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Studies on an inhibitor of in vitro acetoacetate formation : a novel acid phosphohydrolase Caldwell, Ian Carl 1964

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STUDIES ON AN INHIBITOR OF IN VITRO ACETOACETATE FORMATION A NOVEL ACID PHOSPHOHYDROLASE by  IAN CARL CALDWELL M.A., University of B r i t i s h Columbia,  1961  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of Pharmacology  We accept t h i s thesis as conforming to the requ i red standard  THE UNIVERSITY OF BRITISH COLUMBIA October, ]96k  In the  r e q u i r e m e n t s f o r an  British  mission  for reference  for extensive  p u r p o s e s may  be  cation  of  written  Department  of  study,  by  for  the  Library  1964  fulfilment  University  shall  I further  Head o f my  agree for  Columbia,  that  of •  per-  scholarly  Department  that  of  make i t f r e e l y  or  c o p y i n g or  f i n a n c i a l g a i n . s h a l l not  Pharmacology  October 9,  the  this thesis  permission*  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, Canada Date  the  c o p y i n g of  in partial  degree at  I t i s understood  this thesis  w i t h o u t my  that  and  granted  representatives.  this thesis  advanced  Columbia, I agree  available  his  presenting  be  by publi-  allowed  ii  ABSTRACT  The century on  presence  ago.  of acetone  Since that time considerable e f f o r t  k e t o s i s and  ketogenesis.  k e t o n e body.  extracts obtained c h i c k e n , as w e l l  formation  as omasum and  by t h e p r e s e n c e  f o r m a t i o n by b e e f a c t i o n was  begun  inhibitor factor. The  acetoacetate-forming  of  The  the  enzyme A.  product  liver  in v i t r o  subsequently  c h a r a c t e r i z e d by p a p e r and  a  been p u r i f i e d  a c t on  any  acetoacetate  worked out  inhibitor of i t s  by  that  The  independ-  the nature of  the  study. 300-fold  from e x t r a c t s of in vitro  Preliminary studies reveal-  of the enzymatic  is presented  i n a c t i v a t i o n of both of  is  acetoacetate  v a r i o u s p o s s i b l e s i t e s o f a c t i o n on t h e  Evidence  and  t h e mechanism o f a c e t o a c e t a t e f o r m a t i o n .  s y s t e m h a v e been e x a m i n e d .  mation through The  inhibits:  i n t e r e s t , however, t o c l a r i f y  ed t h a t t h e f a c t o r d o e s n o t acetate formation.  in extracts of chicken  T h i s t h e s i s c o n s t i t u t e s such  liver.  liver  l a b o r a t o r y i n t h e h o p e t h a t an u n d e r s t a n d i n g  " i n h i b i t o r " p r o t e i n has  fresh chicken  acetoacetate,  A study of the nature of t h i s  m e c h a n i s m o f a c e t o a c e t a t e f o r m a t i o n was still  to studies  from sheep.  This " i n h i b i t o r " a l s o depresses  liver extracts.  I t was  a  r a b b i t , pigeon  rumen e p i t h e l i u m o b t a i n e d  a c t i o n m i g h t s h e d some l i g h t on  e n t means.  as b e e f , d o g ,  of a f a c t o r which  used.  in t h i s  been d e v o t e d  For t h e s e s t u d i e s , i n v e s t i g a t o r s have used  from v a r i o u s s p e c i e s such  in the assay  has  mechanism f o r t h e f o r m a t i o n o f  A study of acetoacetate formation complicated  r e p o r t e d more than  Recent s t u d i e s i n s e v e r a l l a b o r a t o r i e s have  revealed the p r e c i s e enzymatic the primary  i n d i a b e t i c u r i n e was  components o f  aceto-  i t inhibits acetoacetate  t h e f r e e and  ester ified  i t s a c t i o n on c o e n z y m e A has  been  i o n - e x c h a n g e c h r o m a t o g r a p h y and  forms o f  isolated chemical  forco-  and analysis  i ii  as  3 - d e p h o s p h o c o e n z y m e A.  d e s i g n a t e d as clear that  t h i s enzyme c o u l d  The activity  at  2.5.  The  neither  i n h i b i t o r p r o t e i n can  a "coenzyme A 3 ' - p h o s p h o h y d r o l a s e . "  z y m a t i c mechanism of  pH  The  1  have a f f o r d e d  acetoacetate  no  rate of  enhanced nor  b e t w e e n pH  In r e t r o s p e c t ,  properly i t is  knowledge c o n c e r n i n g the  now en-  formation.  enzyme e x h i b i t s maximal a c t i v i t y a "plateau"  t h u s be m o r e  5.0  and  pH  a t pH  3.6,  6.0,  and  i n a c t i v a t i o n o f c o e n z y m e A by d e p r e s s e d by d i v a l e n t  with  half-maximal  a t some p o i n t  below  the phosphohydrolase is  cation or metal-binding  agents.  A l t h o u g h t h e m o s t h i g h l y p u r i f i e d enzyme f r a c t i o n o b t a i n e d e x h i b i t s p h o s p h o hydrolase a c t i v i t y presented to well  be  against  indicate that  a s p e c i f i c and  a wide v a r i e t y of phosphate e s t e r s , evidence is the  coenzyme A 3 - p h o s p h o h y d r o l a s e a c t i v i t y  u n i q u e enzyme.  1  may  iv  TABLE OF CONTENTS  Page  INTRODUCTION  1  EXPERIMENTAL SECTION  5  Materials  5  Methods  5  I.  Miscellaneous  5  P r e p a r a t i o n a n d A s s a y o f Enzymes  6  A.  Auxiliary  6  B.  Chicken Liver  II.  Enzymes  9  Enzymes  1.  Enzyme a s s a y s  9  2.  P u r i f i c a t i o n o f t h e " i n h i b i t o r enzyme"  12  3.  Isolation  17  of subcellular  fractions  RESULTS I. II. III. IV.  Existence of the Inhibitor  21  Locus o f t h e I n h i b i t o r  2k  Effect  P r e c i s e A c t i o n o f t h e I n h i b i t o r Enzyme  36  T h e Coenzyme A 3 ' - P h o s p h o h y d r o l a s e  kh  DISCUSSION  63  BIBLIOGRAPHY  67  V  L I S T OF  No.  FIGURES  Title  Page  1  Formation  o f a c e t o a c e t a t e by e x t r a c t s o f v a r i o u s t i s s u e s  22  2  Formation  o f a c e t o a c e t a t e by c h i c k e n  23  3  I n h i b i t i o n o f a c e t o a c e t a t e f o r m a t i o n by b e e f l i v e r in t h e presence of chicken l i v e r f r a c t i o n s  4  5  6  7  8  9  10  11  12  13  liver  fractions enzyme  25  E f f e c t o f coenzyme A c o n c e n t r a t i o n oh a c e t o a c e t a t e f o r m a t i o n by b e e f l i v e r enzyme i n t h e p r e s e n c e a n d a b s e n c e o f c h i c k e n 1 i v e r " i n h i b i t o r enzyme"  31  P o s s i b l e s i t e s o f a c t i o n o f t h e i n h i b i t o r enzyme upon c o enzyme A  37  Characterization atography  of t h e reaction product  39  Characterization chromatography  of t h e reaction product  by p a p e r c h r o m -  by  ion-exchange 42  I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n function o f time  liver  I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n function of protein concentration  liver  enzyme a s a 45 enzyme as a 46  C h r o m a t o g r a p h y o f t h e c o e n z y m e A p h o s p h o h y d r o l a s e o n DEAEcellulose  48  I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n f u n c t i o n o f pH  54  liver  enzyme a s a  " F l o w s h e e t " f o r i s o l a t i o n o f s u b c e l l u l a r f r a c t i o n s by differential centrifugation  55  D i s t r i b u t i o n o f p - n i t r o p h e n y l p h o s p h a t e and coenzyme A p h o s p h o h y d r o l a s e a c t i v i t i e s d u r i n g c h r o m a t o g r a p h y o n DEAEcellulose  59a,  vi  L I S T OF TABLES  No.  