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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. M.Sc.  (Hons.),  U n i v e r s i t y o f K a r a c h i , P a k i s t a n , 1965 U n i v e r s i t y o f K a r a c h i , P a k i s t a n , 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  IN THE DEPARTMENT of ZOOLOGY  We accept  THE  t h i s t h e s i s as conforming t o t h e required standard:  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  an advanced degree at the U n i v e r s i t y the L i b r a r y I further  s h a l l make i t  freely  f u l f i l m e n t o f the r e q u i r e m e n t s of B r i t i s h C o l u m b i a , I agree  available for  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  of t h i s thesis f o r written  It  copying of t h i s  i s u n d e r s t o o d that c o p y i n g o r  thesis  f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my  ( TAEIQ MUSTAFA )  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^  M  U  or  publication  permission.  Department of  that  r e f e r e n c e and s t u d y .  f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department by h i s r e p r e s e n t a t i v e s .  for  ABSTRACT C a t a l y t i c and r e g u l a t o r y p r o p e r t i e s o f p y r u v a t e k i n a s e (EC 2.7.1.40) and phosphoenolpyruvate were s t u d i e d .  carboxykinase  (EC 4.1.1.32) from o y s t e r adductor muscle  P a r t i c u l a r a t t e n t i o n was  g i v e n to those p r o p e r t i e s o f the  enzymes which c o u l d h e l p to e x p l a i n the " s w i t c h o v e r " from a e r o b i c to anaerob i c g l u c o s e d e g r a d a t i o n between a e r o ^ = ^ a n a e r o b i c c o n d i t i o n s i n a f a c u l t a t i v e anaerobe,  such as the o y s t e r .  A l l the a v a i l a b l e d a t a can be summarized  as  follows: S i t u a t e d s t r a t e g i c a l l y a t the and a n a e r o b i c metabolism  primary b r a n c h i n g p o i n t between a e r o b i c  a r e the two  enzymes, p y r u v a t e k i n a s e ( f a v o u r e d d u r i n g  a e r o b i o s i s ) and P - e n o l p y r u v a t e c a r b o x y k i n a s e H  +  (favoured d u r i n g a n a e r o b i o s i s ) .  i o n p l a y s a p i v o t a l r o l e i n the c h a n n e l l i n g of P - e n o l p y r u v a t e through  branching point.  When 0  2  i s absent, the pH i s known to drop because  build-up of various acid products.  In the absence  this  of the  o f any o t h e r f a c t o r s ,  this  would l e a d to an e f f e c t i v e a c t i v a t i o n of p - e n o l p y r u v a t e c a r b o x y k i n a s e due to (1) an i n c r e a s e i n a b s o l u t e a c t i v i t i e s , and for p-enolpyruvate.  affinity  A t the same time p y r u v a t e k i n a s e would be i n h i b i t e d  (1) a d e c r e a s e i n the a b s o l u t e a c t i v i t y , and p-enolpyruvate.  (2) an i n c r e a s e i n the  A l a n i n e and ATP  by  (2) a d e c r e a s e i n the a f f i n i t y f o r  i n h i b i t i o n of p y r u v a t e k i n a s e p o t e n t i a t e  these e f f e c t s p a r t i c u l a r l y at low pH.  In c o n t r a s t , a l a n i n e e f f e c t i v e l y  v a t e s p - e n o l p y r u v a t e c a r b o x y k i n a s e by r e v e r s i n g ITP i n h i b i t i o n .  acti-  The more  a c i d i c the a n a e r o b i c system becomes, the more a v i d l y would p - e n o l p y r u v a t e c a r b o x y k i n a s e channel p - e n o l p y r u v a t e towards o x a l o a c e t a t e .  Upon r e t u r n to  a e r o b i c c o n d i t i o n s , the pH would be expected to r i s e a g a i n and a l l o f the above events would be r e v e r s e d . The most important f e a t u r e o f such a r e g u l a t o r y system i s t h a t i t i s a k i n d of a u t o c a t a l y t i c cascade.  Once e i t h e r p y r u v a t e k i n a s e a c t i v a t i o n o r  p-enolpyruvate carboxykinase activation i s i n i t i a t e d , a l l the various regulatory interactions potentiate one another.  There i s l i t t l e doubt that the  specific control components at this point i n the metabolism of molluscan facultative anaerobes are the outcome of selective t a i l o r i n g of the 2 enzymes functioning at this point.  What evolution seems to have done i n molluscs was  to arrange the control characteristics of pyruvate kinase and p-enolpyruvate carboxykinase i n a reciprocal manner so that the two reactions cannot be f u l l y active simultaneously.  Ill  TABLE OF CONTENTS Abstract L i s t of  Page i  Tables  vi  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  a n a e r o b i c way  of  life  The c a r b o x y l a t i o n r e a c t i o n s i n i n v e r t e b r a t e facultative  anaerobes  O x i d a t i o n o f c y t o p l a s m i c NADH Phosphoenolpyruvate of Chapter I I :  4  c r o s s r o a d s and the statement  6 8  the problem  M a t e r i a l s and Methods  12  Animal c o l l e c t i o n  12  and t h e i r h a n d l i n g  P r e p a r a t i o n of mantle and adductor muscle  12  pyruvate kinases P r e p a r a t i o n of m i t o c h o n d r i a  13  P r e p a r a t i o n o f c y t o p l a s m i c phosphoenolpyruvate  13  c a r b o x y k i n a s e , malate dehydrogenase and " m a l i c enzyme" Assay of p y r u v a t e k i n a s e a c t i v i t y  14  Assay  15  o f phosphoenolpyruvate c a r b o x y k i n a s e  (carboxylation reaction) a c t i v i t y Assay  of phosphoenolpyruvate c a r b o x y k i n a s e  15  (decarboxylation reaction) a c t i v i t y Assay of " m a l i c enzyme" a c t i v i t y  16  iv Page Assay  o f malate  and l a c t a t e dehydrogenases  E s t i m a t e s of p r o t e i n  contents  17  Electrophoresis  17  I s o e l e c t r i c focusing  o f p y r u v a t e k i n a s e s and  of phosphoenolpyruvate Chapter  III:  18  carboxykinase  C a t a l y t i c and R e g u l a t o r y P r o p e r t i e s Pyruvate  17  of Oyster  19  Kinases  Introduction  19  Results  21  Electrophoresis  and e l e c t r o f o c u s i n g  21  C a t i o n requirements  21  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 2 m  24  Effect  Effect  of pH and f r u c t o s e - l , 6 - P „ on K of z m  24  p-enolpyruvate ATP  inhibition  Interacting the K  m  28  e f f e c t s o f ATP  and f r u c t o s e - 1 , 6 - P 2 on  of p-enolpyruvate  Search f o r o t h e r modulators  39  Nature o f . a l a n i n e  39  and p h e n y l a l a n i n e i n h i b i t i o n  Discussion Chapter  IV:  32  A3  C a t a l y t i c and R e g u l a t o r y P r o p e r t i e s Phosphoenolpyruvate  of Oyster  47  Carboxykinase  Introduction  47  Results  49  Electrofocusing  49  Page Requirements f o r t h e p - e n o l p y r u v a t e c a r b o x y k i n a s e catalyzed carboxylation  49  reaction  T i s s u e and s u b - c e l l u l a r d i s t r i b u t i o n o f o y s t e r p-enolpyruvate carboxykinase  49  activity  R e v e r s i b i l i t y o f pH e f f e c t  53  I n o s i n e diphosphate s a t u r a t i o n k i n e t i c s  58  P-enolpyruvate s a t u r a t i o n k i n e t i c s  61  j | Cu  Chapter  V:  inhibition kinetics  64  Discussion  67  C a t a l y t i c and R e g u l a t o r y P r o p e r t i e s o f O y s t e r Phosphoenolpyruvate Carboxykinase: I I . Regulation  71  of the Enzyme A c t i v i t y and i t s F u n c t i o n i n Phosphoenolpyruvate  Metabolism  Introduction  71  Results  73  Nature o f i n o s i n e t r i p h o s p h a t e (ITP) i n h i b i t i o n  73  Search f o r o t h e r m e t a b o l i c e f f e c t o r s L-alanine  Chapter V I :  effects  78 82  Discussion  85  Summating Remarks  88  The enzymic c o n t r o l a t p - e n o l p y r u v a t e c r o s s r o a d s  89  Sources o f a l a n i n e and r e d u c i n g e q u i v a l e n t s  92  Other  94  metabolic sources of s u c c i n a t e  P o t e n t i a l m e t a b o l i c sources o f glutamate The y i e l d o f h i g h energy phosphate compounds Chapter  VII:  Literature Cited  96 98  VI LIST OF TABLES Table I I I , 1.  Page E f f e c t o f pH on K  ( p - e n o l p y r u v a t e ) i n absence and  29  presence o f f r u c t o s e - l j G - P ^ I I I , 2.  E f f e c t of ATP and f r u c t o s e - 1 , 6 - P of  I I I , 3.  mantle p y r u v a t e  2  on the K  m  (p-enolpyruvate)  33  kinase  Comparison of the p r o p e r t i e s o f mantle, adductor, r a t  44  muscle and l i v e r p y r u v a t e k i n a s e s IV, 1.  Components of p - e n o l p y r u v a t e c a r b o x y k i n a s e c a t a l y z e d carboxylation  IV, 2.  IV, 3.  reaction  T i s s u e 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 o y s t e r p - e n o l pyruvate  51  52  carboxykinase  Reversibility  o f pH e f f e c t on enzyme a c t i v i t y  56  LIST OF FIGURES Figure III,  1.  E l e c t r o p h o r e t i c r e s o l u t i o n o f o y s t e r mantle, g i l l and adductor p y r u v a t e  I I I , 2.  kinase a c t i v i t y  E l e c t r o f o c u s i n g p a t t e r n o f the mantle  and adductor  pyruvate kinases I I I , 3.  E f f e c t of f r u c t o s e - l 6 P 2 ° -  and adductor p y r u v a t e I I I , 4.  n  t  n  optima o f mantle  e  }  kinase  E f f e c t o f fructose-1,6-P„ on the K /.„.., o f mantle 2 m(ADP) and adductor p y r u v a t e k i n a s e s  I I I , 5.  E f f e c t o f pH and fructose-1,6-P„ on the K o f p - e n o l 2 m p y r u v a t e o f mantle p y r u v a t e k i n a s e  I I I , 6.  E f f e c t o f pH and fructose-1,6-P„ on the K o f p - e n o l 2 m p y r u v a t e o f adductor p y r u v a t e k i n a s e r  I I I , 7.  E f f e c t o f ATP c o n c e n t r a t i o n on the r e a c t i o n r a t e and ATP,.. . d e t e r m i n a t i o n o f mantle i  and adductor p y r u v a t e  k i n a s e s a t d i f f e r e n t pH and p - e n o l p y r u v a t e c o n c e n t r a t i o n s I I I , 8.  Double r e c i p r o c a l p l o t s o f t h e r e a c t i o n v e l o c i t y a g a i n s t p - e n o l p y r u v a t e o f mantle  and adductor p y r u v a t e k i n a s e s a t  d i f f e r e n t ATP c o n c e n t r a t i o n s a t pH 8.5 I I I , 9.  Interacting  e f f e c t s o f ATP and f r u c t o s e - l ^ - P ^ on the  , . f o r t h e mantle enzyme a t pH 8.5 K. , m(p-enolpyruvate) I I I , 10. I n t e r a c t i n g e f f e c t s o f ATP and f r u c t o s e - 1 , 6 - P  2  on the  K , , f o r t h e mantle enzyme a t pH 7.5 m(p-enolpyruvate) x  I I I , 11. E f f e c t o f  L - a l a n i n e c o n c e n t r a t i o n on the r e a c t i o n  rate  and a l a n i n e (K\) d e t e r m i n a t i o n f o r adductor and mantle pyruvate kinases at  two d i f f e r e n t pH values  Vlll  Page I I I , 12.  E f f e c t of p h e n y l a l a n i n e c o n c e n t r a t i o n on the r e a c t i o n  38  r a t e and p h e n y l a l a n i n e (K^) d e t e r m i n a t i o n f o r mantle and adductor p y r u v a t e k i n a s e s a t two d i f f e r e n t pH v a l u e s III,  13.  Double r e c i p r o c a l p l o t s  of the r e a c t i o n  v e l o c i t y against  40  p - e n o l p y r u v a t e of mantle p y r u v a t e k i n a s e i n presence o f a l a n i n e at pH 8.5 I I I , 14.  and  7.5  Double r e c i p r o c a l p l o t s  of the r e a c t i o n  v e l o c i t y of  42  mantle and adductor p y r u v a t e k i n a s e i n presence of p h e n y l a l a n i n e and IV, 1.  Electrofocusing carboxykinase  IV, 2.  p a t t e r n of the adductor p - e n o l p y r u v a t e  50  activity  E f f e c t s of pH on p - e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y i n presence of Zn  IV, 3.  fructose-l^-P^  I |  and  Mn  | |  E f f e c t s of metal i o n a c t i v a t i o n a t d i f f e r e n t pH v a l u e s i n presence of Mn  I |  o r Zn  54  57  | |  I| IV, 4.  E f f e c t s of Mn 6.0  and  on the IDP s a t u r a t i o n  E f f e c t s of Zn at pH  IV, 6.  59  7.0 I |  IV, 5.  k i n e t i c s a t pH  | | and Mn  on the IDP  saturation  kinetics  60  k i n e t i c s at  62  kinetics  63  5.1  E f f e c t s of Mn  on p - e n o l p y r u v a t e s a t u r a t i o n  d i f f e r e n t pH v a l u e s IV, 7.  E f f e c t s o f Zn  on p - e n o l p y r u v a t e s a t u r a t i o n  at d i f f e r e n t pH v a l u e s IV, 8.  I n h i b i t i o n of p - e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y by increasing or Mn  IV, 9.  c o n c e n t r a t i o n of Cu  at pH 5.1  P-enolpyruvate  and  65  i n presence o f Zn  6.0  carboxykinase a c t i v i t y  as a f u n c t i o n  of  66  XX  Page  the  c o n c e n t r a t i o n o f Mn  I |  and Zn  j |  i n the presence or  _| L.  absence o f 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  (1/v  75  I| vs 1/Zn  ) a t v a r y i n g ITP c o n c e n t r a t i o n s .  I j V, 2.  Mn  saturation  V, 3.  I j vs 1/Mn ) a t v a r y i n g ITP c o n c e n t r a t i o n s . IDP s a t u r a t i o n 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  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 I| vs 1/IDP) a t v a r y i n g ITP c o n c e n t r a t i o n s i n presence of Zn  76  V, 4.  IDP s a t u r a t i o n  77  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 I |  vs 1/IDP) a t v a r y i n g ITP c o n c e n t r a t i o n s i n p r e s e n c e o f Mn V, 5.  Double r e c i p r o c a l p l o t s  (1/v vs 1/p-enolpyruvate) a t v a r y i n g  79  ITP c o n c e n t r a t i o n s a t pH 5.1 and 6.0. V, 6.  P-enolpyruvate s a t u r a t i o n plot  k i n e t i c s and i t s double r e c i p r o c a l  (1/v v s 1/p-enolpyruvate) w i t h v a r y i n g I|  of ITP and a l a n i n e w i t h Mn  as the d i v a l e n t  80  concentrations  metal i o n at  pH 6.0. V, 7.  P-enolpyruvate s a t u r a t i o n plot  k i n e t i c s and i t s double r e c i p r o c a l  81  (1/v v s 1/p-enolpyruvate) i n the presence and absence  of 0.25 mM GTP. V, 8.  P-enolpyruvate s a t u r a t i o n  k i n e t i c s and i t s double r e c i p r o c a l I |  p l o t w i t h v a r y i n g c o n c e n t r a t i o n s o f ITP and a l a n i n e w i t h Zn as t h e d i v a l e n t  m e t a l i o n a t pH 6.0.  84  X  ACKNOWLEDGMENTS To my  r e s e a r c h s u p e r v i s o r Dr. P e t e r W.  Hochachka who  i n t r o d u c e d me  the problems of environmental b i o c h e m i s t r y , many thanks a r e extended w i s e , warm and i n t e l l i g e n t s u g g e s t i o n s and alive  throughout  c r i t i c i s m ; these kept my  t h i s study to r e v e a l the m y s t e r i e s of f a c u l t a t i v e  to  for his interests  anaerobio-  sis i n molluscs. I would l i k e to thank the members of my  committee, D r s . W.  S. Hoar,  J . E. P h i l l i p s o f Department o f Zoology and Dr. D. H. Copp of Department o f Physiology f o r t h e i r  comments and c r i t i c i s m .  A l s o , I would l i k e to thank my  c o l l e a g u e s , Thomas Moon and T e r r y Owen f o r t h e i r i n v a l u a b l e h e l p i n c o l l e c t i n g the r e s e a r c h animals. moral support from And,  finally,  I must thank my mother, s i s t e r s and b r o t h e r s f o r t h e i r  12,000 m i l e s away throughout my graduate work a t U. B. C.  the Canadian  I n t e r n a t i o n a l Development Agency supported  through the Canadian Commonwealth S c h o l a r s h i p program, and Research  myself  the N a t i o n a l  C o u n c i l o f Canada p r o v i d e d l o g i s t i c support throughout  this  study.  CHAPTER I : Introduction  1 There i s good g e o c h e m i c a l and b i o l o g i c a l e v i d e n c e s u g g e s t i n g t h a t arose  under r e d u c i n g c o n d i t i o n s  i n t h e t o t a l absence o f m o l e c u l a r  life  oxygen.  U n d e r l y i n g c e l l u l a r m e t a b o l i s m presumably was l i n k e d t o and " d r i v e n b y " substrate-level phosphorylations as  electron acceptors.  0^ became  viiere o r g a n i c s u b s t r a t e , and n o t C^, a c t e d  Because o f i t s h i g h a f f i n i t y f o r e l e c t r o n s , m o l e c u l a r  t h e most i m p o r t a n t e l e c t r o n a c c e p t o r 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 t h e h y d r o s p h e r e and atmosphere. 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  c a p t u r e bond energy as  nucleotide  of n u t r i e n t molecules  I t s high electron avidity  which the organism could  i n "high chemical p o t e n t i a l " form,  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 .  the b a s i c f a b r i c o f i n t e r m e d i a r y m e t a b o l i s m i s f u n d a m e n t a l l y  Y e t t o t h i s day, anaerobic  (e.g.  g l y c o l y s i s , t h e shunt pathway, amino a c i d m e t a b o l i s m , 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 m o l e c u l a r 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 added  onto  embellishments  an a l r e a d y f u n c t i o n a l framework o f a n a e r o b i c m e t a b o l i s m (Wald,  1964). Because  o f t h e use o f  c l a s s i c a l l a b o r a t o r y animals  f o r most b i o c h e m i c a l  s t u d i e s , t h e degree t o w h i c h 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 a n a e r o b i c mechanisms i s not widely  t o s u s t a i n temporary o r i n d e f i n i t e p e r i o d s o f a n o x i a  appreciated.  N e v e r t h e l e s s , i t i s now c l e a r t h a t many i n v e r t e -  b r a t e s a r e f a c u l t a t i v e anaerobes,  capable o f 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 o f 0^ and c a p a b l e o f a c t i v e o x i d a t i v e m e t a b o l i s m i n i t s p r e s e n c e . Examples are p a r t i c u l a r l y Brand,  1966; B r a y n t ,  e v i d e n t t h a t many 1969;  common among w i d e l y d i v e r s i f i e d h e l m i n t h s (Von  1970), b u t d u r i n g r e c e n t  y e a r s i t has a l s o become  m o l l u s c s have comparable m e t a b o l i c c a p a c i t i e s (Hammen,  Chen and Awapara, 1969). Newell  (197o) concluded  t h a t many o f t h e i n t e r t i d a l m o l l u s c s a r e  adapted t o a n a e r o b i o s i s , s u r v i v i n g l o n g  p e r i o d s o f time i n an oxygen f r e e  2 environment.  Mya a r e n a r i a  f o r example s u r v i v e s 8 days w i t h o u t oxygen  ( R i c k e t t s and C a l v i n s , 1948), w h i l e L i t t o r i n a n e r i t o i d e s and _L. p u n c t a t a can survive  a n i t r o g e n atmosphere f o r s e v e r a l days (Patane, 1946, 1955).  cuneata  have been kept i n deoxygenated water f o r 3 weeks w i t h o u t apparent  harm  to  the animal (Chen  b u i l d up pumping  an  oxygen  and Awapara, 1969).  close t h e i r valves Oyster  is  coined i n  like  B i v a l v e s such as M. a r e n a r i a  debt during a n a e r o b i o s i s to  r a t e vhen the t i d e r e t u r n s .  Rangia  be p a i d by i n c r e a s i n g the  Others such as t h e R. cuneata simply  and l i v e w i t h o u t oxygen f o r extended p e r i o d s o f time. Rangia  i n t h i s regard.  The term " f a c u l t a t i v e anaerobe" i s  the l i t e r a t u r e f o r those organisms which can s u s t a i n a n o x i a 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 It  pathway  i s g e n e r a l l y a c c e p t e d t h a t the d e g r a d a t i o n o f the g l u c o s e t o  p y r u v a t e i n many  organisms i s by  r e a c t i o n s , which i s ,  in  a sense,  fermentative processes u n d e r l i e all  types o f c e l l s  oxygen  f o r an a n a e r o b i c way o f l i f e .  way o f t h e Embeden-Meyerhof sequence o f t h e b a s i c way o f l i f e .  a l l o t h e r forms o f metabolism, and v i r t u a l l y  can s u r v i v e f o r p e r i o d s on  (Wald, 1964).  There  G l y c o l y t i c or  g l y c o l y s i s i f deprived of  i s evidence f o r b e l i e v i n g t h a t most o f t h e  i n d i v i d u a l r e a c t i o n s o f t h e Embeden-Meyerhof scheme a r e o p e r a t i v e i n many f a c u l t a t i v e anaerobic i n v e r t e b r a t e s . glucose position  the the  is  Bueding (1962) has made i t c l e a r  that  degraded through the same r e a c t i o n s i n t o p y r u v a t e b u t t h e d i s -  of pyruvate  varies  s u b s t a n t i a l l y from one organism t o another.  The p a r a s i t i c h e l m i n t h s have  been t h e s u b j e c t o f much r e s e a r c h i n t o  nature  The breakdown o f g l y c o g e n i n t o g l u c o s e and  o f t h e i r metabolism.  subsequent  dimunita  g l y c o l y t i c r e a c t i o n s have been i n v e s t i g a t e d i n Hymenolepis  (Read, 1951), A s c a r i s l u m b r i c o i d e s  (Bueding and Saz, 1968),  3 D i r o f i l a r i a immltis these  helminths  except  that l i t t l e  down.  i t was apparent  In l i e u o f  which  (Hutchinson and M c N e i l ,  o r no l a c t a t e c o u l d be measured when g l y c o g e n was broken lactate,  a v a r i e t y o f o t h e r p r o d u c t s were formed, among  anaerobic disappearance  o y s t e r muscle, and the d i s a p p e a r a n c e by the f o r m a t i o n o f  same o b s e r v a t i o n has been  (Bueding,  o f glycogen without  f o r m a t i o n i s n o t l i m i t e d to helminths.  entirely  In a l l o f  that t h e g l y c o l y t i c pathway i s i n o p e r a t i o n ,  s u c c i n a t e and f a t t y a c i d s were i d e n t i f i e d The  for  1970), and o t h e r s .  1962).  a concomitant  lactate  T h i s s i t u a t i o n i s known to o c c u r i n  o f g l u c o s e a n a e r o b i c a l l y i s n o t accounted pyruvate o r l a c t a t e  (Humphrey, 1944).  made i n some f r e s h - w a t e r s n a i l s  The  (von Brand, et^ a l . ,  1950). At t h i s j u n c t u r e i t was e v i d e n t t h a t i n f a c u l t a t i v e anaerobes such as helminths  and o y s t e r s , the f o r m a t i o n o f p y r u v a t e  the u n i v e r s a l l y o c c u r r i n g g l y c o l y t i c p y r u v a t e was a t v a r i a n c e then  some  during  with the  o t h e r p r o v i s i o n must  the o x i d o - r e d u c t i o n s t e p  c o u l d i n f a c t proceed by  route b u t t h e f i n a l d i s p o s i t i o n o f r e a c t i o n scheme.  I f l a c t a t e i s n o t formed  be made f o r t h e r e o x i d a t i o n o f the NADH formed of g l y c o l y s i s .  