Using 1 mM p-enolpyruvate at pH 8.5 both enzymes are less i n h i b i t e d than at pH 7.5. The K\ values for the mantle enzymes are 4 mM and 2.65 mM at pH 8.5 and 7.5 r e s p e c t i v e l y . The adductor enzyme shows a s i m i l a r behaviour, although the K\ values for the adductor pyruvate kinase are lower, about 2.8 mM and 1.9 mM at pH 8.5 and 7.5 r e s p e c t i v e l y . The nature of i n h i b i t i o n i s also d i f f e r e n t . Double r e c i p r o c a l p l o t s of the v e l o c i t y of the pyruvate kinase reaction at d i f f e r e n t ATP concentrations (Fi g . 8-A) i n d i c a t e that the ATP i n h i b i t i o n of the mantle enzyme i s non-competitive with respect to p-enolpyruvate. Thus f o r mantle enzyme ATP decreases the calculated Vmax, but does not a f f e c t the K ., , _ s . m(p-enolpyruvate) This non-competitive i n h i b i t i o n of mantle pyruvate kinase i s r e l a t i v e l y unique having been reported only once previously f o r the mouse brain enzyme by Lowry and Passonneau (1964). Since p-enolpyruvate i n mantle i s not l i k e l y to reach Table 1 E f f e c t of pH on K , , i n absence and presence of f ructose-1,6-P 0.. Values m(p-enolpyruvate) v ' 2 determined from F i g . I l l , 5 and 6. pH 6.5 pH 7.5 pH 8.5 Enzyme form No FDP + FDP No FDP + FDP No FDP + FDP Mantle PyK 5.8 X 10 4M 8 X 10~5M 4.5 X 10 _ 4M 6.6 X 10 5M 1.9 X 10 4M 6.6 X 10~5M Adductor PyK 6.6 X 10 4M 7.4 X 10 5M 2.5 X 10~4M 7.4 X 10 5M 9.0 X 10 5M 7.7 X 10 5M 30 F i g . I l l , 6. E f f e c t of pH and fructose-1,6-P„ on the K of ° 2 m p-enolpyruvate of adductor pyruvate kinase. Reactant contents were the same as described under F i g . 5. 30 <*-31 \ F i g . I l l , 7. E f f e c t of ATP concentration on the reaction rate of mantle and adductor pyruvate kinases at d i f f e r e n t pH and p-enolpyruvate concentrations. Reactant contents were: 50 mM Tr i s - H C l , 6 mM Mg**, 50 mM K +, 0.2 mM ADP, p-enolpyruvate concentrations as indicated, 0.15 mM NADH, increasing concentration of ATP and excess l a c t a t e -4 dehydrogenase. (A) Mantle pyruvate kinase, ( A ) , 5 X 10 , _ 3 p-enolpyruvate, pH 8.5; (•-), 1..X 10 , p-enolpyruvate, pH 7.5; ( A ) , I X 10~ 3, p-enolpyruvate, pH 8.5; (B) adductor pyruvate kinase; ( 0 , A ) 5 X 10 \ _3 p-enolpyruvate, pH 7.5 and 8.5; ( # ) I X 10 , p-enolpyru-_ 3 vate, pH 7.5; CD) 1 X 10 , p-enolpyruvate, pH 8.5. Activity ( A E 3 4 0 / u n i t time) Activity ( A E 3 4 0 / u n i t time) > 001-32 saturating concentrations, these r e s u l t s suggest that ATP i s not an important modulator of mantle enzyme. In contrast, adductor enzyme ( F i g . 8-B) shows competitive i n h i b i t i o n and i s i n h i b i t e d by lower concentration of ATP. Thus, 2 mM ATP increases the K , , ^ . at l e a s t 10 f o l d with l i t t l e or no m(p-enolpyruvate) e f f e c t on the Vmax. Further, i t was noted that the nature of i n h i b i t i o n of I | e i t h e r enzyme could not be altered by increasing or decreasing the Mg con-centration of the assay medium, although the degree of i n h i b i t i o n c l e a r l y depends upon the amount of the Mg present. Interacting e f f e c t s of ATP and fructose-1,6-P Q • Rozengurt et a l . (1969) and Tanaka et a l . (1967) have reported that ATP i n h i b i t i o n of l i v e r pyruvate kinase i s reversed by fructose-1,6-P 2» Studies of this k i n e t i c property with the oyster mantle enzyme at two d i f f e r e n t pH values at varying concentrations of p-enolpyruvate, i n d i c a t e a behaviour s i m i l a r to that of mouse l i v e r pyruvate kinase. Figures 9 and 10 show plo t s of p-enolpyruvate stauration of the mantle enzyme i n the presence of fructose-1,6-P2 and ATP. P-enolpyruvate saturation curves c l e a r l y show that fructose-1, 6-P2 overcomes the ATP i n h i b i t i o n . At pH 8.5 (Fig. 9) 10~ 6 M fructose-1,6-P 2 releases the ATP i n h i b i t i o n (caused by 3 mM ATP) by lowering the K m down to 2/3 of control and increases the Vmax by 2 f o l d ; thus, fructose not only releases the ATP i n h i b i t i o n but overrides i t . At pH 7.5 ( F i g . 10) under s i m i l a r conditions fructose-1,6-P„ lowers the K down to 1/5 of control and z m again doubles the Vmax. In presence of ATP and fructose-1,6-P 2 together the K , -, s i s s i m i l a r at both pH values. These r e s u l t s are b r i e f l y m(p-enolpyruvate) summarized i n Table 2. Table 2 E f f e c t of ATP and fructose-1,6-P 0 on the K , , v mantle pyruvate 2 m(p-enolpyruvate) kinase value determined from F i g . I l l , 9 and 10. pH Control 3 mM ATP 1 pM FDP 3 mM ATP and 1 uM FDP pH 8.5 1.8 X 10 4M 1.8 X 10 4M 1 X 10 4M 1.3 X 10 4M pH 7.5 5 X 10 4M 5 X 10 4M 1.05'X 10 4M 1.3 X 10~4M 34 F i g . I l l , 8. Double r e c i p r o c a l p l o t s of the reaction v e l o c i t y of mantle pyruvate kinase (A) (O, c o n t r o l ; A , 1 mM ATP;#, 2 mM ATP; • , 3 mM ATP; and adductor pyruvate kinase (B) (O, c o n t r o l ; A , 0.5 mM ATP; • , ImM ATP; • , 2 mM ATP) i n presence of d i f f e r e n t ATP concentrations as indicated at pH 8.5. Reactant contents were the same as described under F i g . 5. 3 ^ / [ P E P ] x 1 0 3 M /[pEP]x 1 0 3 M 35 F i g . I l l , 9. Interacting e f f e c t s of ATP and fructose-1,6-P^ on the K / , x f o r the mantle enzyme at pH 8.5. m(p-enolpyruvate) J r Reactant contents were the same as described under F i g . 5, but 3 mM ATP and IX 10~ 6 M fructose-1,6-P 2 were added where indicated. FDP, f r u c t o s e - l , 6 - P 2 ; PyK, pyruvate kinase; PEP, p-enolpyruvate. 35^ »50r ' / [ P E P J x I o ' M 36 F i g . I l l , 10. Interacting e f f e c t s of ATP and fructose-1,6-P 2 on the K / , v for the mantle enzyme at pH 7.5. m(p-enolpyruvate) J v Experimental conditions were the same as described under F i g . 9. 36 «*-37 F i g . I l l , 11. E f f e c t of alanine concentration on the reaction rate and alanine, , determination for (A) adductor, and i (B) mantle pyruvate kinases at two d i f f e r e n t pH values. Reaction contents were: 50 mM Tris-HCl ( A ) , pH 7.5 and ( O ) 8.5 as indicated, 6 mM Mg4"*", 50 mM K +, 0.2 mM ADP, 0.5 mM p-enolpyruvate, 0.15 mM NADH and excess l a c t a t e dehydrogenase. Concentration of alanine was varied as indicated. Activity ( A E 3 4 0 / u n i t time) Activity ( A E 3 4 0 / 5 L t 38 F i g . I l l , 12. E f f e c t of phenylalanine on the reaction rate and phenylalanine, . determination f o r mantle and adductor i pyruvate kinases at two d i f f e r e n t pH values. Experi-mental conditions were the same as described under F i g . 11, but concentrations of phenylalanine were varied as indicated. Mantle pyruvate, ( • ) pH 7.5, (O) pH 8.5 ; '' adductor pyruvate kinase, (A) pH 7.5,-( •) pH 8.5. 39 Search f o r other modulators. Since pyruvate occupies a ce n t r a l crossroads i n oyster tis s u e metabolism, we f e l t i t necessary to study the e f f e c t s of other metabolites on pyruvate kinase a c t i v i t i e s . Of the various compounds tested, 5' AMP, acetylCoA, c i t r a t e , succinate, malate and oxaloacetate have neither stimulatory nor i n h i b i t o r y e f f e c t s on the oyster enzymes. Only L-alanine and phenylalanine were found to a f f e c t the enzyme i n i n h i b i t o r y manner. -4 Using 5 X 10 M p-enopyruvate, both oyster enzymes were found more susceptible to alanine i n h i b i t i o n at pH 7.5 than at 8.5 (Fig. 11). For mantle enzyme, the R\ values f o r alanine i n h i b i t i o n are 7.6 mM and 2.7 mM at pH 8.5 and 7.5 re s p e c t i v e l y . The adductor enzyme i s i n h i b i t e d at lower concentration of alanine (K values 3.6 and 0.6 mM r e s p e c t i v e l y ) . Figure 12 shows mantle and adductor pyruvate kinase a c t i v i t i e s as a function of increasing concen-t r a t i o n of phenylalanine. Phenylalanine i n h i b i t i o n of mantle pyruvate kinase i s pH independent having a R\ value around 6 mM at both pH values examined; again the K\ values f o r the adductor enzyme are somewhat lower. Nature of L-alanine and phenylalanine i n h i b i t i o n . L-alanine i s known to i n h i b i t pyruvate kinase (Weber et al., 1968; Seubert et a l . , 1968; Rozengurt et^ al., 1970) i n a manner competitive with respect to p-enolpyruvate. In contrast, i n the case of oyster pyruvate kinase, alanine i n h i b i t s both enzymes i n a mixed competitive manner with respect to p-enolpyruvate at both pH values examined. As i n the case of ATP i n h i b i t i o n , fructose-1,6-P 2 reverses alanine i n h i b i t i o n . For mantle enzyme, with 6 mM alanine at pH 8.5 ( F i g . 13, upper panel) fructose-1,6-P 2 completely overcomes alanine i n h i b i t i o n . At pH 7.5 under s i m i l a r conditions ( F i g . 13, lower panel) the a c t i v i t y returns to 70% of the c o n t r o l . The nature of phenylalanine d i f -40 F i g . I l l , 13. Double r e c i p r o c a l plots of the reaction v e l o c i t y of mantle pyruvate kinase i n presence of alanine at pH 8.5 and 7.5. Reactant contents were the same as described under F i g . 5, but d i f f e r e n t concentrations of alanine — fi and fructose-1,6-P 2 (2.5 X 10 M) were added: • , 6 mM alanine; A , 4 mM alanine; O , no alanine and fructose-1, 6-P2; I , 6 IDM alanine + fructose-1,6-P 2; • , fructose-1, 6-P„ only. 41 fe r s f o r the 2 enzymes being competitive f o r the mantle enzyme ( F i g . 14, upper panel) and mixed-competitive f o r the adductor enzyme ( F i g . 14, lower panel). For the mantle enzyme, 6 to 10 mM phenylalanine doubles the K , v m(p-enolpyruvate) while Vmax remains almost unaffected. Again fructose-1,6 - P 2 protects the — 6 mantle enzyme against phenylalanine i n h i b i t i o n ; 2.5 X 10 M fructose-1,6-P 2 reverses the i n h i b i t i o n caused by 6 mM phenylalanine. 