I  II  III  IV  V  VI  Title  Page  E f f e c t o f p r e i n c u b a t i n g t h e enzymic assay t h e " i n h i b i t o r enzyme"  components w i t h 27  E f f e c t o f t h e " i n h i b i t o r enzyme" on f o r m a t i o n o f a c e t a t e by r e c o n s t i t u t e d A . S . E . S . p r e p a r a t i o n s  aceto28  E f f e c t o f p r e i n c u b a t i o n o f t h e nonenzymic components o f t h e a s s a y w i t h t h e " i n h i b i t o r enzyme" b e f o r e a d d i t i o n o f t h e b e e f l i v e r enzyme  29  Inactivation enzyme"  32  o f c o e n z y m e A by c h i c k e n  liver  "inhibitor  C o r r e l a t i o n b e t w e e n " i n h i b i t o r e n z y m e " and c o e n z y m e A i n activation activities Evidence  f o r inactivation  of ester ified  c o e n z y m e A by t h e 35  " i n h i b i t o r enzyme" VII VIM IX  X  XI  XII  Composition  o f t h e product  43  of the reaction  P u r i f i c a t i o n o f t h e coenzyme A 3 ' - p h o s p h o h y d r o l a s e E f f e c t o f e x h a u s t i v e d i a l y s i s vs_ EDTA s o l u t i o n i t y o f t h e coenzyme A p h o s p h o h y d r o l a s e Intracellular distribution lase Distribution of acid 1iver f r a c t ions  34  upon  49 activ52  o f t h e coenzyme A phosphohydro-  phosphohydrolase a c t i v i t i e s  56 in chicken  D i s t r i b u t i o n o f p - n i t r o p h e n y l p h o s p h a t e and c o e n z y m e A phosphohydrolases during p u r i f i c a t i o n of t h e " i n h i b i t o r enzyme"  58  60  vi i  ABBREVIATIONS  The acetoacetyl  following abbreviations  h a v e been u s e d :  t h i o e s t e r o f c o e n z y m e A; a c e t y l - C o A and a c e t y l - G , respectively; 2'-,  t h i o e s t e r s o f coenzyme A and g l u t a t h i o n e , AMP,  t h e 2'-,  3'-  the acetyl 3'"  and 5 ' -  enzyme s y s t e m ;  3'(2')-CMP, t h e m i x e d  2 1  and 3  1 -  e s t e r s o f c y t i d i n e ; CoASH, r e d u c e d c o e n z y m e A; D E A E - c e l l u l o s e ,  diethylaminoethyl material  the  and 5 ' - p h o s p h a t e e s t e r s o f a d e n o s i n e ; A.S.E.S., t h e  acetoacetate-synthesizing phosphate  acetoacety1-CoA,  formed  c e l l u l o s e ; ECTEOLA-cellulose, a c e l l u l o s e by r e a c t i n g c e l l u l o s e w i t h  ion-exchange  e p i c h l o r h y d r i n and t r i e t h a n o l -  a m i n e ; EDTA, e t h y l e n e - d i a m i n e t e t r a a c e t i c a c i d ; 3'(2')-GMP, t h e m i x e d and 3 ' - p h o s p h a t e e s t e r s o f g u a n o s i n e ;  GSH, r e d u c e d g l u t a t h i o n e ;  2'-  HMG, p -  h y d r o x y - p - m e t h y l g l u t a r i c a c i d ; HMG-CoA, t h e B - h y d r o x y - p - m e t h y l g l u t a r y l t h i o e s t e r o f c o e n z y m e A; T r i s , t r i s - ( h y d r o x y m e t h y l ) - a m i n o m e t h a n e ; UMP, t h e m i x e d  2'-  and 3'-phosphate e s t e r s o f u r i d i n e .  3 (2 )" ,  1  vi i i  ACKNOWLEDGEMENTS  The a u t h o r w o u l d l i k e t o e x p r e s s h i s g r a t i t u d e t o t h e f o l l o w i n g persons: D r . G e o r g e i . Drummond, D e p a r t m e n t o f P h a r m a c o l o g y , f o r a d v i c e a n d assistance during the course of this project; Dr. James G. F o u l k s , H e a d , D e p a r t m e n t o f P h a r m a c o l o g y , f o r m a k i n g a v a i l a b l e t h e f a c i l i t i e s o f t h e Department; T h e C a n d i d a t e ' s C o m m i t t e e - D r . Drummond; D r . F o u l k s ; D r . J . J . R. C a m p b e l l , D e p a r t m e n t o f D a i r y i n g ; a n d D r . G. M. T e n e r , D e p a r t m e n t o f B i o c h e m i s t r y - f o r a d v i c e and e n c o u r a g e m e n t ; D r . G e o r g e M. L i n g , D e p a r t m e n t o f P h a r m a c o l o g y , f o r p e r s o n a l e n couragement;  sonal  The N a t i o n a l R e s e a r c h C o u n c i l o f Canada, f o r p r o v i d i n g b o t h p e r s u p p o r t and r e s e a r c h s u p p o r t ;  chicken  of  Mr. E l l i s , liver;  P o u l t r y Farm, f o r p r o v i d i n g generous s u p p l i e s o f f r e s h  Mr. F r a m p t o n , Canada f r e s h beef 1iver;  Packers L t d . , f o r p r o v i d i n g generous  M r s . D. W. ( L o v e r n e ) D u n c a n , a h e l p i n g hand when n e e d e d ;  supplies  Department o f Pharmacology, f o r l e n d i n g  Many o t h e r s , t o o n u m e r o u s t o m e n t i o n i n d i v i d u a l l y , who h a v e in t h e completion o f t h i s p r o j e c t .  assisted  1  INTRODUCTION  The  presence  o f a c e t o n e i n d i a b e t i c u r i n e was  c e n t u r y ago  (1).  ketogenesis  i n t h e e n s u i n g y e a r s , but  Much e f f o r t  the p r e c i s e biochemical the primary  has  pathways  b e e n e x p e n d e d on  r e p o r t e d more t h a n  t h e s t u d y o f k e t o s i s and  i t is only w i t h i n the  l a s t decade t h a t  involved in the formation of  k e t o n e body, h a v e been e l u c i d a t e d i n t h i s  acetoacetate,  l a b o r a t o r y (2-4)  and  (5"7).  elsewhere In  I960,  S t e r n and  co-workers at Western Reserve U n i v e r s i t y  land described the formation of acetoacetate from acetyl-CoA various tissues t i o n was  (8).  found  in Cleve-  by e x t r a c t s o f  In m o s t t i s s u e s s t u d i e d , t h e r a t e o f a c e t o a c e t a t e forma-?  e s s e n t i a l l y p r o p o r t i o n a l t o t h e amount o f e x t r a c t a d d e d .  e x c e p t i o n was As  a  in the case of extracts of chicken  the concentration of chicken  l i v e r protein in their  liver  One  notable  acetone powder.  reaction mixture  was  i n c r e a s e d , t h e amount o f a c e t o a c e t a t e f o r m e d u n d e r t h e c o n d i t i o n s o f t h e r o s e and  then d e c l i n e d .  a c e t o a c e t a t e was the presence terfered  in chicken  " i n h i b i t o r " was  not y e t understood.  this  The liver  e x t r a c t s of a secondary  A study of t h i s  l a b o r a t o r y i n t h e hope t h a t  formation.  still  felt  Although  begun i n  into the  i t became a p p a r e n t  s t u d y t h a t such would not  t h a t t h i s o b s e r v a t i o n was  formation  e n z y m e s y s t e m was  i t m i g h t p r o v i d e some i n s i g h t  a t e mechanism o f a c e t o a c e t a t e f o r m a t i o n . In t h e c o u r s e o f t h i s  secondary  to  enzyme s y s t e m w h i c h i n -  d i s c o v e r e d , t h e mechanism o f a c e t o a c e t a t e  early  no  above workers a t t r i b u t e d t h e s e o b s e r v a t i o n s  i n some m a n n e r w i t h t h e e n z y m e ( s ) i n v o l v e d i n a c e t o a c e t a t e  When t h i s was  formed.  At v e r y h i g h p r o t e i n c o n c e n t r a t i o n s , v i r t u a l l y  assay  be t h e c a s e ,  worthy of f u r t h e r study.  intim-  quite  i t was  It i s t h e  pur-  2  pose o f t h i s t h e s i s t o i d e n t i f y and c h a r a c t e r i z e t h i s secondary and  enzyme  system  t o p r o v i d e a n e x p l a n a t i o n f o r t h e a b o v e m e n t i o n e d phenomenon. I t was p r o p o s e d b y L y n e n a n d c o - w o r k e r s (2-4) and o t h e r s  author  extracts  (5~7) t h a t t h e f o r m a t i o n o f a c e t o a c e t a t e b y t i s s u e  i n v o l v e s two enzymes.  T h e s e t w o enzymes a r e t h e HMG-CoA  (3"hydroxy-3-methylglutaryl-CoA  enzyme  (5), and e s t a b l i s h e d by t h e  a c e t o a c e t y l - C o A - l y a s e , EC 4 . 1 . 3 . 5 ;  a l s o known a s HMG-CoA s y n t h a s e )  d e s c r i b e d b y Rudney a n d c o - w o r k e r s  and  (3-hydroxy-3 methylg1utaryl-CoA  t h e HMG-CoA c l e a v a g e  l y a s e , EC 4 . 1 . 3 . 4 ; workers.  