Bueding (1963) c o n s i d e r e d  t h i s problem f o r the p a r a s i t i c worm, A s c a r i s l u m b r i c o i d e s and concluded the  formed  pyruvate  c o u l d r e a c t w i t h CO^ t o i s based  that  form malate and e v e n t u a l l y  fumarate.  This conversion  NAD-linked  " m a l i c enzyme" to c a t a l y z e t h e c a r b o x y l a t i o n r e a c t i o n and a l s o  fumarase (Saz and Hubbard, 1957). Kemetec and Bueding that  on t h e o b s e r v a t i o n t h a t A s c a r i s i has a  In a d d i t i o n to these experimental  (1963) d i s c o v e r e d a l s o  f u n c t i o n s w i t h NADH.  In  i n A s c a r i s a fumarate r e d u c t a s e  o t h e r s u p p o r t i n g experiments,  14 14 (1959), demonstrated t h a t C from l a c t a t e - 3 - C appears carbon  of succinate.  facts,  Saz and V i r d i n e  i n t h e methelyne  Although mechanisms i n v o l v e d i n t h e d i s p o s i t i o n o f  f i n a l g l y c o l y t i c end p r o d u c t s  d i f f e r i n v a r i o u s f a c u l t a t i v e anaerobes,  a  4 c a r b o x y l a t i o n step during is  and the e f f i c i e n t removal ( o r r e o x i d a t i o n ) o f NADH formed  g l y c o l y s i s as a r e s u l t o f t h e o x i d a t i o n o f g l y c e r a l d e h y d e - 3 - p h o s p h a t e  mandatory f o r the c o n t i n u a t i o n of a n a e r o b i c  The  carboxylation reactions i n invertebrate CC^  f i x a t i o n r e a c t i o n s play important,  r o l e s i n a v a r i e t y of metabolic i f not a l l ,  1963;  f a c u l t a t i v e anaerobes. a l t h o u g h somewhat s p e c i a l i z e d ,  areas and i t i s n o t too much t o say t h a t most,  h e t r o t r o p h i c c e l l s a r e dependent on CO^ f i x a t i o n r e a c t i o n s .  Carboxylation (Hammen  glycolysis.  r e a c t i o n s have been shown to  be q u i t e common i n i n v e r t e b r a t e s  and W i l b u r , 1959; Hammen and Osborne, 1959; Awapara and Campbell,  Simpson  and Awapara, 1964).  s u c c i n a t e appears as the main invertebrates  One o f the most common o b s e r v a t i o n s  i s that  p r o d u c t o f c a r b o x y l a t i o n i n a v a r i e t y o f marine  ( i n 14 species r e p r e s e n t i n g  12 p h y l a from P o r i f e r a to _Hemi-  14 chordata) when g i v e n NaHC was  tempting  to  0^ (Hammen and Osborne, 1959),  assume t h a t  At t h a t time, i t  t h e primary s t e p was t h e c a r b o x y l a t i o n o f  propionate-CoA and Hammen and W i l b u r (1959) f i r s t made t h i s s u g g e s t i o n . did  in  f a c t o b t a i n s u c c i n a t e upon i n c u b a t i o n o f p r o p i o n a t e  L a t e r , the f o r m a t i o n  o f s u c c i n a t e as the end p r o d u c t o f a p r i m a r y  t i o n r e a c t i o n became u n t e n a b l e , fixation  were  with  They  o y s t e r mantle. carboxyla-  for organisms known t o form s u c c i n a t e by CO^  found to l a c k p r o p i o n y l - C o A c a r b o x y l a s e  activity  (Simpson and  Awapara, 1964). Hammen (1966), on the b a s i s o f  i n d i c a t e d that malic spectrophotometric  t h e p a t t e r n of l a b e l l i n g o f the a c i d s 14  a c i d was t h e i n i t i a l major product o f NaHC  evidence f o r t h e presence o f a " m a l i c enzyme" i n t h e  s u p e r n a t a n t o f homogenate was demonstrated. tion  0^ f i x a t i o n ;  to homogenate c o n c e n t r a t i o n  The r e d u c t i o n o f NADP i n p r o p o r -  i n d i c a t e s t h a t malate may be formed by t h e  a c t i o n o f a m a l i c enzyme of the type f i r s t  d e s c r i b e d by 0choa,e£ al_.  (1948).  5 Hammen  (1966) c o r r e c t l y  r u l e out  asserts that  the p r e s e n c e o f  the p r e s e n c e o f " m a l i c enzyme" does not  o t h e r c a r b o x y l a t i n g enzymes i n the same t i s s u e ,  i n d e e d t h e r e i s e v i d e n c e f o r two o t h e r s : and W i l b u r , 1959) occurs  in  as mentioned  and  p r i o p i o n y l - C o A c a r b o x y l a s e (Hammen  e a r l i e r , w h i l e phosphoenolpyruvate.carboxykinase  t i s s u e s o f R. cuneata (Simpson and Awapara, 1964), and i n h e l m i n t h s  (Saz and L e s c u r e , 1969). M o l l u s c s have a v e r y b i v a l v e m o l l u s c , ]L  active  cuneata, which i s one o f  enzyme among i n v e r t e b r a t e s enzyme was  phosphoenolpyruvate c a r b o x y k i n a s e .  In one  the r i c h e s t s o u r c e s o f t h i s  (Simpson and Awapara, 1964), the a c t i v i t y of the  found to be on the average t e n times h i g h e r than i n c h i c k e n l i v e r .  A r a p i d c a r b o x y l a t i o n o f phosphoenolpyruvate c o u l d r e a d i l y account f o r the r a p i d f o r m a t i o n of s u c c i n a t e from CO^ i n g a c a r b o x y l a t i o n of p y r u v a t e studies  on  i n the marine organisms w i t h o u t i n v o l v -  o r the n e c e s s i t y of i t s f o r m a t i o n .  R. cuneata r e v e a l e d t h a t i t  of s u c c i n a t e and v e r y l i t t l e Simpson  lactate  produced from g l u c o s e l a r g e amounts  (Simpson and Awapara, 1966).  and Awapara (1966), on the b a s i s o f b a l a n c e s t u d i e s  distribution,  Further  concluded t h a t g l u c o s e i s degraded i n t h i s s p e c i e s  and  isotope  and perhaps  i n o t h e r m o l l u s c s to phosphoenolpyruvate w i t h much o f the phosphoenolpyruvate diverted ultimately  to s u c c i n a t e by r e a c t i n g w i t h CO^  c o n t r a s t , only a s m a l l amount of p y r u v a t e carboxlase  as is  o n l y p r e s e n t i n s m a l l amounts and p y r u v a t e  In o r d e r to d e c i d e which pathway predominates, they  i n c u b a t e d mantle t i s s u e from R. cuneata w i t h glucose-6-C and  with  pyruvate-3-C  In  o f s u c c i n a t e i s formed from the c a r b o x y l a t i o n  the m a l i c enzyme i s absent.  at a r a p i d r a t e .  14  in  another.  p-enolpyruvate reacts with  i n one  experiment,  The e f f i c i e n c y o f s u c c i n a t e formation  i s about ten times g r e a t e r w i t h glucose-6-C From these experiments i t  14  14  as  compared to pyruvate-3-C  14  became reasonable to assume t h a t most o f the C0„ to form o x a l o a c e t a t e and u l t i m a t e l y  succinate.  6  These  workers have  p o s t u l a t e d t h e f o l l o w i n g r e a c t i o n sequence f o r s u c c i n a t e  formation: p-enolpyruvate  C0 ——  Oxaloacetate  Chen and Awapara (1969) found of  the enzymes-catalyzing  r e d u c t i o n o f fumarate,  the s i n g l e  cephalopod  the f o l l o w i n g r e a c t i o n : NADH2  problems  dehydrogenase  Pecten  octopine that c o n t a i n o c t o p i n e dehydro-  However, t h e r e g e n e r a t i o n o f NAD f o r t r i o s e  dehydrogenase i n most  f a c u l t a t i v e anaerobes p r e s e n t s some unusual  i n metabolic organization.  Oxidation  of  c y t o p l a s m i c NADH.  A feature of aerobic glycolysis glyceraldehyde  i t s e l f must  be a b l e  t r a n s p o r t system by  indirect  i n the  E i t h e r t h e NADH  to  or else reducing  t r a n s f e r r e d to the m i t o c h o n d r i a l e l e c t r o n  routes c a l l e d " s h u t t l e s " .  t h a t the r e s p i r a t o r y  inaccessible  i n the mitochondria.  t o p e n e t r a t e a c r o s s the m i t o c h o n d r i a n  e q u i v a l e n t s from t h e NADH must be  found  i s t h a t t h e NADH generated  phosphate dehydrogenase r e a c t i o n i n t h e c y t o p l a s m i s  r e o x i d i z e d a t t h e expense o f oxygen  almost  Octopine  NAD ^>  v  genase a r e f a c u l t a t i v e anaerobes.  1970)  absent, and i n s t e a d o c t o p i n e dehydro-  time i t i s not known whether m o l l u s c s  phosphate  some m o l l u s c s  and t h e b i v a l v e s C a r d i n a l ed ule and  L - a r g i n i n e + pyruvate At t h i s  In  o f o c t o p i n e from p y r u v a t e i n t i s s u e s o f t h e  Sepia o f f i c i n a l i s  maximus v i a  e x c e p t i o n , t h e enzyme c a t a l y z i n g the  (Regnouf and von T h o a i , 1970).  catalyzes the production  Succinate  of carbon from g l u c o s e t o s u c c i n a t e  i s l o c a l i z e d i n the m i t o c h o n d r i a .  dehydrogenase i s shown t o be  genase i s p r e s e n t  NADH — — —  Fumarate  t h a t i n mantle o f R. cuneata a l l b u t one  the o v e r a l l flow  are l o c a l i z e d i n t h e c y t o s o l ;  lactate  NADH — ^ — Malate  2  chain o f  Lehninger  the i s o l a t e d l i v e r  (1951,  mitochondria i s  NADH from t h e e x t e r n a l medium, and a s i m i l a r i n a c c e s -  7 sibility 1962),  has been observed w i t h  and i n s e c t f l i g h t  kidney  (Boxer and D e v l i n , 1961),  m u s c l e ( S a c t o r and D i c k , 1962).  tumor  (Borst,  From experiments i n  14 which  (C  ) n i c o t i n i c a c i d was i n j e c t e d i n t o r a t s , P u r v i s and Lowenstein  (1961), deduced t h a t t h e i n t r a of l i v e r  and e x t r a - m i t o c h o n d r i a l p y r i d i n e n u c l e o t i d e s  do n o t undergo r a p i d e q u i l i b r a t i o n , and they c a l c u l a t e d t h a t NADH  enters l i v e r mitochondria  i nvivo  a t a speed q u i t e inadequate t o account f o r  the r e s p i r a t o r y r a t e o f t h e t i s s u e . m i t o c h o n d r i a a r e impermeable to 1964). from  I t seems, t h e r e f o r e ,  I t i s now a c c e p t e d t h a t v i r t u a l l y a l l  NADH  both i n v i v o and i n v i t r o  (Lehninger,  t h a t r e d u c i n g e q u i v a l e n t s must be t r a n s f e r r e d  t h e cytoplasm to the r e s p i r a t o r y c h a i n by means o t h e r than t h e t r a n s p o r t  of NADH. I t i s e v i d e n t t h a t f o r every m o l e c u l e o f glucose m e t a b o l i z e d a e r o b i c a l l y o r a n a e r o b i c a l l y , two molecules o f NADH a r e formed a c t i o n o f t h e enzyme g l y c e r a l d e h y d e - 3 - p h o s p h a t e t h i s co-enzyme  i s immediately  r e - o x i d i z e d by  another a c c e p t o r , energy p r o d u c t i o n must cease. the  o x i d a t i o n o f c y t o p l a s m i c NADH  mammalian muscle reduced  to  mechanism by shuttle.  and l i v e r ,  l a c t a t e by  and u n l e s s  t h e t r a n s f e r o f e l e c t r o n s to The mechanism i n v o l v e d f o r  t o v a r y from t i s s u e t o t i s s u e .  In  p y r u v a t e a c t s as t h e e l e c t r o n a c c e p t o r , b e i n g  the action  In y e a s t ,  o f l a c t a t e dehydrogenase.  Another known  a l c o h o l dehydrogenase p r o v i d e s a t h i r d mechanism.  i s taken over by  a-glycerophosphate, Embeden-Meyerhof  In  l a c t a t e dehydrogenase has a v e r y low a c t i v i t y , and i t s  d i h y d r o x y a c e t o n e phosphate  into  dehydrogenase,  which NADH can be o x i d i z e d i n l i v e r i s by the m a l a t e / o x a l o a c e t a t e  i n s e c t f l i g h t muscle, function  seems  i n t h e cytoplasm by t h e  t h e s o l u b l e a - g l y c e r p h o s p h a t e dehydrogenase.  Thus,  a c c e p t s e l e c t r o n s from NADH and i s reduced to  w h i l e NAD i s r e s t o r e d f o r p a r t i c i p a t i o n i n t h e main pathway.  The a - g l y c e r o p h o s p h a t e so formed  diffuses  readily  t h e m i t o c h o n d r i a , where i t i s r e o x i d i z e d by the m i t o c h o n d r i a l a - g l y c e r o -  8 phosphate dehydrogenase, t r a n s f e r r i n g e l e c t r o n s through t h e cytochromes to oxygen.  The d i h y d r o x a c e t o n e formed i n t h i s r e a c t i o n d i f f u s e s out o f t h e  mitochondria once more. act  to complete t h e c y c l e , and i s then ready to accept  Thus, the two a - g l y c e r p h o s p h a t e dehydrogenases and t h e i r  as a powerful  initially  electrons  c a t a l y t i c c y c l e whereby r e d u c i n g  equivalents  substrate  derived  from g l y c e r a l dehyde-3-phosphate a r e f e d i n t o t h e m i t o c h o n d r i a l  e l e c t r o n t r a n s p o r t system, thus making p o s s i b l e t h e r a p i d and complete o x i d a t i o n of glucose  t o carbon d i o x i d e and water w i t h i n t h e muscle  cell.  In the mantle t i s s u e o f t h e R. cuneata, Stokes and Awapara (1969) have p o s t u l a t e d t h a t m a l i c dehydrogenase and fumarate r e d u c t a s e r e s p o n s i b l e f o r the o x i d a t i o n of cytoplasmic  NADH.  a r e t h e enzymes  They p o s t u l a t e t h a t the  redox p a i r NAD/NADH i s kept i n t h e o x i d i z e d s t a t e by t h e r e d u c t i o n o f oxaloacetate  and o f fumarate.  The r e d u c t i o n o f o x a l o a c e t a t e  and d e h y d r a t i o n  of malate i s c a t a l y z e d by the enzymes l o c a t e d i n t h e c y t o p l a s m i c but  fumarate i s reduced by NADH i n t h e m i t o c h o n d r i a  mantle, u n l i k e t h e mammalian m i t o c h o n d r i a , NADH.  which i n R. cuneata  appear t o be permeable to t h e  L i t t l e work has been done on t h i s i n o t h e r i n v e r t e b r a t e s .  Phosphoenopyruvate c r o s s r o a d s  and t h e statement o f t h e problem.  From t h e f o r e g o i n g d e s c r i p t i o n i t became c l e a r t h a t d u r i n g in  fraction,  a f a c u l t a t i v e anaerobe l i k e o y s t e r , glucose-6-C  14  anaerobiosis  i s a b e t t e r source o f  14 s u c c i n a t e accumulation  than pyruvate-3-C  , suggesting  s u c c i n a t e branches o f f b e f o r e p y r u v a t e p r o d u c t i o n .  The b r a c k i s h water  clam R. cuneata has phosphoenolpyruvate carboxykinase favour  carboxylation of p-enolpyruvate  v e r s i o n to p y r u v a t e .  I t was r e p o r t e d  t h a t t h e pathway to  to oxaloacetate  activities  r a t h e r than i t s con-  that i n the oyster,  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  t h a t would  Crossostrea  o f phosphoenopyruvate  carboxykinase  9 was  o n l y 5% of the p y r u v a t e k i n a s e a c t i v i t y , which would f a v o u r t h e c o n t i n u a -  t i o n of g l y c o l y s i s  to the s t a g e o f p y r u v a t e  (Hammen, 1969).  This very  o b s e r v a t i o n i s indeed t h e crux o f our problem h e r e , and i t i s t h e r e f o r e n e c e s s a r y to c o n s i d e r b r i e f l y  the p-enolpyruvate crossroads.  In the case of mammalian t i s s u e s which a r e a b l e t o produce g l u c o s e from non-carbohydrate  sources, the pyruvate kinase ( 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 ) r e a c t i o n i s bypassed  by the s u c c e s s i v e a c t i o n o f two enzymes:  pyruvate c a r b o x y l a s e and p - e n o l p y r u v a t e c a r b o x y k i n a s e .  I t i s generally  accepted t h a t t h i s p a i r o f enzymes i n r a t l i v e r generates t h a t w i l l be f u r t h e r m e t a b o l i z e d to g l u c o s e .  the p-enolpyruvate  In t i s s u e s , l i k e r a t l i v e r  ( g l u c o n e o g e n i c ) a l l t h r e e enzymes, e.g., p y r u v a t e k i n a s e , p - e n o l p y r u v a t e c a r b o x y k i n a s e and p y r u v a t e c a r b o x y l a s e occur i n c y t o s o l  (Henning,  Stumpf, and  Ohly, 1966; N o r d l i e , V a r r i c c h i o and H o l t e n , 1965) and t h e a c t i v i t i e s o f these enzymes i f not c o n t r o l l e d c o u l d s e t up a " f u t i l e " c y c l e between pyruvate and p - e n o l p y r u v a t e as f o l l o w s : III  Glucose-6-P  (I) Pyruvate k i n a s e , ( I I ) Pyruvate c a r b o x y l a s e , and ( I I I ) P-enolpyruvate carboxykinase. In a d d i t i o n , such " f u t i l e " c y c l e s o f t e n would f u n c t i o n as n e t ATPases, if  t h e i r o p e r a t i o n were not c o n t r o l l e d .  Although  t h i s mode o f o p e r a t i o n may  appear w a s t e f u l , i t o f f e r s a mechanism f o r r e g u l a t i o n o f both t h e ATP:ADP r a t i o and the r e l a t i o n s h i p between t h e g l y c o l y t i c and g l u c o n e o g e n i c The mechanisms which may r e g u l a t e the p y r u v a t e / p - e n o l p y r u v a t e  flux. cycle are  10 l e s s w e l l c h a r a c t e r i z e d . An acute requirement mentation  f o r the i n h i b i t i o n o r compart-  of p y r u v a t e k i n a s e d u r i n g gluconeogenesis  e x i s t s because the  maximal c a p a c i t y o f t h i s enzyme i n the g l u c o n e o g e n i c t i s s u e exceeds those of p y r u v a t e c a r b o x y l a s e and p - e n o l p y r u v a t e magnitude.  c a r b o x y k i n a s e by an o r d e r o f  In r a t l i v e r , p - e n o l p y r u v a t e f o r m a t i o n occurs i n the c y t o s o l ;  a l s o ATP and a l a n i n e i n h i b i t p y r u v a t e k i n a s e (Tanaka, Harano, Sue, and Morimura, 1967).  However, i n c h i c k e n l i v e r , where p - e n o l p y r u v a t e s y n t h e s i s  o c c u r s w i t h i n the m i t o c h o n d r i a , GTP and ITP a r e i n h i b i t o r y , w h i l e a l a n i n e and ATP have no s i g n i f i c a n t e f f e c t all  these e f f e c t o r s s a t i s f y  ( S c r u t t o n and U t t e r , 1968).  Although  the c r i t e r i o n o f 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 g l u c o n e o g e n i c segments o f t h i s  cycle,  no evidence has been p r e s e n t e d to p r e v e n t r e c y c l i n g a t t h i s l o c u s i n metabolism. In  o y s t e r t i s s u e s , the p - e n o l p y r u v a t e c r o s s r o a d s poses a somewhat  different situation.  U n l i k e the mammalian p - e n o l p y r u v a t e c a r b o x y k i n a s e , i n  o y s t e r , i t s n e t f l u x i s towards the s y n t h e s i s o f o x a l o a c e t a t e , which i s f u r t h e r reduced  to form s u c c i n a t e as the g l y c o l y t i c end p r o d u c t .  upon the e n v i r o n m e n t a l proposed  Depending  c o n d i t i o n s , glycogen d i s s i m i l a t i o n i n o y s t e r i s  as f o l l o w s : I  Pyruvate  C0  2  +  H 0 2  Glucose-6-P ,j==^ P - e n o l p y r u v a t e II  Oxaloacetate — S u c c i n a t e  At the p - e n o l p y r u v a t e b r a n c h - p o i n t , e s s e n t i a l l y available.  (I), i f 0  2  two a l t e r n a t i v e r o u t e s a r e  i s p r e s e n t , t h e p y r u v a t e formed i s " f e d " d i r e c t l y  the Krebs c y c l e i n the u s u a l manner.  However, i f 0  2  i s absent,  p y r u v a t e i s c o n v e r t e d to o x a l o a c e t a t e v i a p - e n o l p y r u v a t e  into  ( I I ) , p-enol-  carboxykinase.  E v i d e n t l y i n such a case the problem of p y r u v a t e / p - e n o l p y r u v a t e  recycling i s  11 absent and p - e n o l p y r u v a t e i s t h e common s u b s t r a t e f o r t h e p y r u v a t e k i n a s e and p-enolpyruvate  carboxykinase.  I f t h e above d a t a a r e c o r r e c t , what a r e t h e  mechanisms a v a i l a b l e to t h e organism p-enolpyruvate  c a r b o x y k i n a s e i n "on and o f f " p o s i t i o n s i n a r e c i p r o c a l manner  so t h a t both a r e n o t f u l l y  a c t i v e simultaneously?  The s t u d i e s r e p o r t e d i n t h i s aspect o f o y s t e r metabolism: roads.  f o r h o l d i n g p y r u v a t e k i n a s e and  thesis deal i n d e t a i l with t h i s  single  enzymic r e g u l a t i o n a t t h e p - e n o l p y r u v a t e  cross-  An u n d e r s t a n d i n g o f t h i s s p e c i f i c c o n t r o l p o i n t , however, has a l l o w e d  a critical  assessment o f s e v e r a l a d d i t i o n a l problems which a r e e v i d e n t i n  the unusual metabolism (NADH/NAD) p o t e n t i a l ,  o f f a c u l t a t i v e anaerobes.  These i n c l u d e (1) t h e redox  (2) pathways o f s u c c i n a t e p r o d u c t i o n , and (3) t h e h i g h  energy phosphate y i e l d . The approach (i)  adopted  t o i n v e s t i g a t e t h i s problem  i s o u t l i n e d as f o l l o w s :  S e l e c t t h e c r i t i c a l p h y s i o l o g i c a l branch p o i n t ( p - e n o l p y r u v a t e  c r o s s r o a d s ) through which c h a n n e l l i n g o f t h e t e r m i n a l g l y c o l y t i c end p r o d u c t s i s c o n t r o l l e d during a e r o b i c ^ anaerobic (ii)  transitions.  I s o l a t e and p a r t i a l l y p u r i f y key enzymes o f t h i s branch p o i n t  ( p y r u v a t e k i n a s e and p - e n o l p y r u v a t e carboxykinase) of two  and i n v e s t i g a t e t h e e f f e c t  pH and o t h e r m e t a b o l i t e s on t h e c a t a l y t i c and r e g u l a t o r y p r o p e r t i e s o f the enzymes.  At t h e o u t s e t , pH was t h e only documented p h y s i o l o g i c a l  factor  known to f l u c t u a t e d u r i n g a e r o - and a n a e r o b i o s i s (Wilbur, 1964). (iii)  C o n s t r u c t a new m e t a b o l i c map which accounts  NAD) b a l a n c e ,  (1) f o r redox  (NADH/  (2) f o r t h e pathways o f s u c c i n a t e p r o d u c t i o n , and (3) f o r t h e  h i g h energy phosphate y i e l d .  CHAPTER I I : M a t e r i a l s and Methods  12 Animal c o l l e c t i o n and t h e i r h a n d l i n g . Oysters  ( C r O s s o s t r e a g i g a s ) were c o l l e c t e d from Chuckanut Bay, B e l l i n g h a m ,  Washington, a t low t i d e s , by the k i n d p e r m i s s i o n of Dr. W a l l a c e Heath o f the Aquaculture  T r a i n i n g Programme, Lummi, Washington.  Animals were brought  to  the l a b o r a t o r y i n an i c e - b o x and opened q u i c k l y to e x c i s e the t i s s u e s . A l l t i s s u e s were washed thoroughly w i t h i c e - c o l d homogenizing medium to remove exogenous a l g a e and o t h e r microorganisms.  A l l the work r e p o r t e d i n t h i s  t h e s i s was c a r r i e d out on mature o y s t e r s and no attempt  was made to c o r r e l a t e  enzyme a c t i v i t i e s w i t h s e x u a l d i f f e r e n c e s o r s e a s o n a l v a r i a t i o n s .  P r e p a r a t i o n of mantle and adductor p y r u v a t e k i n a s e s . Mantle  and adductor  t i s s u e s were homogenized i n a S o r v a l l Omnimixer f o r  1 to 2 min w i t h 3 to 4 volumes of i c e - c o l d 0.01 M T r i s - H C l b u f f e r , pH 7.5, c o n t a i n i n g 2 mM  EDTA.  