42 Fig. I l l , 14. Double reciprocal plots of the reaction velocity of mantle and adductor pyruvate kinases in presence of phenylalanine and fructose-1,6-P 2 at pH 8.5. Reactant contents were the same as described under Fig. 5, but different con-centrations of phenylalanine and fructose-1,6-P 2 were added: • , 10 mM phenylalanine; O > control;B , 6 mM —6 phenylalanine + 2.5 X 10~ M fructose-1,6-P 2; © , 2.5 X —6 10 M fructose-1,6-P 2 only; • , • , 6 mM phenylalanine; A , A , 3mM phenylalanine. \2<L V[PEP] 1 0 3 M DISCUSSION A comparison of the properties of mantle, adductor, r a t muscle and l i v e r pyruvate kinases i s given i n Table I I I . The data i n t h i s study suggest that, as i n the mammalian case, the enzyme pyruvate kinase i n the oyster occur i n tis s u e s p e c i f i c multimolecular forms and that the k i n e t i c properties of each isozyme seem to gear well with the o v e r a l l metabolism of the t i s s u e i n which i t occurs. Thus, the K m values of p-enolpyruvate and ADP f o r mantle pyruvate kinase are 3 and 6 times higher than the corresponding values f o r the adductor pyruvate kinase. Under conditions of gluconeogenesis, when p-enolpyruvate i s being produced from pyruvate and C-4 acids of the Krebs cycle, any s i g n i f i c a n t simultaneous pyruvate kinase a c t i v i t y would serve merely to re c y c l e pyruvate at the expense of ATP (Sols, 1968). In mantle, t h i s kind of r e c y c l i n g would not be favoured at low p-enolpyruvate concentrations because of the high Michaelis constant for the mantle pyruvate kinase. Also, the mantle enzyme i s strongly activated by fructose-1,6-P 2 (causing a large decrease i n K / ., x ) . This may r e f l e c t a p h y s i o l o g i c a l mechanism whereby m(p-enolpyruvate) J r J ° J pyruvate kinase a c t i v i t y can be increased during g l y c o l y s i s and markedly decreased during gluconeogenesis, when fructose-1,6-P 2 concentration may be reduced. An e n t i r e l y analogous s i t u a t i o n occurs i n mammalian t i s s u e s . Thus, i n l i v e r , a major gluconeogenic t i s s u e , the K , .. * for pyruvate ' J ° ° m(p-enolpyruvate) kinase i s an order of magnitude higher than i n the highly g l y c o l y t i c muscle (Sols, 1968; and Reynard et a l l , 1961), and indeed i s comparable to the K , , . f o r the mantle enzyme. In t h i s circumstance, too, p-enol-m(p-enolpyruvate) pyruvate conversion to pyruvate would not be favoured i n the gluconeogenic tis s u e when p-enolpyruvate concentrations are low. Also i n t h i s case, fructose-1,6-P 2 may act as a s p e c i f i c "on-off" switch on the l i v e r enzyme, but i t does not a f f e c t the mammalian muscle enzyme. Thus both l i v e r and mantle 44 Table III Comparison of the properties of mantle, adductor, r a t muscle and l i v e r pyruvate kinases. Oyster Rat Mantle PK Adductor PK Muscle PK Liver PK m (p-enolpyruvate) 2.4 X 10"*M 8 X 10"5M 5 X 10~5M 7 X 10"4M Km(ADP) 6.1 X 10"4M 1.0 X 10~4M 2.1 X lO 'St 2.1 X 10"4M K Potassium 7.7 X 10"3M 7.7 X 10"4M 12.0 X 10"3M 10 X 10"3M a doesn't i n h i b i t doesn't i n h i b i t i n h i b i t s at i n h i b i t s at at high cone. at high cone. high cone. high cone. of K +. of K+. of K+. of K +. K a Magnesium 1.25 X 10"3M 2.2 X 10"3M Electrophoretic slow cathodally slow cathodal- f a s t anodally fast anodally migration moving. l y moving. moving. moving. I s o e l e c t r i c points 6.35 5.6, 6.5 Kinetics Michaelis-Menton Michaelis-Menton Michaelis-Menton Sigmoidal Fructose-l,6-P 2. feedforward feedforward no known feedforward ef f e c t s a c t i v a t o r . a c t i v a t o r . effects.. e f f e c t o r . ATP I n h i b i t i o n i . Nature of i n - non-competitive competitive competitive competitive h i b i t i o n i i . values 4.0 X 10"3M 2.8 X 10"3M 1.7 X 10"2M 5.6 X 10"3M L-alanine i n h i b i t i o n i . Nature of i n h i b i t i o n i i . K^ values Phenylalanine i n h i b i t i o n i . Nature of i n h i b i t i o n i i . K^ values mixed-comp e t i t ive type. protected by FDP. 2.7 X 10"3M competitive type. protected by FDP. 5.8 X 10"3M mixed-competi-t i v e type. competitive type. protected by FDP. no e f f e c t . 2.8 X 10 M 2 X 10 M mixed-competi- no known tiv e type. e f f e c t , protected by FDP. " 3.8 X 10"3M competitive type. protected by FDP. _ 2.5 X 10" M no known e f f e c t . 45 pyruvate kinases seem well adapted f o r function i n a metabolism that involves g l y c o l y t i c and gluconeogenic function within a s i n g l e t i s s u e . The Michaelis constants f o r p-enolpyruvate and ADP for pyruvate kinases of adductor muscle, f i s h muscle, and mammalian muscle are rather s i m i l a r to each other, and as pointed out, are d i s t i n c t l y lower than the mantle and the l i v e r enzymes. Thus, these enzymes would compete favourably f o r quite low concentrations of p-enolpyruvate for conversion to pyruvate. The adductor muscle pyruvate kinase d i f f e r from the mammalian muscle pyruvate kinases, however, i n being strongly feedforward activated by f r u c t o s e - 1 , T h i s has been observed for f i s h muscle as w e l l , and may be a general c h a r a c t e r i s t i c of muscle pyruvate kinase i n poikilothermic organisms (Somero and Hochachka, 1968). The r o l e of fructose-1,6-P 2 protection of both mantle and adductor pyru-vate kinases i s of i n t e r e s t . In a l l cases thus f a r examined, fructose-1,6-P 2 i s able to reverse ATP i n h i b i t i o n of pyruvate kinase. In addition, i n the oyster, fructose-1,6-P 2 protects both enzyme forms against alanine and phenylalanine i n h i b i t i o n . Thus f a r no adequate explanation i s a v a i l a b l e f or these e f f e c t s and t h i s i s c l e a r l y an important area for further research. In mammalian systems, fructose-1,6-P 2 a c t i v a t i o n i s greatest at a l k a l i n e pH values (Rozengurt et a l . , 1969). An opposite pH dependence of the fructose-1,6-P 2 a c t i v a t i o n of the oyster pyruvate kinases i s observed. For both enzymes, fructose-1,6-P„ lowers the K , , ^ , and t h i s e f f e c t 3 ' ' 2 m(p-enolpyruvate) i s p a r t i c u l a r l y s t r i k i n g at lower pH values (at pH 6.5 the K m i s reduced from 5.8 X 10~ 4 M to 8 X I O - 5 M; at pH 8.5 the K i s reduced from 1.9 X 10~ A M to m 6.6 X 10 -^ M). In consequence, i n the presence of fructose-1,6-P 2 the K , ., N i s e s s e n t i a l l y pH independent. m(p-enolpyruvate) The adductor enzyme appears to be under t i g h t ATP regulation. Thus, 46 2 mM ATP, a value probably within the p h y s i o l o g i c a l range (Williamson e t a l . , 1967) , causes about a 10 f o l d increase i n the K . n . . Under con-' m(p-enolpyruvate) d i t i o n s of low p-enolpyruvate concentrations, i t i s evident that adductor pyruvate kinase would be unusually s e n s i t i v e to ATP. In this c h a r a c t e r i s t i c , the adductor enzyme resembles mammalian muscle pyruvate kinase (Reynard et_ a l . , 1961), and adipose pyruvate kinase (Pogson, 1968) a l l of which have s i m i l a r K\ values, but i t d i f f e r s from mammalian brain pyruvate kinase (Lowry and Passonneau, 1964) and the mantle enzyme. In both of the l a t t e r ATP i n h i b i t i o n i s noncompetitive. Because of the high K-j^TP) ^ 0 r t * i e m a n t-'- e enzyme, and because ATP does not a l t e r the apparent K , .. . , ATP would not be m(p-enolpyruvate) an e f f i c i e n t i n h i b i t o r of this enzyme. In this connection, i t i s i n t e r e s t i n g that both mantle enzyme and mammal-ian brain pyruvate kinases (Schwark et a l . , 1970; and Weber, 1969) are com-p e t i t i v e l y i n h i b i t e d by phenylalanine and the K_ values are again s i m i l a r f o r the enzymes from the 2 tiss u e types. In the mantle, pyruvate kinase a c t i v i t y i s f a i r l y s e n s i t i v e to phenylalanine control since phenylalanine (at R\ con-centrations) produces quite large increases i n the K , , N . Since r n ° m(p-enolpyruvate) phenylalanine concentrations are known to be unusually high i n mollusc tissues (Virkar e_t a l . , 1970) this amino acid may be an important p h y s i o l o g i -c a l feedback i n h i b i t o r of pyruvate kinase a c t i v i t y i n t h i s t i s s u e as i t i s i n mammalian brai n . CHAPTER IV: C a t a l y t i c and Regulatory Properties of Oyst Phosphoenolpyruvate Carboxykinase: I. C e l l u l a r d i s t r i b u t i o n and the ef f e c t s of and metal ions on the enzyme a c t i v i t y . 47 INTRODUCTION It i s now well documented that many i n t e r t i d a l b i valve molluscs are f a c u l t a t i v e anaerobes. Under anaerobic conditions, glucose i s catabolized by the reactions of g l y c o l y s i s and the Krebs cycle, but under axonic conditions, l a c t a t e , the usual end product of anaerobic g l y c o l y s i s i n vertebrates, does not accumulate. Instead, the end product of anaerobic glucose catabolism i s succinate (Hammen, 1969). Although there has been some dispute over the precise metabolic pathways involved i n anaerobiosis, Simpson and Awapara (1966) have presented convincing evidence that p-enolpyruvate i s carboxylated to oxaloacetate, with the subsequent r e v e r s a l of a portion of the Krebs cycle (oxaloacetate > malate > fumarate —> succinate) leading to succinate as the f i n a l end product of anaerobic glucose d i s s i m i l a t i o n . Since pyruvate carboxy-lase i s not detectable i n these organisms, the ( ^ - f i x a t i o n step i s apparently catalyzed by p-enolpyruvate carboxykinase (ITP, GTP: oxaloacetate carboxy-l a s e (transphosphorylating); I.U.B.E.C. 4.1.1.32): oxaloacetate + ITP (or GTP) p-enolpyruvate + IDP (or GDP) + C0 2 These metabolic capacities of i n t e r t i d a l bivalves r a i s e some e s s e n t i a l regulatory requirements at the p-enolpyruvate branching point. In the f i r s t place, p-enolpyruvate carboxykinase (PEPCK) reaction i n vertebrate tissues normally functions i n the decarboxylating d i r e c t i o n , during gluconeogensis i n the l i v e r and kidneys (Scrutton and Utter, 1968), during glyceroneogenesis i n adipose ti s s u e (Meyuhas, Boshwitz and Reshef, 1971), and during operation of the malate-oxaloacetate cycle i n s k e l e t a l white muscle (Opie and Newsholme, 1967). In contrast, i n the molluscs there i s an e s s e n t i a l requirement for PEPCK function i n the CO^-fixing d i r e c t i o n , and t h i s would be p a r t i c u l a r l y important i n s k e l e t a l muscle. Secondly, under these conditions, p-enolpyruvate i s a common substrate 48 f o r both the pyruvate kinase reaction, which i s favoured during aerobic meta-bolism (Mustafa and Hochachka, 1971), and f o r the PEPCK reaction, which i s favoured during anaerobiosis. Hence, i t i s clear that the c a t a l y t i c a c t i v i t i e s of PEPCK and pyruvate kinase must be c l o s e l y integrated f o r e f f i c i e n t channel-l i n g of carbon through this metabolic branchpoint, during aerobic anaerobic t r a n s i t i o n s . And f i n a l l y , tissues and f l u i d s of oyster have unusually high I | | | Zn and Cu concentrations, which may influence the cation cofactor requirements of PEPCK. For these reasons, I i n i t i a t e d a d e t a i l e d k i n e t i c study of the PEPCK catalyzed carboxylation reaction i n oyster adductor muscle. This chapter establishes (a) the c e l l u l a r d i s t r i b u t i o n of oyster phosphoenolpyruvate carboxykinase, (b) the behaviour of oyster PEPCK with I | [ | respect to Mn , Zn and IDP concentrations, (c) pH modulation of the [ | | | K / , % i n the presence of Mn and Zn , and (d) the r e l a t i v e m(p-enolpyruvate) I | | | enzyme a c t i v i t i e s with Mn and Zn under d i f f e r e n t experimental conditions. 