Thef i r s t  the condensation  enzyme  condensing  _  (9-13),  acetoacetate-  a l s o known a s HMG-CoA l y a s e ) d e s c r i b e d b y Coon a n d c o o f t h e s e e n z y m e s , t h e HMG-CoA c o n d e n s i n g  o f o n e mole each o f a c e t y l - C o A  enzyme, c a t a l y z e s  and a c e t o a c e t y l - C o A  t o form  o n e m o l e o f HMG-CoA a n d l i b e r a t e s o n e m o l e o f f r e e c o e n z y m e A ( R e a c t i o n T h e s e c o n d o f t h e s e e n z y m e s , t h e HMG-CoA c l e a v a g e  1).  enzyme, c a t a l y z e s t h e  c l e a v a g e o f HMG-CoA t o f o r m o n e m o l e o f f r e e a c e t o a c e t a t e a n d o n e m o l e o f (Reaction 2 ) .  acetyl-CoA  CH3-C-SC0A  +  acetyl-CoA  CH -C-CH -C-SCoA 3  +  2  H 0 2  acetoacetyl-CoA  9  H  *•  fl  CH -C-CH -C-SCoA 3  2  +  CoASH  (1)  +  0 CH -C-SCoA  (2)  CH -C00" 2  HMG-CoA OH ^0 CH -C-CH -C-SCoA 3  2  »-  1  CH-COO:* 2  HMG-CoA  0 CH -C-CH -C00" 3  2  acetoacetate  3  acetyl-CoA  3  The  n e t r e s u l t o f t h e s e q u e n t i a l o p e r a t i o n o f t h e s e t w o enzymes i s t h e h y d r o -  lysis and  o f one mole o f acetoacetyl-CoA,  producing  one mole each o f a c e t o a c e t a t e  c o e n z y m e A. In t h e a s s a y  s y s t e m u s e d by t h e C l e v e l a n d g r o u p , a c e t y l - C o A  acetyl-CoA were generated  from acetyl  o f coenzyme A i n t h e presence bacterial  extract served  orthophosphate  p h o s p h a t e and c a t a l y t i c  and a c e t o -  concentrations  o f an e x t r a c t o f C l o s t r i d i u m k l u y v e r i .  The  as a s o u r c e o f p h o s p h a t e t r a n s a c e t y l a s e ( a c e t y l - C o A :  a c e t y l t r a n s f e r a s e , EC 2 . 3 . 1 . 8 ) ( R e a c t i o n 3 ) a n d p - k e t o t h i o l a s e  (acetyl-CoA:acetyl-CoA  2 CH -C-0-P-0" 3  +  C - a c y l t r a n s f e r a s e , EC 2 . 3 . 1 . 9 ) ( R e a c t i o n  2 CoASH  =^=^=  2 ChyC-SCoA  +  k).  2 P.  (3)  acetyl-CoA acetyl  phosphate  0  0  2 CH -C-SCoA 3  acetyl-CoA  An  examination  0 CH -C^CH -d'-SCoA 3  2  +  o f t h e sequence o f r e a c t i o n s involved i n t h e assay  t h e formation of acetoacetate  Similarly,  in chicken  liver extracts  i n a number o f w a y s .  b e a p r o t e o l y t i c e n z y m e r e l e a s e d when t h e c e l l s tion of the extracts.  (h)  acetoacetyl-CoA  s y s t e m shows t h a t t h e " i n h i b i t o r " e n z y m e p r e s e n t might prevent  CoASH  a r e broken during  It could prepara-  i t c o u l d combine w i t h one o r more o f t h e  enzymes t o g i v e an i n a c t i v e c o m p l e x , p a r a l l e l i n g t h e s i t u a t i o n s e e n w i t h trypsin  and t h e v a r i o u s n a t u r a l l y - o c c u r r i n g t r y p s i n  inhibitors  (16). In-  a c t i v a t i o n o f t h e enzyme(s) i n v o l v e d In t h e r a t e - l i m i t i n g s t e p o f a c e t o a c e t a t e f o r m a t i o n by e i t h e r o f t h e s e means w o u l d  result  i n t h e observed  Inhibitor effect.  On t h e o t h e r h a n d , t h e " i n h i b i t o r e n z y m e " c o u l d  a c e t o a c e t a t e f o r m a t i o n by m o d i f y i n g assay  o n e o r more o f t h e e s s e n t i a l  components o r one o f t h e i n t e r m e d i a t e s formed  obvious  p o s s i b i l i t y would be t h a t  hydrolase 07).  (EC 3 . 1 . 2 . 5 ) o f c h i c k e n  Lipmann  i n e x t r a c t s o f horse  however, s i n c e a c e t y l The  liver  w i t h t h e HMG-CoA  step  in acetoacetate formation, the I t c o u l d a l s o b e an  (EC 3 * 6 . 1 . 7 ) , s i m i l a r t o t h a t d e s c r i b e d b y (18).  phosphate i spresent  This p o s s i b i l i t y i n t h e assay  i n h i b i t o r enzyme c o u l d a l s o be an a c e t y l - C o A  i s r a t h e r remote,  i n large excess.  hydrolase  (EC 3 . 1 * 2 . 1 ) .  I t c o u l d b e a c t i n g upon c o e n z y m e A i n some manner - f o r e x a m p l e , be and  identical with t h epeptidase described co-workers  (19),  in avian l i v e r  functions c a t a l y t i c a l l y  could  e x t r a c t s by Novel 1 i  result  i n depressed  acetoacetate formation.  i n some o t h e r w a y .  explanations for t h e inhibitor effect w i l l In t h i s t h e s i s , e v i d e n c e w i l l acetoacetate formation  S i n c e coenzyme A  i n t h e a s s a y method u s e d , any removal o f coenzyme A  a l s o produce i t s e f f e c t  of t h e presence  i t could  h y d r o l y z i n g t h e amide l i n k a g e between t h e p a n t o t h e n i c  a c i d a n d p - m e r c a p t o e t h y l a m i n e m o i e t i e s o f c o e n z y m e A.  would n a t u r a l l y  One  l i v e r d e s c r i b e d by Coon a n d c o - w o r k e r s  c l e a v a g e o f HMG-CoA ( R e a c t i o n 2 ) , w o u l d b e p r e v e n t e d . phosphate phosphohydrolase  nonenzymic  in t h eprocess.  i t might be i d e n t i c a l  I f t h i s were t h e case, t h e f i n a l  acetyl  prevent  i n t h e e x t r a c t s o f a novel  be shown t h a t t h e i n h i b i t o r e f f e c t  Many o f t h e s e p o s s i b l e  be d i s c u s s e d l a t e r  be p r e s e n t e d  i n v i t r o by c h i c k e n  liver  The i n h i b i t o r  i n more d e t a i l .  that the inhibition of  extracts i sa direct  acid phosphohydrolase.  result  It w i l l  i s d u e t o t h e h y d r o l y s i s o f t h e 3'-phospho*  m o n o e s t e r l i n k a g e o f c o e n z y m e A, r e s u l t i n g a l l y - ? n a c t i v e 3 ' - d e p h o s p h o c o e n z y m e A.  i n t h e formation o f t h e enzymic-  5  EXPERIMENTAL SECTION  Materials  Coenzyme A  ( A g r a d e , " 90 p e r c e n t p u r e ) , a " B i o c h e m i c a B o e h r i n g e r " n  p r o d u c t , and d i s o d i u m p - n i t r o p h e n y l the  phosphate  ("A g r a d e " ) w e r e o b t a i n e d f r o m  C a l i f o r n i a C o r p o r a t i o n f o r B i o c h e m i c a l Research, Los A n g e l e s ,  California.  Dried c u l t u r e s o f C l o s t r i d i u m k l u y v e r i were o b t a i n e d from Worthington chemical  Co., F r e e h o l d , N . J .  obtained from Carl  Ion-exchange  S c h l e i c h e r and S c h u e l l  eelluloses  Bio-  ( " S e l e c t a c e l s " ) were  C o . , K e e n e , N.H.  Authentic  3'  -  d e p h o s p h o c o e n z y m e A was t h e g i f t o f D r . J.G. M o f f a t t , S y n t e x  Institute for  Molecular Biology, Palo Alto, California.  g e l was p r e -  p a r e d by t h e m e t h o d o f K e i l i n pared  Calcium phosphate  and H a r t r e e ( 2 0 ) . A c e t y l  by t h e a c e t y l a t i o n o f o r t h o p h o s p h a t e w i t h a c e t i c a n h y d r i d e i n a q u e o u s  p y r i d i n e a s d e s c r i b e d by S t a d t m a n ( 2 1 ) . A c e t y l the  p h o s p h a t e was p r e -  m e t h o d o f S i m o n and S h e m i n  t h i o e s t e r s w e r e p r e p a r e d by  ( 2 2 ) , i n which t h e reduced t h i o l  i s reacted  w i t h a 4 0 t o 5 0 p e r c e n t e x c e s s o f a c e t i c a n h y d r i d e a t pH 7.5.  In a  manner, a c e t o a c e t y l  and d i k e t e n e  t h i o e s t e r s were prepared from reduced t h i o l  as d e s c r i b e d by L y n e n e t a l _ . ( 5 ) , a n d HMG HMG  a n h y d r i d e as d e s c r i b e d  similar  t h i o e s t e r s from reduced t h i o l  by H i l z e t a1_. (23).  and  Other m a t e r i a l s used were  r e a g e n t g r a d e and w e r e o b t a i n e d f r o m v a r i o u s c o m m e r c i a l  sources.  Methods I.  