The homogenates were s t i r r e d  f o r 1 h r a t 4° and then  c e n t r i f u g e d a t 12,000 X g f o r 15 min and the p e l l e t was d i s c a r d e d . s u p e r n a t a n t s were f i l t e r e d  through  g l a s s wool and then brought  t i o n w i t h s o l i d ammonium s u l p h a t e and s t i r r e d  f o r 1 hr at 4°.  The  to 40% s a t u r a The s u s p e n s i o n  were then c e n t r i f u g e d as above, the p e l l e t s were d i s c a r d e d , and the supern a t a n t were brought  to 75% s a t u r a t i o n w i t h s o l i d ammonium s u l p h a t e .  1 hr with s t i r r i n g ,  the s o l u t i o n s were c e n t r i f u g e d a t 37,000 X g f o r 20 min.  The p e l l e t s were d i s s o l v e d i n a minimal pH 7.5.  After  volume o f 0.01 M T r i s - H C l b u f f e r ,  The d i s s o l v e d p e l l e t s were f u r t h e r c e n t r i f u g e d a t 84,000 X g f o r  90 min i n r e f r i g e r a t e d Beckman model L p r e p a r a t i v e u l t r a c e n t r i f u g e to remove glycogen and the h i g h speed kinases.  supernatant were used as the sources o f p y r u v a t e  P o r t i o n s of enzymes were d i a l y z e d b e f o r e use a g a i n s t 0.05 M  HCl b u f f e r , pH 7.5.  Tris-  The enzyme was s t a b l e a t 0-4° f o r a few days and i f  f r o z e n , was s t a b l e f o r s e v e r a l weeks w i t h o u t b r i n g i n g any change i n the K  13 of the s u b s t r a t e p - e n o l p y r u v a t e .  Mantle  enzyme was  somewhat u n s t a b l e to  d i a l y s i s , showing a l o s s of a c t i v i t y of approximately  10% w i t h i n 2 hours  of  dialyzing.  P r e p a r a t i o n of m i t o c h o n d r i a . 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 o f the enzymes, m i t o c h o n d r i a were p r e p a r e d by an a d a p t a t i o n of the method o f Hogboom (1965). and adductor  t i s s u e s were o b t a i n e d and washed i n a c o l d b u f f e r medium pH  at 4° c o n t a i n i n g 0.02  M T r i s - H C l , 1 mM  o p e r a t i o n s were conducted f o r 1 to 1.5  min,  homogenate was min  at 650 X g.  M g C l , 0.025 M s u c r o s e .  at 0° to 4 ° .  the t i s s u e was  through  cheese c l o t h . RC  2-B  7,000 X g.  The  The p e l l e t  d i s c a r d e d and  from the p r e c e d i n g  and washed i n the homogenizing medium.  After  c y c l e s of washing and c e n t r i f u g i n g , the m i t o c h o n d r i a l p e l l e t was c e n t r i f u g a t i o n i n the homogenizing medium a t 24,000 X g. p u r i t y of m i t o c h o n d r i a i n the p e l l e t was scopy.  crude  c e n t r i f u g e f o r 10  F o l l o w i n g c e n t r i f u g a t i o n , the t i s s u e p u l p was  resuspended  7.2  A f t e r g r i n d i n g i n the S o r v a l l Omnimixer  filtered  c e n t r i f u g e d at  gill  A l l subsequent  2  c e n t r i f u g e d i n a S o r v a l l superspeed  the supernatant was step was  Mantle,  several  o b t a i n e d by  The presence  and  confirmed w i t h phase c o n t r a s t m i c r o -  The s u s p e n s i o n o f m i t o c h o n d r i a so o b t a i n e d was  t h r e e times, then c e n t r i f u g e d and the supernatant was  f r o z e n and  thawed  used as the s o u r c e of  the m i t o c h o n d r i a l enzyme.  P r e p a r a t i o n of c y t o p l a s m i c phosphoenolpyruvate As no a c t i v i t y was  carboxykinase.  found i n the m i t o c h o n d r i a l f r a c t i o n , t i s s u e s were  homogenized i n a S o r v a l Omnimixer f o r 1 to 2 min w i t h 4 to 5 volumes of i c e c o l d 0.01  M T r i s - H C l b u f f e r , pH  7.2  c e n t r i f u g e d a t 12,000 X g f o r 15 min  c o n t a i n i n g 2 mM and  EDTA.  the p e l l e t was  The homogenate  discarded.  The  was  14 s u p e r n a t a n t was a d j u s t e d t o pH 5.5 w i t h 0.1 M a c e t i c a c i d ; a f t e r t i o n a t 37,000 X g f o r 7 min,  centrifuga-  the p r e c i p i t a t e was d i s c a r d e d and t h e super-  n a t a n t was n e u t r a l i z e d w i t h 0.1 M KHCO^.  The n e u t r a l i z e d s u p e r n a t a n t was then  brought  to 40% s a t u r a t i o n w i t h s o l i d ammonium s u l p h a t e and s t i r r e d f o r 1 h r  at 4 ° .  The s u s p e n s i o n was then c e n t r i f u g e d as above, t h e p e l l e t was d i s c a r d e d ,  and the s u p e r n a t a n t was brought After 1 hr with s t i r r i n g , 15 min.  to 75% s a t u r a t i o n w i t h s o l i d ammonium s u l p h a t e .  the s u s p e n s i o n was c e n t r i f u g e d a t 37,000 X g f o r  The p e l l e t was d i s s o l v e d i n a minimal volume o f 0.01 M T r i s - H C l  b u f f e r , pH 7.2.  The d i s s o l v e d p e l l e t was f u r t h e r c e n t r i f u g e d a t 41,300 X g  f o r 15 min and the h i g h speed s u p e r n a t a n t was used as t h e s o u r c e f o r phosphoenolp y r u v a t e c a r b o x y k i n a s e (PEPCK).  P o r t i o n s o f enzyme were d i a l y z e d b e f o r e use  a g a i n s t c o l d 0.05 M T r i s - H C l b u f f e r , pH 7.2.  The enzyme was s t a b l e a t 0 ° t o  4° f o r a few days and i f f r o z e n was s t a b l e f o r s e v e r a l weeks.  Above p r e p a r a -  t i o n was a l s o used as the s o u r c e o f malate dehydrogenase and " m a l i c enzyme".  Assay o f p y r u v a t e k i n a s e a c t i v i t y . Pyruvate k i n a s e was assayed by t h e method o f BUcher and P f l e i d e r  (1959).  Pyruvate f o r m a t i o n was coupled to l a c t a t e dehydrogenase and t h e r a t e o f p y r u v a t e k i n a s e a c t i v i t y was measured as t h e decrease i n E ^ Q due t o NADH. T r i s - H C l b u f f e r s were used i n a l l assay r e a c t i o n s .  Standard assay  c o n t a i n e d t h e f o l l o w i n g i n a f i n a l volume o f 2 m l :  50 mM T r i s - H C l b u f f e r ,  Mg  mixtures  , K , ADP, p - e n o l p y r u v a t e , NADH and excess o f d i a l y z e d Sigma l a c t a t e  dehydrogenase a t c o n c e n t r a t i o n s s p e c i f i e d i n t h e f i g u r e l e g e n d s . c o n c e n t r a t i o n s f o r each o f t h e r e a c t a n t s f o r both mantle  Saturating  and adductor enzymes  were 6 mM Mg" ", 50 mM K , 0.2 mM ADP, 1.4 mM p - e n o l p y r u v a t e , 5 X 1 0 1-1  +  _ 5  M  f r u c t o s e - 1 , 6 - P , 5 mM ATP, 3 mM L - a l a n i n e (adductor p y r u v a t e k i n a s e ) , 8 mM 2  L-alanine  (mantle p y r u v a t e k i n a s e ) , and 10 mM p h e n y l a l a n i n e .  A l l reactions  15 were s t a r t e d by t h e a d d i t i o n o f p y r u v a t e k i n a s e p r e p a r a t i o n . were performed c  A l l experiments  a t 20° s i n c e K , , , was found temperature m(p-enolpyruvate)  ent over a temperature  independ-  range o f 5-30°.  Assay o f the phosphoenolpyruvate  carboxykinase (carboxylation r e a c t i o n ) a c t i v i t y .  The enzyme was assayed i n the d i r e c t i o n o f o x a l o a c t a t e s y n t h e s i s by t h e method o f U t t e r and K u r a h a s h i  (1954), O x a l o a c e t a t e f o r m a t i o n was coupled t o  excess q u a n t i t i e s o f d i a l y z e d malate dehydrogenase,  and t h e r a t e o f p - e n o l p y r u -  v a t e c a r b o x y k i n a s e a c t i v i t y was f o l l o w e d as t h e decrease i n E ^ Q due t o NADH. Sodium a c e t a t e o r T r i s - M a l e a t e b u f f e r s were used i n a l l assay r e a c t i o n s t o cover a wide pH range.  Standard assay m i x t u r e c o n t a i n e d t h e f o l l o w i n g i n a I |  f i n a l volume of 1 ml: diphosphate  , o r Zn  , Inosine  (IDP), p - e n o l p y r u v a t e , NaHCO^, NADH and an excess o f d i a l y z e d  p i g h e a r t malate legends.  50 mM T r i s - M a l e a t e b u f f e r , Mn  | |  Sigma  dehydrogenase a t c o n c e n t r a t i o n s s p e c i f i e d i n t h e f i g u r e  S a t u r a t i n g c o n c e n t r a t i o n s f o r each o f t h e r e a c t a n t s f o r t h e  p - e n o l p y r u v a t e c a r b o x y k i n a s e r e a c t i o n were: 1 mM IDP, and 1.4 mM p - e n o l p y r u v a t e .  10 mM NaHCO^, 1 mM Mn  A l l experiments  at 25°.  Assay o f phosphoenolpyruvate D e c a r b o x y l a t i o n assay  carboxykinase (decarboxylation r e a c t i o n ) a c t i v i t y . ( s y n t h e s i s o f p - e n o l p y r u v a t e ) was measured by t h e  method d e s c r i b e d by N o r d l i e and L a r d y v a t e formed  ,  A l l r e a c t i o n s were i n i t i a t e d by t h e  a d d i t i o n of t h e p - e n o l p y r u v a t e c a r b o x y k i n a s e p r e p a r a t i o n . were performed  , o r Zn  (1963) w i t h s l i g h t m o d i f i c a t i o n s .  P-enolpyru-  as a r e s u l t o f the d e c a r b o x y l a t i o n r e a c t i o n was measured  e n z y m i c a l l y by the method o f BUcher and P f l e i d e r e r c o n t a i n e d i n a f i n a l volume o f 1 ml: o x a l o a c e t a t e , 1 mM Mn  (1959).  The assay medium  0.05 mM T r i s - M a l e a t e b u f f e r , 120 uM  , 2 0 uM I n o s i n e t r i p h o s p h a t e ( I T P ) ,  16 KF.  T h i s m i x t u r e was p r e i n c u b a t e d f o r 5 min a t 25°, a f t e r which p - e n o l p y r u v a t e  c a r b o x y k i n a s e was added.  The r e a c t i o n was stopped by adding a p p r o x i m a t e l y  1 mg of potassium b o r o h y d r a t e  (KBH^).  The r e a c t i o n tubes were kept i n an i c e  b a t h and 0.2 ml of i c e - c o l d 6% HCIO^ was added.  Denatured  p r o t e i n was removed  by c e n t r i f u g a t i o n and s u p e r n a t a n t was n e u t r a l i z e d by t h e a d d i t i o n o f 3 M T r i s . The volume o f the n e u t r a l i z e d s u p e r n a t a n t was brought 0.05  t o 2 ml by adding o f  mM T r i s - M a l e a t e b u f f e r pH 7.6, 50 mM K , 1 mM ADP, 1 mM Mn"*", 0.15 mM +  NADH and excess l a c t a t e dehydrogenase. a d d i t i o n o f c o n s t a n t amount  1 -  The r e a c t i o n was i n i t i a t e d by t h e  o f Sigma r a b b i t p y r u v a t e k i n a s e .  The p - e n o l p y r u -  v a t e c o n c e n t r a t i o n can be c a l c u l a t e d from t h e d e c r e a s e i n E „ . a f t e r t h e 340 /r  a d d i t i o n of pyruvate kinase. I | D e c a r b o x y l a t i o n r e a c t i o n was assayed between pH 5.1 and 8.2, Mn  | [  , Zn  I j or Mg  .  A l l the components o f t h e d e c a r b o x y l a t i o n assay were t e s t e d by  d e l e t i n g o r adding, by i n c r e a s i n g o r d e c r e a s i n g t h e c o n c e n t r a t i o n s o f t h e r e a c t a n t s a t a l l pH v a l u e s .  The e f f e c t o f a c e t y l C o A was a l s o t e s t e d .  In no  i n s t a n c e , c o u l d s i g n i f i c a n t p - e n o l p y r u v a t e s y n t h e s i s be d e t e c t e d w i t h t h e assay system.  Low and v a r i a b l e a c t i v i t y was d e t e c t e d by measuring  from p - e n o l p y r u v a t e by t h e method o f Seubert and Huth (1965).  P^ r e l e a s e d  Further studies  are r e q u i r e d t o assess t h e p r o p e r t i e s o f t h e d e c a r b o x y l a t i o n r e a c t i o n . Assay  o f t h e " m a l i c enzyme" a c t i v i t y . " M a l i c enzyme" a c t i v i t y was measured by t h e method o f Ochoa- (1965). Rate o f  enzyme a c t i v i t y was measured by the i n c r e a s e o r d e c r e a s e i n absorbance a t due  to the r e d u c t i o n o f NADP o r o x i d a t i o n o f NADPH.  Standard  m i x t u r e c o n t a i n e d t h e f o l l o w i n g i n a f i n a l volume o f 1 ml:  assay  50 mM T r i s - M a l e a t e  I | b u f f e r , pH 8.2, 1 mM Mn  , 1 mM m a l i c a c i d and 0.15 mM NADP.  were i n i t i a t e d by t h e a d d i t i o n o f enzyme p r e p a r a t i o n .  A l l reactions  CHAPTER I I I : C a t a l y t i c and R e g u l a t o r y P r o p e r t i e s of O y s t e r Pyruvate  Kinases  17  Assay o f malate  and l a c t a t e dehydrogenases  .  M a l a t e and l a c t a t e dehydrogenase a c t i v i t i e s were measured by t h e methods d e s c r i b e d by Ochoa ( 1 9 6 5 ) and Romberg  (1965).  R e d u c t i o n o f o x a l o a c e t a t e and  p y r u v a t e was measured i n a medium c o n t a i n i n g 5 0 mM T r i s - H C l b u f f e r , pH 7 . 5 , 1 mM o f e i t h e r s u b s t r a t e , 0 . 1 5 mM NADH and enzyme p r e p a r a t i o n . was  determined  The a c t i v i t y  from t h e d e c r e a s e i n e x t i n c t i o n a t E ^ Q (*see page 1 8 ) .  Estimates of p r o t e i n content. The p r o t e i n c o n t e n t o f a l l enzyme p r e p a r a t i o n s wherever r e q u i r e d were determined  by the method o f Lowry, Rosebrough, F a r r and R a n d a l l ( 1 9 5 1 ) . A l l  samples were compared w i t h a s t a n d a r d curve determined  from 0 t o 1 0 0 ugm/ml  b o v i n e serum albumen p r o t e i n .  Electrophoresis. Mantle, g i l l ,  and adductor muscle p y r u v a t e k i n a s e s were p r e p a r e d and  studied e l e c t r o p h o r e t i c a l l y p r e s e n t i n the o y s t e r .  to determine whether t i s s u e - s p e c i f i c forms a r e  E l e c t r o p h o r e s i s was c a r r i e d out on c e l l u l o s e p o l y a c e -  t a t e s t r i p s a t a p o t e n t i a l g r a d i e n t o f 1 7 v/cm f o r 2 h r a t 4 ° i n b u f f e r c o n t a i n i n g 0.5 M s u c r o s e 0 . 0 1 M T r i s - H C l , pH 7 . 5 and 0 . 0 0 1 M f r u c t o s e - l , 6 - P . 2  Samples were a p p l i e d to t h e c e n t e r o f the s t r i p . P y r u v a t e k i n a s e a c t i v i t y was d e t e c t e d by c o u p l i n g w i t h t h e l a c t i c dehydrogenase system.  The r e g i o n p r o d u c i n g NADH o x i d a t i o n was v i s u a l l y  observed by the l o s s o f f l u o r e s c e n c e . w i t h 3 4 0 mu l i g h t .  I t was r e c o r d e d by exposing t h e s t r i p s  The c e l l u l o s e a c e t a t e s t r i p s  t o be assayed were p l a c e d on  an agar f i l m c o n t a i n i n g 8 mg/ml Nobel agar, 5 0 mM T r i s - H C l , pH 7 . 5 , 2 mM EDTA, 2 0 mM Mg ", 6 0 mM X , 2 mM p - e n o l p y r u v a t e , 1 mM ADP, 1 mM NADH, 0.2 mM 44  +  f r u c t o s e - l , 6 - P „ and excess sigma d i a l y z e d LDH.  Development was a l l o w e d t o  18 proceed f o r 1 to 3 min a t room temperature.  Pyruvate k i n a s e a c t i v i t y was  r e c o r d e d by c o n t a c t p r i n t i n g on Kodak p h o t o g r a p h i c e n l a r g i n g paper u s i n g a 340 mu l i g h t s o u r c e . incident do n o t .  Areas o f enzymatic  activity  (low NADH) t r a n s m i t t h e  l i g h t and expose t h e p h o t o g r a p h i c paper w h i l e o t h e r areas ( h i g h NADH) These experiments were performed  i n c o l l a b o r a t i o n w i t h Dr. W a l t e r  Susor o f C a l i f o r n i a M e d i c a l Center, San F r a n c i s c o ,  California.  Electrofocusing. Technique  o f e l e c t r o f o c u s i n g was used t o determine whether isozymes  h a v i n g d i f f e r e n t p i v a l u e s were p r e s e n t . performed  by the method o f Haglund  (1967).  E l e c t r o f o c u s i n g experiments F o r p y r u v a t e k i n a s e both  were mantle  and adductor enzymes were r u n a t pH 5 to 8 g r a d i e n t (LKB-8133) a t 900 v o l t s f o r 53 hours.  F o r phosphoenolpyruvate  c a r b o x y k i n a s e , a pH 3 t o 10 g r a d i e n t  (LKB-8141) was used a t a p o t e n t i a l o f 300 v o l t s .  The temperature  of the  apparatus was m a i n t a i n e d a t 4 ° f o r t h e e n t i r e p e r i o d o f t h e experiments. Small f r a c t i o n s o f 15 drops each were c o l l e c t e d . all  For pyruvate kinase a c t i v i t y  f r a c t i o n s were assayed i n presence o f 2.5 X 10  Phosphoenolpyruvate  6  fructose-1,6-P  2  a t pH 8.5.  c a r b o x y k i n a s e a c t i v i t y was measured i n t h e p r e s e n c e o f  I | Zn  a t pH v a l u e 5.1.  *A11 the s p e c t r o p h o t o m e t r i c assays were r e p e a t e d 2 t o 3 times; change i n O.D. was r e p r o d u c i b l e between 90-100% i n a l l c a s e s . r  K measurements o f d i f f e r e n t m  m e t a b o l i t e s f o r one enzyme p r e p a r a t i o n i f r e p e a t e d were r e p r o d u c i b l e b u t v a r i e d between 3-5% when compared w i t h d i f f e r e n t enzyme p r e p a r a t i o n s .  19 INTRODUCTION In t h e o r y p y r u v a t e o c c u p i e s a c e n t r a l c r o s s r o a d s p o s i t i o n i n energy metabolism  s i n c e i t may be m e t a b o l i z e d by a number o f d i f f e r e n t pathways.  l e a s t f i v e such pathways a r e p r e s e n t i n o y s t e r t i s s u e s  (Hammen, 1969).  At  In  o y s t e r adductor muscle the major source o f p y r u v a t e i s s t o r a g e g l y c o g e n . A c c o r d i n g to Hammen (1969), the p y r u v a t e , which i s produced  i n high quantities  d u r i n g p e r i o d s o f a n a e r o b i o s i s , can have s e v e r a l m e t a b o l i c f a t e s , t h e most important one b e i n g c o n v e r s i o n to m a l i c a c i d .  The l a t t e r i s r e a d i l y  metabol-  i z e d to s u c c i n a t e , which i s a major end p r o d u c t o f a n a e r o b i c metabolism i n oyster.  L a c t a t e i n c o n t r a s t does not accumulate  (Hammen, 1969).  c o n d i t i o n s p y r u v a t e can be o x i d i z e d by the u s u a l Krebs mantle,  Under a e r o b i c  cycle reactions.  In  on the o t h e r hand, the p y r u v a t e b r a n c h i n g p o i n t i s more complex and  the degree muscle.  t o which these pathways o p e r a t e d i f f e r s from t h a t i n t h e adductor  During a n a e r o b i o s i s glucose-6-C  14  i s a b e t t e r source o f s u c c i n a t e  14 than i s pyruvate-3-C  , s u g g e s t i n g t h a t the pathway t o s u c c i n a t e branches o f f  before pyruvate production.  Simpson and Awapara (1966) suggest t h a t  l a t i o n o f p - e n o l p y r u v a t e by phosphoenolpyruvate mechanism l e a d i n g t o s u c c i n a t e a c c u m u l a t i o n . muscle, mantle  w e l l as from Krebs most organisms be bypassed  cycle intermediates.  i s physiologically  c a r b o x y k i n a s e i s t h e major  A l s o , i n c o n t r a s t t o adductor  i s a major g l u c o n e o g e n i c t i s s u e .  p y r u v a t e and p - e n o l p y r u v a t e may be produced  Under g l u c o n e o g e n i c  from non-carbohydrate  conditions,  sources as  The p y r u v a t e k i n a s e s t e p , which i n  irreversible  ( S c r u t t o n and U t t e r , 1968) may  d u r i n g g l u c o s e ( o r glycogen) s y n t h e s i s by the p - e n o l p y r u v a t e  c a r b o x y k i n a s e which generate p - e n o l p y r u v a t e f o r f u r t h e r metabolism In mantle  carboxy-  to glucose.  o f most m o l l u s c s examined, however, p y r u v a t e k i n a s e a c t i v i t y  a p p a r e n t l y exceeds p - e n o l p y r u v a t e c a r b o x y k i n a s e by a f a c t o r o f 10 (Hammen, 1969).  Hence i f the d a t a a r e c o r r e c t t h e r e a r e s p e c i a l requirements f o r  h o l d i n g p y r u v a t e k i n a s e i n a "shut o f f " c o n f o r m a t i o n i n t h i s  tissue.  From the above c o n s i d e r a t i o n i t i s e v i d e n t t h a t the requirements f o r the r e g u l a t i o n of p y r u v a t e k i n a s e r e a c t i o n i n adductor and mantle quite d i s t i n c t  from each o t h e r .  One  obvious way  o f meeting  t i s s u e s are  these d i f f e r e n t  requirements i s the e l a b o r a t i o n of t i s s u e s p e c i f i c v a r i a n t s o f the enzyme. T h e r e f o r e t h i s study was for different  i n i t i a t e d by examining mantle  e l e c t r o p h o r e t i c forms of p y r u v a t e k i n a s e .  and adductor  tissues  21  RESULTS E l e c t r o p h o r e s i s and  electrofocusing.  E l e c t r o p h o r e t i c r e s o l u t i o n of p y r u v a t e k i n a s e a c t i v i t y i n t h r e e d i f f e r e n t t i s s u e s i s shown i n F i g . 1.  Mantle p y r u v a t e k i n a s e a c t i v i t y appears as a  s i n g l e band, moving towards the cathode, p y r u v a t e k i n a s e i n the g i l l displays a similar electrophoretic mobility.  tissue  Adductor p y r u v a t e k i n a s e ,  showing a d i f f e r e n t p a t t e r n , moves as a " d o u b l e t " towards the cathode. e l e c t r o p h o r e t i c d i f f e r e n c e s were confirmed by e l e c t r o f o c u s i n g (Fig. 2).  The  experiments  Mantle p y r u v a t e k i n a s e appears as a s i n g l e major a c t i v i t y peak w i t h  a p i v a l u e o f 6.35.  In c o n t r a s t , the adductor p y r u v a t e k i n a s e appears  d i s t i n c t peaks h a v i n g p i v a l u e s o f 5.6  and 6.5.  F u r t h e r i t was  noted  as  two  that  mantle p y r u v a t e k i n a s e i s more u n s t a b l e than the adductor p y r u v a t e k i n a s e . s h o u l d be mentioned  here t h a t i n a l l of the k i n e t i c experiments  adductor p y r u v a t e k i n a s e the two  components were not s e p a r a t e d because  t i s s u e i s s m a l l and i t i s d i f f i c u l t  Cation  performed  It on  the  to o b t a i n l a r g e q u a n t i t i e s o f m a t e r i a l .  requirements.  In common w i t h p y r u v a t e k i n a s e s from o t h e r s p e c i e s , o y s t e r p y r u v a t e j | k i n a s e s show a b s o l u t e requirements  f o r d i v a l e n t and monovalent c a t i o n s .  can s a t i s f y  (Ka v a l u e s a t pH 8.5  f o r mantle satisfy  the former requirement  and adductor p y r u v a t e k i n a s e s r e s p e c t i v e l y ) and K  the l a t t e r requirement  systems).  are 1.25  High K  +  (Ka f o r K  +  a t pH 8.5  +  i s 7.7 mM  Mg  and 2.27  o r NH^  +  mM  can  f o r both enzyme  c o n c e n t r a t i o n s i n h i b i t e d the o y s t e r enzymes, i n c o n t r a s t  to p y r u v a t e k i n a s e s from o t h e r sources c o n c e n t r a t i o n o f up to 10 mM  (Carminatti et a l . ,  do not a f f e c t these enzymes.  1968).  Cu  at  22  Fig. I l l ,  1.  Electrophoretic  r e s o l u t i o n of o y s t e r mantle, g i l l ,  adductor p y r u v a t e k i n a s e a c t i v i t y . electrophoresis Susor and out  on  7.5  Rutter  o f 17 v o l t s per  at 4 ° ) , and  applied  (1968).  