49 RESULTS Electrofocusing. I s o e l e c t r i c focusing of p-enolpyruvate carboxykinase indicates a s i n g l e major component with enzyme a c t i v i t y . Under our conditions, the i s o e l e c t r i c point i s about 6.64 (Fig. 1). I t was noted that prolonged electrofocusing leads to enzyme i n a c t i v a t i o n . Requirement for the p-enolpyruvate carboxykinase catalyzed carboxylation  reaction. The requirements for the p-enolpyruvate carboxykinase catalyzed carboxy-l a t i o n are summarized i n Table I. Absolute requirements for p-enolpyruvate, divalent ion, nucleotide diphosphate and bicarbonate are indicated. Neither I | ADP nor IMP can replace IDP (or GDP). The enzyme i n the presence of Mg checked at d i f f e r e n t IDP concentrations and pH values shows e s s e n t i a l l y no a c t i v i t y . The p-enolpyruvate carboxykinase preparation used does not have any l a c t a t e dehydrogenase a c t i v i t y while endogenous malate dehydrogenase (MDH) and "malic" enzyme a c t i v i t i e s were found to be present. Neither of these enzymes i n t e r f e r e with the assay system used i n t h i s study, since MDH i s needed i n large amounts for coupling oxaloacetate to malate formation, while oxaloacetate decarboxylation by "malic" enzyme i s NADP dependent. Under our experimental conditions, p-enolpyruvate carobxykinase a c t i v i t y could not be detected i n these t i s s u e s . Tissue and s u b - c e l l u l a r d i s t r i b u t i o n of oyster p-enolpyruvate carboxykinase. Results of studies of mitochondrial and cytoplasmic p-enolpyruvate car-boxykinase l e v e l s i n various oyster tissues assayed immediately a f t e r preparation are given i n Table II. A l l assays were repeated at l e a s t twice. 50 Fig. IV, 1. Electrofocusing pattern of the adductor p-enolpyruvate carboxykinase. For experimental procedures see "Materials and Methods". Activity is plotted against fraction numbers; the numerical values plotted on the abscissa indicate the pH of the selected fractions. Reactant concentra-tions are the same as in Fig. 2. 51 Table I Components of p-enolpyruvate carboxykinase catalyzed carboxylation reations. Complete reaction mixture contained 50 mM Tris-Maleate b u f f e r , pH 6.0, 1 mM Mn4"1", 10 mM KHC03, 1 mM IDP, 1 mM p-enolpyruvate, 0.15 mM NADH and excess Sigma MDH. Reactants deleted from or added to complete assay A c t i v i t y AE 0,„/unit time 340 % A c t i v i t y of complete assay None (complete) p-enolpyruvate, deleted I | Mn , deleted j | j | Mn , deleted, Mg added, 1 mM I | | | Mn deleted, Zn added, 1 mM K + added, 1 mM or 10 mM IDP, deleted IDP, deleted, IMP added 1 mM IDP, deleted, ADP added 1 mM IDP, deleted, GDP added 1 mM KHC03, deleted Malate dehydrogenase, deleted p-enolpyruvate carboxykinase deleted 0.46 0.00 0.00 0.00 0.30 0.42 0.00 0.00 0.00 0.24 0.08 0.38 0.00 100 0.00 0.00 0.00 65.2 91.3 0.00 0.00 0.00 52.0 1.70 82.6 0.00 52 Table II Tissue and s u b c e l l u l a r d i s t r i b u t i o n of oyster p-enolpyruvate carboxykinase. Reactant concentrations and assay conditions were e s s e n t i a l l y the same as referred i n Table I. Mitochondrial f r a c t i o n s were also checked at pH I | | j 7.0 and 7.4 i n presence of eit h e r Mn and Mg . A c t i v i t y i s expressed as A E 0 / n / u n i t time. Tissues, s u b c e l l u l a r f r a c t i o n A c t i v i t y Mitochondrial p-enolpyruvate carboxykinase (a) Mantle (b) G i l l (c) Adductor muscle Cytoplasmic p-enolpyruvate carboxykinase (High speed supernatant) (a) Mantle (b) G i l l (c) Adductor muscle 40 to 75% saturated cytoplasmic p-enolpyruvate carboxykinase (a) Mantle (b) G i l l (c) Adductor muscle undetectable undetectable undetectable n e g l i g i b l e n e g l i g i b l e 0.40 0.10 0.20 0.8 53 No a c t i v i t y was found i n any of the mitochondrial preparations of g i l l , mantle and adductor t i s s u e s . In each case a l l of the a c t i v i t y appeared i n the soluble f r a c t i o n . Levels of the enzymic a c t i v i t i e s i n mantle and g i l l t i s s u e s , compared to the a c t i v i t y i n adductor muscle were extremely low. I t i s i n f e r r e d from these r e s u l t s that only cytoplasmic p-enolpyruvate carboxy-kinase i s present i n oyster t i s s u e . E f f e c t s of pH on p-enolpyruvate carboxykinase a c t i v i t y . Figure 2 shows the a c t i v i t y of p-enolpyruvate carboxykinase assayed i n I | | | the d i r e c t i o n of oxaloacetate synthesis i n the presence of Mn and Zn as a function of pH. pH measurements were made j u s t before and a f t e r termina-t i o n of the reaction. No change i n pH during the reaction was noted i n any instance. The pH a c t i v i t y p r o f i l e s obtained i n the presence of e i t h e r metal ion were s i m i l a r i n general pattern. However, both the p-enolpyruvate concen-t r a t i o n and the cation cofactor determine the precise pH optimum. Thus i n the I | presence of Zn and low p-enolpyruvate concentrations, the pH optimum i s about I | pH 6.0. In the presence of Zn and saturating p-enolpyruvate concentration I | maximal a c t i v i t y appears around pH 5.1 while i n the presence of Mn the a c t i v i t y i s maximal at pH 6.0. Values of enzyme a c t i v i t i e s measured i n the presence of sodium acetate buffer at below pH 5 were corrected for the i n h i b i t o r y e f f e c t of Na +. (The a c t i v i t y i n the sodium acetate buffer, at pH 5 i s only 60% of the a c t i v i t y i n Tris-Maleate buffer at pH 5). On the basis I | | | of the r e s u l t s presented i n the F i g . 2 subsequent studies with Mn or Zn divalent ions were made at the d i f f e r e n t pH optima. R e v e r s i b i l i t y of pH e f f e c t . To rule out the p o s s i b i l i t y that the reduced a c t i v i t y at a l k a l i n e pH 54 F i g . IV, 2. E f f e c t s of pH on p-enolpyruvate carboxykinase a c t i v i t y ; o) i n presence of Zn and ( • ) i n presence of Mn , ( — ) high and ( ) low concentrations of p-enolpyruvate. Reaction contents were 50 mM Tris-Maleate buffer at I | | | d i f f e r e n t pH values, 1 mM Mn or Zn as indicated, 1 mM IDP, 10 mM KHC03, 1 mM p-enolpyruvate, 0.15 mM NADH and excess malate dehydrogenase. ( A c t i v i t y at the f i r s t closed c i r c l e was assayed i n sodium acetate buffer at pH 4.8 which accounts for the steepness of the curve.) Activity (AE 3 4 0 /uni t time) 55 value (pH 7.0 and above) i s not due to the destruction of the active s i t e , experiments were c a r r i e d out on the r e v e r s i b i l i t y of pH e f f e c t s . Change i n the pH was brought by d i a l y z i n g a small amount of enzyme against Tris-Maleate I | | | buff e r at d i f f e r e n t pH values containing e i t h e r Mn or Zn . P i l o t experi-ments established that the pH changes i n the enzyme s o l u t i o n produced by t h i s technique occurred within 90 min of d i a l y z i n g at 4°. I t can be observed (Table III) that preincubating the enzyme at d i f f e r e n t pH does not bring any i r r e v e r s i b l e change i n the enzyme a c t i v i t y i n the presence of e i t h e r metal ion. [ | | | The r a t i o of Mn /Zn activated enzyme c a t a l y s i s f a l l s i n a narrow range for a l l pH treatments (Table I I I ) . Metal ion requirement. Since p-enolpyruvate carboxykinase have been shown to require a divalent metal ion-nucleotide complex, we f e l t i t necessary to study t h i s requirement at d i f f e r e n t pH values. Metal ion s p e c i f i t y was checked i n Tris-Maleate b u f f e r , at pH 5, 6,7 and 8, containing 1 mM metal ion, 1 mM p-enolpyruvate, I | 1 mM IDP, 0.15 mM NADH and axcess sigma MDH. In preliminary experiments Mn , I j | [ j | | j | | [ | Mg , Zn , Co , Ca , Fe and Cu ions were tested at a l l pH values. Only I | _| |_ | [ _j |_ | [ Mn , Zn and Co form active metal-nucleotide complexes: Mg , Ca and I | | | Fe neither a c t i v a t e nor i n h i b i t the enzyme, while Cu i n h i b i t s the enzyme i n presence of any of the ac t i v e metal ion complexes and at a l l the pH values tested. To further i n v e s t i g a t e the metal ion a c t i v a t i o n of the enzyme, we determined enzyme a c t i v i t y as a function of metal ion concentration at three d i f f e r e n t pH values. In the absence of preincubation p-enolpyruvate carboxy-kinase shows greater a c t i v i t y i n the presence of Mn at a l l the pH values j | tested (Fig. 3-A). However, maximal a c t i v a t i o n i n presence of Zn occurs at Table I I I R e v e r s i b i l i t y of pH e f f e c t on enzyme a c t i v i t y . S t arting enzyme was neut r a l i z e d by 0.1 mM KHCO^. 1 ml enzyme was dialyzed against 100 ml of Tris-Maleate buffer (at pH value s p e c i f i e d below) I | | j containing e i t h e r 10 mM Zn or Mn . Enzyme a c t i v i t y was assayed at the respective optimal pH f o r e i t h e r metal ion. The assay system contained Tris-Maleate b u f f e r , 1 mM metal ion, 10 mM KHCO^, 1 mM p-enolpyruvate, 1 mM IDP, 0.15 mM NADH, and excess dialyzed Sigma MDH, and the reaction was i n i t i a t e d by addition of the pH treated enzyme pH Treatment of the Enzyme 8 -h. pH 7 no metal ion i n the di a l y z i n g b u f f e r pH 7 > 7, pH 7 » 5.4, pH 7 v 5.4-j,6, pH 7 * 5.4-*6 -T»7, pH 7 s> 5 . 4 - » 6 - * 7 - * 7 . 5 , pH 7 — * 5 . 4 ->7, pH 7 — » 5.4 -*7-jv5.4, A c t i v i t y A E 3 4 0 / u n i t time pH 5.1 (Zn++) 0.75 0.675 0.85 0.82 0.60 0.55 0.60 0.65 pH 6.0 (Mn44") 0.53 0.53 0.55 0.51 0.46 0.40 0 . 5 0 ; 0.51 Ratio of Mn^/Zn 4 4" activated Enzyme 0.705 0.785 0.647 0.621 0.766 0.722 0.837 0.784 57 F i g . IV, 3. E f f e c t s of metal ion a c t i v a t i o n at d i f f e r e n t pH values; I | | | 3A i n presence of Mn and 3B i n presence of Zn . ( O ) pH 5.1, ( • ) pH 6.0, and ( A ) PH 7.0. Reactant contents were 50 mM Tris-Maleate b u f f e r , increasing concentrations of the metal ion, 1 mM IDP, 1 mM p-enolpyruvate, 10 mM KHCO^, 0.15 mM NADH and excess malate dehydrogenase. In 3A, S. n q. at pH 5.1, 6.0 and 7.0 are 0.52 mM, 0.15 mM and 0.15 mM. In 3B, S, ... at pH 5.1 and 6.0 are 0.15 mM w. ->) and 0.29 mM. Activity (AE3 4 0/unit time) Activity (AE3 4 0/unit time) 58 I | about pH 5.1 while with Mn i t occurs at pH 6.0. Both metal ions, above 1 mM concentration at a l l pH values tested were found to be s l i g h t l y i n h i b i t o r y . I | | | The r a t i o , at 1 mM metal ion, Mn stimulated a c t i v i t y / Z n stimulated a c t i v i t y , increases with increasing pH being 1.1, 2.0 and 14.3 at pH 5.1, 6.0 and 7.0 r e s p e c t i v e l y . As the metal ion curves were not t r u l y hyperbolic, S , N values were calculated by H i l l treatment of the data, w. J ) I | In the pH range of 5-6, both the shape of the Zn saturation k i n e t i c s and the ^ value depend upon pH. Thus at pH 5.1 the ^ value i s about two f o l d greater compared to S ^ ^ at pH 6.0 ( F i g . 3-B). In contrast, the Mn saturation k i n e t i c s appear to be somewhat more complex ( F i g . 