M i s c e l l a n e o u s Methods Acetyl  p h o s p h a t e was d e t e r m i n e d by t h e h y d r o x a m i c  Lipmann and T u t t l e  a c i d method o f  ( 2 4 ) . Coenzyme A s o l u t i o n s w e r e f r e s h l y p r e p a r e d a s r e -  q u i r e d a n d s t a n d a r d i z e d by a m o d i f i c a t i o n o f t h e s t o i c h i o m e t r i c  phosphate  6 t r a n s a c e t y l a s e m e t h o d o f S t a d t m a n (25).  Protein concentrations of  f r a c t i o n s w e r e d e t e r m i n e d by t h r e e m e t h o d s . tions with very  For t u r b i d  (26)  was u s e d .  of o p t i c a l l y c l e a r f r a c t i o n s were determined m e t h o d o f W a r b u r g and C h r i s t i a n ultraviolet 280  fractions, or frac-  high p r o t e i n concentrations, the c o l o r i m e t r i c  b i u r e t m e t h o d o f G o r n a l 1 et_ a l _ .  radiation  (27),  by a r o m a t i c  which  enzyme  modified  The p r o t e i n c o n c e n t r a t i o n s  by t h e  spectrophotometric  i s b a s e d upon t h e a b s o r p t i o n o f  amino a c y l  residues  mu, w i t h a c o r r e c t i o n f o r n u c l e i c a c i d p r e s e n t  in the proteins at  from t h e o t p i c a l  density  a t 260  mu.  ample,  i n e f f l u e n t s from chromatographic columns - were determined from t h e  optical  The p r o t e i n c o n c e n t r a t i o n s o f v e r y  d e n s i t i e s a t 215  mu a n d 225  mu a s d e s c r i b e d  B o v i n e s e r u m a l b u m i n was u s e d a s a p r o t e i n  11.  by M u r p h y a n d K i e s ( 2 8 ) .  standard.  P r e p a r a t i o n a n d A s s a y o f Enzymes Enzyme a c t i v i t i e s ,  enzyme u n i t recommended Union o f B i o c h e m i s t r y zyme w h i c h w i l l m i n u t e a t 30* of  d i l u t e s o l u t i o n s - f o r ex-  activities  where noted,  are expressed  i n terms o f t h e  by t h e C o m m i s s i o n o n Enzymes o f t h e I n t e r n a t i o n a l  (29).  This  unit  is defined  a s t h a t amount  o f any e n -  c a t a l y z e t h e t r a n s f o r m a t i o n o f one umole o f s u b s t r a t e p e r  under s p e c i f i e d  inconvenient  ific  except  conditions.  numbers, a c t i v i t i e s a r e expressed  A. A u x ? 1 i a r y  Where t h i s w o u l d r e q u i r e t h e u s e  a r e expressed  in milliunits  (mU).  Spec-  a s u n i t s o r m i l l i u n i t s p e r mg o f p r o t e i n .  Enzymes  C r u d e e x t r a c t s o f C l o s t r i d i u m k l u y v e r i . used as a s o u r c e o f p h o s p h a t e t r a n s a c e t y l a s e , were prepared b u f f e r as p r e v i o u s l y d e s c r i b e d by  by a u t o l y s i s o f t h e d r i e d c e l l s (2).  P h o s p h a t e t r a n s a c e t y l a s e was m e a s u r e d  a m o d i f i c a t i o n o f t h e m e t h o d o f S t a d t m a n (30),  than  5 u n i t s , o f c o e n z y m e A.  in phosphate  using  10 m u m o l e s ,  rather  7 The  acetoacetate-synthesizing  were prepared from beef a c t i v i t y was (For  an  (4),  in the  thione,  m e a s u r e d by  explanation  of  50  as  described  a modification  the  basis  Introduction.) 8.1,  b u f f e r , pH  liver  The  of  with  kluyver?  acetylase);  and  this  by  the  tubes were c h i l l e d  0.5  ml  per  centrifuging  addition  of  ml  the  cold.  (containing  ucible,  i s as  (31).  with  0°.  To  6 per  to  hydrochloride gluta5  system,  consist-  the  r e a c t i o n was  Suitable  0.5  After  ml. 60  The  reaction  the  a c e t o a c e t a t e by  38°,  addition  D e n a t u r e d p r o t e i n was  a l i q u o t s of  trans-  m i n u t e s at  s t o p p e d by  and  of  removed  supernatant  solution  a modification is extremely  of  the  reprod-  follows: the  deproteinized  cent t r i c h l o r o a c e t i c acid  a n i l i n e reagent. minutes, the  The  5.1,  and  ml  by  the  k e p t a t 0°  10  minutes.  Ethyl  3.5  ml  of Walker's d i a z o t i z e d i n a w a t e r bath at  in ice f o r 3 to 5 minutes.  a d d i t i o n o f 6.0  were then a c i d i f i e d  ml  acetate  ml)  was  at  of  M  p-nitro 30°.  The  of 5 N h y d r o c h l o r i c (4.0  0.50  diluted to  in rapid succession,  tubes were then placed  tubes were c h i l l e d  for 8 to  3.0  s o l u t i o n was  in a glass-stoppered t e s t tube  t h i s d i l u t e d a l i q u o t were added,  s o d i u m a c e t a t e b u f f e r , pH  20  (1)  equations  of T r i s  group.  2 units of phosphate  This modified procedure, which  A s u i t a b l e a l i q u o t of ml  Cleveland  umoles; reduced  volume o f  fraction.  w e r e t h e n removed f o r d e t e r m i n a t i o n o f method o f W a l k e r  A.S.E.S.  immediately b e f o r e use,  liver  i n i c e and  The  10 u m o l e s , c o e n z y m e A, 0.0125 u m o l e s ,  cent t r i c h l o r o a c e t i c a c i d . in the  the  assay method, see  enzyme f r a c t i o n i n a f i n a l  initiated  by  o f t h e method o f  1 umole; a t h i o e s t e r - g e n e r a t i n g  e x t r a c t , 0.03  was  ert a l _ . (8).  assay system consisted  ing of d i l i t h i u m a c e t y l phosphate, Clostridium  (A.S.E.S.) f r a c t i o n s  Stern  potassium bicarbonate  umoles; magnesium c h l o r i d e ,  12  by  u m o l e s ; p o t a s s i u m c h l o r i d e , 2.5  neutralized  of  enzyme s y s t e m  After solutions  acid,  and  then added,  and  8 the  formazan  d e r i v a t i v e was q u a n t i t a t i v e l y  by v i g o r o u s s h a k i n g . at  4 5 0 mu.  extracted  into theorganic  layer  T h e a b s o r b a n c e o f t h e o r g a n i c l a y e r was t h e n m e a s u r e d  U n d e r t h e a b o v e c o n d i t i o n s , 0.1 u m o l e o f a c e t o a c e t a t e w i l l  uce an o p t i c a l  d e n s i t y o f O.36  (d = 0.5 c m ) .  c o l o u r d e v e l o p e d was p r o p o r t i o n a l  Using t h i s  prod-  procedure, t h e  t o t h e amount o f a c e t o a c e t a t e p r e s e n t  o v e r t h e r a n g e 0.05 t o 0.55 u m o l e . The  HMG-CoA c o n d e n s i n g was p u r i f i e d  and  Ferguson  (12).  ate  fractionation,  f r o m y e a s t a c c o r d i n g t o Rudney  T h e p u r i f i c a t i o n was c a r r i e d t h r o u g h t h e ammonium  a d s o r p t i o n upon and e l u t i o n f r o m c a l c i u m p h o s p h a t e g e l ,  t r e a t m e n t w i t h p r o t a m i n e s u l f a t e a n d p r e c i p i t a t i o n w i t h ammonium The  final  two s t e p s - t r e a t m e n t w i t h  w i t h c a l c i u m phosphate  g e l - which  i o d o a c e t a m i d e and a second  sulfate. treatment  t h e s e a u t h o r s e m p l o y e d t o r e m o v e p-keto-  t h i o l a s e were o m i t t e d , s i n c e they were unnecessary resulted  sulf-  i n v e r y l o w r e c o v e r i e s o f enzyme.  f o r o u r p u r p o s e s , and  The above a u t h o r s r e p o r t e d t h a t e  this  e n z y m e was v e r y u n s t a b l e i f s t o r e d a s a s o l u t i o n , e v e n a t -20 , a n d  t h a t t h e enzyme was o n l y s l i g h t l y m o r e s t a b l e under s a t u r a t e d  ammonium s u l f a t e s o l u t i o n s .  