containing  to the  0.5  0.001  Procedure f o r  a c t i v i t y s t a i n i n g are a c c o r d i n g Electrophoresis  c e l l u l o s e polyacetate  gradient buffer  and  and  cm  carried  s t r i p s at a p o t e n t i a l f o r 3 hours at 4° i n a  M s u c r o s e , 0.01  M Tris-HCl  M fructose-l^-P^.  c e n t e r of the  was  to  strips.  Samples  (pH are  MANTLE  GILL ADDUCTOR  23  Fig. I l l ,  2.  E l e c t r o f o c u s i n g p a t t e r n o f the mantle and  adductor p y r u v a t e  kinases.  "Materials  Methods".  For e x p e r i m e n t a l Activity  the n u m e r i c a l the pH  values  procedures see  i s p l o t t e d against  and  f r a c t i o n numbers;  p l o t t e d on the a b s c i s s a i n d i c a t e  of s e l e c t e d f r a c t i o n s .  Reactant  are the same as d e s c r i b e d under F i g . 3.  concentrations  Activity ( A E  3 4 0  /unit time)  24 E f f e c t o f 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  pH optima a t pH 8.5 b u t the shape o f the a c t i v i t y  b o t h forms show a  2  curves d i f f e r f o r b o t h , w i t h  adductor p y r u v a t e k i n a s e b e i n g more s e n s i t i v e t o pH changes. of f r u c t o s e - 1 , 6 - P , the a c t i v i t y  o f the mantle  2  In t h e presence  enzyme i s a p p r o x i m a t e l y c o n s t a n t  between pH 7.5 and 9, w h i l e the pH optimum f o r the adductor enzyme i s d i s p l a c e d towards pH 7.  Effect of fructose-1,6-P  2  on ADP s a t u r a t i o n c u r v e s .  The e f f e c t o f i n c r e a s i n g ADP c o n c e n t r a t i o n a t a f i x e d c o n c e n t r a t i o n on the a c t i v i t y o f 2 enzymes i n the absence fructose-1,6-P  p-enolpyruvate and presence o f  a t pH 8.5 a r e g i v e n i n F i g . 4 (upper p a n e l ) a l o n g w i t h  2  double  -4 (lower p a n e l ) . Both enzymes r e q u i r e 2 X 10 M ADP f o r -4 a t 1 X 10 M p - e n o l p y r u v a t e . F o r both enzymes the curves f o r  reciprocal plots maximal a c t i v i t y  the r e a c t i o n v e l o c i t y a g a i n s t ADP i n the presence and absence  of fructose-1,  F o r mantle p y r u v a t e k i n a s e t h e K / . ^ v m(ADP) -4 i s 6.1 X 10 M, w h i l e f r u c t o s e - 1 , 6 - P  6-P„ have the M i c h a e l i s - M e n t o n form. 2 i n the absence  of f r u c t o s e - l , 6 - P  2  2  i n c r e a s e s the K , „ s a t l e a s t by 6 f o l d . m(ADP) /  6-P  2  A  T  has no e f f e c t on the  K m  ^^rjp)  E f f e c t o f pH and fructose-1,6»P'  2  In contrast to t h i s ,  fructose-1,  ° f adductor p y r u v a t e k i n a s e .  on the  of_ p - e n o l p y r u v a t e .  P - e n o l p y r u v a t e s a t u r a t i o n curves a t d i f f e r e n t pH v a l u e s i n t h e presence and absence  of fructose-1,6-P  reciprocal plots  i n c r e a s e s markedly 2  a r e shown i n F i g . 5, 6 ( l e f t p a n e l ) , double  (right panel).  minimum K i n the absence m  fructose-1,6-P  2  F o r mantle  enzyme ( F i g . 5, middle p a n e l ) , the  o f f r u c t o s e - 1 , 6 - P appears a t pH 8.5 and t h e K 2 ^ m 0  r  below pH 8.5 b e i n g maximum a t pH 6.5.  the  f o r the mantle  enzyme i s markedly  I n t h e presence o f lowered and remains  25  Fig. I l l ,  3.  E f f e c t of f r u c t o s e - l , 6 - P 2 on t h e pH optima fructose-l,6-P ; 2  Reaction  no  • , A , 2.5 X 1 0 ~ M f r u c t o s e - 1 , 6 - P ) .  contents were:  6  2  50 mM T r i s - H C l b u f f e r a t  d i f f e r e n t pH v a l u e s , 6 mM Mg**, 50 mM K , 0.2 mM ADP, +  0.5  mM p-enopyruvate, 0.15 mM NADH and excess o f  l a c t a t e dehydrogenase.  PyK, pyruvate  kinase.  26  F i g . I l l , 4. ' &  E f f e c t o f fructose-1,6-P„ on t h e K o f ADP o f mantle 2 m and adductor p y r u v a t e k i n a s e s .  Reactant c o n t e n t s were:  50 mM T r i s - H C l b u f f e r , pH 8.5, 6 mM Mg""*", 50 mM K , 1  +  0.1 mM p - e n o l p y r u v a t e , i n c r e a s i n g c o n c e n t r a t i o n s o f ADP, •  0.15 mM NADH and excess l a c t a t e  , mantle p y r u v a t e k i n a s e ; A,  dehydrogenase;  adductor  pyruvate  —6 k i n a s e ; • , A , w i t h 2.5 X 10 M fructose-1,6-P,.  27  Fig. I l l ,  5.  E f f e c t o f pH and f r u c t o s e - 1 , 6 - ? ^ on t h e K v a t e o f mantle p y r u v a t e k i n a s e . 50 mM T r i s - H C l , NADH, i n c r e a s i n g excess  Reactant  m  of p-enolpyruc o n t e n t s were:  6 mM Mg**, 50 mM K , 0.2 mM ADP, 0.15 mM +  c o n c e n t r a t i o n s o f p - e n o l p y r u v a t e and  l a c t a t e dehydrogenase.  2 7 <*-  28 c o n s t a n t over a pH range o f 6.5 for  to 8.5.  the adductor p y r u v a t e k i n a s e .  to those r e p o r t e d by Rozengurt  kinase.  Rozengurt  susceptibility  response  These r e s u l t s summarized i n T a b l e 1, are i n  contrast  demonstrated  F i g . 6 shows the same k i n d of  eit al_. (1969) f o r mouse l i v e r  pyruvate  t h a t as the pH o f the assay medium i s lowered,  to f r u c t o s e - 1 , 6 - P  a c t i v a t i o n correspondingly decreases.  2  In  case of o y s t e r p y r u v a t e k i n a s e s both enzymes are much more s u s c e p t i b l e to f r u c t o s e - 1 , 6 - P ^ a c t i v a t i o n a t a c i d i c pH than a t a l k a l i n e  A d e n o s i n e - t r i p h o s p h a t e (ATP)  inhibition.  L i k e p y r u v a t e k i n a s e s from o t h e r s o u r c e s Tanaka e t a l . , 1967) Fig.  ATP  pH.  (Rozengurt e t a l . , 1969;  and  a l s o i n h i b i t s both forms o f the o y s t e r enzymes.  7 ( l e f t p a n e l ) the e f f e c t o f ATP  at d i f f e r e n t pH v a l u e s and  In  p-enolpyruvate  c o n c e n t r a t i o n s i s shown f o r both enzymes, a l o n g w i t h Dixon p l o t of the d a t a (right panel).  While ATP  i n h i b i t s enzymes a t both pH v a l u e s examined, s e v e r a l  pH d i f f e r e n c e s can be noted. are  less inhibited  4 mM  and 2.65  mM  Using 1 mM  than a t pH 7.5.  at pH 8.5  and 7.5  p - e n o l p y r u v a t e a t pH 8.5  The K\ v a l u e s f o r the mantle respectively.  b o t h enzymes enzymes are  The adductor enzyme shows a  s i m i l a r b e h a v i o u r , a l t h o u g h the K\ v a l u e s f o r the adductor p y r u v a t e k i n a s e are lower, about  2.8 mM  and 1.9  mM  a t pH 8.5  and 7.5  The n a t u r e of i n h i b i t i o n i s a l s o d i f f e r e n t .  respectively. 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 p y r u v a t e k i n a s e r e a c t i o n a t d i f f e r e n t ATP c o n c e n t r a t i o n s (Fig.  8-A)  i n d i c a t e t h a t the ATP  i n h i b i t i o n of the mantle  c o m p e t i t i v e w i t h r e s p e c t to p - e n o l p y r u v a t e .  Thus f o r mantle enzyme ATP  decreases the c a l c u l a t e d Vmax, but does not a f f e c t the K  T h i s n o n - c o m p e t i t i v e i n h i b i t i o n o f mantle  enzyme i s non-  ., , _ s. m(p-enolpyruvate)  pyruvate kinase i s r e l a t i v e l y  unique  h a v i n g been r e p o r t e d o n l y once p r e v i o u s l y f o r the mouse b r a i n enzyme by Lowry and Passonneau (1964).  S i n c e p - e n o l p y r u v a t e i n mantle  i s not l i k e l y  to r e a c h  Table 1 E f f e c t of pH on K  , , m(p-enolpyruvate)  determined from F i g . I l l ,  5 and  i n absence and p r e s e n c e o f f ructose-1,6-P .. ' 2 0  v  Values  6.  pH 6.5  pH 7.5  pH  8.5  Enzyme form No FDP  + FDP  Mantle PyK  5.8 X 10 M  Adductor PyK  6.6  4  X 10 M 4  No FDP  8 X 10~ M  4.5 X 1 0 M  7.4 X 10 M  2.5 X 10~ M  5  5  _4  4  + FDP  6.6  7.4  No FDP  X 10 M 5  X 10 M 5  1.9 X 10 M 4  9.0  X 10 M 5  +  6.6  X  FDP  10~ M  7.7 X 10  5  5  M  30  Fig. 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  o f adductor p y r u v a t e k i n a s e .  Reactant  contents were the same as d e s c r i b e d under F i g . 5.  30 <*-  31 \  Fig. I l l ,  7.  E f f e c t of ATP c o n c e n t r a t i o n on t h e r e a c t i o n r a t e o f mantle and adductor p y r u v a t e k i n a s e s a t d i f f e r e n t pH and penolpyruvate concentrations.  Reactant  contents were:  50 mM T r i s - H C l , 6 mM Mg**, 50 mM K , 0.2 mM ADP, +  p-enolpyruvate  c o n c e n t r a t i o n s as i n d i c a t e d , 0.15 mM NADH,  i n c r e a s i n g c o n c e n t r a t i o n o f ATP and excess dehydrogenase.  (A) Mantle p y r u v a t e k i n a s e , ( A ) ,  _3  p - e n o l p y r u v a t e , pH 8.5; (•-), 1..X 10 pH 7.5; ( A ) ,  lactate  I X 10~ , 3  , p-enolpyruvate,  p - e n o l p y r u v a t e , pH 8.5;  (B) adductor p y r u v a t e k i n a s e ; ( 0 , A )  5 X 10  _3  p - e n o l p y r u v a t e , pH 7.5 and 8.5; ( # ) I X 10  _3  v a t e , pH 7.5; CD)  -4 5 X 10 ,  1 X 10  \ , p-enolpyru-  , p - e n o l p y r u v a t e , pH 8.5.  Activity  (AE  3 4 0  /unit  time)  Activity  (AE  3 4 0  /unit  time)  >  001-  32 s a t u r a t i n g c o n c e n t r a t i o n s , these r e s u l t s suggest modulator o f mantle enzyme. competitive  t h a t ATP i s n o t an  I n c o n t r a s t , adductor  important  enzyme ( F i g . 8-B) shows  i n h i b i t i o n and i s i n h i b i t e d by lower c o n c e n t r a t i o n o f ATP.  Thus,  2 mM ATP i n c r e a s e s the K , , ^ . a t l e a s t 10 f o l d w i t h l i t t l e o r no m(p-enolpyruvate) e f f e c t on the Vmax. F u r t h e r , i t was noted t h a t the n a t u r e o f i n h i b i t i o n o f I| e i t h e r enzyme c o u l d n o t be a l t e r e d by i n c r e a s i n g o r d e c r e a s i n g c e n t r a t i o n o f the assay medium, although depends upon the amount o f the Mg  t h e Mg  the degree o f i n h i b i t i o n  con-  clearly  present.  I n t e r a c t i n g e f f e c t s o f ATP and f r u c t o s e - 1 , 6 - P Q • Rozengurt et a l . (1969) and Tanaka e t a l . (1967) have r e p o r t e d t h a t ATP i n h i b i t i o n of l i v e r pyruvate  k i n a s e i s r e v e r s e d by fructose-1,6-P » 2  of t h i s k i n e t i c p r o p e r t y w i t h values  the o y s t e r mantle enzyme a t two d i f f e r e n t pH  at varying concentrations of p-enolpyruvate,  s i m i l a r t o t h a t o f mouse l i v e r p y r u v a t e of p - e n o l p y r u v a t e 1,6-P2 and ATP. 6-P  2  kinase.  indicate a  behaviour  F i g u r e s 9 and 10 show p l o t s  s t a u r a t i o n o f the mantle enzyme i n t h e p r e s e n c e o f f r u c t o s e P-enolpyruvate  s a t u r a t i o n curves  overcomes the ATP i n h i b i t i o n .  r e l e a s e s the ATP i n h i b i t i o n  At pH 8.5  c l e a r l y show t h a t f r u c t o s e - 1 ,  ( F i g . 9) 1 0 ~  6  (caused by 3 mM ATP) by l o w e r i n g  2/3 o f c o n t r o l and i n c r e a s e s the Vmax by 2 f o l d ; releases  Studies  M  fructose-1,6-P the K  m  down to  thus, f r u c t o s e not o n l y  the ATP i n h i b i t i o n but o v e r r i d e s i t . At pH 7.5 ( F i g . 10) under  s i m i l a r c o n d i t i o n s fructose-1,6-P„ lowers the K down t o 1/5 o f c o n t r o l and z m a g a i n doubles the Vmax. In presence o f ATP and f r u c t o s e - 1 , 6 - P together the 2  K , -, s i s s i m i l a r a t both pH v a l u e s . m(p-enolpyruvate) summarized i n T a b l e  2.  These r e s u l t s a r e b r i e f l y  2  Table 2 E f f e c t of ATP  and  fructose-1,6-P  k i n a s e v a l u e determined  pH  on the K , , v mantle 2 m(p-enolpyruvate) from F i g . I l l , 9 and 10.  Control  0  3 mM  ATP  1 pM  pyruvate  FDP  3 mM  ATP and  1 uM  pH 8.5  pH  7.5  1.8  X 10 M 4  5 X 10 M 4  1.8  X 10 M  1 X 10 M  1.3  X 10  5 X 10 M  1.05'X 10 M  1.3  X  4  4  4  4  FDP  4  M  10~ M 4  34  Fig. I l l ,  8.  Double r e c i p r o c a l p l o t s o f t h e r e a c t i o n pyruvate kinase ATP; •  (A) (O,  v e l o c i t y o f mantle  c o n t r o l ; A , 1 mM A T P ; # , 2 mM  , 3 mM ATP; and adductor p y r u v a t e k i n a s e (B)  (O,  c o n t r o l ; A , 0.5 mM ATP; • , ImM ATP; • , 2 mM ATP) i n p r e s e n c e of d i f f e r e n t ATP c o n c e n t r a t i o n s as i n d i c a t e d at pH 8.5.  Reactant c o n t e n t s were the same as d e s c r i b e d  under F i g . 5.  3 ^  /[PEP]x10 M 3  /[pEP]x 1 0 M 3  35  Fig. I l l ,  9.  I n t e r a c t i n g e f f e c t s o f ATP and f r u c t o s e - 1 , 6 - P ^ on the K / , f o r the mantle enzyme a t pH 8.5. m(p-enolpyruvate) J  x  r  Reactant contents were the same as d e s c r i b e d under F i g . 5, but 3 mM ATP and I X 1 0 ~ added where i n d i c a t e d . p y r u v a t e k i n a s e ; PEP,  6  M fructose-1,6-P  2  FDP, f r u c t o s e - l , 6 - P ; PyK, 2  p-enolpyruvate.  were  35^  »50r  '/[PEPJxIo'M  36  Fig. I l l ,  10.  I n t e r a c t i n g e f f e c t s o f ATP and f r u c t o s e - 1 , 6 - P  2  on the  K / , v f o r the mantle enzyme a t pH 7.5. m(p-enolpyruvate) J  v  E x p e r i m e n t a l c o n d i t i o n s were the same as d e s c r i b e d under F i g . 9.  36 «*-  37  Fig. I l l ,  11.  E f f e c t o f a l a n i n e c o n c e n t r a t i o n on t h e r e a c t i o n r a t e and  alanine,  , d e t e r m i n a t i o n f o r (A) adductor, and i  (B) mantle p y r u v a t e k i n a s e s a t two d i f f e r e n t pH v a l u e s . R e a c t i o n contents were: and ADP,  pH 7.5  ( O ) 8.5 as i n d i c a t e d , 6 mM Mg"*", 50 mM K , 0.2 mM 4  0.5 mM p - e n o l p y r u v a t e ,  l a c t a t e dehydrogenase. was  50 mM T r i s - H C l ( A ) ,  v a r i e d as i n d i c a t e d .  +  0.15 mM NADH and excess  Concentration  of alanine  Activity  5  L  (AE  3 4 0  /unit  Activity  time)  t  (AE  3 4 0  /  38  Fig. I l l ,  12.  E f f e c t of p h e n y l a l a n i n e on the r e a c t i o n r a t e and phenylalanine,  . d e t e r m i n a t i o n f o r mantle and  adductor  i p y r u v a t e k i n a s e s a t two d i f f e r e n t pH v a l u e s .  Experi-  mental c o n d i t i o n s were the same as d e s c r i b e d under F i g . 11, but c o n c e n t r a t i o n s of p h e n y l a l a n i n e were v a r i e d as i n d i c a t e d . (O)  pH 8.5 ;  ( • ) pH 8.5.  Mantle p y r u v a t e ,  '' adductor  ( • ) pH 7.5,  pyruvate kinase, (A)  pH 7.5,-  39 Search  f o r other  modulators.  Since pyruvate we f e l t  occupies  i t necessary  a central  to study  kinase a c t i v i t i e s .  crossroads i n oyster tissue  the e f f e c t s  metabolism,  o f o t h e r m e t a b o l i t e s on p y r u v a t e  Of the v a r i o u s compounds t e s t e d , 5' AMP,  acetylCoA,  citrate,  s u c c i n a t e , malate and o x a l o a c e t a t e have n e i t h e r s t i m u l a t o r y n o r i n h i b i t o r y effects  on the o y s t e r enzymes.  to a f f e c t  Only  the enzyme i n i n h i b i t o r y  L - a l a n i n e and p h e n y l a l a n i n e were found  manner.  -4 U s i n g 5 X 10  M p-enopyruvate, both o y s t e r enzymes were found more  s u s c e p t i b l e to a l a n i n e i n h i b i t i o n a t pH 7.5 than a t 8.5 ( F i g . 11).  F o r mantle  enzyme, the R\ v a l u e s f o r a l a n i n e i n h i b i t i o n a r e 7.6 mM and 2.7 mM a t pH 8.5 and of and  7.5 r e s p e c t i v e l y . a l a n i n e (K adductor  tration  The adductor  enzyme i s i n h i b i t e d  v a l u e s 3.6 and 0.6 mM r e s p e c t i v e l y ) .  pyruvate  concentration  F i g u r e 12 shows mantle  k i n a s e a c t i v i t i e s as a f u n c t i o n o f i n c r e a s i n g concen-  of phenylalanine.  i s pH independent h a v i n g  P h e n y l a l a n i n e i n h i b i t i o n o f mantle p y r u v a t e a R\ v a l u e around 6 mM a t both pH v a l u e s  a g a i n the K\ v a l u e s f o r t h e adductor Nature o f L - a l a n i n e and p h e n y l a l a n i n e  enzyme a r e somewhat  kinase  examined;  lower.  inhibition.  L - a l a n i n e i s known to i n h i b i t p y r u v a t e Seubert  a t lower  k i n a s e (Weber e t al., 1968;  et a l . , 1968; Rozengurt et^ al., 1970) i n a manner c o m p e t i t i v e w i t h  r e s p e c t to p - e n o l p y r u v a t e .  In c o n t r a s t , i n the case o f o y s t e r p y r u v a t e  kinase,  a l a n i n e i n h i b i t s both enzymes i n a mixed c o m p e t i t i v e manner w i t h r e s p e c t t o p-enolpyruvate fructose-1,6-P  a t both pH v a l u e s examined. 2  reverses alanine i n h i b i t i o n .  a l a n i n e a t pH 8.5 ( F i g . 13, upper panel) alanine i n h i b i t i o n .  As i n the case o f ATP i n h i b i t i o n , F o r mantle enzyme, w i t h 6 mM  fructose-1,6-P  At pH 7.5 under s i m i l a r  the a c t i v i t y r e t u r n s to 70% o f the c o n t r o l .  2  completely  overcomes  c o n d i t i o n s ( F i g . 13, lower  panel)  The n a t u r e o f p h e n y l a l a n i n e d i f -  40  Fig. I l l ,  13.  Double r e c i p r o c a l p l o t s o f the r e a c t i o n v e l o c i t y of mantle p y r u v a t e k i n a s e and  7.5.  i n p r e s e n c e o f a l a n i n e a t pH 8.5  Reactant contents  were the same as d e s c r i b e d  under F i g . 5, but d i f f e r e n t c o n c e n t r a t i o n s  of a l a n i n e  — fi  and  fructose-1,6-P  alanine; A 6-P ; I 2  2  (2.5 X 10  M) were added: • , 6 mM  , 4 mM a l a n i n e ; O , no a l a n i n e and f r u c t o s e - 1 ,  , 6 IDM a l a n i n e + f r u c t o s e - 1 , 6 - P ;  6-P„ o n l y .  2  •  , fructose-1,  41  f e r s f o r the 2 enzymes b e i n g c o m p e t i t i v e f o r t h e mantle enzyme ( F i g . 14, upper panel)  and mixed-competitive  f o r the adductor  enzyme ( F i g . 14, lower  F o r the mantle enzyme, 6 t o 10 mM p h e n y l a l a n i n e doubles  panel).  the K ,  v  m(p-enolpyruvate) w h i l e Vmax remains almost  unaffected.  Again  f r u c t o s e - 1 , 6 - P 2 p r o t e c t s the —6  mantle enzyme a g a i n s t p h e n y l a l a n i n e i n h i b i t i o n ; r e v e r s e s the i n h i b i t i o n  caused  by 6 mM  2.5 X 10  phenylalanine.  M fructose-1,6-P  2  42  F i g . I l l , 14.  Double reciprocal plots of the reaction v e l o c i t y of mantle and adductor pyruvate kinases i n presence of phenylalanine and fructose-1,6-P  2  at pH 8.5.  Reactant contents were  the same as described under F i g . 5, but d i f f e r e n t concentrations of phenylalanine and fructose-1,6-P  2  were  added: • , 10 mM phenylalanine; O > c o n t r o l ; B  , 6 mM  —6 phenylalanine + 2.5 X 10~  M fructose-1,6-P ; © , 2.5 X 2  —6 10  M fructose-1,6-P  2  only; •  A , A , 3mM phenylalanine.  , • , 6 mM phenylalanine;  \2<L  V[PEP] 1 0 M 3  DISCUSSION A comparison of the p r o p e r t i e s of mantle, adductor, pyruvate kinases i s given i n Table I I I .  r a t muscle and  liver  The d a t a i n t h i s study suggest  that,  as i n the mammalian case, the enzyme p y r u v a t e k i n a s e i n the o y s t e r o c c u r i n t i s s u e s p e c i f i c m u l t i m o l e c u l a r forms and  t h a t the k i n e t i c p r o p e r t i e s of each  isozyme seem to gear w e l l w i t h the o v e r a l l metabolism of the t i s s u e i n which it  occurs.  Thus, the K  k i n a s e are 3 and  simultaneous  v a l u e s of p - e n o l p y r u v a t e  and ADP  pyruvate adductor  Under c o n d i t i o n s of g l u c o n e o g e n e s i s , when p - e n o l p y r u v a t e i s  from p y r u v a t e and C-4  a c i d s of the Krebs c y c l e , any  p y r u v a t e k i n a s e a c t i v i t y would s e r v e merely  at the expense of ATP  ( S o l s , 1968).  i s s t r o n g l y a c t i v a t e d by f r u c t o s e - 1 , 6 - P x  T h i s may  J  pyruvate kinase a c t i v i t y  pyruvate  c o n c e n t r a t i o n s because of the h i g h  M i c h a e l i s c o n s t a n t f o r the mantle p y r u v a t e k i n a s e .  / ., ) . m(p-enolpyruvate)  to r e c y c l e  significant  In mantle, t h i s k i n d of r e c y c l i n g would  not be f a v o u r e d a t low p - e n o l p y r u v a t e  K  f o r mantle  6 times h i g h e r than the c o r r e s p o n d i n g v a l u e s f o r the  pyruvate kinase. b e i n g produced  m  reflect  A l s o , the mantle enzyme  ( c a u s i n g a l a r g e decrease i n  2  a p h y s i o l o g i c a l mechanism whereby ° r  J  J  can be i n c r e a s e d d u r i n g g l y c o l y s i s and markedly  decreased  d u r i n g g l u c o n e o g e n e s i s , when f r u c t o s e - 1 , 6 - P  reduced.  An e n t i r e l y analogous  i n l i v e r , a major gluconeogenic ' ° ° J  2  c o n c e n t r a t i o n may  s i t u a t i o n o c c u r s i n mammalian t i s s u e s . t i s s u e , the K  be Thus,  , .. * f o r pyruvate m(p-enolpyruvate)  k i n a s e i s an o r d e r of magnitude h i g h e r than i n the h i g h l y g l y c o l y t i c muscle ( S o l s , 1968; K  and Reynard et a l l ,  1961), and  , , . f o r the mantle enzyme. m(p-enolpyruvate)  pyruvate  In t h i s c i r c u m s t a n c e ,  c o n v e r s i o n to p y r u v a t e would not be f a v o u r e d i n the  t i s s u e when p - e n o l p y r u v a t e fructose-1,6-P it  indeed i s comparable to the  2  may  does not a f f e c t  c o n c e n t r a t i o n s are low.  too, p-enol-  gluconeogenic  Also i n this  case,  a c t as a s p e c i f i c " o n - o f f " s w i t c h on the l i v e r enzyme, but the mammalian muscle enzyme.  Thus b o t h l i v e r and mantle  44 Table I I I Comparison o f the p r o p e r t i e s o f mantle, adductor, l i v e r pyruvate  r a t muscle and  kinases.  Oyster Mantle PK  Rat Adductor PK  Muscle PK  Liver  2.4 X 10"*M  8 X 10" M  5 X 10~ M  7 X 10" M  6.1 X 10" M  1.0 X 10~ M  2.1 X lO'St  2.1 X 10" M  7.7 X 10" M doesn't i n h i b i t at high cone. of K .  7.7 X 10" M doesn't i n h i b i t at h i g h cone. of K+.  12.0 X 10" M i n h i b i t s at h i g h cone. of K+.  10 X 10" M i n h i b i t s at h i g h cone. of K .  1.25 X 10" M  2.2 X 10" M  Electrophoretic migration  slow c a t h o d a l l y moving.  slow c a t h o d a l l y moving.  fast anodally moving.  fast anodally moving.  Isoelectric  6.35  5.6,  Michaelis-Menton  MichaelisMenton  MichaelisMenton  Sigmoidal  Fructose-l,6-P . effects  feedforward activator.  feedforward activator.  no known effects..  feedforward effector.  ATP i.  non-competitive  competitive  competitive  competitive  4.0 X 10" M  2.8 X 10" M  1.7 X 10" M  5.6 X 10" M  mixed-comp e t i t i v e type.  mixed-competicompetitive t i v e type. type. p r o t e c t e d by FDP. no e f f e c t .  m (p-enolpyruvate)  4  K  m(ADP)  K  a  Potassium  3  +  K  a  Magnesium  3  points  Kinetics  2  ii.  Inhibition Nature o f i n hibition values  L-alanine i n h i b i t i o n i. Nature o f inhibition  3  p r o t e c t e d by FDP. ii.  