3-A) and the pH dependencies of the S^Q ^ values are more pronounced. Thus at pH 6.0 the S , N value i s only about 1/4 of the values at pH 5.1. This pH dependency i n the presence of Mn i s opposite to pH dependency i n the I j | | presence of Zn . Thus the apparent enzyme-Mn a f f i n i t y i s greater at pH 6.0 I | than at pH 5.1 while apparent enzyme-Zn a f f i n i t y i s greater at pH 5.1 than at pH 6.0. Inosine diphosphate (IDP) saturation k i n e t i c s . IDP saturation curves are shown f o r p-enolpyruvate carboxykinase cata-l y s i s i n the presence of Mn and Zn at d i f f e r e n t pH values (Figs. 4, 5). At pH 6 and 7 (4-A,B) i n the presence of Mn , IDP saturation k i n e t i c s obey Michaelis-Menton consideration under a l l conditions tested. Values of S, (0.5) are i n the range of 0.03 mM to 0.06 mM, and these are l a r g e l y independent of pH. There i s no evidence of substrate i n h i b i t i o n at high (up to 2 mM) IDP l e v e l s . I | | | The e f f e c t s of Zn and Mn on the IDP saturation k i n e t i c s at pH 5.1 59 F i g . IV, 4. E f f e c t of Mn on the IDP saturation k i n e t i c s at pH 6.0 (4A) and pH 7.0 (4B). ( • ) 1 mM Mn44" and 1 mM p-enolpyruvate, ( A) 0.5 mM Mn and 1 mM p-enolpyruvate, I j ( O ) 1 mM Mn and 0.5 mM p-enolpyruvate. Other condi-tions as i n F i g . 3. Activity (AE340/unit time) 60 F i g . IV, 5. E f f e c t s of Zn and Mn on the IDP saturation k i n e t i c s I | at pH 5.1, 4A i n presence of Zn and 4B i n presence I | of Mn . ( • ) 1 mM metal ion and 1 mM p-enolpyruvate, ( A ) 0.5 mM metal ion and 1 mM p-enolpyruvate, ( O) 1 mM metal ion and 0.5 mM p-enolpyruvate. Other conditons as i n F i g . 3. Activity (AE3 4 0/unit t ; m e ) Activity (AE 3 4 0/unit time) 61 I | are compared i n F i g s . 5-A,B. In the presence of Zn the saturation curves are s l i g h t l y sigmoidal. H i l l p l o t s y i e l d _n values of about 1.5. The absolute values of S, n ^ f o r IDP are e n t i r e l y comparable i n the presence of Zn and (.0. J) I | Mn . I t i s evident from the data that the adductor muscle p-enolpyruvate carboxykinase i s able to compete for l i m i t i n g IDP concentration with s i m i l a r effectiveness when eit h e r Mn or Zn i s present as the c a t i o n i c cofactor P-enolpyruvate saturation k i n e t i c s . P-enolpyruvate saturation curves for p-enolpyruvate carboxykinase I | | | c a t a l y s i s at d i f f e r e n t pH values i n the presence of Mn and Zn are shown I | i n F i g s . 6 and 7. In the presence of Mn , p-enolpyruvate saturation k i n e t i c s are complex at a l l pH values examined. As i s evident, under the conditions tested the enzyme does not f u l l y saturate even at 2 mM concentration of p-enolpyruvate (Fig. 6). This aberrant behaviour of the enzyme i n the presence of Mn tends to r u l e out t h i s cation as the jLn vivo cofactor, and precludes accurate estimate of the a f f i n i t y constant f o r p-enolpyruvate. I | In contrast, p-enolpyruvate saturation k i n e t i c s i n the presence of Zn (F i g . 7) e s s e n t i a l l y follow Michaelis-Menton patterns. Equally s i g n i f i c a n t , the apparent K m values are at least 1 order of magnitude l e s s than observed I | | | f o r the enzyme i n the presence of Mn . Moreover i n the presence of Zn the apparent K , ~ , at pH 6.0 i s about 1/3 the value at pH 5.1 (Fig. r r m(p-enolpyruvate) 7 ). Thus, although the pH optimum f o r the enzyme under saturating p-enolpy-ruvate concentrations i s about 5.1, i t i s evident that i n the presence of I | Zn i t competes quite favourably for l i m i t i n g p-enolpyruvate at higher pH values (see F i g s . 2, 7). 62 F i g . IV, 6. Ef f e c t s of Mn on p-enolpyruvate saturation k i n e t i c s at d i f f e r e n t pH values, ( • ) pH 5.1, ( A ) pH 6.0, and ( O ) pH 7.0. Reactant contents were: 50 mM T r i s -Maleate buffer, 1 mM Mn4"1", 1 mM IDP, 10 mM KHC03, increasing concentrations of p-enolpyruvate, 0.15 mM NADH and excess malate dehydrogenase. 63 F i g . IV, 7. E f f e c t s of Zn on p-enolpyruvate saturation k i n e t i c s at d i f f e r e n t pH values, ( • ) pH 5.1, ( A ) pH 6.0, and ( O ) pH 7.0. Reactant contents were 50 mM Tris-Maleate buffer 1 mM Zn , 1 mM IDP, 10 mM KHC0 3 > increasing con-centrations of p-enolpyruvate, 0.15 mM NADH and excess malate dehydrogenase. K , n values at pH ° m(p-enolpyruvate) 5.1 and 6.0 are 0.5 mM and 0.18 mM. Activity (AE 3 4 0/unit time) 64 I | Cu i n h i b i t i o n k i n e t i c s . I | No reports are a v a i l a b l e on Cu i n h i b i t i o n of p-enolpyruvate carboxykin-I | ase a c t i v i t i e s . We found that Cu i n h i b i t i o n of the oyster enzyme i s qu a n t i t a t i v e l y dependent on the c a t i o n i c cofactor present i n the assay system and i s pH independent i n the presence of Zn . The i n h i b i t i o n per cent of I | enzyme by Cu at pH 5.1 and 6.0 and f i x e d concentrations of c a t i o n i c cofactor I | and p-enolpyruvate are shown i n F i g . 8. In the presence of Zn ( F i g . 8, I | open c i r c l e s and squares), the enzyme i s much less s e n s i t i v e to Cu i n h i b i t i o n I | (apparent K\ i s about 60 pM at both pH 5.1 and 6.0); with Mn as the metal I | ion ( F ig. 8 , closed c i r c l e s and squares); the s e n s i t i v i t y to Cu increases by nearly an order of magnitude (the apparent K\ values now are 6 to 100 uM at pH 5.1 and 6.0 r e s p e c t i v e l y ) . I | [ | P-enolpyruvate carboxykinase a c t i v i t y as a function of Mn and Zn I | saturation i n the presence of d i f f e r e n t Cu concentrations at pH 6.0 are I | shown i n Figs. 9-A,B. In presence of Mn (9-A) both the Vmax and the a f f i n i t y I | | | | | constant f o r Mn are alte r e d by Cu . Thus, Cu (40 p M ) increases the S ^ ^ I | values of Mn by several f o l d over the cont r o l S ^ ^ values. At the same I | time 40 yuM Cu reduces the Vmax to about 20 per cent of control value at I | 2 mM Mn I | | | In contrast, i n the presence of Zn ( F i g . 9-B) Cu reduces the Vmax but does not s i g n i f i c a n t l y a f f e c t the a f f i n i t y f o r the metal i o n . Thus I | ^(0 5) v a x u e s i n absence and i n the presence of Cu (at 40 or 60 nM) were ++ i d e n t i c a l (0.23 mM) although 60 JUM Cu reduces the Vmax to 40 per cent of I | | | control rates at 2 mM Zn . It i s evident from these r e s u l t s that Cu i n -I | | | h i b i t i o n i s less e f f e c t i v e i n presence of Zn than i n the presence of Mn , since only the maximum c a t a l y t i c rate i s affected while the ^ f o r the cation i s unaltered. 65 F i g . IV, 8. I n h i b i t i o n of p-enolpyruvate carboxykinase a c t i v i t y by I | | | increasing concentrations of Cu i n presence of Zn (open symbols) or Mn (closed symbols) at ( O) pH 5.1, and ( • ) pH 6.0. Reaction contents were: 50 mM T r i s -j | | | Maleate b u f f e r , 1 mM Mn or Zn as indicated, 1 mM IDP, 10 mM KHCO.,, 1 mM p-enolpyruvate, increasing concentration of Cu , 0.15 mM NADH and excess malate dehydrogenase. T 66 F i g . IV, 9. P-enolpyruvate carboxykinase a c t i v i t y as a function of I | | ] the concentration of Mn and Zn i n the presence or absence of Cu* 4. In 9A, ( • ) c o n t r o l , ( A ) 10 uM Cu4"*", and ( O ) 40 juM Cu44". In 9B, ( • ) c o n t r o l , ( O ) 40 JJM I | j | Cu and ( A ) 60 uM Cu . Reactant contents were 50 mM Tris-Maleate buffer pH 6.0, increasing concentrations of the metal ion as i n i d i c a t e d , 1 mM IDP, 1 mM p-enolpyruvate, 10 mM KHC0», 0.15 mM NADH and excess malate dehydrogenase. Activity (AE3 4 0/unit time) Activity (AE 3 4 0/unit time) ON r5 6 7 DISCUSSION A fundamental question arises from the above studies concerning the concentrations of metal ions within the i n t r a c e l l u l a r m i l i e u . Do these i n fact occur i n concentration ranges which would a f f e c t PEPCK a c t i v i t y i n vivo? The problem of estimating the i n t r a c e l l u l a r concentration of free metal ions i s d i f f i c u l t and few d e f i n i t i v e studies are a v a i l a b l e (Thiers and Va l l e e , 1957). The only a v a i l a b l e data on the c e l l u l a r d i s t r i b u t i o n of metal ions i n oyster are those of Galstoff (1964). From these i t appears that the tis s u e j | | | | j | [ content of Fe , Cu , Zn and Mn fluctuates i n oyster tissues on a season-I [ a l b a s i s . Of these cations, Zn occurs i n by f a r the highest concentrations, I | | j being approximately 1000 times more abundant than Mn . Cu concentrations I | also are always much higher than Mn , p a r t i c u l a r l y during the winter season (G a l s t o f f , 1964). Since PEPCK i n vertebrate systems requires a divalent I | | | cation that i s thought to be f u l f i l l e d In vivo by Mn or Mg (Holten and I | Nordlie, 1965), i t i s of p a r t i c u l a r i n t e r e s t to note that i n the oyster, Zn appears to be the more l i k e l y candidate f o r this function. Although oyster I | | | muscle PEPCK i s active i n the presence of e i t h e r Mn or Zn , the u t i l i z a t i o n I | of Zn as the _in vivo cofactor would appear to present two d i s t i n c t advantages: i n the f i r s t place, the p-enolpyruvate saturation of the enzyme I | i s aberrant i n the presence of Mn , but appears to follow c l a s s i c a l Michaelis-Menten k i n e t i c s d i s p l a y i n g a much lower apparent K , ^ N i n the m (p-eno lpy ruva t e ) I | presence of Zn , and secondly, the apparent a f f i n i t y of the PEPCK f o r I [ | | p-enolpyruvate i s greatly reduced by Cu i f Mn i s used as the cofactor, I | | [ while the a f f i n i t y i s l a r g e l y unaffected by Cu i f Zn i s the cofactor, even i f the Vmax i s s t i l l reduced. In addition, as already mentioned, the abundance I | | j of Zn i s nearly 3 orders of magnitude greater than the abundance of Mn , I | and t h i s f actor alone would tend to favour Zn as the probable i n vivo 68 cofactor, even i f high proportions of these cations occurred i n the bound form. In vertebrates, the s u b c e l l u l a r d i s t r i b u t i o n of PEPCK appears to depend upon the tissues and the species examined. In adipose tissues PEPCK i s a s t r i c t l y cytoplasmic enzyme (Meyuhas, Boshwitz and Resef, 1971). In gluconeogenic t i s s u e s , such as l i v e r , the enzyme i s found i n the cytoplasm i n the rat and mouse, i n the mitochondria i n rabbit and chicken, and i n both cytoplasm and mitochondria i n the guinea pig (Scrutton and Utter, 1968). In the oyster, e s s e n t i a l l y a l l the PEPCK a c t i v i t y i s to be found i n the c y t o s o l , a s i t u a t i o n comparable to that observed i n p a r a s i t i c helminths (Saz, 1971). This l o c a l i z a t i o n appears to f i t w e l l the functions of PEPCK i n the anaerobic metabolism of these organisms. In invertebrate