we h a v e f o u n d t h a t at  i f water d i s t i l l e d  i fstored  as a f r o z e n  In c o n t r a s t  f r o m an a l l - g l a s s  a l l stages o f t h ep u r i f i c a t i o n procedure, t h e f i n a l  to this  paste  report,  apparatus  i s used  preparation  is stable  for  several  m o n t h s a t - 2 0 ° . T h e HMG-CoA c o n d e n s i n g enzyme was a s s a y e d b y  its  ability  t o c a t a l y z e t h e f o r m a t i o n o f a c e t o a c e t a t e i n t h e s t a n d a r d A.S.E.S.  assay system  i n t h e p r e s e n c e o f e x c e s s HMG-CoA c l e a v a g e e n z y m e f r o m  beef  liver. The  HMG-CoA c l e a v a g e enzyme was p r e p a r e d f r o m b e e f  p o w d e r by t h e m e t h o d o f L y n e n et_ a K  liver  ( 5 ) , o m i t t i n g t h e acetone  which affords c o n s i d e r a b l e p u r i f i c a t i o n  acetone fractionation,  b u t l o w r e c o v e r y o f t h e enzyme.  The  9 enzyme was a s s a y e d in t h e standard condensing  by i t s a b i l i t y  A.S.E.S. a s s a y  enzyme f r o m  B. T h e C h i c k e n 1.  Enzyme  t o catalyze the formation of acetoacetate  system  in the presence  Enzymes  assays enzyme" a s s a y .  "Inhibitor  a t e d by m e a s u r i n g t h e e x t e n t t o w h i c h c h i c k e n f o r m a t i o n o f a c e t o a c e t a t e by b e e f  conditions of the standard liver  fraction  ate.  T h e r e a c t i o n was  estim-  1 i v e r f r a c t i o n s were a b l e t o  l i v e r A.S.E.S. p r e p a r a t i o n s .  t o c a t a l y z e t h e f o r m a t i o n o f 0.50 initiated  t h e r e a c t i o n was s t o p p e d  One " i n h i b i t o r  e n z y m e " a c t i v i t y was  The  A.S.E.S. a s s a y w e r e e m p l o y e d , w i t h s u f f i c i e n t t o 0.55 u m o l e of  by a d d i t i o n o f t h e b e e f  l o w e d w i t h i n 15 s e c o n d s by t h e c h i c k e n 38°,  HMG-CoA  yeast.  Liver  The " i n h i b i t o r  inhibit  o f excess  liver fraction.  liver  beef  acetoacet-  fraction,  fol-  A f t e r 60 m i n u t e s a t  and a c e t o a c e t a t e d e t e r m i n e d  in t h e usual  manner.  u n i t " was d e f i n e d a s t h a t amount o f enzyme w h i c h u n d e r t h e  s p e c i f i e d c o n d i t i o n s p r o d u c e s a 50 p e r c e n t  reduction  in the rate of aceto-  acetate formation. Coenzyme A p h o s p h o h y d r o l a s e . potassium  The r e a c t i o n m i x t u r e c o n s i s t e d o f  s u c c i n a t e b u f f e r , pH 6.0, kO u m o l e s ; c y s t e i n e , 10 u m o l e s ;  c o e n z y m e A, 5 0 m u m o l e s ; a n d e n z y m e , 0.5 t o 1.5 m i l l i u n i t s , o f 0.5 m l . with  10 mM  Where n e c e s s a r y , potassium  o f t h e enzyme.  After  A l l components except  t o 30° and t h e r e a c t i o n was  15 m i n u t e s t h e t u b e s  b a t h f o r 5 m i n u t e s and t h e n  in a final  chilled  initiated  were p l a c e d  the  by a d d i t i o n  in a boiling  water  i n i c e . The s o l u t i o n s were a d j u s t e d t o  approximately  pH 8 by t h e a d d i t i o n o f 0.5 ml o f 0.2 M p o t a s s i u m  and  p r o t e i n was r e m o v e d by c e n t r i f u g a t i o n  denatured  volume  t h e enzyme f r a c t i o n s w e r e a p p r o p r i a t e l y d i l u t e d  s u c c i n a t e b u f f e r , pH 6.0.  enzyme w e r e e q u i l i b r a t e d  reduced  bicarbonate,  i f necessary.  Duplicate  10 standards  ( m i n u s e n z y m e ) and  duplicate controls  were c a r r i e d through the procedure Residual  c o e n z y m e A was  s u p e r n a t a n t s o l u t i o n by (32).  This  gradation  method  t h e n determined  a modification  i s b a s e d upon t h e  i n s u i t a b l e a l i q u o t s o f the  o f t h e method d e s c r i b e d  ability  o f c o e n z y m e A,  arsenolysis  presence of phosphate t r a n s a c e t y l a s e of T r i s hydrochloride  and  of  arsenate.  b u f f e r , pH  8.1,  20  enzyme A s o l u t i o n t o be  assayed  in a f i n a l  by  but  Novel 1i  not  its  acetyl phosphate in The  reaction  volume of  0.3 0.7  the  10  u n i t ; and ml.  de-  mixture  umoles; c y s t e i n e ,  phosphate, 6 umoles; phosphate t r a n s a c e t y l a s e ,  acetyl  enzyme)  simultaneously.  products, to catalyze the  consisted  ( m i n u s c o e n z y m e A and  umoles;  the  co-  After the  assay  initiated  by  O  e q u i l i b r a t e d t o 30  m i x t u r e was the  a d d i t i o n of  equilibrated  0.3  ml  t o 30°.  of  O.I67  After  p h o s p h a t e d e t e r m i n e d by  Tuttle  (24).  Enzyme a c t i v i t y  liver  M p o t a s s i u m a r s e n a t e , pH  the was  r e a c t i o n was  8.1,  h y d r o x a m i c a c i d method o f defined  in terms of  previously  s t o p p e d and  the  phosphate phosphohydrolase.  The  residual  Lipmann  standard  a b i l i t y of  f r a c t i o n s t o h y d r o l y z e t h e p h o s p h o m o n o e s t e r bond o f  p h o s p h a t e was 6.0,  a final with  r e a c t i o n was  and  units  above). p-Nitrophenyl  pH  arsenolysis  15 m i n u t e s , t h e  acetyl  (see  , the  40  estimated  10 mM  0.5  ml.  The  potassium succinate  p h o s p h a t e , 5 u m o l e s ; and  b u f f e r , pH  a d d i t i o n of  the  enzyme f r a c t i o n .  was  s t o p p e d by  the  a d d i t i o n of  substrate  buffer,  enzyme f r a c t i o n i n  enzyme f r a c t i o n s w e r e d i l u t e d when n e c e s s a r y  by  c o n s i s t i n g of  p-nitrophenyl  in a system c o n s i s t i n g of potassium succinate  umoles; p - n i t r o p h e n y l  volume of  chicken  2.0  6.0.  The  r e a c t i o n was  A f t e r t e n m i n u t e s at 30*, ml  o f 0.05  w i t h o u t e n z y m e and  c a r r i e d through the procedure concurrently.  of  the  N sodium h y d r o x i d e .  initiated reaction Controls  enzyme w i t h o u t s u b s t r a t e The  amount o f  were  p-nitrophenyl  11 p h o s p h a t e h y d r o l y z e d was  calculated  i o n a t 405  p-nitrophenylate  mu  from the absorbance  i n a Beckman DU  these c o n d i t i o n s , the rate of the reaction  of the  resultant  spectrophotometer.  is proportional  Under  t o t h e amount o f  p r o t e i n added p r o v i d i n g t h a t not more t h a n t h r e e p e r c e n t o f t h e s u b s t r a t e is u t i l i z e d .  In c o n t r a s t t o t h e r e p o r t o f Neuhaus and  a u t h o r found neither  that p-nitrophenyl phosphate phosphohydrolase  e n h a n c e d by m a g n e s i u m  ence o f magnesium to the gradual  "Mixed  ion nor  inhibited  ion in t h e r e a c t i o n m i x t u r e  appearance of t u r b i d i t y  p r e c i p i t a t i o n of  tions  Byrne  by EDTA.  (see Reaction  (5))  was  The  a t a l k a l i n e pH,  activity  was  In f a c t , t h e p r e s -  p r o b a b l y due  to  due  the  carbonate.  "mixed t h i o l a s e " a c t i v i t y o f  determined  the  interfered w i t h the assay,  i n s o l u b l e magnesium h y d r o x i d e o f  t h i o l a s e " assay.  (33),  liver  s p e c t r o p h o t o m e t r i c a l l y by  frac-  measuring  t h e d i s a p p e a r a n c e o f t h e e n o l a t e i o n a b s o r p t i o n band o f a c e t o a c e t y l - C o A a t 310  (34)  mu  in the presence of g l u t a t h i o n e .  sisted of T r i s  h y d r o c h l o r i d e b u f f e r , pH  umole; reduced  g l u t a t h i o n e , 1.5  of  1.5  ml.  tained  Cuvette  7.5,  The  complete  100  umoles; a c e t o a c e t y l - C o A ,  u m o l e s ; enzyme; and w a t e r  a l l components except  g l u t a t h i o n e ; and  acetoacetyl-CoA.  Cuvette  i a b l e r a t e s under t h e c o n d i t i o n s o f t h e assay by a d d i t i o n o f g l u t a t h i o n e , and  and  mu  II a l t e r n a t e l y .  density  in cuvettes  reaction.  