K^ v a l u e s  5  4  3  4  3  +  3  6.5  3  2.8 X 10 2.7 X 10" M  4  5  4  PK  M  2  2 X 10  M  3  competitive type. p r o t e c t e d by FDP. _ 2.5 X 10" M  3  Phenylalanine inhibition i. Nature o f inhibition ii.  K^ v a l u e s  competitive type. p r o t e c t e d by FDP. 5.8 X 10" M 3  mixed-competino known t i v e type. effect, p r o t e c t e d by FDP. " 3.8 X 10"3M  no known effect.  45 p y r u v a t e k i n a s e s seem w e l l adapted  f o r f u n c t i o n i n a metabolism  g l y c o l y t i c and g l u c o n e o g e n i c f u n c t i o n w i t h i n a s i n g l e  f i s h muscle,  f o r pyruvate kinases  and mammalian muscle are r a t h e r s i m i l a r to  each o t h e r , and as p o i n t e d out, a r e d i s t i n c t l y l i v e r enzymes.  involves  tissue.  The M i c h a e l i s c o n s t a n t s f o r p - e n o l p y r u v a t e and ADP of adductor muscle,  that  lower than the mantle  and  Thus, these enzymes would compete f a v o u r a b l y f o r q u i t e  c o n c e n t r a t i o n s o f p - e n o l p y r u v a t e f o r c o n v e r s i o n to p y r u v a t e .  The  the  low  adductor  muscle p y r u v a t e k i n a s e d i f f e r from the mammalian muscle p y r u v a t e k i n a s e s , however, i n b e i n g s t r o n g l y f e e d f o r w a r d a c t i v a t e d by f r u c t o s e - 1 , T h i s has been observed f o r f i s h muscle as w e l l , and may  be a g e n e r a l c h a r a c t e r i s t i c of  muscle p y r u v a t e k i n a s e i n p o i k i l o t h e r m i c organisms  (Somero and Hochachka,  1968). The r o l e of f r u c t o s e - 1 , 6 - P v a t e k i n a s e s i s of i n t e r e s t . i s a b l e to r e v e r s e ATP oyster, fructose-1,6-P  2  p r o t e c t i o n o f both mantle  In a l l cases thus f a r examined, f r u c t o s e - 1 , 6 - P  i n h i b i t i o n of p y r u v a t e k i n a s e . 2  and adductor p y r u 2  In a d d i t i o n , i n the  p r o t e c t s both enzyme forms a g a i n s t a l a n i n e and  phenylalanine i n h i b i t i o n .  Thus f a r no adequate  explanation i s available for  these e f f e c t s and t h i s i s c l e a r l y an important a r e a f o r f u r t h e r r e s e a r c h . In mammalian systems,  fructose-1,6-P  pH v a l u e s (Rozengurt et a l . , 1969). fructose-1,6-P  2  2  a c t i v a t i o n i s g r e a t e s t at a l k a l i n e  An o p p o s i t e pH dependence of the  a c t i v a t i o n of the o y s t e r p y r u v a t e k i n a s e s i s observed.  b o t h enzymes, fructose-1,6-P„ lowers the K , , ^ , and t h i s ' ' 2 m(p-enolpyruvate) 3  is particularly 5.8  X 10~  6.6  X 10 ^  K  -  4  s t r i k i n g a t lower pH v a l u e s ( a t pH 6.5  M to 8 X I O M).  - 5  M;  a t pH 8.5  In consequence,  m  m  effect  i s reduced  i s reduced from 1.9  X 10~  i n the presence o f f r u c t o s e - 1 , 6 - P  , ., N i s e s s e n t i a l l y pH m(p-enolpyruvate) The adductor enzyme appears  the K  the K  For  2  A  from M to  the  independent.  to be under t i g h t ATP  regulation.  Thus,  46 2 mM ATP, a v a l u e p r o b a b l y w i t h i n t h e p h y s i o l o g i c a l range ( W i l l i a m s o n 1967) , causes about a 10 f o l d i n c r e a s e i n t h e K . .. ' m(p-enolpyruvate) n  d i t i o n s o f low p - e n o l p y r u v a t e pyruvate  concentrations, i t i s evident that  k i n a s e would be u n u s u a l l y s e n s i t i v e t o ATP.  the adductor  In t h i s  etal.,  Under conadductor  characteristic,  enzyme resembles mammalian muscle p y r u v a t e k i n a s e (Reynard  1961), and a d i p o s e p y r u v a t e k i n a s e  (Pogson, 1968) a l l o f which have s i m i l a r  K\ v a l u e s , but i t d i f f e r s from mammalian b r a i n p y r u v a t e k i n a s e Passonneau, 1964) and the mantle enzyme. i s noncompetitive.  et_ a l . ,  In both o f t h e l a t t e r ATP i n h i b i t i o n  Because o f t h e h i g h K-j^TP) ^  because ATP does not a l t e r the apparent  (Lowry and  0 r  t  *  i e  m a n t  -'-  e  enzyme, and  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 o f t h i s enzyme. In t h i s c o n n e c t i o n , i t i s i n t e r e s t i n g i a n b r a i n pyruvate petitively  kinases  (Schwark e t a l . , 1970; and Weber, 1969) a r e com-  i n h i b i t e d by p h e n y l a l a n i n e and the K_ v a l u e s a r e a g a i n s i m i l a r f o r  the enzymes from the 2 t i s s u e t y p e s . is  fairly  t h a t both mantle enzyme and mammal-  In the mantle, p y r u v a t e k i n a s e  s e n s i t i v e to phenylalanine c o n t r o l s i n c e phenylalanine  c e n t r a t i o n s ) produces q u i t e l a r g e i n c r e a s e s i n t h e K , r  n  °  ,  activity  ( a t R\ conN  .  Since  m(p-enolpyruvate)  p h e n y l a l a n i n e c o n c e n t r a t i o n s a r e known to be u n u s u a l l y h i g h i n m o l l u s c tissues  ( V i r k a r e_t a l . , 1970) t h i s amino a c i d may be an important  c a l feedback i n h i b i t o r o f p y r u v a t e mammalian b r a i n .  kinase a c t i v i t y  physiologi-  i n t h i s t i s s u e as i t i s i n  CHAPTER IV: Catalytic  and R e g u l a t o r y P r o p e r t i e s o f Oyst  Phosphoenolpyruvate I.  Carboxykinase:  C e l l u l a r d i s t r i b u t i o n and the e f f e c t s of and m e t a l i o n s on the enzyme a c t i v i t y .  47 INTRODUCTION I t i s now  w e l l documented t h a t many i n t e r t i d a l b i v a l v e m o l l u s c s are  f a c u l t a t i v e anaerobes.  Under a n a e r o b i c c o n d i t i o n s , g l u c o s e i s c a t a b o l i z e d by  the r e a c t i o n s o f g l y c o l y s i s and lactate,  the Krebs  c y c l e , but under a x o n i c  conditions,  the u s u a l end p r o d u c t o f a n a e r o b i c g l y c o l y s i s i n v e r t e b r a t e s , does  not accumulate.  I n s t e a d , the end p r o d u c t o f a n a e r o b i c g l u c o s e c a t a b o l i s m i s  s u c c i n a t e (Hammen, 1969).  A l t h o u g h t h e r e has been some d i s p u t e over  the  p r e c i s e m e t a b o l i c pathways i n v o l v e d i n a n a e r o b i o s i s , Simpson and Awapara (1966) have p r e s e n t e d c o n v i n c i n g evidence t h a t p - e n o l p y r u v a t e i s c a r b o x y l a t e d to o x a l o a c e t a t e , w i t h the subsequent (oxaloacetate  > malate  r e v e r s a l of a p o r t i o n of the Krebs  > fumarate — >  s u c c i n a t e ) l e a d i n g to s u c c i n a t e as the  f i n a l end p r o d u c t of a n a e r o b i c g l u c o s e d i s s i m i l a t i o n . l a s e i s not d e t e c t a b l e i n these organisms, c a t a l y z e d by p - e n o l p y r u v a t e lase  cycle  carboxykinase  Since pyruvate  carboxy-  the ( ^ - f i x a t i o n step i s a p p a r e n t l y (ITP, GTP:  o x a l o a c e t a t e carboxy-  ( t r a n s p h o s p h o r y l a t i n g ) ; I.U.B.E.C. 4.1.1.32): o x a l o a c e t a t e + ITP  (or GTP)  p - e n o l p y r u v a t e + IDP  (or GDP)  +  C0  2  These m e t a b o l i c c a p a c i t i e s of i n t e r t i d a l b i v a l v e s r a i s e some e s s e n t i a l r e g u l a t o r y requirements place, p-enolpyruvate  a t the p - e n o l p y r u v a t e b r a n c h i n g p o i n t .  carboxykinase  In the  first  (PEPCK) r e a c t i o n i n v e r t e b r a t e t i s s u e s  n o r m a l l y f u n c t i o n s i n the d e c a r b o x y l a t i n g d i r e c t i o n , d u r i n g g l u c o n e o g e n s i s i n the l i v e r and kidneys i n adipose t i s s u e  ( S c r u t t o n and U t t e r , 1968), d u r i n g g l y c e r o n e o g e n e s i s  (Meyuhas, Boshwitz  and Reshef,  1971), and d u r i n g o p e r a t i o n  o f the m a l a t e - o x a l o a c e t a t e c y c l e i n s k e l e t a l white muscle (Opie and Newsholme, 1967).  In c o n t r a s t , i n the m o l l u s c s t h e r e i s an e s s e n t i a l requirement  PEPCK f u n c t i o n i n the C O ^ - f i x i n g d i r e c t i o n , and important  t h i s would be  for  particularly  i n s k e l e t a l muscle.  Secondly, under these c o n d i t i o n s , p - e n o l p y r u v a t e i s a common s u b s t r a t e  48 f o r both the p y r u v a t e k i n a s e r e a c t i o n , which i s f a v o u r e d d u r i n g a e r o b i c metabolism  (Mustafa and Hochachka, 1971), and f o r the PEPCK r e a c t i o n , which i s  favoured during a n a e r o b i o s i s .  Hence, i t i s c l e a r t h a t t h e c a t a l y t i c  activities  of PEPCK and p y r u v a t e k i n a s e must be c l o s e l y i n t e g r a t e d f o r e f f i c i e n t l i n g o f carbon through t h i s m e t a b o l i c b r a n c h p o i n t , d u r i n g a e r o b i c transitions. I | Zn  And f i n a l l y ,  anaerobic  t i s s u e s and f l u i d s o f o y s t e r have u n u s u a l l y h i g h  | | and Cu  requirements  c o n c e n t r a t i o n s , which may i n f l u e n c e t h e c a t i o n o f PEPCK.  study o f the PEPCK muscle.  I | r e s p e c t to Mn  cofactor  F o r these r e a s o n s , I i n i t i a t e d a d e t a i l e d  kinetic  c a t a l y z e d c a r b o x y l a t i o n r e a c t i o n i n o y s t e r adductor  T h i s chapter e s t a b l i s h e s  phosphoenolpyruvate  K  channel-  carboxykinase,  (a) t h e c e l l u l a r d i s t r i b u t i o n o f o y s t e r (b) the b e h a v i o u r o f o y s t e r PEPCK w i t h  [ | , Zn  and IDP c o n c e n t r a t i o n s , (c) pH modulation [|  | |  o f the  / , % i n the presence o f 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 w i t h Mn and Zn under d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s .  49 RESULTS Electrofocusing. I s o e l e c t r i c f o c u s i n g of p - e n o l p y r u v a t e c a r b o x y k i n a s e i n d i c a t e s a s i n g l e major component w i t h enzyme a c t i v i t y . p o i n t i s about 6.64  ( F i g . 1).  I t was  Under our c o n d i t i o n s , the noted t h a t p r o l o n g e d  isoelectric  electrofocusing  l e a d s to enzyme i n a c t i v a t i o n .  Requirement f o r the p - e n o l p y r u v a t e  carboxykinase c a t a l y z e d c a r b o x y l a t i o n  reaction. The requirements  f o r the p - e n o l p y r u v a t e c a r b o x y k i n a s e c a t a l y z e d carboxy-  l a t i o n are summarized i n T a b l e I.  A b s o l u t e requirements  d i v a l e n t i o n , n u c l e o t i d e diphosphate  f o r p-enolpyruvate,  and b i c a r b o n a t e a r e i n d i c a t e d .  Neither I|  ADP  nor IMP  checked  can r e p l a c e IDP  at d i f f e r e n t IDP  activity.  (or GDP).  The enzyme i n the presence o f Mg  c o n c e n t r a t i o n s and pH v a l u e s shows e s s e n t i a l l y  no  The p - e n o l p y r u v a t e c a r b o x y k i n a s e p r e p a r a t i o n used does not have any  l a c t a t e dehydrogenase a c t i v i t y w h i l e endogenous malate dehydrogenase (MDH) " m a l i c " enzyme a c t i v i t i e s were found to be p r e s e n t .  and  N e i t h e r o f these enzymes  i n t e r f e r e w i t h the assay system used i n t h i s study, s i n c e MDH  i s needed i n  l a r g e amounts f o r c o u p l i n g o x a l o a c e t a t e t o malate f o r m a t i o n , w h i l e o x a l o a c e t a t e d e c a r b o x y l a t i o n by " m a l i c " enzyme i s NADP dependent.  Under our  experimental  c o n d i t i o n s , p - e n o l p y r u v a t e c a r o b x y k i n a s e a c t i v i t y c o u l d not be d e t e c t e d i n these  tissues.  T i s s u e and s u b - c e l l u l a r d i s t r i b u t i o n o f o y s t e r p - e n o l p y r u v a t e R e s u l t s of s t u d i e s of m i t o c h o n d r i a l and boxykinase  carboxykinase.  cytoplasmic p-enolpyruvate car-  l e v e l s i n v a r i o u s o y s t e r t i s s u e s assayed immediately  p r e p a r a t i o n are g i v e n i n T a b l e I I .  after  A l l assays were r e p e a t e d a t l e a s t  twice.  50  F i g . IV, 1.  Electrofocusing pattern of the adductor p-enolpyruvate carboxykinase. and Methods".  For experimental procedures see "Materials A c t i v i t y i s plotted against f r a c t i o n numbers;  the numerical values plotted on the abscissa indicate the pH of the selected f r a c t i o n s . tions are the same as i n F i g . 2.  Reactant concentra-  51 Table I Components o f p - e n o l p y r u v a t e c a r b o x y k i n a s e c a t a l y z e d  carboxylation  reations. Complete r e a c t i o n m i x t u r e c o n t a i n e d  50 mM T r i s - M a l e a t e b u f f e r , pH 6.0,  1 mM Mn " ", 10 mM KHC0 , 1 mM IDP, 1 mM p - e n o l p y r u v a t e , 0.15 mM NADH and 4  1  3  excess Sigma MDH.  Reactants d e l e t e d from o r added to complete assay  Activity AE ,„/unit time 340 0  None (complete)  % A c t i v i t y of complete assay  0.46  100  0.00  0.00  I | Mn , deleted  0.00  0.00  j| j | Mn , d e l e t e d , Mg added, 1 mM  0.00  0.00  p-enolpyruvate,  Mn K  +  I |  d e l e t e d , Zn  deleted  | |  added, 1 mM  added, 1 mM o r 10 mM  0.30  65.2  0.42  91.3  0.00  0.00  IMP added 1 mM  0.00  0.00  IDP,  d e l e t e d , ADP added 1 mM  0.00  0.00  IDP,  d e l e t e d , GDP added 1 mM  0.24  IDP,  deleted  IDP,  deleted,  KHC0 , d e l e t e d  0.08  M a l a t e dehydrogenase, d e l e t e d  0.38  3  52.0 1.70 82.6  p-enolpyruvate carboxykinase deleted  0.00  0.00  52 Table I I T i s s u e and s u b c e l l u l a r d i s t r i b u t i o n o f o y s t e r p - e n o l p y r u v a t e carboxykinase. Reactant  c o n c e n t r a t i o n s and assay c o n d i t i o n s were e s s e n t i a l l y  as r e f e r r e d i n T a b l e I.  M i t o c h o n d r i a l f r a c t i o n s were a l s o checked I |  7.0 and 7.4 i n presence AE  0 / n  /unit  of e i t h e r Mn  the same a t pH  |j and Mg  .  Activity  i s expressed as  time.  Tissues, subcellular  fraction  M i t o c h o n d r i a l p-enolpyruvate  Activity  carboxykinase  (a) Mantle  undetectable  (b)  undetectable  (c)  Gill Adductor  Cytoplasmic p - e n o l p y r u v a t e (High speed  undetectable  muscle carboxykinase  supernatant)  (a) Mantle  negligible  (b)  Gill  negligible  (c)  Adductor  muscle  0.40  40 to 75% s a t u r a t e d c y t o p l a s m i c p-enolpyruvate  carboxykinase  (a) Mantle  0.10  (b)  Gill  0.20  (c)  Adductor  muscle  0.8  53 No a c t i v i t y was found i n any o f the m i t o c h o n d r i a l p r e p a r a t i o n s o f g i l l , mantle  and adductor t i s s u e s .  the s o l u b l e f r a c t i o n .  In each case a l l o f the a c t i v i t y appeared i n  L e v e l s o f the enzymic  t i s s u e s , compared to the a c t i v i t y is  inferred  activities  i n mantle  and g i l l  i n adductor muscle were extremely low. I t  from these r e s u l t s t h a t o n l y c y t o p l a s m i c p - e n o l p y r u v a t e  carboxy-  kinase i s present i n oyster t i s s u e .  E f f e c t s o f pH on p - e n o l p y r u v a t e c a r b o x y k i n a s e  activity.  F i g u r e 2 shows the a c t i v i t y o f p - e n o l p y r u v a t e c a r b o x y k i n a s e assayed i n I | the d i r e c t i o n o f o x a l o a c e t a t e s y n t h e s i s i n t h e presence o f Mn as a f u n c t i o n o f pH. t i o n o f the r e a c t i o n . instance. ion  | | and Zn  pH measurements were made j u s t b e f o r e and a f t e r  termina-  No change i n pH d u r i n g t h e r e a c t i o n was noted i n any  The pH a c t i v i t y p r o f i l e s o b t a i n e d i n t h e presence o f e i t h e r metal  were s i m i l a r i n g e n e r a l p a t t e r n .  However, both t h e p - e n o l p y r u v a t e  t r a t i o n and the c a t i o n c o f a c t o r determine t h e p r e c i s e pH optimum.  concen-  Thus i n the  I | presence o f Zn pH 6.0.  and low p - e n o l p y r u v a t e c o n c e n t r a t i o n s , the pH optimum i s about I | In the presence of Zn and s a t u r a t i n g p - e n o l p y r u v a t e c o n c e n t r a t i o n I |  maximal a c t i v i t y  appears  around pH 5.1 w h i l e i n the presence o f Mn  a c t i v i t y i s maximal a t pH 6.0.  the  Values o f enzyme a c t i v i t i e s measured i n the  presence o f sodium a c e t a t e b u f f e r a t below pH 5 were c o r r e c t e d f o r 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  5 i s o n l y 60% o f the a c t i v i t y  of  i n the sodium a c e t a t e b u f f e r , a t pH  i n T r i s - M a l e a t e b u f f e r a t pH 5 ) .  the r e s u l t s p r e s e n t e d i n the F i g . 2 subsequent  On t h e b a s i s I | | |  s t u d i e s w i t h Mn  o r Zn  d i v a l e n t i o n s were made a t the d i f f e r e n t pH optima. Reversibility  of pH e f f e c t .  To r u l e out the p o s s i b i l i t y  t h a t the reduced a c t i v i t y a t a l k a l i n e pH  54  Fig.  IV, 2.  E f f e c t s of pH on p - e n o l p y r u v a t e c a r b o x y k i n a s e o) i n presence of Zn (—)  h i g h and  (  and  ( • ) i n presence o f Mn  Tris-Maleate buffer at I |  d i f f e r e n t pH v a l u e s , 1 mM IDP,  10 mM  Mn  KHC0 , 1 mM 3  | | o r Zn  dehydrogenase.  c l o s e d c i r c l e was  assayed  which accounts  as  indicated,  p - e n o l p y r u v a t e , 0.15  and excess malate  pH 4.8  ,  ) low c o n c e n t r a t i o n s o f p - e n o l p y r u v a t e .  R e a c t i o n contents were 50 mM  1 mM  activity;  mM  ( A c t i v i t y a t the  NADH first  i n sodium a c e t a t e b u f f e r a t  f o r the steepness  of the  curve.)  Activity  (AE  340  / u n i t time)  55 value  (pH 7.0 and above) i s not due t o the d e s t r u c t i o n o f the a c t i v e s i t e ,  experiments were c a r r i e d out on t h e r e v e r s i b i l i t y o f 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 s m a l l amount o f enzyme a g a i n s t I| b u f f e r a t d i f f e r e n t pH v a l u e s  c o n t a i n i n g e i t h e r Mn  Tris-Maleate  | | o r Zn  .  Pilot  experi-  ments e s t a b l i s h e d t h a t the pH changes i n the enzyme s o l u t i o n produced by t h i s technique  o c c u r r e d w i t h i n 90 min o f d i a l y z i n g a t 4 ° .  (Table I I I ) that p r e i n c u b a t i n g  t h e enzyme a t d i f f e r e n t pH does n o t b r i n g 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 [| The  ratio  o f Mn  Metal  a c t i v a t e d enzyme c a t a l y s i s f a l l s  i n a narrow range f o r  (Table I I I ) .  i o n requirement. Since p-enolpyruvate  carboxykinase  m e t a l i o n - n u c l e o t i d e complex, we f e l t at  i n the presence o f e i t h e r metal i o n .  | |  /Zn  a l l pH treatments  I t can be observed  d i f f e r e n t pH v a l u e s .  Metal  have been shown t o r e q u i r e a d i v a l e n t  i t necessary  t o study  Mg Mn Fe  Ij I | I |  IDP, 0.15 mM NADH and axcess sigma MDH. , Zn , Zn  |[  , Co  _| |_  j|  , Ca  and Co  |[  requirement  i o n s p e c i f i t y was checked i n T r i s - M a l e a t e  b u f f e r , a t pH 5, 6,7 and 8, c o n t a i n i n g 1 mM m e t a l i o n , 1 mM 1 mM  this  p-enolpyruvate,  In p r e l i m i n a r y experiments Mn  | j || [ | , Fe and Cu ions were t e s t e d a t a l l pH v a l u e s . form a c t i v e m e t a l - n u c l e o t i d e  n e i t h e r a c t i v a t e nor i n h i b i t  complexes: Mg  t h e enzyme, w h i l e Cu  | |  I | ,  Only  _j |_ | [ , Ca and  inhibits  the enzyme  i n p r e s e n c e o f any o f the a c t i v e m e t a l i o n complexes and a t a l l the pH v a l u e s tested. To f u r t h e r i n v e s t i g a t e the metal i o n a c t i v a t i o n o f t h e enzyme, we determined enzyme a c t i v i t y as a f u n c t i o n o f metal i o n c o n c e n t r a t i o n a t t h r e e d i f f e r e n t pH v a l u e s .  In the absence o f p r e i n c u b a t i o n p - e n o l p y r u v a t e  k i n a s e shows g r e a t e r a c t i v i t y tested  ( F i g . 3-A).  i n the p r e s e n c e o f Mn  carboxy-  a t a l l t h e pH v a l u e s  However, maximal a c t i v a t i o n i n p r e s e n c e o f Zn  j |  occurs a t  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 a r t i n g enzyme was n e u t r a l i z e d by 0 . 1 mM KHCO^. dialyzed against  1 0 0 ml o f T r i s - M a l e a t e I |  containing  e i t h e r 1 0 mM Zn  buffer  ( a t pH v a l u e s p e c i f i e d below)  | j o r Mn  .  Enzyme a c t i v i t y was assayed a t the  r e s p e c t i v e o p t i m a l pH f o r e i t h e r metal i o n . Tris-Maleate  1 ml enzyme was  The assay system  contained  b u f f e r , 1 mM m e t a l i o n , 1 0 mM KHCO^, 1 mM p - e n o l p y r u v a t e , 1 mM  IDP, 0 . 1 5 mM NADH, and excess d i a l y z e d Sigma MDH, and the r e a c t i o n was i n i t i a t e d by a d d i t i o n of the pH t r e a t e d  pH Treatment o f the Enzyme  enzyme  Activity AE /unit time pH 5 . 1 (Zn++)  44  3 4 0  pH 7 no m e t a l i o n i n the dialyzing  Ratio of Mn^/Zn " activated Enzyme  pH 6.0 (Mn ") 44  0.75  0.53  0.705  buffer  pH 7  > 7,  0.675  0.53  0.785  pH 7  » 5.4,  0.85  0.55  0.647  pH  7  v 5.4-j,6,  0.82  0.51  0.621  pH  7  * 5 . 4 - * 6 -T»7,  0.60  0.46  0.766  pH  7  s> 5 . 4 - » 6 - * 7 - * 7 . 5 ,  0.55  0.40  0.722  8-  pH  7  — * 5 . 4 ->7,  0.60  0.50;  0.837  h.  pH  7  — » 5.4 -*7-jv5.4,  0.65  0.51  0.784  57  F i g . IV, 3.  E f f e c t s of metal i o n a c t i v a t i o n a t d i f f e r e n t pH v a l u e s ; 3A i n presence of Mn pH 5.1,  I |  ( • ) pH 6.0,  were 50 mM  and  KHCO^, 0.15 S.  n  and 0.15  q  ( A  mM  P  7.0.  ( O  )  Reactant c o n t e n t s  mM.  1 mM  p - e n o l p y r u v a t e , 10  mM  NADH and excess malate dehydrogenase.  . a t pH 5.1, mM.  IDP,  6.0  In 3B, S,  and 7.0  are 0.52  ... a t pH 5.1  w. ->) and 0.29  ) H  | | .  Tris-Maleate buffer, increasing concentrations  of the metal i o n , 1 mM  3A,  and 3B i n presence o f Zn  mM,  and 6.0  0.15  In mM  are 0.15  mM  Activity (AE /unit time) 340  Activity (AE  340  /unit time)  58 about pH 5.1 w h i l e w i t h Mn  I|  i t occurs a t pH 6.0.  Both metal i o n s , above 1  c o n c e n t r a t i o n a t a l l pH v a l u e s t e s t e d were found t o be s l i g h t l y The  r a t i o , at 1 mM  activity, and  7.0  S, w.  J)  N  metal i o n , Mn  respectively.  2.0  | |  inhibitory.  