f a c u l t a t i v e anaerobes such as the oyster, the capacity for i n d e f i n i t e s u r v i v a l i n the t o t a l absence of has involved a number of adaptations i n the pathway of anaerobic glucose d i s s i m i l a t i o n . One such adaptation has been the evolutionary " d e l e t i o n " of the enzyme, l a c t a t e dehydro-genase, a " d e l e t i o n " which can be viewed as a means of avoiding the deleterious e f f e c t s of the anaerobic accumulation of l a c t a t e . For g l y c o l y t i c a c t i v i t y to continue i n d e f i n i t e l y i n the absence of the l a c t a t e dehydrogenase reaction, however, pr o v i s i o n must be made for the regeneration of NAD f o r the t r i o s e phosphate i n very high a c t i v i t y and which i s very much i n the "mainstream" of carbon flow i n this organism, serves to regenerate NAD through a coupled reduction of oxaloacetate to malate. The oxaloacetate for t h i s reaction i n the cytoplasm i s supplied by the PEPCK catalyzed carboxylation of p-enolpyru-vate. Since oxaloacetate does not move f r e e l y across the mitochondrial b a r r i e r (Scrutton and Utter, 1968), i t i s evident that PEPCK function must be l a r g e l y l o c a l i z e d i n the cytoplasm of the tissues i n a l l invertebrate f a c u l t a t i v e anaerobes r e l y i n g on this reaction scheme. The data i n the Table II are 69 consistent with these considerations, and an i d e n t i c a l s i t u a t i o n has been described f o r another mollusc (Chen and Awapara, 1969) and several helminth parasites (Saz, 1971). As i t i s l o c a l i z e d i n the cy t o s o l , PEPCK competes d i r e c t l y with pyruvate kinase for the common substrate p-enolpyruvate, and i t i s cl e a r that the channelling of p-enolpyruvate towards oxaloacetate or towards pyruvate must be a cl o s e l y regulated process in vivo. Some of the p o t e n t i a l regulatory i n t e r a c t i o n s between PEPCK and pyruvate kinase are discussed i n the next chapter; s u f f i c e to mention at th i s point that the a f f i n i t y of PEPCK for p-enolpyruvate depends c r i t i c a l l y upon the microenvironment. At low pH (below 6.5) and p a r t i c u l a r l y i n the presence of Zn as a cofactor, PEPCK can re a d i l y "out-compete" pyruvate kinase f o r l i m i t i n g p-enolpyruvate, while the reverse i s obviously the case at higher pH values (Mustafa and Hochachka, I | 1971). Thus at pH 6.0 i n the presence of Zn the apparent K , , r r m(p-enolpyruvate) f o r PEPCK i s about 0.2 mM; under the same conditions of H + concentration, adductor muscle pyruvate kinase i s e s s e n t i a l l y i n a c t i v e i n the absence of fructose-1,6-P 2 and thus could not serve as an alternate pathway f o r p-enol-pyruvate metabolism. In contrast, at pH values over pH 7.0, the pyruvate kinase a f f i n i t y f o r p-enolpyruvate i s r e l a t i v e l y high, while PEPCK a c t i v i t y i s reduced to less than 5% of rates at optimal pH. As we have stressed, the metabolic s i t u a t i o n at the p-enolpyruvate branching point i s rather unique to f a c u l t a t i v e anaerobes such as the oyster. A comparable competitive s i t u a t i o n , however, could a r i s e i n gluconeogenic tissues of vertebrates which also possess both pyruvate kinase and phosphoenol-pyruvate carboxykinase. The A G° for the PEPCK CO^-fixing reaction i s small and negative (about -1 Kcal/mole); hence, the d i r e c t i o n of net c a t a l y t i c function i n vivo presumably i s l a r g e l y determined by adjustments i n the 70 apparent K values f o r p-enolpyruvate and oxaloacetate. In vertebrate l i v e r , the competitive s i t u a t i o n between pyruvate kinase and phosphoenolpyruvate carboxykinase appears to be avoided by the elaboration of a PEPCK which has about a 10 f o l d greater a f f i n i t y f o r oxaloacetate than f o r p-enolpyruvate (Holten and Nordlie, 1965; B a l l a r d , 1970). Under conditions of l i m i t i n g oxaloacetate and p-enolpyruvate, t h i s enzyme would therefore favour net c a t a l y t i c function i n the d i r e c t i o n of p-enolpyruvate synthesis, and this would be further favoured by the e f f i c i e n t removal of p-enolpyruvate during gluconeogenesis. The opposite s i t u a t i o n appears to be required i n the invertebrate anaerobes. Thus i n PEPCK of A s c a r i s , the r e l a t i v e a f f i n i t y constants f o r p-enolpyruvate and oxaloacetate have been adjusted so as to strongly favour p-enolpyruvate carboxylation to oxaloacetate (Saz, 1971). Although a s i m i l a r s i t u a t i o n would be anticipated i n the oyster, the control of p-enolpyruvate metabolism appears to be more complex since, as a f a c u l t a -t i v e anaerobe, this organism must be able to "switch over" from anaerobiosis (favouring PEPCK a c t i v i t y ) to aerobiosis (favouring pyruvate kinase a c t i v i t y ) i n r e l a t i v e l y short time periods. Further aspects of t h i s regulatory problem are discussed i n the following-chapter. CHAPTER V: C a t a l y t i c and Regulatory Properties of Oyster Phosphoenolpyruvate Carboxykinase: I I . Regulation of the enzyme a c t i v i t y and i t s function i n phosphoenolpyruvate metabolism. 71 INTRODUCTION Recent reports have shown that phosphoenolpyruvate carboxykinase plays an important regulatory r o l e i n gluconeogenesis i n l i v e r and kidney of mammals (Scrutton and Utter, 1968). In conjunction with pyruvate carboxylase, phosphoenolpyruvate carboxykinase (PEPCK) provides a d i r e c t route of p-enolpyruvate synthesis which bypasses the p h y s i o l o g i c a l l y i r r e v e r s i b l e pyruvate kinase reaction: Pyruvate + ATP + C0 2 » Oxaloacetate + ADP + P ± (I) Oxaloacetate + ITP (or GTP) ^ k P-enolpyruvate + IDP (or GDP) + HC0 3 (II) Although the evidence f o r the involvement of these two reactions i n gluconeogenesis i s quite convincing (Scrutton and Utter, 1968; Seubert and Huth, 1965), e a r l i e r studies of the k i n e t i c properties of PEPCK puzzled over the high ^ m ( o x a i o a c e t a t e ) • This value was about 1 order of magnitude higher than expected i n vivo oxaloacetate concentrations (Holten and Nordlie, 1965). The s i t u a t i o n was cleared up at l e a s t p a r t i a l l y i n a recent study by B a l l a r d (1970), who noted that under c a r e f u l l y c o n t r o l l e d assay conditions at pH 7.0, the apparent K^Q,^ was i n the pM range i n which in vivo oxaloacetate concentrations are thought to f l u c t u a t e . Since Ballard's (1970) assays were performed at pH 7.0 rather than at more a l k a l i n e pH optimum we reasoned that H + i s an important metabolic s i g n a l f o r a c t i v a t i o n of this enzyme. This would account f o r the w e l l known observation of increased gluconeogenesis during mild acidosis (Flores and Alleyene, 1971). Flores and Alleyene (1971) indeed have described a rapid increase i n PEPCK a c t i v i t y following the development of a mild acidosis but t h e i r data do not specify the nature of the a c t i v a t i o n . In invertebrate f a c u l t a t i v e anaerobes, the " p h y s i o l o g i c a l poise" of the PEPCK during anaerobiosis appears to be i n the carboxylating d i r e c t i o n (Saz, 72 1971). The oxaloacetate formed i s ultimately converted to succinate. The accumulation of succinate during the development of anaerobiosis i n i n t e r t i d a l molluscs, such as the oyster, apparently occurs concomittantly with the development of a mild acidosis (Wilbur, 1964). These observations coupled with the documented acid pH optimum for PEPCK i n the oyster (Mustafa and Hochachka, 1972) and i n other invertebrates (Saz, 1971), suggested to us that H + i s c r i t i c a l l y involved i n the control of oyster PEPCK as well as i n mammalian systems, even though the d i r e c t i o n of c a t a l y s i s under p h y s i o l o g i c a l conditions i s reversed. For these reasons, and because PEPCK i n the oyster appears to function i n d i r e c t competition with pyruvate kinase for the common substrate, p-enolpyruvate, we were interes t e d i n examining further regulatory properties of oyster phosphoenolpyruvate carboxykinase. 73 RESULTS Nature of inosine triphosphate (ITP) i n h i b i t i o n . As phosphoenolpyruvate carboxykinase i s considered to function on the mainstream of the anaerobic glucose catabolism i n the oyster muscle, we anticipated that the a c t i v i t y of the enzyme would be integrated with the energy status of the c e l l . Our i n v e s t i g a t i o n was therefore i n i t i a t e d by examining the e f f e c t s of ITP, GTP, and ATP on the PEPCK catalyzed carboxyla-tion of p-enolpyruvate. E a r l i e r data have indicated that the c a t a l y t i c properties of oyster PEPCK depend c r i t i c a l l y upon the metal ion cofactor; hence the e f f e c t of various ITP concentrations were studied i n the presence of I | | | both Zn and Mn at t h e i r respective pH optima (Figs. 1, 2). In both instances a competitive type of ITP i n h i b i t i o n was obtained with respect to eit h e r metal ion. In the presence of e i t h e r metal ion, 0.25 mM ITP increases the apparent K^ values of the metal ions by 10 f o l d but the calculated Vmax values remain unchanged. ITP i n h i b i t i o n studies i n r e l a t i o n to metal ion complexes with impure enzyme preparations are subject to the l i m i t a t i o n that the r e s u l t s may be influenced by non s p e c i f i c binding of the metal ions by contaminating proteins. To overcome t h i s l i m i t a t i o n p a r t i a l l y , saturating concentrations of the metal ions were used i n a l l l a t e r experiments. I | In the presence of Zn with ITP as the i n h i b i t o r and IDP as the va r i a b l e substrate, a l i n e a r non-competitive i n h i b i t i o n pattern was obtained ( F i g . 