0.4  volume  the reference cuvette containI I s e r v e d as a  f o r the spontaneous d e a c y l a t i o n of acetoacetyl-CoA, which  s o r p t i o n a t 310  to a final  con-  I c o n t a i n e d a l l components o f t h e a s s a y ; c u v e t t e II c o n -  ed a l l c o m p o n e n t s e x c e p t  iated  assay system  was  r e c o r d e d a t 30  The I and  (35).  The  occurs at  T h i s a s s a y m e t h o d was  apprec-  r e a c t i o n was  the disappearance of enolate ion second  intervals,  t a k e n as a m e a s u r e o f t h e " m i x e d only approximate,  due  initab-  reading cuvettes I  d i f f e r e n c e between t h e r a t e s o f change o f II was  control  optical thiolase"  to the occurrence  of  12  competing  reactions.  T h i s has been more f u l l y  S-Acetylglutathione thioesterase.  discussed elsewhere (35).  T h i s enzymic  a c t i v i t y was m e a s u r -  ed s p e c t r o p h o t o m e t r i c a l l y by t h e m e t h o d o f Drummond a n d S t e r n ( 3 6 ) . method  i n v o l v e s t h e measurement o f t h e d i s a p p e a r a n c e o f t h e t h i o e s t e r  a b s o r p t i o n a t 2k0 mu cuvettes  i n a Beckman m o d e l DU s p e c t r o p h o t o m e t e r  (d = 0.5 c m ) .  and w a t e r t o a f i n a l  The r e f e r e n c e c u v e t t e c o n t a i n e d a l l  T h e r e a c t i o n was i n i t i a t e d  enzyme, and t h e c h a n g e i n o p t i c a l  hydro-  S - a c e t y l g l u t a t h i o n e , 0.5 u m o l e ; enzyme;  v o l u m e o f 1.5 m l .  components except s u b s t r a t e .  bond  in quartz  The c o m p l e t e assay system c o n s i s t e d o f T r i s  c h l o r i d e b u f f e r , pH 7.5,100 u m o l e s ;  ond  This  by a d d i t i o n o f  d e n s i t y a t 2h0 mu was r e c o r d e d a t 30 s e c -  intervals. Non-specific acid phosphohydrolase.  activity  Non-specific  i n c h i c k e n l i v e r f r a c t i o n s was d e t e r m i n e d  phosphohydrolase  from t h e a b i l i t y  f r a c t i o n s t o h y d r o l y z e a number o f p h o s p h o m o n o e s t e r s .  of the  The assay system  con-  s i s t e d o f p o t a s s i u m s u c c i n a t e b u f f e r o f t h e a p p r o p r i a t e p H , 100 u m o l e s ;  sub-  strate,  10 u m o l e s ;  enzyme; a n d w a t e r t o a f i n a l  a c t i o n was i n i t i a t e d  v o l u m e o f 1.0 m l .  by a d d i t i o n o f t h e enzyme f r a c t i o n .  After  The r e -  15 m i n u t e s  a t 3 0 * , t h e r e a c t i o n was s t o p p e d by t h e a d d i t i o n o f 1.0 ml o f 2 0 p e r c e n t trichloroacetic  acid.  The tubes were c h i l l e d  i n i c e f o r 10 m i n u t e s , a n d  d e n a t u r e d p r o t e i n was removed by c e n t r i f u g a t i o n .  The o r t h o p h o s p h a t e  1.0 ml a l i q u o t o f t h e s u p e r n a t a n t s o l u t i o n was t h e n d e t e r m i n e d  in a  by t h e m e t h o d  o f Dryer e t al.. ( 3 7 ) . 2.  Purification of the "inhibitor  A p r o c e d u r e fcr t h e p a r t i a l from e x t r a c t s o f c h i c k e n l i v e r t o r y by D r . G . I . Drummond.  enzyme"  purification of the "inhibitor  had been d e v i s e d p r e v i o u s l y  This procedure  involved s a l t  in this  enzyme" labora-  fractionation,  heat  13  t r e a t m e n t a t a c i d pH, removal o f i n e r t p r o t e i n w i t h precipitation with presence of zinc with had  ammonium s u l f a t e a n d f r a c t i o n a t i o n w i t h  ions.  v a r i a b l e recovery  This  with  respect  of total  w h i c h was f i n a l l y Chicken packed  in the earlier  t o recovery  phosphate g e l , ethanol  p r o c e d u r e gave a s i x - t o t e n - f o l d activity.  of activity.  The f o l l o w i n g p r o c e d u r e  as soon as p o s s i b l e a f t e r  t o the laboratory.  from b r o i l i n g o r f r y i n g chickens from  A l l subsequent  required.  months w i t h o u t Extract ion.  small with  operations  L i v e r s were ob-  of the extracts  rather  Workup o f t h e l i v e r s was u s u a l l y begun l e s s t h a n o n e h o u r a f t e r  until  several  slaughter  whenever p o s s i b l e , s i n c e t h e l i p i d  l a y i n g h e n s made p r o c e s s i n g  removal from t h e c h i c k e n s . frozen  i s t h eone  a d o p t e d f o r t h e p u r i f i c a t i o n o f t h e enzyme.  in ice for transport  difficult.  that  f r a c t i o n a t i o n procedure were u n s a t i s f a c t o r y  l i v e r s were obtained  content of l i v e r s  purification,  i t was f o u n d  were performed a t o r near 0 , except where noted o t h e r w i s e . tained  in the  A f t e r t h e n a t u r e o f t h e enzyme  been e s t a b l i s h e d and a r e l i a b l e d i r e c t a s s a y d e v i s e d ,  some o f t h e s t e p s  and  calcium  pieces  When t h i s was n o t f e a s i b l e , t h e l i v e r s  The l i v e r s c o u l d  be f r o z e n  and s t o r e d  were  a t -20* f o r  l o s s o f enzyme.  The l i v e r s were f r e e d o f c o n n e c t i v e  and d i v i d e d  i n t o 130 g p o r t i o n s .  2 0 0 ml o f 0.2 M p o t a s s i u m b i c a r b o n a t e  c y s t e i n e f o r approximately one minute  tissue, cut into  E a c h p o r t i o n was h o m o g e n i z e d  b u f f e r , pH 8 . 2 , c o n t a i n i n g  i n a Waring blendor.  5 mjl  The c o a r s e  h o m o g e n a t e was t h e n h o m o g e n i z e d f o r f i v e m i n u t e s m o r e i n a S e r v a l l Omnimixer with further ing  t h e s t a i n l e s s s t e e l chamber  immersed  100 ml o f t h e b i c a r b o n a t e - c y s t e i n e  was c o n t i n u e d  f o rf i v e minutes.  layers of cheesecloth  and c e n t r i f u g e d  i n a bath o f ice water.  A  b u f f e r was a d d e d a n d h o m o g e n i z -  T h e h o m o g e n a t e was s t r a i n e d t h r o u g h t w o f o r 6 0 m i n u t e s a t 13,000 x £ i n a  14  Servall  angle centrifuge.  solution  - "Initial  remove l i p i d . could  e x t r a c t " - was s t r a i n e d  cold  salt  stirred mechanically. saturation with  tained The  The o p a l e s c e n t  ammonium s u l f a t e  initial  e x t r a c t was d i l u t e d  concentration  extract  o f 3 0 mg/ml a n d  was b r o u g h t t o 3 0 p e r c e n t  a t pH 7.5 t o 7.8 by o c c a s i o n a l  and c e n t r i f u g e d  The e x t r a c t  addition o f s o l i d potassium  f o r 60 m i n u t e s a f t e r a d d i t i o n  f o r 45- m i n u t e s a t 10,000 x £ .  with  o f 45 t o 5 0  ( 1 6 . 8 g f o r e a c h 100 ml o f d i l u t e d  f o r 0° a c c o r d i n g t o N o l t m a n n e t a j _ . ( 3 8 ) ) .  thoroughly extracted ate  The e n t i r e  water t o a p r o t e i n  s u s p e n s i o n was s t i r r e d  pleted  (400 g o f t i s s u e )  ammonium s u l f a t e by t h e a d d i t i o n , o v e r a p e r i o d  minutes, o f s o l i d calculated  livers  a t one time.  fractionation.  glass-distilled  The supernatant  through a pad o f c h e e s e c l o t h t o  Approximately twelve t o fourteen  be p r o c e s s e d c o n v e n i e n t l y First  with  T h e p r e c i p i t a t e was d i s c a r d e d .  a 30 p e r c e n t s a t u r a t e d  extract;  was m a i n bicarbonate.  o f s a l t was com-  T h e p r e c i p i t a t e was  s o l u t i o n o f ammonium  sulf-  ( a p p r o x i m a t e l y 0.1 v o l u m e , b a s e d o n t h e V o l u m e o f t h e d i l u t e d e x t r a c t ) .  After standing  f o r 30 m i n u t e s t h i s  at 20,000 x £.  s u s p e n s i o n was c e n t r i f u g e d  T h e p r e c i p i t a t e was d i s c a r d e d .  