stimulated  and 14.3  As the metal i o n curves were not t r u l y  v a l u e s were c a l c u l a t e d by H i l l  a t pH 5.1,  6.0  hyperbolic,  treatment o f the d a t a , I|  and the  ^  both the shape of the Zn  v a l u e depend upon pH.  Thus a t pH 5.1  f o l d g r e a t e r compared to S ^  two  the Mn  s a t u r a t i o n k i n e t i c s appear  the pH dependencies 6.0  stimulated activity/Zn  i n c r e a s e s w i t h i n c r e a s i n g pH b e i n g 1.1,  In the pH range o f 5-6,  about  I |  mM  the S ,  N  o f the S^Q  ^  ^  a t pH 6.0  saturation the  ^  ( F i g . 3-B).  value i s In c o n t r a s t ,  to be somewhat more complex ( F i g . 3-A)  v a l u e s are more pronounced.  v a l u e i s o n l y about  kinetics  1/4  and  Thus a t pH  of the v a l u e s a t pH 5.1.  T h i s pH  dependency i n the presence of Mn i s o p p o s i t e 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 g r e a t e r a t pH I| than at pH 5.1 w h i l e apparent enzyme-Zn at pH  affinity  i s g r e a t e r a t pH 5.1  6.0  than  6.0.  I n o s i n e diphosphate  (IDP) s a t u r a t i o n  kinetics.  IDP s a t u r a t i o n curves are shown f o r p - e n o l p y r u v a t e c a r b o x y k i n a s e c a t a lysis  i n the presence o f Mn  At pH 6 and  and Zn  a t d i f f e r e n t pH v a l u e s ( F i g s . 4, 5 ) .  7 (4-A,B) i n the presence of Mn  , IDP s a t u r a t i o n k i n e t i c s obey  M i c h a e l i s - M e n t o n c o n s i d e r a t i o n under a l l c o n d i t i o n s t e s t e d .  are i n the range of 0.03 pH.  mM  to 0.06  mM,  Values of  S, (0.5)  and these a r e l a r g e l y independent  There i s no evidence of s u b s t r a t e i n h i b i t i o n a t h i g h (up to 2 mM)  IDP  levels. The e f f e c t s o f Zn  I |  and Mn  | |  on the IDP s a t u r a t i o n k i n e t i c s a t pH  5.1  of  59  F i g . IV, 4.  E f f e c t of Mn 6.0  on the IDP  (4A) and pH 7.0  p-enolpyruvate,  ( A)  (4B). 0.5  saturation ( • mM  Mn  ) 1 mM  kinetics  a t pH  Mn " and 1 44  and 1 mM  mM  p-enolpyruvate,  I j ( O  )  tions  1 mM  Mn  and 0.5  as i n F i g . 3.  mM  p-enolpyruvate.  Other c o n d i -  Activity (AE /unit time) 340  60  F i g . IV, 5.  E f f e c t s o f Zn  and Mn  on the IDP s a t u r a t i o n  kinetics  I | at pH 5.1,  4A i n presence  of Zn  and 4B i n presence  I | of Mn  .  ( A)  0.5  1 mM  ( • ) 1 mM mM  metal  metal i o n and  metal i o n and 0.5  as i n F i g . 3.  mM  i o n and 1 mM 1 mM  p-enolpyruvate,  p-enolpyruvate,  p-enolpyruvate.  Other  (  O) conditons  Activity  (AE /unit ; ) 340  t  me  Activity (AE  340  /unit time)  61 are compared i n F i g s . 5-A,B.  In the presence o f Zn  I|  the s a t u r a t i o n  are s l i g h t l y s i g m o i d a l .  H i l l p l o t s y i e l d _n v a l u e s of about  v a l u e s of S, ^ f o r IDP (.0. J) I|  a r e e n t i r e l y comparable i n the presence o f Zn  n  Mn  .  1.5.  curves  The  absolute and  I t i s e v i d e n t from the d a t a t h a t the adductor muscle p - e n o l p y r u v a t e  c a r b o x y k i n a s e i s a b l e to compete f o r l i m i t i n g IDP e f f e c t i v e n e s s when e i t h e r Mn  P-enolpyruvate  saturation  P-enolpyruvate  o r Zn  concentration with  i s p r e s e n t as the c a t i o n i c  similar  cofactor  kinetics.  s a t u r a t i o n curves f o r p - e n o l p y r u v a t e  c a t a l y s i s a t d i f f e r e n t pH v a l u e s i n the presence o f Mn  carboxykinase  I |  and Zn  | |  a r e shown  I| i n F i g s . 6 and  7.  In the p r e s e n c e of Mn  a r e complex a t a l l pH v a l u e s examined. t e s t e d the enzyme does not f u l l y p-enolpyruvate  ( F i g . 6).  presence o f Mn  , p-enolpyruvate s a t u r a t i o n  kinetics  As i s e v i d e n t , under the c o n d i t i o n s  s a t u r a t e even a t 2 mM  c o n c e n t r a t i o n of  T h i s a b e r r a n t b e h a v i o u r o f the enzyme i n the  tends to r u l e out t h i s c a t i o n as the jLn v i v o c o f a c t o r ,  p r e c l u d e s a c c u r a t e e s t i m a t e o f the a f f i n i t y  and  constant f o r p-enolpyruvate. I|  In c o n t r a s t , p - e n o l p y r u v a t e s a t u r a t i o n 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 the apparent  v a l u e s are a t l e a s t 1 I f o r the enzyme i n the presence of Mn apparent K , ~ , a t pH 6.0 m(p-enolpyruvate) r  7  K  follow Michaelis-Menton p a t t e r n s .  m  r  ).  Equally s i g n i f i c a n t ,  o r d e r o f magnitude l e s s than observed | | | . Moreover i n the presence o f Zn the i s about 1/3 the v a l u e a t pH 5.1 ( F i g .  Thus, although the pH optimum f o r the enzyme under s a t u r a t i n g  r u v a t e c o n c e n t r a t i o n s i s about  5.1,  p-enolpy-  i t i s e v i d e n t t h a t i n the p r e s e n c e  of  I| Zn  i t competes q u i t e f a v o u r a b l y f o r l i m i t i n g p - e n o l p y r u v a t e a t h i g h e r pH  values  (see F i g s .  2, 7 ) .  62  F i g . IV, 6.  E f f e c t s o f Mn  on p - e n o l p y r u v a t e s a t u r a t i o n  at d i f f e r e n t pH v a l u e s , ( • ) pH 5.1, ( A ) and  ( O ) pH 7.0.  Maleate b u f f e r , increasing  Reactant  contents were:  kinetics pH 6.0, 50 mM  Tris-  1 mM Mn " ", 1 mM IDP, 10 mM KHC0 , 4  1  3  c o n c e n t r a t i o n s o f p - e n o l p y r u v a t e , 0.15 mM  NADH and excess malate  dehydrogenase.  63  F i g . IV, 7.  E f f e c t s o f Zn  on p - e n o l p y r u v a t e s a t u r a t i o n k i n e t i c s a t  d i f f e r e n t pH v a l u e s , ( • ( O)  pH 7.0.  b u f f e r 1 mM  Zn  Reactant , 1 mM  ) pH 5.1,  ( A ) pH 6.0,  c o n t e n t s were 50 mM IDP,  10 mM  KHC0  c e n t r a t i o n s o f p - e n o l p y r u v a t e , 0.15 malate 5.1  dehydrogenase. °  and 6.0  are 0.5  mM  K  mM  3>  and  Tris-Maleate  increasing  NADH and  excess  , values at m(p-enolpyruvate)  and 0.18  n  mM.  con-  pH  Activity (AE  340  /unit time)  64 I|  Cu  inhibition  kinetics.  No r e p o r t s a r e a v a i l a b l e on Cu ase a c t i v i t i e s .  We found t h a t Cu  I|  I|  i n h i b i t i o n of p-enolpyruvate carboxykin-  i n h i b i t i o n o f t h e o y s t e r enzyme i s  q u a n t i t a t i v e l y dependent on t h e c a t i o n i c c o f a c t o r p r e s e n t i n t h e assay and i s pH independent  i n t h e presence o f Zn  .  system  The i n h i b i t i o n p e r cent o f  I|  enzyme by Cu  a t pH 5.1 and 6.0 and f i x e d c o n c e n t r a t i o n s o f c a t i o n i c  cofactor  I|  and p - e n o l p y r u v a t e a r e shown i n F i g . 8.  In t h e presence o f Zn  ( F i g . 8, I|  open c i r c l e s and s q u a r e s ) , the enzyme i s much l e s s s e n s i t i v e t o Cu  inhibition  I|  (apparent K\ i s about 60 pM a t both pH 5.1 and 6.0); w i t h Mn  as t h e metal I|  ion  ( F i g . 8 , c l o s e d c i r c l e s and s q u a r e s ) ; t h e s e n s i t i v i t y  t o Cu  increases  by n e a r l y an o r d e r o f magnitude ( t h e apparent K\ v a l u e s now a r e 6 t o 100 uM a t pH 5.1 and 6.0 r e s p e c t i v e l y ) . P - e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y as a f u n c t i o n o f Mn s a t u r a t i o n i n the presence o f d i f f e r e n t Cu shown i n F i g s . 9-A,B. c o n s t a n t f o r Mn  I |  In presence o f Mn  a r e a l t e r e d by Cu  ||  .  I|  I|  I |  [ |  and Zn  c o n c e n t r a t i o n s a t pH 6.0 a r e  (9-A) b o t h t h e Vmax and t h e a f f i n i t y  Thus, Cu  | |  (40 p M ) i n c r e a s e s t h e S ^  I|  v a l u e s o f Mn  by s e v e r a l f o l d over t h e c o n t r o l S ^ ^  values.  A t t h e same  I|  time 40 yuM Cu  reduces t h e Vmax to about 20 p e r cent o f c o n t r o l v a l u e a t  I|  2 mM Mn In c o n t r a s t , i n t h e presence o f Zn but does n o t s i g n i f i c a n t l y a f f e c t  I |  ( F i g . 9-B) Cu  the a f f i n i t y  | |  reduces t h e Vmax  f o r the metal i o n .  Thus  I|  ^(0 5)  v  a  x  u  e  s  i  n  absence and i n t h e presence o f Cu  ( a t 40 o r 60 nM) were  ++  identical  (0.23 mM) a l t h o u g h 60 JUM Cu  c o n t r o l r a t e s a t 2 mM Zn  I |  .  reduces t h e Vmax t o 40 p e r cent o f  I t i s e v i d e n t from these r e s u l t s t h a t Cu  h i b i t i o n i s l e s s e f f e c t i v e i n presence o f Zn  I |  in-  than i n t h e presence o f Mn  s i n c e o n l y the maximum c a t a l y t i c r a t e i s a f f e c t e d w h i l e t h e cation i s unaltered.  | |  ^  | |  ,  f o r the  ^  65  Fig.  IV, 8.  I n h i b i t i o n o f p - e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y by i n c r e a s i n g c o n c e n t r a t i o n s o f Cu (open symbols) o r Mn and  ( • ) pH 6.0.  I|  i n presence o f Zn  ( c l o s e d symbols) a t ( O)  R e a c t i o n c o n t e n t s were: j|  Maleate b u f f e r , 1 mM Mn  50 mM  | |  pH 5.1, Tris-  | | o r Zn  as i n d i c a t e d , 1 mM IDP,  10 mM KHCO.,, 1 mM p - e n o l p y r u v a t e , i n c r e a s i n g c o n c e n t r a t i o n of  Cu  , 0.15 mM NADH and excess malate  dehydrogenase.  T  66  F i g . IV, 9.  P - e n o l p y r u v a t e c a r b o x y k i n a s e a c t i v i t y as a f u n c t i o n o f I |  the c o n c e n t r a t i o n o f Mn absence  of C u * . 4  In 9A,  |]  and Zn ( •  i n the presence o r  ) control,  ( A ) 10 uM Cu"*", 4  and ( O ) 40 juM Cu ". 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 44  T r i s - M a l e a t e b u f f e r pH 6.0,  i n c r e a s i n g c o n c e n t r a t i o n s of  the metal i o n as i n i d i c a t e d , 1 mM 10 mM  KHC0», 0.15  mM  IDP,  1 mM  NADH and excess malate  p-enolpyruvate, dehydrogenase.  Activity (AE /unit time) 340  Activity (AE  340  /unit time)  ON  r  5  6 7 DISCUSSION A fundamental q u e s t i o n concentrations occur The  a r i s e s from t h e above s t u d i e s c o n c e r n i n g t h e  o f metal i o n s w i t h i n t h e i n t r a c e l l u l a r m i l i e u .  Do these i n f a c t  i n c o n c e n t r a t i o n ranges which would a f f e c t PEPCK a c t i v i t y  i n vivo?  problem o f e s t i m a t i n g the i n t r a c e l l u l a r c o n c e n t r a t i o n o f f r e e m e t a l i o n s  is difficult 1957).  and few d e f i n i t i v e s t u d i e s a r e a v a i l a b l e ( T h i e r s and V a l l e e ,  The o n l y a v a i l a b l e data on t h e c e l l u l a r d i s t r i b u t i o n o f metal i o n s i n  o y s t e r a r e those content  o f Fe  j|  of G a l s t o f f (1964).  , Cu  From these  i t appears t h a t t h e t i s s u e  | | |j | [ , Zn and Mn f l u c t u a t e s i n o y s t e r t i s s u e s on a seasonI[  al basis. being  Of these  c a t i o n s , Zn  approximately  i n by f a r t h e h i g h e s t  1000 times more abundant than Mn  a l s o a r e always much h i g h e r ( G a l s t o f f , 1964).  occurs  than Mn  I|  I |  .  Cu  | j  concentrations, concentrations  , p a r t i c u l a r l y during the winter  season  S i n c e PEPCK i n v e r t e b r a t e systems r e q u i r e s a d i v a l e n t I |  c a t i o n t h a t i s thought to be f u l f i l l e d  | |  In v i v o by Mn  o r Mg  ( H o l t e n and  N o r d l i e , 1965), i t i s o f p a r t i c u l a r i n t e r e s t t o note t h a t i n the o y s t e r , Zn appears to be the more l i k e l y  candidate  f o r this function. I |  muscle PEPCK i s a c t i v e i n the p r e s e n c e o f e i t h e r Mn  Although  I|  oyster  | | o r Zn  , the u t i l i z a t i o n  I| of Zn  as the _in v i v o c o f a c t o r would appear to p r e s e n t  advantages: i s aberrant  i n the f i r s t p l a c e , the p - e n o l p y r u v a t e i n the p r e s e n c e o f Mn  I|  two d i s t i n c t  s a t u r a t i o n o f t h e enzyme  , b u t appears to f o l l o w c l a s s i c a l M i c h a e l i s -  Menten k i n e t i c s d i s p l a y i n g a much lower apparent K , ^ i n the m (p-eno l p y ruva t e ) I| presence o f Zn , and s e c o n d l y , the apparent a f f i n i t y o f t h e PEPCK f o r I [ | | p - e n o l p y r u v a t e i s g r e a t l y reduced by Cu i f Mn i s used as the c o f a c t o r , N  while if  i s l a r g e l y u n a f f e c t e d by Cu  the Vmax i s s t i l l  of Zn and  the a f f i n i t y  I |  reduced.  I |  i f Zn  | [  i s t h e c o f a c t o r , even  I n a d d i t i o n , as a l r e a d y mentioned, t h e abundance  i s n e a r l y 3 o r d e r s o f magnitude g r e a t e r than t h e abundance o f Mn  t h i s f a c t o r alone would tend  to favour  Zn  I|  as t h e p r o b a b l e  i n vivo  | j ,  68 c o f a c t o r , even i f h i g h p r o p o r t i o n s o f these c a t i o n s o c c u r r e d i n the bound In v e r t e b r a t e s , the s u b c e l l u l a r d i s t r i b u t i o n o f PEPCK appears depend upon the t i s s u e s and is a strictly  the s p e c i e s examined.  c y t o p l a s m i c enzyme (Meyuhas, Boshwitz  gluconeogenic t i s s u e s , such as l i v e r ,  form.  to  In adipose t i s s u e s PEPCK and Resef, 1971).  In  the enzyme i s found i n the cytoplasm i n  the r a t and mouse, i n the m i t o c h o n d r i a i n r a b b i t and c h i c k e n , and i n both cytoplasm and m i t o c h o n d r i a i n the guinea p i g ( S c r u t t o n and U t t e r , 1968). the o y s t e r , e s s e n t i a l l y a l l the PEPCK  activity  a s i t u a t i o n comparable to t h a t observed T h i s l o c a l i z a t i o n appears metabolism  of these  i s to be found i n the  i n p a r a s i t i c helminths  In  cytosol,  (Saz, 1971).  to f i t w e l l the f u n c t i o n s o f PEPCK i n the a n a e r o b i c  organisms.  In i n v e r t e b r a t e f a c u l t a t i v e anaerobes such as the o y s t e r , the c a p a c i t y f o r 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 i n v o l v e d a number o f  a d a p t a t i o n s i n the pathway of a n a e r o b i c g l u c o s e d i s s i m i l a t i o n .  One  such  a d a p t a t i o n has been the e v o l u t i o n a r y " d e l e t i o n " o f the enzyme, l a c t a t e genase, a " d e l e t i o n " which can be viewed  dehydro-  as a means o f a v o i d i n g the d e l e t e r i o u s  e f f e c t s o f the a n a e r o b i c a c c u m u l a t i o n o f l a c t a t e .  For g l y c o l y t i c  activity  to c o n t i n u e i n d e f i n i t e l y i n the absence o f the l a c t a t e dehydrogenase r e a c t i o n , however, p r o v i s i o n must be made f o r the r e g e n e r a t i o n o f NAD phosphate i n v e r y h i g h a c t i v i t y of carbon f l o w i n t h i s organism,  f o r the  triose  and which i s v e r y much i n the "mainstream" serves to r e g e n e r a t e NAD  r e d u c t i o n of o x a l o a c e t a t e to malate.  through a coupled  The o x a l o a c e t a t e f o r t h i s r e a c t i o n i n  the cytoplasm i s s u p p l i e d by the PEPCK c a t a l y z e d c a r b o x y l a t i o n of p - e n o l p y r u vate.  S i n c e o x a l o a c e t a t e does not move f r e e l y a c r o s s the m i t o c h o n d r i a l b a r r i e r  ( S c r u t t o n and U t t e r , 1968), i t i s e v i d e n t t h a t PEPCK f u n c t i o n must be  largely  l o c a l i z e d i n the cytoplasm of the t i s s u e s i n a l l i n v e r t e b r a t e f a c u l t a t i v e anaerobes r e l y i n g on t h i s r e a c t i o n scheme.  The d a t a i n the T a b l e I I are  69 c o n s i s t e n t w i t h these c o n s i d e r a t i o n s , and an i d e n t i c a l s i t u a t i o n has been d e s c r i b e d f o r another m o l l u s c (Chen and Awapara, 1969) and s e v e r a l h e l m i n t h parasites  (Saz, 1971).  As i t i s l o c a l i z e d i n t h e c y t o s o l , PEPCK competes d i r e c t l y w i t h p y r u v a t e k i n a s e f o r the common s u b s t r a t e p - e n o l p y r u v a t e , and i t i s c l e a r t h a t t h e c h a n n e l l i n g o f p - e n o l p y r u v a t e towards o x a l o a c e t a t e o r towards p y r u v a t e must be a c l o s e l y r e g u l a t e d p r o c e s s in v i v o .  Some o f t h e 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 p y r u v a t e k i n a s e a r e d i s c u s s e d i n t h e next c h a p t e r ; s u f f i c e to mention  a t t h i s p o i n t t h a t t h e a f f i n i t y o f PEPCK f o r  p - e n o l p y r u v a t e depends c r i t i c a l l y (below 6.5) and p a r t i c u l a r l y  upon t h e microenvironment.  i n t h e presence o f Zn  At low pH  as a c o f a c t o r , PEPCK can  r e a d i l y "out-compete" p y r u v a t e k i n a s e f o r l i m i t i n g p - e n o l p y r u v a t e , w h i l e t h e r e v e r s e i s o b v i o u s l y t h e case a t h i g h e r pH v a l u e s (Mustafa and Hochachka, I|  1971).  Thus a t pH 6.0 i n t h e presence o f Zn r  r  t h e apparent K , , m(p-enolpyruvate)  f o r PEPCK i s about 0.2 mM; under t h e same c o n d i t i o n s o f H  +  concentration,  adductor muscle p y r u v a t e k i n a s e i s e s s e n t i a l l y i n a c t i v e i n t h e absence o f fructose-1,6-P  2  and thus c o u l d n o t s e r v e as an a l t e r n a t e pathway f o r p - e n o l -  p y r u v a t e metabolism. kinase a f f i n i t y i s reduced  In c o n t r a s t , a t pH v a l u e s over pH 7.0, t h e p y r u v a t e  f o r p - e n o l p y r u v a t e i s r e l a t i v e l y h i g h , w h i l e PEPCK a c t i v i t y  to l e s s than 5% o f r a t e s a t o p t i m a l pH.  As we have s t r e s s e d , t h e m e t a b o l i c s i t u a t i o n a t t h e p - e n o l p y r u v a t e b r a n c h i n g p o i n t i s r a t h e r unique  to f a c u l t a t i v e anaerobes  such as t h e o y s t e r .  A comparable c o m p e t i t i v e s i t u a t i o n , however, c o u l d a r i s e i n g l u c o n e o g e n i c t i s s u e s o f v e r t e b r a t e s which a l s o possess both p y r u v a t e k i n a s e and phosphoenolpyruvate carboxykinase.  The A G° f o r t h e PEPCK C O ^ - f i x i n g r e a c t i o n i s s m a l l  and n e g a t i v e (about -1 K c a l / m o l e ) ; hence, t h e d i r e c t i o n o f n e t c a t a l y t i c f u n c t i o n i n v i v o 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 o x a l o a c e t a t e .  In v e r t e b r a t e l i v e r ,  the c o m p e t i t i v e s i t u a t i o n between p y r u v a t e k i n a s e and phosphoenolpyruvate carboxykinase  appears t o be avoided by the e l a b o r a t i o n o f a PEPCK which has  about a 10 f o l d g r e a t e r a f f i n i t y  f o r o x a l o a c e t a t e than f o r p - e n o l p y r u v a t e  ( H o l t e n and N o r d l i e , 1965; B a l l a r d , 1970). o x a l o a c e t a t e and p - e n o l p y r u v a t e ,  Under c o n d i t i o n s o f l i m i t i n g  t h i s enzyme would t h e r e f o r e f a v o u r n e t  c a t a l y t i c f u n c t i o n i n the d i r e c t i o n o f p - e n o l p y r u v a t e  s y n t h e s i s , and t h i s  would be f u r t h e r f a v o u r e d by the e f f i c i e n t removal o f p - e n o l p y r u v a t e gluconeogenesis.  The o p p o s i t e s i t u a t i o n appears to be r e q u i r e d i n t h e  i n v e r t e b r a t e anaerobes.  Thus i n PEPCK o f A s c a r i s , t h e r e l a t i v e  constants f o r p-enolpyruvate  affinity  and o x a l o a c e t a t e have been a d j u s t e d so as to  s t r o n g l y favour p-enolpyruvate Although  during  carboxylation to oxaloacetate  ( S a z , 1971).  a s i m i l a r s i t u a t i o n would be a n t i c i p a t e d i n t h e o y s t e r , t h e c o n t r o l  of p - e n o l p y r u v a t e  metabolism appears to be more complex s i n c e , as a f a c u l t a -  t i v e anaerobe, t h i s organism must be a b l e t o " s w i t c h o v e r " from a n a e r o b i o s i s ( f a v o u r i n g PEPCK a c t i v i t y ) t o a e r o b i o s i s ( f a v o u r i n g p y r u v a t e k i n a s e in relatively  s h o r t time p e r i o d s .  activity)  F u r t h e r a s p e c t s o f t h i s r e g u l a t o r y problem  are d i s c u s s e d i n the f o l l o w i n g - c h a p t e r .  CHAPTER V: C a t a l y t i c and R e g u l a t o r y P r o p e r t i e s of O y s t e r Phosphoenolpyruvate II.  Carboxykinase:  R e g u l a t i o n o f t h e enzyme a c t i v i t y i n phosphoenolpyruvate  and i t s f u n c t i o n  metabolism.  71 INTRODUCTION Recent r e p o r t s have shown t h a t phosphoenolpyruvate carboxykinase an important  r e g u l a t o r y r o l e i n gluconeogenesis  ( S c r u t t o n and U t t e r , 1968).  In c o n j u n c t i o n w i t h p y r u v a t e  phosphoenolpyruvate carboxykinase p-enolpyruvate  i n l i v e r and kidney  (PEPCK) p r o v i d e s  plays  of mammals  carboxylase,  a d i r e c t route  of  s y n t h e s i s 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 r e a c t i o n : P y r u v a t e + ATP Oxaloacetate  + C0  + ITP  » Oxaloacetate  2  (or GTP) ^  Although the evidence gluconeogenesis  k  + ADP  + P  P - e n o l p y r u v a t e + IDP  f o r the involvement of these  i s quite convincing  (I)  ±  ( o r GDP) two  + HC0  reactions i n  ( S c r u t t o n and U t t e r , 1968;  Seubert  and  Huth, 1965), e a r l i e r s t u d i e s of the k i n e t i c p r o p e r t i e s of PEPCK p u z z l e d the h i g h ^  m  (  o  x  a  i  o  a  c  e  t  a  t  e  ) •  T h i s v a l u e was  than expected i n v i v o o x a l o a c e t a t e The  s i t u a t i o n was  (1970), who  c l e a r e d up  the apparent K^Q,^ concentrations performed at pH H  +  was  at l e a s t p a r t i a l l y  i n the pM  i s an important  ( H o l t e n and N o r d l i e , 1965).  i n a r e c e n t study by B a l l a r d  c o n t r o l l e d assay c o n d i t i o n s at pH  range i n which in v i v o  are thought to f l u c t u a t e . 7.0  S i n c e B a l l a r d ' s (1970) assays were  would account f o r the w e l l known o b s e r v a t i o n of i n c r e a s e d d u r i n g m i l d a c i d o s i s ( F l o r e s and A l l e y e n e , 1971).  This  gluconeogenesis  F l o r e s and A l l e y e n e  indeed have d e s c r i b e d a r a p i d i n c r e a s e i n PEPCK a c t i v i t y  the  reasoned t h a t  s i g n a l f o r a c t i v a t i o n of t h i s enzyme.  