3). Thus i n presence of Zn as the metal ion,ITP decreases the calculated Vmax several f o l d i t . does not a f f e c t the K . At a l l concentrations of ITP tested m the K m(jj)p) remains unchanged at about 0.06 mM. Thus under probable physio-I | l o g i c a l conditions (with high Zn concentration), the presence of ITP does not appear to reduce PEPCK a f f i n i t y f o r l i m i t i n g IDP. In contrast, i n the I [ presence of Mn a l i n e a r , mixed-competitive ITP i n h i b i t i o n pattern was "74 F i g . V, 1. Zn saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v vs 1/Zn ) at varying ITP concentrations. (•) c o n t r o l , ( A ) 0.25 mM ITP, ( A ) 0.5 mM ITP, and ( O ) 1 mM ITP. Reactant concentrations were 50 mM Tris-Maleate buffer pH 5.1, increasing concentrations of Zn , 1 mM IDP, 1 mM p-enolpyruvate, 10 mM KHC03, 0.15 mM NADH, d i f f e r e n t ITP concentrations as indicated and excess malate dehydrogenase. Activity (AE-j^/unit time) 75 F i g . V, 2. Mn saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v vs 1/Mn ) at varying ITP concentrations. (O ) c o n t r o l , ( A ) 0.25 mM ITP, and ( A ) 0.5 mM ITP. Reactant concentra-tions were 50 mM Tris-Maleate buffer pH 6.1, increasing j | concentrations of Mn , 1 mM IDP, 1 mM p-enolpyruvate, 10 mM KHC03, 0.15 mM NADH, d i f f e r e n t ITP concentrations as indicated and excess malate dehydrogenase. Activity (AEg^Q /unit time) 76 F i g . V, 3. IDP saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v vs 1/IDP) at varying ITP concentrations i n presence of Zn 4 4". ( • ) co n t r o l , ( • ) 0.25 mM ITP, ( A ) 0.5 mM ITP, and ( O ) 1 mM ITP. Reactant concentrations were 50 mM I [ Tris-Maleate buffer pH 5.1, 1 mM Zn , increasing concentra-ti o n of IDP, 1 mM p-enolpyruvate, 10 mM KHC03, 0.15 mM NADH and malate dehydrogenase. A c t i v i t y ( A E g 4 0 / unit time) 77 F i g . V, 4. IDP saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t ( l / v vs 1/IDP) at varying ITP concentrations i n presence Mn44". ( • ) co n t r o l , and ( A ) 0.25 mM ITP. Reactant con-I | centrations were 50 mM Tris-Maleate buffer pH 6.0, 1 mM Mn , increasing concentrations of IDP, 1 mM p-enolpyruvate, 10 mM KHCO , 0.15 mM NADH and malate dehydrogenase. Activity (AE3 4 Q/unit time) 78 obtained ( F i g . 4). In this instance 0.25 mM ITP increases the K /TTNT,> m(IDP) several f o l d and decreases the calculated Vmax to about 50 per cent of the control values. Thus, even under these conditions, ITP-IDP i n t e r a c t i o n s with PEPCK are not competitive i n the oyster, whereas they appear to be s t r i c t l y competitive i n the case of chicken l i v e r mitochondrial PEPCK ( ' F e l i c i o l i , Barsacchi and Ipata, 1970). The e f f e c t of ITP on p-enolpyruvate saturation k i n e t i c s appears to be unique to the oyster enzyme as w e l l . In the presence of e i t h e r metal ion, ITP i n h i b i t i o n i s competitive with respect to p-enolpyruvate. With Zn , at pH 5.1 or 6.0, 0.5 mM ITP increases the K , 1 . , by about 2 f o l d r m(p-enolpyruvate) (Fig. 5 ). At 1 mM ITP, i n h i b i t i o n of the enzyme i s so severe that measure-I | ment becomes d i f f i c u l t . With Mn as the metal ion cofactor, oyster PEPCK i s extremely s e n s i t i v e to ITP. Thus 0.25 mM ITP increases the K , ., ^ . m(p-enolpyruvate) at l e a s t by 5 f o l d ( F ig. 6 ). These r e s u l t s i n d i c a t e that the PEPCK catalyzed carboxylation of p-enolpyruvate i n oyster adductor muscle i s remarkably s e n s i t i v e to product i n h i b i t i o n by ITP. Since ITP a l t e r s the capacity of the PEPCK to compete f o r p-enolpyruvate, ITP may be an important determinant of the per cent p a r t i c i p a t i o n of the PEPCK and pyruvate kinase reactions. The energy status of the c e l l may also be " r e f l e c t e d " by the concentra-tions of GTP and ATP. (GDP, but not ADP, can be a substrate f o r th i s enzyme.) In the presence of ei t h e r IDP or GDP, GTP i n h i b i t s the p-enolpyruvate carboxykinase reaction at both pH values. Its i n h i b i t i o n pattern with r e l a -t i o n to p-enolpyruvate shows a l i n e a r mixed-competitive type of i n h i b i t i o n ( F i g . 7 ). ATP, i n contrast, does not a f f e c t the oyster adductor PEPCK. Search for other metabolite e f f e c t o r s . Although phosphoenolpyruvate carboxykinase appears to be established as 79 F i g . V, 5. Double r e c i p r o c a l plots (1/v vs 1/p-enolpyruvate) at varying ITP concentrations at pH 5.1 (5A) and at pH 6.0 (5B). Reactant concentrations were 50 mM Tris-Maleate I j buffer, 1 mM Zn , increasing concentrations of p-enol-pyruvate, 1 mM IDP, 10 mM KHC03, 0.15 mM NADH d i f f e r e n t concentrations of ITP as indicated and excess malate dehydrogenase. In 5A, at 0 mM ITP ( • ), 0.25 mM ITP ( A ) 0.5 mM ITP ( A ), the K , . _ , values ' m(p-enolpyruvate) are 0.44 mM, 0.57 mM, and 0.8 mM r e s p e c t i v e l y . In 5B, at 0.0 mM ITP ( O ) , 0.25 mM ITP ( # ), and 0.5 mM ITP ( A ), the K , , _ , are 0.22 mM, 0.33 mM and m(p-enolpyruvate) 0.41 mM r e s p e c t i v e l y . 80 F i g . V, 6. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t (1/v vs 1/p-enolpyruvate) with varying concentrations j | of ITP and alanine with Mn as the divalent metal ion at pH 6.0; ( • ) c o n t r o l , ( O ) 4 mM alanine, ( • ) 4 mM alanine and 0.25 mM ITP, ( A ) 0.25 mM ITP, and ( A ) 0.5 mM ITP. Reactant concentrations and assay conditions as described i n F i g . 5. K , , N values are m(p-enolpyruvate) ( • ) 0.5 mM, ( O ) 0.5 mM, ( • ) 1 mM, and ( A ) 2.5 mM. 80 ^  31 F i g . V, 7. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o -c a l p l o t (1/v vs 1/p-enolpyruvate) i n the presence ( A ) and absence ( • ) of 0.25 mM GTP. Reactant concentrations I | were 50 mM Tris-Maleate buffer pH 6.0, 1 mM Zn , 1 mM IDP, increasing concentration of p-enolpyruvate, 10 mM KHCO^, 0.15 mM NADH and excess malate dehydrogenase. K , -| ^ , values are ( • ) 0.2 mM and ( A ) 0.5 mM. m(p-enolpyruvate) 82 a key enzyme i n the regulation of glucose metabolism i n vertebrates (Scrutton and Utter, 1968), l i t t l e information has been a v a i l a b l e on p o t e n t i a l regulatory mechanisms. However, two negative e f f e c t o r s are w e l l documented; these are 5' AMP (Holten and Nordlie, 1965) and malic acid ( B a l l a r d , 1970). The malate i n h i b i t i o n requires very high (40 mM) malate concentrations and probably i s not of major p h y s i o l o g i c a l s i g n i f i c a n c e . The 5' AMP i n h i b i t i o n , on the other hand, could serve to i n h i b i t PEPCK function i n gluconeogenesis under energy depleted (low ATP, high AMP) conditions and th i s e f f e c t could be potentiated by IDP product i n h i b i t i o n (FelUtcJU, , Barsacchi and Ipata, 1970). Most previous attempts to f i n d e f f e c t o r s f o r th i s enzyme have concentrated on a search f o r i n h i b i t o r s of the PEPCK catalyzed decarboxylation r e a c t i o n . In contrast, we i n i t i a t e d a search f o r p o s i t i v e e f f e c t o r s of the oyster PEPCK catalyzed carboxylation of p-enolpyruvate. We made a de t a i l e d survey of the e f f e c t s of the various metabolites at two d i f f e r e n t concentrations of the substrate p-enolpyruvate (1 mM and 0.5 mM) at pH 6.0 with Zn as the divalent metal ion. Of the various metabolites tested, 1 mM concentrations of fructose-6-phosphate, fructose-1,6-diphosphate, acetylCoA, c i t r a t e , malate, succinate, fumarate, 5' AMP, IMP, ADP, phenylala-nine and tryptophane have neither appreciable stimulatory nor i n h i b i t o r y e f f e c t s on the enzyme a c t i v i t y . Only L-alanine was shown to have a stimula-tory e f f e c t at low p-enolpyruvate concentration and markedly d e - i n h i b i t ITP (or GTP) i n h i b i t e d p-enolpyruvate carboxykinase. L-alanine e f f e c t s . In our previous studies (Mustafa and Hochachka, 1971) , we found that alanine i n h i b i t s oyster pyruvate kinase i n a manner competitive with respect to p-enolpyruvate. I t was therefore of i n t e r e s t to examine the e f f e c t s of t h i s amino acid on p-enolpyruvate carboxykinase. With Zn , alanine lowers the K , N to about 75% of control value at pH 6.0 m(p-enolpyruvate) and decreases the Vmax by about the same amount (Fig. 8). In terms of regulation of PEPCK a c t i v i t y , these e f f e c t s of alanine appear to be le s s s i g n i f i c a n t than i t s e f f e c t s on ITP i n h i b i t i o n . L-alanine strongly counter-acts the i n h i b i t i o n of PEPCK by ITP, but the precise degree of r e v e r s a l depends upon the nature of the cation cofactor. I | In the presence of Zn at pH 6.0 the i n h i b i t i o n caused by 0.25 mM ITP i s completely reversed by 4 mM L-alanine; a K , , ^ , of 0.4 mM J m(p-enolpyruvate) i n the presence of ITP i s completely returned to control (0.2 mM) value by addition of alanine ( F i g . 8 ). Comparable r e s u l t s are obtained at pH 5.0. With Mn , at pH 6.0 alanine produces a small but consistent increase i n c a t a l y t i c rate at a l l p-enolpyruvate concentrations. However, there i s no s i g n i f i c a n t change i n the a f f i n i t y constant for p-enolpyruvate. Perhaps, I | because of the greater ITP i n h i b i t i o n i n the presence of Mn , alanine does not completely reverse the e f f e c t of ITP on PEPCK c a t a l y s i s . The r e v e r s a l i s nevertheless large. At 0.25 mM ITP, a K . .. , of 2.5 mM i s ° m(p-enolpyruvate) reduced by 4 mM alanine to 1 mM ( F i g . 6 ). From these data i t i s evident that alanine supplies an e f f e c t i v e mechanism for r e v e r s a l of ITP i n h i b i t i o n I | of p-enolpyruvate carboxykinase c a t a l y s i s i n the presence of e i t h e r Mn or 84 F i g . V, 8. P-enolpyruvate saturation k i n e t i c s and i t s double r e c i p r o c a l p l o t with varying concentration of ITP and I | alanine with Zn as the divalent metal ion at pH 6.0. ( • ) c o n t r o l , ( O ) 4 mM alanine, ( • ) 4 mM alanine and 0.25 mM ITP, ( • ) 0.25 mM ITP, and ( A ) 0.5 mM ITP. Reactant concnetrations and assay conditions as described i n F i g . 