f o r 20 m i n u t e s  The two s u p e r n a t a n t  solutions  w e r e combined and t h e volume n o t e d . T h i s c o m b i n e d s u p e r n a t a n t s o l u t i o n was b r o u g h t t o 4 0 p e r c e n t tion with ml  ammonium s u l f a t e b y a d d i t i o n o f t h e s o l i d  of solution)  as d e s c r i b e d  fuged as b e f o r e .  above.  salt  with  and c e n t r i -  0.1 v o l u m e o f 4 0 p e r  cent saturated  ammonium s u l f a t e s o l u t i o n a n d c e n t r i f u g e d .  was  as f r a c t i o n AS-I 30-40.  designated  ( 5 . 8 g f o r e a c h 100  T h e s u s p e n s i o n was s t i r r e d  T h e p r e c i p i t a t e was e x t r a c t e d  satura-  The p r e c i p i t a t e  The two s u p e r n a t a n t s o l u t i o n s  were  combined and t h e v o l u m e n o t e d . The  c l e a r r e d s o l u t i o n was b r o u g h t t o 8 0 p e r c e n t s a t u r a t i o n  by  addi-  15 t i o n of s o l i d manner The  indicated.  The  The minimal  stirred  100 ml  of s o l u t i o n )  and c e n t r i f u g e d  T h e p r e c i p i t a t e was  in the  as b e f o r e . designated  as  40-80) were d i s s o l v e d  in  40-80.  volumes o f  d i a l y z e d vs 6  10 mM  (AS-1  Tris  liters  stage  3 0 - 4 0 and A S - I  h y d r o c h l o r i d e b u f f e r , pH  7.5.  Each  o f t h e same b u f f e r f o r 12 t o 16 h o u r s .  t h e n removed by  At t h i s divided  g f o r each  discarded.  two p r e c i p i t a t e s  m a t e r i a l was  (26.3  s u s p e n s i o n was  s u p e r n a t a n t s o l u t i o n was  f r a c t i o n AS-I  was  ammonium s u l f a t e  fraction Insoluble  centrifugation.  i n t h e p u r i f i c a t i o n p r o c e d u r e , t h e p r e p a r a t i o n was  i n t o two p o r t i o n s .  s t e p became u n m a n a g e a b l e .  I f t h i s was One  not done, t h e volumes  p o r t i o n was  in the  next  p r o c e s s e d as d e s c r i b e d below;  the  e  o t h e r was  stored  Gel distilled 5.5  (0°,  acid.  water  (20 mg  The  The  briefly  in the  AS-I  and  sufficient  glass-  brought  to  pH  acetic  c a l c i u m phos-  a l l o w e d t o s t a n d f o r 15  a gel/  minutes  washed w i t h c o l d  added t o t h e s u p e r n a t a n t  solu-  The  was  supernatant s o l u t i o n  a g a i n added  (60 mg  g e l f o r each  The  s u s p e n s i o n was  40-80 f r a c t i o n ) .  and t h e p r e c i p i t a t e w a s h e d as b e f o r e .  carded.  The  the careful  and  in a t h i n stream t o produce  trifuged  two  p e r ml  T h e p r e c i p i t a t e was  then d i s c a r d e d .  initial  10 mg  a t 0°  and t h e w a s h s o l u t i o n  g e l was  diluted with cold  dropwise addition of 2 N  s u s p e n s i o n was  c a l c i u m phosphate  of protein  4 0 - 8 0 was  added  a t 5,000 x £ .  p r e c i p i t a t e was  and  stirred  s o l i d s p e r m l ) was  r a t i o o f 0.35.  stirred  f r a c t i o n AS-I  to a p r o t e i n concentration of  g l a s s - d i s t i l l e d water  mg  The  needed.  c l o u d y s u s p e n s i o n was  centrifuged  tion.  until  g l a s s e l e c t r o d e ) by t h e c a r e f u l  phate gel  and  treatment.  The  protein  a t -20  T h e p r e c i p i t a t e was  supernatant s o l u t i o n s were combined, brought  a d d i t i o n o f 5 N. p o t a s s i u m h y d r o x i d e and  t o pH  b u f f e r e d by t h e  100 cen-  then 7.5  disby  addition  16 o f 0.03 v o l u m e o f M T r f s h y d r o c h l o r i d e  b u f f e r , pH 7.5.  t h e n b r o u g h t t o 90 p e r c e n t s a t u r a t i o n w i t h the  solid  utes.  salt  (61.5  and  T h e s u s p e n s i o n was s t i r r e d  for  o f 60 m i n -  f o r 60 t o 9 0 m i n u t e s a n d c e n t r i f u g e d  T h e p r e c i p i t a t e was d i s s o l v e d  10 mM p o t a s s i u m s u c c i n a t e dialyzed  ammonium s u l f a t e by a d d i t i o n o f  g f o r e a c h 100 ml o f s o l u t i o n ) o v e r a p e r i o d  60 m i n u t e s a t 10,000 x £ . of  T h i s s o l u t i o n was  b u f f e r , pH 6.0, c o n t a i n i n g  16 h o u r s vs_ 6 l i t e r s  i n a minimal  for volume  1 mM p o t a s s i u m EDTA,  o f t h e same b u f f e r .  The d i a l y z e d  solu-  t i o n was d e s i g n a t e d a s f r a c t i o n A S - 1 I . Ethanol succinate  fractions.  S u f f i c i e n t M m a g n e s i u m c h l o r i d e and M p o t a s s i u m  b u f f e r , pH 6.0, w e r e a d d e d t o t h e AS-11 f r a c t i o n t o p r o d u c e  concentrations mechanically  o f 0.10 a n d 0.05 M, r e s p e c t i v e l y . i na water-ethanol-dry  T h i s s o l u t i o n was s t i r r e d  i c e b a t h a t -6*,  and s u f f i c i e n t  o f 15 p e r c e n t  chilled  ethanol  (-20°) a d d e d t o g i v e a f i n a l  concentration  (v/v).  T h e s o l u t i o n was t r a n s f e r r e d  t o a -12° b a t h , a n d m o r e c h i l l e d was 2k p e r c e n t  added u n t i l  t h e ethanol  was  a t -12° f o r 15 m i n u t e s , a n d c e n t r i f u g e d  stirred  concentration  final  (v/v).  ethanol ethanol  The suspension  15 m i n u t e s a t 13,000  for  x £ and -10°. In a s i m i l a r m a n n e r , f r a c t i o n s w e r e c o l l e c t e d a t 3 5 , k3, o  per  cent ethanol  (v/v)  o  a t -15 , "15  p r e c i p i t a t e was d i s s o l v e d  for  o f t h e same b u f f e r .  four  changes.  fresh buffer  , "20  o  a n d -20 , r e s p e c t i v e l y .  Each  a t 0° i n a m i n i m a l v o l u m e o f 10 mM p o t a s s i u m  c i n a t e b u f f e r , pH 6.0, c o n t a i n i n g liters  o  5 5 a n d 6k  1 mM p o t a s s i u m EDTA, a n d d i a l y z e d  T h e b u f f e r was r e p l a c e d  by f r e s h b u f f e r  A t t h i s t i m e , t h e b u f f e r was r e p l a c e d  a n d d i a l y s i s was c o n t i n u e d f o r  16 h o u r s .  was  removed by c e n t r i f u g a t i o n .  the  f r a c t i o n p r e c i p i t a t i n g b e t w e e n 35 a n d k$ p e r c e n t  suc-  vs_ 3 hourly,  by 7 l i t e r s o f Insoluble  material  T h e b u l k o f t h e enzyme was u s u a l l y f o u n d i n ethanol.  17 Chromatography. w i t h 5 mM  DEAE-eellulose  was  T r i s h y d r o c h l o r i d e b u f f e r , pH  7.2,  prepared  f o r use,  c o n t a i n i n g 1 mM  equilibrated EDTA and  packed  i n t o j a c k e t e d g l a s s c o l u m n s u n d e r h a n d - b u l b p r e s s u r e as d e s c r i b e d by son  and  Sober  (39).  columns were e q u i l i b r a t e d t o 0  The  f r a c t i o n c o l l e c t o r f o r s e v e r a l hours b e f o r e use.  The  prepared  vs_ 6  f o r c h r o m a t o g r a p h y by o v e r n i g h t d i a l y s i s  b u f f e r used t o p r e p a r e  t h e column.  The  e l u t e d w i t h t h e above b u f f e r .  pooled  and  t h e s o l u t i o n was  s o l u t i o n was 10  mM  then  potassium  initial  s u c c i n a t e b u f f e r , pH fraction  extract.  when s t o r e d a t -20 no e f f e c t  .  The  and 6.0,  represents  final  by  was  The  concentrated of  over  thawing  appears t o have l i t t l e procedure.  fractions by d i f f e r e n t i a l c e n t r i f u g a t i o n The and  procedure  given below i s  Hogeboom ( 4 0 ) f o r t h e  (nuclei, mitochondria,  iso-  microsomes  t h e m e t h o d o f Sawant e t a l _ . ( 4 1 ) f o r t h e  isola-  lysosomes. The  l i v e r was  o f s l a u g h t e r and to the  run  EDTA.  stage of the p u r i f i c a t i o n  l a t i o n of the usual s u b c e l l u l a r f r a c t i o n s  t i o n of  column  t o 300-fold p u r i f i c a t i o n  in sucrose s o l u t i o n s .  