development of a m i l d a c i d o s i s but  7.0,  oxaloacetate  r a t h e r than at more a l k a l i n e pH optimum we metabolic  over  about 1 o r d e r of magnitude h i g h e r  concentrations  noted t h a t under c a r e f u l l y  (II)  3  (1971)  f o l l o w i n g the  t h e i r d a t a do not s p e c i f y the n a t u r e  of  activation. In i n v e r t e b r a t e 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 p o i s e " of  PEPCK d u r i n g a n a e r o b i o s i s  appears to be i n the c a r b o x y l a t i n g d i r e c t i o n  the  (Saz,  72 1971).  The o x a l o a c e t a t e formed i s u l t i m a t e l y c o n v e r t e d  accumulation  to s u c c i n a t e .  of s u c c i n a t e d u r i n g the development of a n a e r o b i o s i s i n i n t e r t i d a l  m o l l u s c s , such as the o y s t e r , a p p a r e n t l y occurs c o n c o m i t t a n t l y w i t h development of a m i l d a c i d o s i s with  (Wilbur, 1964). These o b s e r v a t i o n s  the  coupled  the documented a c i d pH optimum f o r PEPCK i n the o y s t e r (Mustafa  Hochachka, 1972)  and  H  i n v o l v e d i n the c o n t r o l of o y s t e r PEPCK  +  The  is critically  i n o t h e r i n v e r t e b r a t e s (Saz, 1971), suggested  and  to us t h a t  as w e l l 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,  appears to f u n c t i o n i n d i r e c t  and because PEPCK i n the o y s t e r  c o m p e t i t i o n w i t h p y r u v a t e k i n a s e f o r the  common s u b s t r a t e , p - e n o l p y r u v a t e ,  we were i n t e r e s t e d i n examining f u r t h e r  r e g u l a t o r y p r o p e r t i e s of o y s t e r phosphoenolpyruvate  carboxykinase.  73 RESULTS Nature o f i n o s i n e t r i p h o s p h a t e  (ITP)  inhibition.  As phosphoenolpyruvate c a r b o x y k i n a s e i s c o n s i d e r e d mainstream o f t h e a n a e r o b i c  glucose  catabolism  to f u n c t i o n on the  i n t h e o y s t e r muscle, we  a n t i c i p a t e d t h a t t h e a c t i v i t y o f t h e enzyme would be i n t e g r a t e d w i t h t h e energy s t a t u s of t h e c e l l . examining the e f f e c t s  Our i n v e s t i g a t i o n was t h e r e f o r e i n i t i a t e d by  of ITP, GTP, and ATP on t h e PEPCK c a t a l y z e d  tion of p-enolpyruvate.  E a r l i e r data have i n d i c a t e d t h a t the c a t a l y t i c  p r o p e r t i e s o f o y s t e r PEPCK depend c r i t i c a l l y hence t h e e f f e c t I |  of various  upon t h e m e t a l i o n c o f a c t o r ;  ITP c o n c e n t r a t i o n s were s t u d i e d i n t h e presence of  | |  both Zn  and Mn  instances  a competitive  e i t h e r metal i o n .  a t t h e i r r e s p e c t i v e pH optima ( F i g s .  type o f ITP i n h i b i t i o n was o b t a i n e d w i t h  the r e s u l t s  respect to  o f t h e m e t a l i o n s by 10 f o l d b u t t h e c a l c u l a t e d Vmax  remain unchanged.  complexes w i t h  1, 2 ) . I n both  In the p r e s e n c e o f e i t h e r m e t a l i o n , 0.25 mM ITP i n c r e a s e s  the apparent K^ v a l u e s values  carboxyla-  ITP i n h i b i t i o n s t u d i e s i n r e l a t i o n  impure enzyme p r e p a r a t i o n s  are subject  t o metal i o n  to t h e l i m i t a t i o n  may be i n f l u e n c e d by non s p e c i f i c b i n d i n g o f t h e metal i o n s by  contaminating p r o t e i n s . concentrations  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 , s a t u r a t i n g  o f the metal i o n s were used i n a l l l a t e r I |  In the p r e s e n c e o f Zn  with  experiments.  ITP as t h e i n h i b i t o r and IDP as t h e v a r i a b l e  s u b s t r a t e , a l i n e a r n o n - c o m p e t i t i v e i n h i b i t i o n p a t t e r n was o b t a i n e d Thus i n p r e s e n c e o f Zn several fold  that  as t h e m e t a l ion,ITP  (Fig. 3).  decreases t h e c a l c u l a t e d Vmax  i t . does n o t a f f e c t  t h e K . A t a l l c o n c e n t r a t i o n s o f ITP t e s t e d m the K ( j j ) p ) remains unchanged a t about 0.06 mM. Thus under p r o b a b l e p h y s i o I | l o g i c a l c o n d i t i o n s (with h i g h Zn c o n c e n t r a t i o n ) , t h e p r e s e n c e o f ITP does m  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 c o n t r a s t , i n t h e  I[ presence o f Mn  a l i n e a r , m i x e d - c o m p e t i t i v e ITP i n h i b i t i o n p a t t e r n was  "74  F i g . V,  1.  Zn  s a t u r a t i o n k i n e t i c s and  i t s double r e c i p r o c a l  (1/v vs 1/Zn  ) at v a r y i n g ITP c o n c e n t r a t i o n s .  ( A  ITP,  ) 0.25  mM  ( A)  0.5  mM  ITP, and  Reactant c o n c e n t r a t i o n s were 50 mM 5.1,  KHC0 , 0.15 3  O  (•)  ) 1 mM  control, ITP.  T r i s - M a l e a t e b u f f e r pH  i n c r e a s i n g c o n c e n t r a t i o n s of Zn  p - e n o l p y r u v a t e , 10 mM  (  plot  mM  , 1 mM  IDP,  1  NADH, d i f f e r e n t  mM ITP  c o n c e n t r a t i o n s as i n d i c a t e d and excess malate dehydrogenase.  Activity (AE-j^/unit time)  75  F i g . V, 2.  Mn  s a t u r a t i o n k i n e t i c s and  (1/v vs 1/Mn ( A )  0.25  i t s double r e c i p r o c a l  ) a t v a r y i n g ITP mM  ITP, and  t i o n s were 50 mM  ( A )  concentrations.  0.5  mM  ITP.  T r i s - M a l e a t e b u f f e r pH  (O  Reactant 6.1,  plot )  control,  concentra-  increasing  j | c o n c e n t r a t i o n s of Mn KHC0 , 0.15 3  and excess  mM  , 1 mM  IDP,  1 mM  p-enolpyruvate,  10  mM  NADH, d i f f e r e n t ITP c o n c e n t r a t i o n s as i n d i c a t e d  malate dehydrogenase.  Activity (AEg^Q /unit time)  76  F i g . V, 3.  IDP s a t u r a t i o n k i n e t i c s and i t s double  reciprocal  plot  (1/v vs 1/IDP) a t v a r y i n g ITP c o n c e n t r a t i o n s i n presence of Zn ". 44  and  ( • ) control,  ( O ) 1 mM  ITP.  ( • ) 0.25 mM ITP, ( A )  Reactant  0.5 mM ITP,  c o n c e n t r a t i o n s were 50 mM I [  T r i s - M a l e a t e b u f f e r pH 5.1, 1 mM Zn t i o n o f IDP, 1 mM p - e n o l p y r u v a t e , and malate dehydrogenase.  , i n c r e a s i n g concentra-  10 mM KHC0 , 0.15 mM NADH 3  Activity  (AEg  4 0  / unit time)  77  F i g . V, 4.  IDP s a t u r a t i o n k i n e t i c s and i t s double r e c i p r o c a l  plot  ( l / v vs 1/IDP) a t v a r y i n g ITP c o n c e n t r a t i o n s i n presence Mn ". 44  ( • ) c o n t r o l , and ( A ) 0.25 mM ITP.  Reactant conI |  c e n t r a t i o n s were 50 mM T r i s - M a l e a t e b u f f e r pH 6.0, 1 mM Mn i n c r e a s i n g c o n c e n t r a t i o n s o f IDP, 1 mM  p-enolpyruvate,  10 mM KHCO , 0.15 mM NADH and malate dehydrogenase.  ,  Activity (AE  34Q  /unit time)  78 obtained  ( F i g . 4 ) . In t h i s i n s t a n c e 0.25 mM ITP i n c r e a s e s t h e K ,> m(IDP) /TTNT  s e v e r a l f o l d and decreases t h e c a l c u l a t e d Vmax to about control values.  50 p e r cent of the  Thus, even under these c o n d i t i o n s , ITP-IDP i n t e r a c t i o n s  w i t h PEPCK a r e n o t c o m p e t i t i v e i n t h e o y s t e r , whereas they appear strictly  t o be  c o m p e t i t i v e i n t h e case o f c h i c k e n l i v e r m i t o c h o n d r i a l PEPCK  ( ' F e l i c i o l i , B a r s a c c h i and I p a t a , 1970). The e f f e c t o f ITP on p - e n o l p y r u v a t e s a t u r a t i o n k i n e t i c s appears unique  t o the o y s t e r enzyme as w e l l .  In t h e presence o f e i t h e r metal i o n ,  ITP i n h i b i t i o n i s c o m p e t i t i v e w i t h r e s p e c t to p - e n o l p y r u v a t e .  With Zn  pH 5.1 or 6.0, 0.5 mM ITP i n c r e a s e s t h e K , . , by about m(p-enolpyruvate) 1  r  (Fig.  5 ).  , at  2 fold  At 1 mM ITP, i n h i b i t i o n o f the enzyme i s so s e v e r e t h a t measure-  ment becomes d i f f i c u l t .  With Mn  extremely s e n s i t i v e t o ITP. at  t o be  l e a s t by 5 f o l d  ( F i g . 6 ).  I |  as t h e metal i o n c o f a c t o r , o y s t e r PEPCK i s  Thus 0.25 mM ITP i n c r e a s e s t h e K , ., ^ . m(p-enolpyruvate) These r e s u l t s i n d i c a t e t h a t t h e PEPCK c a t a l y z e d  c a r b o x y l a t i o n o f p - e n o l p y r u v a t e i n o y s t e r adductor muscle i s remarkably s e n s i t i v e t o p r o d u c t i n h i b i t i o n by ITP.  S i n c e ITP a l t e r s t h e c a p a c i t y o f t h e  PEPCK t o compete f o r p - e n o l p y r u v a t e , ITP may be an important determinant o f the p e r cent p a r t i c i p a t i o n o f t h e PEPCK and p y r u v a t e k i n a s e r e a c t i o n s . The energy s t a t u s o f t h e c e l l may a l s o be " r e f l e c t e d " by t h e c o n c e n t r a t i o n s o f GTP and ATP. In  (GDP, but n o t ADP, can be a s u b s t r a t e f o r t h i s enzyme.)  the presence o f e i t h e r IDP o r GDP, GTP i n h i b i t s t h e p - e n o l p y r u v a t e  c a r b o x y k i n a s e r e a c t i o n a t both pH v a l u e s .  I t s i n h i b i t i o n pattern with  t i o n t o p - e n o l p y r u v a t e shows a l i n e a r m i x e d - c o m p e t i t i v e (Fig.  7 ).  ATP, i n c o n t r a s t , does n o t a f f e c t  rela-  type o f i n h i b i t i o n  t h e o y s t e r adductor PEPCK.  Search f o r o t h e r m e t a b o l i t e e f f e c t o r s . Although phosphoenolpyruvate  c a r b o x y k i n a s e appears  t o be e s t a b l i s h e d as  79  F i g . V, 5.  Double r e c i p r o c a l p l o t s (1/v vs 1/p-enolpyruvate) a t v a r y i n g ITP c o n c e n t r a t i o n s a t pH 5.1 (5B).  (5A) and a t pH  Reactant c o n c e n t r a t i o n s were 50 mM  6.0  Tris-Maleate  I j b u f f e r , 1 mM  Zn  p y r u v a t e , 1 mM  , i n c r e a s i n g concentrations of p-enolIDP, 10 mM  KHC0 , 0.15 mM 3  NADH d i f f e r e n t  c o n c e n t r a t i o n s o f ITP as i n d i c a t e d and excess malate dehydrogenase. ( A )  In 5A, a t 0 mM  ITP ( • ) , 0.25 mM  ITP  ITP ( A ) , the K , . _ , values ' m(p-enolpyruvate) a r e 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  0.41  0.5 mM  ) , the K , , _ , a r e 0.22 m(p-enolpyruvate) mM r e s p e c t i v e l y .  mM,  0.33 mM  and  80  F i g . V,  6.  P-enolpyruvate s a t u r a t i o n k i n e t i c s and plot  (1/v  i t s double r e c i p r o c a l  vs 1/p-enolpyruvate) w i t h v a r y i n g  concentrations  j | of ITP  and  a l a n i n e w i t h Mn  at pH 6.0;  mM  ITP.  ) c o n t r o l , ( O ) 4 mM  ( •  a l a n i n e and  as the d i v a l e n t metal i o n  0.25  mM  ITP,  ( A  ) 0.25  Reactant c o n c e n t r a t i o n s  described  i n F i g . 5.  ( • ) 0.5  mM,  (  O )  K  and  alanine, mM  ITP,  ( •  and  ) 4  ( A )  assay c o n d i t i o n s  , , v a l u e s are m(p-enolpyruvate) 0.5 mM, ( • ) 1 mM, and ( A )  mM 0.5 as  N  2.5  mM.  80 ^  31  F i g . V, 7.  P - e n o l p y r u v a t e s a t u r a t i o n k i n e t i c s and i t s double r e c i p r o cal plot  (1/v vs 1/p-enolpyruvate) i n the presence ( A  and absence  ( • ) of 0.25  mM  GTP.  )  Reactant c o n c e n t r a t i o n s I|  were 50 mM  T r i s - M a l e a t e b u f f e r pH 6.0,  1 mM  Zn  , 1  mM  IDP, i n c r e a s i n g c o n c e n t r a t i o n o f p - e n o l p y r u v a t e , 10  mM  KHCO^, 0.15 mM NADH and excess malate dehydrogenase. K , -| ^ , v a l u e s are ( • ) 0.2 mM and ( A ) 0.5 m(p-enolpyruvate)  mM.  82 a key enzyme i n the r e g u l a t i o n o f g l u c o s e metabolism i n v e r t e b r a t e s and U t t e r , 1968), l i t t l e mechanisms. 5' AMP  i n f o r m a t i o n has been a v a i l a b l e on p o t e n t i a l r e g u l a t o r y  However, two n e g a t i v e e f f e c t o r s a r e w e l l documented; these a r e  ( H o l t e n and N o r d l i e , 1965) and m a l i c a c i d  i n h i b i t i o n r e q u i r e s very h i g h  ( B a l l a r d , 1970).  The 5' AMP i n h i b i t i o n , on t h e o t h e r  hand, c o u l d s e r v e t o i n h i b i t PEPCK f u n c t i o n i n gluconeogenesis  by  The malate  (40 mM) malate c o n c e n t r a t i o n s and probably i s  not o f 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 .  depleted  (Scrutton  under energy  (low ATP, h i g h AMP) c o n d i t i o n s and t h i s e f f e c t c o u l d be p o t e n t i a t e d  IDP product  inhibition  (FelUtcJU,  , B a r s a c c h i and I p a t a , 1970).  p r e v i o u s attempts to f i n d e f f e c t o r s f o r t h i s enzyme have c o n c e n t r a t e d  Most on a  s e a r c h f o r i n h i b i t o r s o f the PEPCK c a t a l y z e d d e c a r b o x y l a t i o n r e a c t i o n . c o n t r a s t , we i n i t i a t e d  a search  catalyzed c a r b o x y l a t i o n of  f o r p o s i t i v e e f f e c t o r s o f t h e o y s t e r PEPCK  p-enolpyruvate.  We made a d e t a i l e d survey two  o f the e f f e c t s o f t h e v a r i o u s m e t a b o l i t e s a t  d i f f e r e n t c o n c e n t r a t i o n s o f the s u b s t r a t e p - e n o l p y r u v a t e  at pH 6.0 w i t h Zn  as the d i v a l e n t metal i o n .  metabolites  IMP, ADP, p h e n y l a l a -  have n e i t h e r a p p r e c i a b l e s t i m u l a t o r y n o r 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 . t o r y e f f e c t a t low p - e n o l p y r u v a t e (or GTP) i n h i b i t e d p - e n o l p y r u v a t e  L-alanine  mM)  fructose-1,6-diphosphate,  c i t r a t e , malate, s u c c i n a t e , fumarate, 5' AMP,  n i n e and tryptophane  (1 mM and 0.5  Of the v a r i o u s  t e s t e d , 1 mM c o n c e n t r a t i o n s o f f r u c t o s e - 6 - p h o s p h a t e , acetylCoA,  In  Only L - a l a n i n e was shown t o have a s t i m u l a c o n c e n t r a t i o n and markedly d e - i n h i b i t ITP carboxykinase.  effects.  In our p r e v i o u s s t u d i e s (Mustafa alanine i n h i b i t s oyster pyruvate to p - e n o l p y r u v a t e .  and Hochachka, 1971) , we found  k i n a s e i n a manner c o m p e t i t i v e w i t h  that respect  I t was t h e r e f o r e o f i n t e r e s t t o examine t h e e f f e c t s o f  t h i s amino a c i d the K  on p - e n o l p y r u v a t e c a r b o x y k i n a s e .  , N to about m(p-enolpyruvate)  and decreases  the Vmax by about  r e g u l a t i o n of PEPCK a c t i v i t y , significant  With Zn  75% o f c o n t r o l v a l u e a t  pH  , a l a n i n e lowers 6.0  the same amount ( F i g . 8 ) .  In terms of  these e f f e c t s o f a l a n i n e appear to be  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  less counter-  a c t s the i n h i b i t i o n of PEPCK by ITP, but the p r e c i s e degree o f r e v e r s a l depends upon the n a t u r e o f the c a t i o n In the presence of Zn is  I|  cofactor.  a t pH 6.0  completely r e v e r s e d by 4 mM J  the i n h i b i t i o n caused by 0.25  L-alanine; a K ,  , ^ , o f 0.4 m(p-enolpyruvate)  i n the presence o f ITP i s completely r e t u r n e d t o c o n t r o l a d d i t i o n o f a l a n i n e ( F i g . 8 ). With Mn  , a t pH 6.0  (0.2 mM)  a l a n i n e produces  ITP mM  v a l u e by  Comparable r e s u l t s are o b t a i n e d a t pH  5.0.  a s m a l l but c o n s i s t e n t i n c r e a s e i n  c a t a l y t i c r a t e at a l l p-enolpyruvate c o n c e n t r a t i o n s . significant  mM  However, t h e r e i s no  change i n the a f f i n i t y  constant f o r p-enolpyruvate. Perhaps, I| because of the g r e a t e r ITP i n h i b i t i o n i n the presence of Mn , a l a n i n e does not completely r e v e r s e the e f f e c t of ITP on PEPCK c a t a l y s i s . nevertheless large. ° reduced by 4 mM  At 0.25  mM  a l a n i n e to 1 mM  The r e v e r s a l i s  ITP, a K .  .. , o f 2.5 mM m(p-enolpyruvate)  (Fig. 6  ).  From these d a t a i t i s e v i d e n t  t h a t a l a n i n e s u p p l i e s an e f f e c t i v e mechanism f o r r e v e r s a l o f ITP of p - e n o l p y r u v a t e  is  inhibition  c a r b o x y k i n a s e c a t a l y s i s i n the presence o f e i t h e r Mn  I|  or  84  F i g . V,  8.  P - e n o l p y r u v a t e s a t u r a t i o n k i n e t i c s and r e c i p r o c a l p l o t with varying  i t s double  concentration  of ITP  and  I| alanine with  Zn  as  ( •  ) control, ( O  and  0.25  ITP.  mM  ) 4 mM  ( •  alanine,  ) 0.25  mM  Reactant c o n c n e t r a t i o n s  described  ( O ) 0.22 0.4  ITP,  the d i v a l e n t m e t a l i o n a t pH  mM.  i n F i g . 5.  mM,  (  O  K  ( • ) 4 mM  ITP, and  and  6.0.  alanine  ( A ) 0.5  mM  assay c o n d i t i o n s  as  , .. ^ values m(p-enolpyruvate)  are  )  (  ) 0.16  mM,  (#  0.21  mM,  and  A )  85 DISCUSSION The  a c t i v i t i e s of many enzymes i n v o l v e d i n c e l l u l a r energy metabolism  are governed at l e a s t i n p a r t by  energy s t a t u s o f the c e l l .  m e t a b o l i c " s i g n a l s " are the a d e n y l a t e s : inhibitory h i g h AMP  to the enzymes i n v o l v e d  (equivalent  to low  ATP)  h i g h ATP  The  usual  concentrations  often  i n energy metabolism.  o f t e n are s t i m u l a t o r y  o y s t e r muscle, p - e n o l p y r u v a t e can be m e t a b o l i z e d by p y r u v a t e , by a p y r u v a t e k i n a s e (ii)  to o x a l o a c e t a t e  generate a h i g h kinase; by  ITP  by  a PEPCK c a r b o x y l a t i o n  i n the case of PEPCK) and In t h i s  the o t h e r  (Atkinson,  two  reaction.  Both  hand,  1968).  pathways:  catalyzed transphosphorylation  energy phosphate compound (ATP  these compounds.  On  are  In  ( i ) to  r e a c t i o n , or reactions  i n the case of p y r u v a t e  both are s u b j e c t  sense, both enzymes are  to product  inhibition  i n accordance w i t h  Atkinson's  (1968) energy charge concept.  contribute  to the p h y s i o l o g i c a l " p o i s e " of the p - e n o l p y r u v a t e branch p o i n t ,  they do n o t ,  of themselves, d i s p l a y adequate s p e c i f i c i t y  t r a n s i t i o n from a e r o b i c  f u n c t i o n under a e r o b i c  anaerobiosis).  The  be s u p p l i e d by H  the  two  +  conditions  to PEPCK f u n c t i o n  s p e c i f i c i t y r e q u i r e d , as f a r as we  demonstrable r e g u l a t o r y and  to account f o r  to a n a e r o b i c metabolism ( i . e . f o r the t r a n s i t i o n  pyruvate kinase  PEPCK and  Whereas these mechanisms undoubtedly  p r o p e r t i e s of these two  alanine.  pyruvate kinase  during  can judge from  the  enzyme systems, appears to  Furthermore, from the r e g u l a t o r y  to these two  from  responses of  e f f e c t o r compounds, i t appears  that  enzymes have been s e l e c t e d to f u n c t i o n l a r g e l y on an e i t h e r / o r b a s i s  r a t h e r than to f u n c t i o n s i m u l t a n e o u s l y .  The  nub  of the argument i s summarized  below. I t i s w i d e l y h e l d t h a t under a n a e r o b i c c o n d i t i o n s , m o l l u s c a n b i v a l v e s s u s t a i n s u b s t a n t i a l a c i d i f i c a t i o n o f t h e i r t i s s u e s and 1964).  T h i s drop i n pH  ,  fluids  (Wilbur,  appears to p l a y a p i v o t a l r o l e i n the c h a n n e l l i n g  of  86 p-enolpyruvate  from the p y r u v a t e k i n a s e r e a c t i o n and towards PEPCK, because  of the pH p r o f i l e s  f o r PEPCK (Mustafa and Hochachka, 1972a) and p y r u v a t e  k i n a s e (Mustafa and Hochachka, 1971) a r e e s s e n t i a l l y n o n - o v e r l a p p i n g .  In  consequence, i n the absence o f any o t h e r f a c t o r d e c r e a s i n g pH l e a d s t o an automatic PEPCK.  i n h i b i t i o n of p y r u v a t e k i n a s e w i t h a concomitant  a c t i v a t i o n of  At the same time, L - a l a n i n e which i s known to accumulate a l o n g w i t h  s u c c i n a t e under a n a e r o b i c c o n d i t i o n s , p o t e n t i a l l y i n h i b i t s p y r u v a t e (by i n c r e a s i n g the K , ^ and d e c r e a s i n g t h e Vmax). m(p-enolpyruvate) J  s  G  particularly  6  kinase  It is  i n s t r u c t i v e t h a t t h e L - a l a n i n e i n h i b i t i o n i s p o t e n t i a t e d by de-  c r e a s i n g pH: f o r adductor muscle p y r u v a t e k i n a s e , t h e K. , . , a t pH 7.5 l(L-alanxne) /T  r  i s o n l y 1/6 t h e K^ v a l u e observed 1971). kinase.  a t t h e o p t i m a l pH 8.5 (Mustafa and Hochachka,  Indeed, low pH l i k e w i s e p o t e n t i a t e s ATP i n h i b i t i o n o f p y r u v a t e In marked c o n t r a s t , t h e primary  e f f e c t s o f L - a l a n i n e on PEPCK appear  to be ( i ) a r e v e r s a l o f 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  acti-  v a t i o n a t low p - e n o l p y r u v a t e c o n c e n t r a t i o n s due t o a r e d u c t i o n i n t h e apparent K , , . Both these e f f e c t s o f L - a l a n i n e on PEPCK o c c u r m(p-enolpyruvate) at pH ranges  (pH 5-6) i n which p y r u v a t e k i n a s e a c t i v i t y i s v e r y low and i n  which L - a l a n i n e i n h i b i t i o n o f p y r u v a t e k i n a s e i s u n u s u a l l y extreme.  From  these d a t a we a r e l e d t o the c o n c l u s i o n t h a t the d e c r e a s i n g pH and i n c r e a s i n g L - a l a n i n e c o n c e n t r a t i o n , both o f which o c c u r i n a e r o b i c ^£anaerobic t i o n , cause an e x p o n e n t i a l i n c r e a s e i n the PEPCK c a t a l y t i c r a t e , w i t h an e x p o n e n t i a l decrease  transi-  concurrent  i n the pyruvate kinase c a t a l y t i c r a t e .  This  would appear to be an adequate arrangement f o r c h a n n e l l i n g p - e n o l p y r u v a t e towards o x a l o a c e t a t e , which u l t i m a t e l y accumulates  as s u c c i n a t e .  But i f the  p y r u v a t e k i n a s e path to p y r u v a t e i s b l o c k e d , what i s t h e s o u r c e o f t h e La l a n i n e which accumulates  a l o n g w i t h s u c c i n a t e d u r i n g a n a e r o b i c metabolism?  Two p o s s i b i l i t i e s suggest  themselves.  ( i ) I n our e a r l i e r s t u d i e s on  87 oyster pyruvate  kinases  (Mustafa  and Hochachka, 1971), we  f r u c t o s e - 1 , 6 - P 2 r e v e r s e s the e f f e c t s of a l a n i n e , ATP, l e s s e r extent, H . +  Hence, t h i s c o u l d s u p p l y  m a i n t a i n i n g some p y r u v a t e pyruvate  noted  and  to a somewhat  the o y s t e r w i t h a mechanism f o r  k i n a s e f u n c t i o n under a n a e r o b i c  produced c o u l d i n t u r n be  transaminated  i n d i c a t e d i n the p r e v i o u s paper (Mustafa  that  conditions.  to a l a n i n e .  ( i i ) As  The we  and Hochachka, 1972a), o y s t e r muscle  c o n t a i n s v e r y h i g h a c t i v i t i e s o f NADH-linked m a l i c dehydrogenase and NADPl i n k e d " m a l i c enzyme".  These enzymes o c c u r i n much h i g h e r s p e c i f i c  than do e i t h e r PEPCK or p y r u v a t e  kinase.  A h i g h malate dehydrogenase  ty i s assumed to f u n c t i o n i n the maintenance o f low  oxaloacetate  t i o n s thus p r e v e n t i n g s i g n i f i c a n t r e v e r s a l o f PEPCK a c t i v i t y The  r e v e r s i b l e but 20 mM  the a f f i n i t y  (Saz, 1971).  generate  constants  f o r pyruvate  and  CC^  production.  The  a r e so h i g h  (10  mM  f u n c t i o n s o n l y i n the  " m a l i c enzyme" r e a c t i o n then  could  the p y r u v a t e which upon t r a n s a m i n a t i o n accumulates as L - a l a n i n e .  T h i s indeed 1971).  1969;  In our hands o y s t e r " m a l i c enzyme" i s f u l l y  r e s p e c t i v e l y ) t h a t in v i v o the enzyme p r o b a b l y  d i r e c t i o n of pyruvate  activi-  concentra-  f u n c t i o n o f " m a l i c enzyme" r e c e n t l y has been i n d i s p u t e (Hammen,  Simpson and Awapara, 1966).  and  activities  i s the documented f u n c t i o n o f m a l i c enzyme i n h e l m i n t h s  (Saz,  CHAPTER V I : Summating Remarks  88 These s t u d i e s on o y s t e r muscle p y r u v a t e k i n a s e and p - e n o l p y r u v a t e boxykinase metabolic  have suggested  a number of c o n c l u s i o n s c o n c e r n i n g both  c o n t r o l of the enzymes and  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 t h i s study,  car-  the  the u n d e r l y i n g p o s s i b l e enzyme a d a p t a t i o n  d u r i n g aero- and a n a e r o b i o s i s .  To  conclude  a number of these w i l l be d i s c u s s e d as w e l l as p r o b a b l e s o l u t i o n s  (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 o u t s t a n d i n g problems such as  (1) sources  sources  of s u c c i n a t e , and  map  of a l a n i n e and r e d u c i n g e q u i v a l e n t s , (2) o t h e r (3) the y i e l d of h i g h energy compounds.  metabolic A metabolic  f o r p r o b a b l e pathways of a n a e r o b i c i n t e r m e d i a r y metabolism i n m o l l u s c a n  f a c u l t a t i v e anaerobes i s  developed.  I t i s a w i d e l y h e l d view i n contemporary b i o c h e m i s t r y t h a t the r a t e of a multi-enzyme r e a c t i o n sequence i s l a r g e l y  overall  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 s u s c e p t i b l e to r e g u l a t i o n by a c t i v a t i o n o r i n h i b i t i o n of enzyme by s p e c i f i c m e t a b o l i t e s . pathway may  be governed by  The r a t e s of such  F u r t h e r , the r a t e o f any  metabolic  t h a t enzyme i n the pathway whose r a t e i s minimal.  c o n t r o l l i n g s t e p s are determined  by  amount of the enzyme, (2) the amount of s u b s t r a t e , and effectors  given  the  three f a c t o r s :  (1)  the  (3) the q u a n t i t i e s of  ( p o s i t i v e and n e g a t i v e ) .  From these s t u d i e s of the r e g u l a t o r y p r o p e r t i e s of the o y s t e r enzymes and v a r i o u s examples i n contemporary l i t e r a t u r e , i t appears t h a t even i n a simple f o r m u l a t i o n a m u l t i t u d e of p o s s i b i l i t i e s e x i s t f o r the c o n t r o l of a metabolic process:  (1)  each pathway i s comprised  of many steps and r e q u i r e -  ments, (2) a s i n g l e component or s u b s t r a t e can p l a y a number of d i v e r s e r o l e s i n r e v e r s a l of the s t e p s , (3) each s t e p can be c o n t r o l l e d by changes i n each of t h r e e components - enzyme, s u b s t r a t e and  effectors,  (4) each component  be m o d i f i e d by i n t e r a c t i o n w i t h o t h e r s w i t h i n the c e l l environment, (5) the components ( a r c h i t e c t u r e ) and  and  the environment of the c e l l can  be  may  89 r e s p o n s i b l e f o r the c o n c e n t r a t i o n of any one  component (enzyme, s u b s t r a t e and  e f f e c t o r s ) v a r y i n g i n the o p p o s i t e d i r e c t i o n i n the same c e l l . There i s no  reason why  c o n t r o l s h o u l d be e x e r t e d o n l y by c o n c e n t r a t i o n  changes i n e i t h e r the enzyme o r i n i t s s u b s t r a t e s o r e f f e c t o r s ; nor are t h e r e reasons at  t h a t c o n t r o l s h o u l d be e x e r t e d e x c l u s i v e l y a t one enzyme r a t h e r than  several.  Rather,  i t seems a t the p r e s e n t t h a t many m e t a b o l i c paths  r e g u l a t e d by more than one enzyme and by a v a r i e t y of f a c t o r s and actions. of  c r o s s r o a d s i n o y s t e r adductor  by the o v e r a l l scheme of a n a e r o b i c metabolism i n these  The enzymic c o n t r o l of p - e n o l p y r u v a t e  t i s s u e as w e l l as organisms.  enzymic pathways are  Under a e r o b i c c o n d i t i o n s , PEP  p y r u v a t e i s f a v o u r e d , w h i l e under a n a e r o b i c c o n d i t i o n s , PEP i s favoured.  to s u c c i n a t e .  at  Stokes  the PEP  PEP  and Awapara, 1968).  i s c o n v e r t e d to  as a l a n i n e (Chen and Awapara,  I f t h i s scheme i s c o r r e c t , i t suggests  b r a n c h i n g p o i n t under a n a e r o b i c c o n d i t i o n s , 50% of the PEP  be c a r b o x y l a t e d to OXA  that t h i s i s u n l i k e l y . c o n t r o l of the two  A n a l y s i s of the a v a i l a b l e d a t a  But more s i g n i f i c a n t l y ,  a t the PEP  (PK)  suggests  In the f i r s t p l a c e , i t would r e q u i r e extremely  enzymes competing f o r PEP  that  should  w h i l e 50% s h o u l d be a v a i l a b l e f o r p y r u v a t e k i n a s e  c a t a l y z e d c o n v e r s i o n to p y r u v a t e .  are  Awapara and h i s co-workers  t h a t i n b i v a l v e m o l l u s c s , the o t h e r mole of PEP  p y r u v a t e which upon t r a n s a m i n a t i o n accumulates 1969;  c a r b o x y l a t i o n to  (NADH) r e d u c i n g e q u i v a l e n t s a r e r e q u i r e d f o r , and  d u r i n g , c o n v e r s i o n of OXA  suggested  c o n v e r s i o n to  A c c o r d i n g to the scheme i n F i g . 1, f o r one mole of  formed from g l u c o s e , two utilized  inter-  crossroads.  crux o f the c o n t r o l problem here i s t h a t two  a v a i l a b l e f o r PEP metabolism.  OXA  their  These g e n e r a l i z a t i o n s a r e w e l l i l l u s t r a t e d by enzymic r e g u l a t i o n  the phosphoenolpyruvate  The  are  tight  branching point.  from demonstrable r e g u l a t o r y p r o p e r t i e s of the  two  90  F i g . V I , 1.  P r o b a b l e pathways o f a n a e r o b i c i n t e r m e d i a r y metabolism i n molluscan f a c u l t a t i v e  anaerobes.  90«L  G  6  arginine  P  T F  6  P  V FDP) I  ornithine +urea  7  NAD  V  triose-P  NADH^ \ NAD proline P  NADH |-~--&-ATP  5  ^  3-PG  I  proline IDP  /  E H V ^ N A D H *'  P 5 C  mo/oe .'  IT " 3 i  NADH-^ NAD-^' • . malute^->-|pyruvaie| NADP NADPH Y  r  l ' ^l g/on n  -^NADH  Cytoplasm  -::  o ^etoglutGrar3 :  NAD-d .  NADH>^ succinylCoA GDP  ^  (fumarate] •  OXA  -5*- pyruvate-^qlulamate  NAD  {*-  L- fumarate^^-^l^ccTnolg Mitochondrion  GTP  91 enzymes i n v o l v e d , i t appears t h a t p y r u v a t e k i n a s e and r e c i p r o c a l c o n t r o l , e i t h e r / o r b a s i s ; they do not simultaneously. The  The  nub  PEPCK o p e r a t e on  a  appear a b l e to f u n c t i o n  of t h i s argument can be summarized as  follows.  a c t i v i t i e s of many enzymes i n v o l v e d i n c e l l u l a r energy metabolism  are governed at l e a s t i n p a r t by metabolic  the energy s t a t u s o f the c e l l .  " s i g n a l s " are the a d e n y l a t e s :  h i g h ATP  concentrations  i n h i b i t o r y to the enzymes i n v o l v e d i n energy metabolism. h i g h AMP  levels  (equivalent  to low ATP)  In o y s t e r muscle, both the PK  On  o f t e n are s t i m u l a t o r y  catalyzed transphosphorylation  The  unusual  often  are  the o t h e r hand, (Atkinson,  1968).  r e a c t i o n and  the  PEPCK c a t a l y z e d c a r b o x y l a t i o n r e a c t i o n generate a h i g h energy phosphate compound (ATP subject 1971;  i n the case of PK;  ITP  to product i n h i b i t i o n by  Mustafa and  or GTP  these compounds (Mustafa and  Hochachka, 1972b).  accordance w i t h A t k i n s o n ' s  i n the case of PEPCK) and both Hochachka,  In t h i s sense, both enzymes behave i n  (Atkinson,  1968)  energy charge concept.  Whereas  these mechanisms undoubtedly c o n t r i b u t e to the p h y s i o l o g i c a l " p o i s e " of PEP  r e q u i r e d can be s u p p l i e d by H It i s widely  +  and  to a n a e r o b i c  metabolism.  T h i s drop i n pH  h e l d t h a t under a n a e r o b i c  of any  PK  other  conditions, molluscan b i v a l v e s  r e a c t i o n and  fluids  (Wilbur,  1964).  f a c t o r , decreasing  pH  a concomitant a c t i v a t i o n of PEPCK.  /  P T r p  s  and  for  In consequence, i n the absence  l e a d s to an automatic i n h i b i t i o n of PK  with  At the same time, L - a l a n i n e which accumu-  s u c c i n a t e under a n a e r o b i c  (by i n c r e a s i n g the K  of  towards PEPCK, because the p_H p r o f i l e s  are e s s e n t i a l l y n o n - o v e r l a p p i n g .  l a t e s along w i t h  specificity  appears to us to p l a y a p i v o t a l r o l e i n the c h a n n e l l i n g  away from the PK  PEPCK and  The  to  L-alanine.  s u s t a i n 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 t i s s u e s and  PEP  the  branch p o i n t , they do n o t , of themselves, d i s p l a y adequate s p e c i f i c i t y  account f o r t r a n s i t i o n from a e r o b i c  are  decreasing  conditions potently i n h i b i t s the maximum c a t a l y t i c r a t e ) .  PK 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 t h a t the L - a l a n i n e i n h i b i t i o n i s p o t e n t i a t e d by d e c r e a s i n g pH;  f o r adductor  the K_^ v a l u e observed In sharp  muscle PK  at the o p t i m a l pH  c o n t r a s t , the primary  a r e v e r s a l of any p-enolpyruvate  the K ^ ^ i  i  n e  )  a t  P  H  7.5  c o n c e n t r a t i o n s due  to a r e d u c t i o n i n the apparent  i s v e r y low and  kinases  i n the PK a c t i v i t y .  themselves.  IV and V, (i)  two  ^anaerobic concurrent  T h i s would appear to be  with  an  towards o x a l o a c e t a t e .  p o s s i b l e metabolic  sources  In our e a r l i e r s t u d i e s on o y s t e r  (Mustafa and Hochachka, 1971), we and,  m  reducing equivalents.  i n d i c a t e d i n Chapters  of a l a n i n e , ATP,  ^ ^pgp^  The net e f f e c t of d e c r e a s i n g  adequate arrangement f o r c h a n n e l l i n g p - e n o l p y r u v a t e  a l a n i n e suggest  low  i n which L - a l a n i n e  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 i n c r e a s e i n the PEPCK a c t i v i t y  As  (i)  Both these e f f e c t s o f L - a l a n i n e on PEPCK  i n c r e a s i n g L - a l a n i n e c o n c e n t r a t i o n d u r i n g the a e r o b i c " '  Sources of a l a n i n e and  1/6  ( i i ) a s l i g h t a c t i v a t i o n at  i n h i b i t i o n of PK i s u n u s u a l l y extreme.  an e x p o n e n t i a l decrease  i s only  (Mustafa and Hochachka, 1971).  r e s i d u a l ITP i n h i b i t i o n and  occur a t pH ranges i n which PK a c t i v i t y  pH and  8.5  a n :  e f f e c t s of L - a l a n i n e on PEPCK appear to be  (Mustafa and Hochachka, 1972a, b ) .  and ATP  a  noted  t h a t FDP  to a somewhat l e s s e r e x t e n t , H . +  pyruvate  r e v e r s e s the Such FDP  of  effects  effects  c o u l d supply the b i v a l v e w i t h a mechanism f o r m a i n t a i n i n g some p y r u v a t e k i n a s e f u n c t i o n under a n a e r o b i c t u r n be  transaminated  (ii)  The  pyruvate produced c o u l d i n  to a l a n i n e .  O y s t e r muscle c o n t a i n s v e r y h i g h a c t i v i t i e s of NADH-linked m a l i c  dehydrogenase (MDH) data).  conditions.  and NADP-linked " m a l i c enzyme" (Mustafa,  unpublished  These enzymes occur i n much h i g h e r s p e c i f i c a c t i v i t i e s  e i t h e r PEPCK or PK.  A h i g h c y t o p l a s m i c MDH  activity  than  do  i s assumed to f u n c t i o n  93 (a) i n the maintenance of low o x a l o a c e t a t e c o n c e n t r a t i o n s thus s i g n i f i c a n t r e v e r s a l o f PEPCK a c t i v i t y  preventing  and (b) i n r e g e n e r a t i n g NAD f o r t h e  t r i o s e phosphate dehydrogenase (TDH) r e a c t i o n (Saz, 1971).  While t h e f u n c t i o n  o f "malic enzyme" r e c e n t l y has been i n d i s p u t e (Hammen, 1969; Chen and Awapara, 1969), i n our hands, o y s t e r "malic enzyme" i s f u l l y however, the a f f i n i t y unpublished  f o r pyruvate  and CO^ a r e so h i g h  data) t h a t i n v i v o t h e enzyme p r o b a b l y  d i r e c t i o n o f pyruvate pyruvate  constants  production.  which upon t r a n s a m i n a t i o n  reversible; (Mustafa,  f u n c t i o n s o n l y i n the  T h i s r e a c t i o n then can generate the accumulates as L - a l a n i n e  (Stokes and  Awapara, 1968). A second advantage to the organism c o u l d d e r i v e from t h e u t i l i z a t i o n of m a l i c enzyme as the source o f p y r u v a t e .  As i n d i c a t e d i n F i g u r e 1, two  r e d u c i n g e q u i v a l e n t s a r e produced i n the t r i o s e phosphate dehydrogenase r e a c t i o n f o r each mole o f g l u c o s e ;  the NAD f o r t h i s r e a c t i o n i s  i n the c y t o s o l by the MDH c a t a l y z e d r e d u c t i o n o f OXA t o malate.  regenerated However,  f o l l o w i n g malate — ^ f u m a r a t e c o n v e r s i o n , t h e fumarate r e d u c t i o n t o s u c c i n a t e r e q u i r e s an a d d i t i o n a l r e d u c i n g e q u i v a l e n t  ( F i g . 1 ) . Reducing e q u i v a l e n t s  c o u l d be s u p p l i e d by m a l i c enzyme, which generates as products  of t h e r e a c t i o n .  T h i s indeed  CC^, p y r u v a t e  i s the e s t a b l i s h e d f u n c t i o n of  m a l i c enzyme i n the o b l i g a t e anaerobe, A s c a r i s l u m b r i c o i d e s However, i n the l a t t e r case, w i t h i n the m i t o c h o n d r i a ,  (Saz, 1971).  the enzyme i s NAD-linked and generates  which can then be u t i l i z e d  a l s o o c c u r r i n g i n the m i t o c h o n d r i a .  NADH  i n fumarate r e d u c t i o n ,  I n m o l l u s c s , m a l i c enzyme i s l o c a t e d  l a r g e l y i n t h e c y t o s o l and i s NADP-linked. t h i s r e a c t i o n would have to be converted mitochondria  and NADPH  Hence, t h e NADPH generated by  t o NADH and t r a n s p o r t e d i n t o the  i f i t were t o d e l i v e r r e d u c i n g power to t h e m i t o c h o n d r i a l  succinoxidase.  Although  t h i s may occur i n m o l l u s c s  (Chen and Awapara, 1969),  94 it  i s more p r o b a b l e  utilized  t h a t the NADPH produced by c y t o p l a s m i c m a l i c enzyme i s  i n r e d u c t i v e steps of f a t t y a c i d b i o s y n t h e s i s .  f a c t , known t o accumulate d u r i n g a n a e r o b i o s i s  F a t t y a c i d s are, i n  i n o t h e r f a c u l t a t i v e anaerobes  (Von Brand, 1 9 6 6 ) , and the m a l i c enzyme c o u l d be i m p l i c a t e d i n a s i m i l a r cytoplasmic  process  i n bivalves.  The r e d u c i n g  equivalents required f o r  fumarate r e d u c t i o n to s u c c i n a t e presumably a r e generated by o t h e r mitochondr i a l oxidation-reduction reactions. candidate  As w i l l become e v i d e n t below, the c h i e f  f o r this job i s a-ketoglutarate  Other m e t a b o l i c  sources  dehydrogenase ( F i g . 1 ) .  of succinate.  Whatever the predominant r o u t e o f p y r u v a t e f o r m a t i o n i n b i v a l v e s , the primary m e t a b o l i c alanine, according  during  anaerobiosis  f a t e o f p y r u v a t e i s t r a n s a m i n a t i o n to  to the r e a c t i o n > a-ketoglutarate + alanine  p y r u v a t e + glutamate  Hence, i f our c o n s i d e r a t i o n s a r e c o r r e c t , a - k e t o g l u t a r a t e produced i n s t o c h i o m e t r i c q u a n t i t i e s . demonstrated i n m o l l u s c s .  experimentally  In any event, i t would be d i f f i c u l t  i n i n t a c t or s e m i - i n t a c t p r e p a r a t i o n s OXA produced from g l u c o s e  T h i s has n o t been  (a-KGA) s h o u l d be  f o r two reasons.  t o demonstrate  In the f i r s t  c o u l d a l s o r e a c t w i t h glutamate a c c o r d i n g  place, t o the  reaction OXA + glutamate  > a-ketoglutarate + aspartate  T h i s r e a c t i o n would a f f e c t a-KGA l e v e l s and may account f o r the h i g h of a s p a r t a t e found i n o y s t e r t i s s u e s ( F l o r k i n , 1 9 6 6 ) . organisms possess the enzymes capable succinate for  represents  another major pathway  of succinate during anaerobiosis  pathway may be p a r t i c u l a r l y important  these  of converting a-ketoglutarate to  (Hammen, 1969) and t h i s indeed  the accumulation  Secondly,  levels  ( F i g . 1).  This  s i n c e a v a r i e t y o f amino a c i d s  could  95  "feed" into i t v i a transamination reactions amino a c i d + a-KGA In most organisms,  > a-KGA produced  t i o n i s r e c o n v e r t e d t o glutamate  glutamate + keto  d u r i n g pyruvate-glutamate  by glutamate  transamina-  dehydrogenase (GDH), a r e a c t i o n  which u t i l i z e s NADH.  I n f a c u l t a t i v e anaerobes,  i s v e r y low (Campbell  and Bishop, 1970)  a c t i v i t i e s are very high.  acid  t h e s p e c i f i c a c t i v i t y o f GDH  w h i l e a - k e t o g l u t a r a t e dehydrogenase  F o r these reasons 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 would p r o b a b l y outcompete t h e GDH r e a c t i o n f o r t h e common s u b s t r a t e , a-KGA.  Under these c o n d i t i o n s , t h e g l u t a m a t e - a l a n i n e transaminase  s e r v e s to  channel a-KGA d i r e c t l y towards a - k e t o g l u t a r a t e dehydrogenase.  F u n c t i o n a l s i g n i f i c a n c e o f two r o u t e s to s u c c i n a t e . The f a c u l t a t i v e anaerobe g a i n s a c r i t i c a l e n e r g e t i c advantage by u t i l i z ing t h i s route: a-KGA  +  t h e o v e r a l l a - k e t o g l u t a r a t e dehydrogenase r e a c t i o n , CoASH  +  NAD  s u c c i n y l CoA  +  C0  2  s e t s t h e s t a g e f o r t h e c o n v e r s i o n o f t h i o l e s t e r bond energy triphosphate.  +  NADH into nucleoside  The r e a c t i o n , c a t a l y z e d by s u c c i n i c t h i o k i n a s e , i s h i g h l y  e x e r g o n i c and can u t i l i z e e i t h e r GDP o r IDP as c o s u b s t r a t e , g e n e r a t i n g GTP o r ITP.  T h i s energy y i e l d i n g r e a c t i o n i s u t i l i z e d as an a n a e r o b i c mechanism f o r  s u p p l a n t i n g a e r o b i c metabolism  i n c e r t a i n mammalian t i s s u e s  presumably has been s e l e c t e d f o r an analogous  (Cohen, 1968), and  function i n facultative  anaerobic  invertebrates. In t h e event  t h a t t h e organism  utilizes  the a - k e t o g l u t a r a t e dehydrogenase  pathway, some p r o v i s i o n must be made f o r the r e g e n e r a t i o n o f NAD r e q u i r e d f o r the r e a c t i o n .  The most l i k e l y c a n d i d a t e f o r the j o b i s fumarate r e d u c t a s e ,  which couples the o x i d a t i o n o f NADH w i t h t h e r e d u c t i o n o f fumarate to s u c c i n a t e  96 (Fig.  1).  In a d d i t i o n , fumarate reductase  mitochondrion  f o r t h e d e l i v e r y o f NAD  i s properly positioned i n  to the a - k e t o g l u t a r a t e  the  dehydrogenase  reaction. From t h e s e  c o n s i d e r a t i o n s , one  can view t h e u n i q u e pathway o f  anaerobic  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 a n a e r o b e s as a means f o r " p r i m i n g " f l o w o f g l u t a m a t e —>a-KGA pyruvate  >succinylCoA—^.succinate,  f o r the transaminase  r e a c t i o n and  yield To  molluscs  of h i g h date, has  a l aspects.  energy phosphate  although  From t h e s e  the organism g a i n s upon g l y c o l y s i s  of anaerobic  a t t e n t i o n has  i t appears t h a t by  energy p r o d u c t i o n .  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 obtained.  and  generated,  transphosphorylation,  Cohen (1968) has  the s u c c i n i c glycolysis  those which r e l y  I n the  classical  a-ketoglutarate (1)  acetic  solely  glycolytic is  and  1 mole  3 moles of h i g h  energy coupling  the phosphoglycerate  t h i o k i n a s e , and  (3) t h e (5)  kinase pyruvate  succinic  thiokin-  c a l c u l a t e d t h a t i n terms of h i g h energy phosphate  t h i o k i n a s e pathway by  i n t h e mammalian k i d n e y ,  itself and  a-  dehydrogenase.  ( 2 ) t h e PEPCK c a r b o x y l a t i o n o f P E P ,  t r a n s p h o s p h o r y l a t i o n , (4)  metabolism  a s s u m i n g an o b l i g a t e r e d o x  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  ase.  this  g a i n o f 2 m o l e s o f ATP  a net g a i n of at l e a s t  t r i p h o s p h a t e can be  between fumarate reductase  kinase  utilizing  I n the f a c u l t a t i v e anaerobe, f o r 1 mole of g l u c o s e  ketoglutarate metabolized, nucleoside  metabolism i n b i v a l v e  been g i v e n to i t s f u n c t i o n -  a d i s t i n c t e n e r g e t i c advantage over  f o r anaerobic  through  compounds.  little  results  supplying  dehydrogenase r e a c t i o n ( F i g . 1).  the p e c u l i a r n a t u r e  been r e c o g n i z e d ,  (1)  (2) r e g e n e r a t i n g NAD  fumarate r e d u c t i o n f o r the a - k e t o g l u t a r a t e  The  by  the  i s a b o u t 50%  as e f f i c i e n t  i t w o u l d a p p e a r no  yield  as  less important  in  the f a c u l t a t i v e a n a e r o b i o s i s  of  invertebrates.  CHAPTER V I I : Literature  Cited  98 Atkinson, D. 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