5. K , .. ^ values are m(p-enolpyruvate) ( O ) 0.22 mM, ( O ) 0.16 mM, ( # ) 0.21 mM, and ( A ) 0.4 mM. 85 DISCUSSION The a c t i v i t i e s of many enzymes involved i n c e l l u l a r energy metabolism are governed at l e a s t i n part by energy status of the c e l l . The usual metabolic " s i g n a l s " are the adenylates: high ATP concentrations often are i n h i b i t o r y to the enzymes involved i n energy metabolism. On the other hand, high AMP (equivalent to low ATP) often are stimulatory (Atkinson, 1968). In oyster muscle, p-enolpyruvate can be metabolized by two pathways: ( i ) to pyruvate, by a pyruvate kinase catalyzed transphosphorylation reaction, or ( i i ) to oxaloacetate by a PEPCK carboxylation reaction. Both reactions generate a high energy phosphate compound (ATP i n the case of pyruvate kinase; ITP i n the case of PEPCK) and both are subject to product i n h i b i t i o n by these compounds. In t h i s sense, both enzymes are i n accordance with Atkinson's (1968) energy charge concept. Whereas these mechanisms undoubtedly contribute to the p h y s i o l o g i c a l "poise" of the p-enolpyruvate branch point, they do not, of themselves, display adequate s p e c i f i c i t y to account for t r a n s i t i o n from aerobic to anaerobic metabolism ( i . e . f o r the t r a n s i t i o n from pyruvate kinase function under aerobic conditions to PEPCK function during anaerobiosis). The s p e c i f i c i t y required, as f a r as we can judge from the demonstrable regulatory properties of these two enzyme systems, appears to be supplied by H + and alanine. Furthermore, from the regulatory responses of PEPCK and pyruvate kinase to these two e f f e c t o r compounds, i t appears that the two enzymes have been selected to function l a r g e l y on an e i t h e r / o r basis , rather than to function simultaneously. The nub of the argument i s summarized below. I t i s widely held that under anaerobic conditions, molluscan bivalves sustain s u b s t a n t i a l a c i d i f i c a t i o n of t h e i r tissues and f l u i d s (Wilbur, 1964). This drop i n pH appears to play a p i v o t a l r o l e i n the channelling of 86 p-enolpyruvate from the pyruvate kinase reaction and towards PEPCK, because of the pH p r o f i l e s for PEPCK (Mustafa and Hochachka, 1972a) and pyruvate kinase (Mustafa and Hochachka, 1971) are e s s e n t i a l l y non-overlapping. In consequence, i n the absence of any other f a c t o r decreasing pH leads to an automatic i n h i b i t i o n of pyruvate kinase with a concomitant a c t i v a t i o n of PEPCK. At the same time, L-alanine which i s known to accumulate along with succinate under anaerobic conditions, p o t e n t i a l l y i n h i b i t s pyruvate kinase (by increasing the K , ^ s and decreasing the Vmax). I t i s J G m(p-enolpyruvate) 6 p a r t i c u l a r l y i n s t r u c t i v e that the L-alanine i n h i b i t i o n i s potentiated by de-creasing pH: for adductor muscle pyruvate kinase, the K. / T , . , at pH 7.5 l(L-alanxne) r i s only 1/6 the K^ value observed at the optimal pH 8.5 (Mustafa and Hochachka, 1971). Indeed, low pH likewise potentiates ATP i n h i b i t i o n of pyruvate kinase. In marked contrast, the primary e f f e c t s of L-alanine on PEPCK appear to be ( i ) a rever s a l of any r e s i d u a l ITP i n h i b i t i o n , and ( i i ) a s l i g h t a c t i -vation at low p-enolpyruvate concentrations due to a reduction i n the apparent K , , . Both these e f f e c t s of L-alanine on PEPCK occur m(p-enolpyruvate) at pH ranges (pH 5-6) i n which pyruvate kinase a c t i v i t y i s very low and i n which L-alanine i n h i b i t i o n of pyruvate kinase i s unusually extreme. From these data we are led to the conclusion that the decreasing pH and increasing L-alanine concentration, both of which occur i n aerobic ^ £anaerobic t r a n s i -t i o n , cause an exponential increase i n the PEPCK c a t a l y t i c rate, concurrent with an exponential decrease i n the pyruvate kinase c a t a l y t i c rate. This would appear to be an adequate arrangement f or channelling p-enolpyruvate towards oxaloacetate, which ultimately accumulates as succinate. But i f the pyruvate kinase path to pyruvate i s blocked, what i s the source of the L-alanine which accumulates along with succinate during anaerobic metabolism? Two p o s s i b i l i t i e s suggest themselves. ( i ) In our e a r l i e r studies on 87 oyster pyruvate kinases (Mustafa and Hochachka, 1971), we noted that fructose-1,6-P2 reverses the e f f e c t s of alanine, ATP, and to a somewhat le s s e r extent, H +. Hence, t h i s could supply the oyster with a mechanism for maintaining some pyruvate kinase function under anaerobic conditions. The pyruvate produced could i n turn be transaminated to alanine. ( i i ) As we indicated i n the previous paper (Mustafa and Hochachka, 1972a), oyster muscle contains very high a c t i v i t i e s of NADH-linked malic dehydrogenase and NADP-linked "malic enzyme". These enzymes occur i n much higher s p e c i f i c a c t i v i t i e s than do e i t h e r PEPCK or pyruvate kinase. A high malate dehydrogenase a c t i v i -ty i s assumed to function i n the maintenance of low oxaloacetate concentra-tions thus preventing s i g n i f i c a n t r e v e r s a l of PEPCK a c t i v i t y (Saz, 1971). The function of "malic enzyme" recently has been i n dispute (Hammen, 1969; Simpson and Awapara, 1966). In our hands oyster "malic enzyme" i s f u l l y r e v e r s i b l e but the a f f i n i t y constants f o r pyruvate and CC^ are so high (10 mM and 20 mM respectively) that in vivo the enzyme probably functions only i n the d i r e c t i o n of pyruvate production. The "malic enzyme" reaction then could generate the pyruvate which upon transamination accumulates as L-alanine. This indeed i s the documented function of malic enzyme i n helminths (Saz, 1971). CHAPTER VI: Summating Remarks 88 These studies on oyster muscle pyruvate kinase and p-enolpyruvate car-boxykinase have suggested a number of conclusions concerning both the metabolic control of the enzymes and the underlying possible enzyme adaptation to f l u c t u a t i o n s i n 0^ a v a i l a b i l i t y during aero- and anaerobiosis. To conclude t h i s study, a number of these w i l l be discussed as well as probable solutions (based on our data or on the e x i s t i n g l i t e r a t u r e ) to outstanding problems such as (1) sources of alanine and reducing equivalents, (2) other metabolic sources of succinate, and (3) the y i e l d of high energy compounds. A metabolic map f o r probable pathways of anaerobic intermediary metabolism i n molluscan f a c u l t a t i v e anaerobes i s developed. It i s a widely held view i n contemporary biochemistry that the o v e r a l l rate of a multi-enzyme reaction sequence i s l a r g e l y c o n t r o l l e d at a s i n g l e s i t e which i s susceptible to regulation by a c t i v a t i o n or i n h i b i t i o n of the enzyme by s p e c i f i c metabolites. Further, the rate of any given metabolic pathway may be governed by that enzyme i n the pathway whose rate i s minimal. The rates of such c o n t r o l l i n g steps are determined by three f a c t o r s : (1) the amount of the enzyme, (2) the amount of substrate, and (3) the quantities of e f f e c t o r s ( p o s i t i v e and negative). From these studies of the regulatory properties of the oyster enzymes and various examples i n contemporary l i t e r a t u r e , i t appears that even i n a simple formulation a multitude of p o s s i b i l i t i e s e x i s t f or the control of a metabolic process: (1) each pathway i s comprised of many steps and require-ments, (2) a s i n g l e component or substrate can play a number of diverse roles i n r e v e r s a l of the steps, (3) each step can be c o n t r o l l e d by changes i n each of three components - enzyme, substrate and e f f e c t o r s , (4) each component may be modified by i n t e r a c t i o n with others within the c e l l environment, and (5) the components (architecture) and the environment of the c e l l can be 89 responsible f o r the concentration of any one component (enzyme, substrate and ef f e c t o r s ) varying i n the opposite d i r e c t i o n i n the same c e l l . There i s no reason why control should be exerted only by concentration changes i n e i t h e r the enzyme or i n i t s substrates or e f f e c t o r s ; nor are there reasons that control should be exerted excl u s i v e l y at one enzyme rather than at s everal. Rather, i t seems at the present that many metabolic paths are regulated by more than one enzyme and by a v a r i e t y of factors and t h e i r i n t e r -actions. These generalizations are w e l l i l l u s t r a t e d by enzymic regulation of the phosphoenolpyruvate crossroads i n oyster adductor t i s s u e as w e l l as by the o v e r a l l scheme of anaerobic metabolism i n these organisms. The enzymic control of p-enolpyruvate crossroads. The crux of the control problem here i s that two enzymic pathways are a v a i l a b l e f or PEP metabolism. Under aerobic conditions, PEP conversion to pyruvate i s favoured, while under anaerobic conditions, PEP carboxylation to OXA i s favoured. According to the scheme i n F i g . 1, f o r one mole of PEP formed from glucose, two (NADH) reducing equivalents are required f o r , and are u t i l i z e d during, conversion of OXA to succinate. Awapara and h i s co-workers suggested that i n bivalve molluscs, the other mole of PEP i s converted to pyruvate which upon transamination accumulates as alanine (Chen and Awapara, 1969; Stokes and Awapara, 1968). If t h i s scheme i s correct, i t suggests that at the PEP branching point under anaerobic conditions, 50% of the PEP should be carboxylated to OXA while 50% should be a v a i l a b l e f o r pyruvate kinase (PK) catalyzed conversion to pyruvate. Analysis of the a v a i l a b l e data suggests that t h i s i s u n l i k e l y . In the f i r s t place, i t would require extremely t i g h t c o n t r o l of the two enzymes competing for PEP at the PEP branching point. But more s i g n i f i c a n t l y , from demonstrable regulatory properties of the two 90 F i g . VI, 1. Probable pathways of anaerobic intermediary metabolism i n molluscan f a c u l t a t i v e anaerobes. 90«L I NAD NADH G 6 P T F 6 P V FDP) 7 triose-P arginine |-~--&-ATP 3-PG ornithine +urea V P 5 ^ NADH^ \ NAD proline IDP E H NADH-^ NAD-^' Y r • . malute^->-|pyruvaie| NADP NADPH (fumarate] • - 5 * - pyruvate-^qlulamate I proline / V^NADH *' P 5 C mo/o.'e IT " 3 i OXA lg/on'n l^ - : :o : ^etoglutGrar3 NAD-d . NADH>^ ^ succinylCoA GDP -^ NADH NAD {*-GTP L- fumarate^ ^-^l^ccTnolg Cytoplasm Mitochondrion 91 enzymes involved, i t appears that pyruvate kinase and PEPCK operate on a r e c i p r o c a l c o n t r o l , either/or basis; they do not appear able to function  simultaneously. The nub of this argument can be summarized as follows. The a c t i v i t i e s of many enzymes involved i n c e l l u l a r energy metabolism are governed at l e a s t i n part by the energy status of the c e l l . The unusual metabolic " s i g n a l s " are the adenylates: high ATP concentrations often are i n h i b i t o r y to the enzymes involved i n energy metabolism. On the other hand, high AMP l e v e l s (equivalent to low ATP) often are stimulatory (Atkinson, 1968). In oyster muscle, both the PK catalyzed transphosphorylation r e a c t i o n and the PEPCK catalyzed carboxylation reaction generate a high energy phosphate compound (ATP i n the case of PK; ITP or GTP i n the case of PEPCK) and both are subject to product i n h i b i t i o n by these compounds (Mustafa and Hochachka, 1971; Mustafa and Hochachka, 1972b). In this sense, both enzymes behave i n accordance with Atkinson's (Atkinson, 1968) energy charge concept. Whereas these mechanisms undoubtedly contribute to the p h y s i o l o g i c a l "poise" of the PEP branch point, they do not, of themselves, display adequate s p e c i f i c i t y to account f o r t r a n s i t i o n from aerobic to anaerobic metabolism. The s p e c i f i c i t y required can be supplied by H + and L-alanine. It i s widely held that under anaerobic conditions, molluscan bivalves sustain s u b s t a n t i a l a c i d i f i c a t i o n of t h e i r tissues and f l u i d s (Wilbur, 1964). This drop i n pH appears to us to play a p i v o t a l r o l e i n the channelling of PEP away from the PK reaction and towards PEPCK, because the p_H p r o f i l e s f or  PEPCK and PK are e s s e n t i a l l y non-overlapping. In consequence, i n the absence of any other f a c t o r , decreasing pH leads to an automatic i n h i b i t i o n of PK with a concomitant a c t i v a t i o n of PEPCK. At the same time, L-alanine which accumu-lates along with succinate under anaerobic conditions potently i n h i b i t s PK (by increasing the K / P T r p s and decreasing the maximum c a t a l y t i c r a t e ) . It 92 i s p a r t i c u l a r l y i n s t r u c t i v e that the L-alanine i n h i b i t i o n i s potentiated by decreasing pH; for adductor muscle PK the K ^ ^ a i a n : i n e ) a t P H 7.5 i s only 1/6 the K_^  value observed at the optimal pH 8.5 (Mustafa and Hochachka, 1971). In sharp contrast, the primary e f f e c t s of L-alanine on PEPCK appear to be (i) a r e v e r s a l of any r e s i d u a l ITP i n h i b i t i o n and ( i i ) a s l i g h t a c t i v a t i o n at low p-enolpyruvate concentrations due to a reduction i n the apparent ^ m^pgp^ (Mustafa and Hochachka, 1972a, b). Both these e f f e c t s of L-alanine on PEPCK occur at pH ranges i n which PK a c t i v i t y i s very low and i n which L-alanine and ATP i n h i b i t i o n of PK i s unusually extreme. The net e f f e c t of decreasing pH and increasing L-alanine concentration during the aerobic"' ^anaerobic t r a n s i t i o n i s an a u t o c a t a l y t i c increase i n the PEPCK a c t i v i t y concurrent with an exponential decrease i n the PK a c t i v i t y . This would appear to be an adequate arrangement for channelling p-enolpyruvate towards oxaloacetate. Sources of alanine and reducing equivalents. As indicated i n Chapters IV and V, two possible metabolic sources of alanine suggest themselves. ( i ) In our e a r l i e r studies on oyster pyruvate kinases (Mustafa and Hochachka, 1971), we noted that FDP reverses the e f f e c t s of alanine, ATP, and, to a somewhat l e s s e r extent, H +. Such FDP e f f e c t s could supply the bivalve with a mechanism for maintaining some pyruvate kinase function under anaerobic conditions. The pyruvate produced could i n turn be transaminated to alanine. ( i i ) Oyster muscle contains very high a c t i v i t i e s of NADH-linked malic dehydrogenase (MDH) and NADP-linked "malic enzyme" (Mustafa, unpublished data). These enzymes occur i n much higher s p e c i f i c a c t i v i t i e s than do ei t h e r PEPCK or PK. A high cytoplasmic MDH a c t i v i t y i s assumed to function 93 (a) i n the maintenance of low oxaloacetate concentrations thus preventing s i g n i f i c a n t r e v e r s a l of PEPCK a c t i v i t y and (b) i n regenerating NAD for the t r i o s e phosphate dehydrogenase (TDH) rea c t i o n (Saz, 1971). While the function of "malic enzyme" recently has been i n dispute (Hammen, 1969; Chen and Awapara, 1969), i n our hands, oyster "malic enzyme" i s f u l l y r e v e r s i b l e ; however, the a f f i n i t y constants f o r pyruvate and CO^ are so high (Mustafa, unpublished data) that i n vivo the enzyme probably functions only i n the d i r e c t i o n of pyruvate production. This reaction then can generate the pyruvate which upon transamination accumulates as L-alanine (Stokes and Awapara, 1968). A second advantage to the organism could derive from the u t i l i z a t i o n of malic enzyme as the source of pyruvate. As indicated i n Figure 1, two reducing equivalents are produced i n the t r i o s e phosphate dehydrogenase reaction for each mole of glucose; the NAD f o r this r eaction i s regenerated i n the cytosol by the MDH catalyzed reduction of OXA to malate. However, following malate —^fumarate conversion, the fumarate reduction to succinate requires an a d d i t i o n a l reducing equivalent (Fig. 1). Reducing equivalents could be supplied by malic enzyme, which generates CC^, pyruvate and NADPH as products of the reaction. This indeed i s the established function of malic enzyme i n the obligate anaerobe, Ascaris lumbricoides (Saz, 1971). However, i n the l a t t e r case, the enzyme i s NAD-linked and generates NADH within the mitochondria, which can then be u t i l i z e d i n fumarate reduction, also occurring i n the mitochondria. In molluscs, malic enzyme i s located l a r g e l y i n the cytosol and i s NADP-linked. Hence, the NADPH generated by thi s reaction would have to be converted to NADH and transported i n t o the mitochondria i f i t were to d e l i v e r reducing power to the mitochondrial succinoxidase. Although t h i s may occur i n molluscs (Chen and Awapara, 1969), 94 i t i s more probable that the NADPH produced by cytoplasmic malic enzyme i s u t i l i z e d i n reductive steps of f a t t y acid biosynthesis. Fatty acids are, i n f a c t , known to accumulate during anaerobiosis i n other f a c u l t a t i v e anaerobes (Von Brand, 1966) , and the malic enzyme could be implicated i n a s i m i l a r cytoplasmic process i n bivalves. The reducing equivalents required f o r fumarate reduction to succinate presumably are generated by other mitochond-r i a l oxidation-reduction reactions. As w i l l become evident below, the chief candidate f o r t h i s job i s a-ketoglutarate dehydrogenase ( F i g . 1 ) . Other metabolic sources of succinate. Whatever the predominant route of pyruvate formation during anaerobiosis i n bivalves, the primary metabolic f a t e of pyruvate i s transamination to alanine, according to the reaction pyruvate + glutamate > a-ketoglutarate + alanine Hence, i f our considerations are correct, a-ketoglutarate (a-KGA) should be produced i n stochiometric q u a n t i t i e s . This has not been experimentally demonstrated i n molluscs. In any event, i t would be d i f f i c u l t to demonstrate i n i n t a c t or semi-intact preparations f o r two reasons. In the f i r s t place, OXA produced from glucose could also react with glutamate according to the reaction OXA + glutamate > a-ketoglutarate + aspartate This reaction would a f f e c t a-KGA le v e l s and may account f o r the high l e v e l s of aspartate found i n oyster tissues ( F l o r k i n , 1966) . Secondly, these organisms possess the enzymes capable of converting a-ketoglutarate to succinate (Hammen, 1969) and th i s indeed represents another major pathway  for the accumulation of succinate during anaerobiosis ( F i g . 1). This pathway may be p a r t i c u l a r l y important since a v a r i e t y of amino acids could 95 "feed" into i t v i a transamination reactions amino acid + a-KGA > glutamate + keto acid In most organisms, a-KGA produced during pyruvate-glutamate transamina-t i o n i s reconverted to glutamate by glutamate dehydrogenase (GDH), a reaction which u t i l i z e s NADH. In f a c u l t a t i v e anaerobes, the s p e c i f i c a c t i v i t y of GDH i s very low (Campbell and Bishop, 1970) while a-ketoglutarate dehydrogenase a c t i v i t i e s are very high. For these reasons the a-ketoglutarate dehydrogenase reaction would probably outcompete the GDH reaction f or the common substrate, a-KGA. Under these conditions, the glutamate-alanine transaminase serves to channel a-KGA d i r e c t l y towards a-ketoglutarate dehydrogenase. Functional s i g n i f i c a n c e of two routes to succinate. The f a c u l t a t i v e anaerobe gains a c r i t i c a l energetic advantage by u t i l i z -ing t h i s route: the o v e r a l l a-ketoglutarate dehydrogenase reaction, a-KGA + CoASH + NAD succinyl CoA + C0 2 + NADH sets the stage for the conversion of t h i o l e s t e r bond energy into nucleoside triphosphate. The reaction, catalyzed by s u c c i n i c thiokinase, i s highly exergonic and can u t i l i z e e i t h e r GDP or IDP as cosubstrate, generating GTP or ITP. This energy y i e l d i n g reaction i s u t i l i z e d as an anaerobic mechanism f o r supplanting aerobic metabolism i n c e r t a i n mammalian tissues (Cohen, 1968), and presumably has been selected f or an analogous function i n f a c u l t a t i v e anaerobic invertebrates. In the event that the organism u t i l i z e s the a-ketoglutarate dehydrogenase pathway, some pr o v i s i o n must be made f o r the regeneration of NAD required for the reaction. The most l i k e l y candidate f o r the job i s fumarate reductase, which couples the oxidation of NADH with the reduction of fumarate to succinate 96 ( F i g . 1 ) . I n a d d i t i o n , f u m a r a t e r e d u c t a s e i s p r o p e r l y p o s i t i o n e d i n t h e m i t o c h o n d r i o n f o r t h e d e l i v e r y o f NAD t o the a - k e t o g l u t a r a t e dehydrogenase r e a c t i o n . From t h e s e c o n s i d e r a t i o n s , one can v i e w t h e u n i q u e pathway o f a n a e r o b i c g l u c o s e m e t a b o l i s m i n f a c u l t a t i v e anaerobes as a means f o r " p r i m i n g " t h e f l o w o f g l u t a m a t e —>a-KGA > s u c c i n y l C o A — ^ . s u c c i n a t e , by (1) s u p p l y i n g p y r u v a t e f o r t h e t r a n s a m i n a s e r e a c t i o n and (2) r e g e n e r a t i n g NAD t h r o u g h f u m a r a t e r e d u c t i o n f o r t h e a - k e t o g l u t a r a t e dehydrogenase r e a c t i o n ( F i g . 1 ) . The y i e l d o f h i g h energy phosphate compounds. To d a t e , a l t h o u g h the p e c u l i a r n a t u r e o f a n a e r o b i c m e t a b o l i s m i n b i v a l v e m o l l u s c s has been r e c o g n i z e d , l i t t l e a t t e n t i o n has been g i v e n t o i t s f u n c t i o n -a l a s p e c t s . From t h e s e r e s u l t s i t appears t h a t by u t i l i z i n g t h i s m e t a b o l i s m the o r g a n i s m g a i n s a d i s t i n c t e n e r g e t i c advantage o v e r t h o s e w h i c h r e l y s o l e l y upon g l y c o l y s i s f o r a n a e r o b i c energy p r o d u c t i o n . I n the c l a s s i c a l g l y c o l y t i c scheme, f o r 1 mole o f g l u c o s e m e n t i o n e d , a n e t g a i n o f 2 moles o f ATP i s o b t a i n e d . I n t h e f a c u l t a t i v e a n aerobe, f o r 1 mole o f g l u c o s e and 1 mole a-k e t o g l u t a r a t e m e t a b o l i z e d , a n e t g a i n o f a t l e a s t 3 moles o f h i g h energy n u c l e o s i d e t r i p h o s p h a t e can be g e n e r a t e d , assuming an o b l i g a t e r e d o x c o u p l i n g between f u m a r a t e r e d u c t a s e and a - k e t o g l u t a r a t e dehydrogenase. Known e n e r g y - y i e l d i n g r e a c t i o n s a r e (1) the p h o s p h o g l y c e r a t e k i n a s e t r a n s p h o s p h o r y l a t i o n , (2) t h e PEPCK c a r b o x y l a t i o n o f PEP, (3) t h e p y r u v a t e k i n a s e t r a n s p h o s p h o r y l a t i o n , (4) a c e t i c t h i o k i n a s e , and (5) s u c c i n i c t h i o k i n -a s e . 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