t h e s o l u b l e p h a s e ) and  The  to  i s s t a b l e f o r a t l e a s t t h r e e months  a c o m p r o m i s e between t h e method o f S c h n e i d e r  and  lyophilization.  c o n t a i n i n g 1 mM  S u b c e l l u l a r f r a c t i o n s were i s o l a t e d o f homogenates p r e p a r e d  allowed  d i a l y z e d o v e r n i g h t vs 3 l i t e r s  R e p e a t e d f r e e z i n g and  Isolation of s u b c e l l u l a r  was  l i t e r s o f t h e same  1 ml p e r m i n u t e .  a 200-  fraction  u p o n t h e enzyme a t any  3.  6.0  fraction  f r a c t i o n s c o n t a i n i n g t h e enzyme w e r e  concentrated  a d j u s t e d t o pH  This f i n a l the  The  refrigerated  ethanol  enzyme f r a c t i o n was  i n t o t h e column at a r a t e o f a p p r o x i m a t e l y then  in a  Peter-  obtained  immediately  laboratory.  from a b r o i l i n g chicken w i t h i n f i v e minutes  immersed  i n c o l d 0.25  M sucrose  for transport  A l l subsequent o p e r a t i o n s were c a r r i e d out  at or  near  or  18  0°.  Workup o f t h e l i v e r was b e g u n w i t h i n 20 m i n u t e s Homogenate.  The l i v e r  ( a p p r o x i m a t e l y 20 g ) was f r e e d o f c o n n e c t i v e  t i s s u e , c u t i n t o s m a l l p i e c e s and h o m o g e n i z e d of  0.25  of death.  M s u c r o s e c o n t a i n i n g 2 mM  Tris  i n p o r t i o n s w i t h f o u r volumes  h y d r o c h l o r i d e b u f f e r , pH 7.5,  P o t t e r - E l v j e h m homogenizer f i t t e d w i t h a T e f l o n p e s t l e . d i l u t e d w i t h 0.25 s t r a i n e d through  cells, ing  M s u c r o s e t o g i v e a 10 p e r c e n t  T h e h o m o g e n a t e was  (w/v) h o m o g e n a t e and  two l a y e r s o f c h e e s e c l o t h .  "Nuclear" fraction.  A crude "nuclear" f r a c t i o n  unbroken  and c e l l u l a r d e b r i s ) was o b t a i n e d by  liver cells  f o r 10 m i n u t e s  aside.  a t 750 x £ .  The s u p e r n a t a n t  (including  solution  T h e p r e c i p i t a t e was w a s h e d t w i c e by s u s p e n d i n g  s u c r o s e each  p r e c i p i t a t e was s u s p e n d e d  Mitochondrial  fraction.  The o p a l e s c e n t s u p e r n a t a n t  i n 50 ml o f 0 . 2 5  i n 0.25  solution  i n 5 0 ml 0 . 2 5  The twice-washed  The s u p e r n a t a n t s o l u t i o n  suspended  -  w 2  S] and t h e t w o a t 5,000 x £ . layer"  t i m e and r e c e n t r i f u g i n g a s b e f o r e . i n 0.25  M sucrose - Fraction  The s u p e r n a t a n t s o l u t i o n S  2  - was s e t a s i d e .  M  a t 16,300  The p r e c i p i t a t e  i n 80 ml o f 0.7 M s u c r o s e and c e n t r i f u g e d f o r 30 m i n u t e s  was  a t 6,000  o x £ and 0 . centrifuged  T h e p e l l e t was d i s c a r d e d . f o r 30 m i n u t e s  tion discarded.  T h e r e m a i n i n g s u s p e n s i o n was  a t 14,000 x £ a n d 0 ° , and t h e s u p e r n a t a n t  T h e p e l l e t was w a s h e d t w i c e by r e s u s p e n d i n g  w 2  and t h e t w o  m i t o c h o n d r i a l w a s h e s w e r e c o m b i n e d and c e n t r i f u g e d f o r 2 0 m i n u t e s x £.  N  T h e p r e c i p i t a t e was w a s h e d t w i c e by  M s u c r o s e each  fraction."  as b e f o r e .  (including the bulk of the " f l u f f y  p r e c i p i t a t e was s u s p e n d e d  "Lysosome-rich  M  M sucrose - Fraction  The s u p e r n a t a n t s o l u t i o n  r e m o v e d and s e t a s i d e - S 2 .  resuspending  centrifug-  - S j - was s e t  n u c l e a r w a s h e s w e r e c o m b i n e d and c e n t r i f u g e d f o r 10 m i n u t e s  ( 4 2 ) ) was  blood  t i m e w i t h t h e a i d o f t h e h o m o g e n i z e r and c e n t r i f u g i n g  The twice-washed  in a  then solu-  i n 4 0 ml o f  19  0.7  M s u c r o s e each  i n 0.7  s e d i m e n t i n g as b e f o r e .  M sucrose - Fraction Microsomal  for  t i m e and  i n 0.25  fraction.  The  supernatant  M sucrose - Fraction  Soluble fraction. t i o n of t h e microsomal Fraction  The  The  The  was c e n t r i f u g e d  t i g h t l y - p a c k e d p e l l e t was  supernatant s o l u t i o n  fraction  resus-  remaining a f t e r  sedimenta-  represents the soluble portion of the c e l l  -  is given  in t h e " f l o w s h e e t " form  in  12. o f t h e s u b c e l l u l a r f r a c t i o n s w e r e f r o z e n and  To p r e p a r e t h e f r a c t i o n s  were mixed w i t h s u f f i c i e n t 6.0,  solution  S/^  All night.  suspended  P.  A summary o f t h e f r a c t i o n a t i o n Figure  p e l l e t was  LRF.  90 m i n u t e s a t 66,000 x £ and 0 .  pended  The  t o produce f i n a l  Triton  s t o r e d a t -20*  f o r enzyme a s s a y s , t h e t h a w e d X-100  p e r c e n t and  0.02  d e t e r g e n t - t r e a t e d f r a c t i o n s w e r e a l l o w e d t o s t a n d a t 0*  before the v a r i o u s enzymatic  suspensions  and p o t a s s i u m s u c c i n a t e b u f f e r ,  c o n c e n t r a t i o n s o f 0.2  a c t i v i t i e s were  determined.  over-  M, f o r 30  pH  respectively. minutes  Received without pape(s)  F i l m e d as  0  received.  University Microfilms, Inc.  21  RESULTS  I.  Existence of the  Inhibitor  The formation of acetoacetate in v i t r o by extracts of l i v e r from most species  is proportional to the amount of t i s s u e protein added.  From  Figure 1 it can be seen that the rate of acetoacetate formation by pigeon l i v e r enzymes exhibits d i r e c t dependence upon protein concentration under the conditions of the assay.  The same is true in the case of beef l i v e r .  This is not true, however, in the case of chicken l i v e r .  As Figure 1 shows,  the rate of acetoacetate formation by an extract of chicken l i v e r is not proportional to protein concentration when r e l a t i v e l y large amounts of protein are added.  This indicates the presence of an inhibitory factor in  extracts of chicken l i v e r .  When a crude extract of chicken l i v e r is sub-  jected to s a l t f r a c t i o n a t i o n , this becomes even more apparent.  Figure 2  shows that the enzyme(s) responsible for acetoacetate formation can be separated from the inhibitory f a c t o r .  The f r a c t i o n p r e c i p i t a t i n g between the  limits of 3 0 and kO per cent saturation with ammonium s u l f a t e contains the A.S.E.S., r e l a t i v e l y f r e e of the inhibitory f a c t o r .  The f r a c t i o n p r e c i p i t a t -  ing between the limits of kO and 70 per cent saturation with ammonium s u l f a t e also exhibits some A'.S.E.S. a c t i v i t y , but is r e l a t i v e l y r i c h in the i n h i b i t ory f a c t o r . Preliminary studies of the inhibitor in t h i s Or. G.I. that t h i s  laboratory, begun by  Drummond and continued by the author, were based on the observation inhibitor was able to prevent the formation of acetoacetate by  beef l i v e r enzyme f r a c t i o n s .  An assay system was devised on the basis of  this observation, for the simple reason that this was the only property of  22  20  4-0  PROTEIN (mg)  Figure 1. Formation of acetoacetate by extracts of various t i s s u e s . The standard conditions of the A.S.E.S. assay were employed. -• *r beef l i v e r extract; *• *, pigeon l i v e r extract; * x, chicken l i v e r extract. The extracts were prepared in bicarbonate-cysteine buffer as described for chicken l i v e r in the Experimental Section.  23  2-0 PROTEIN  40 (mg)  F i g u r e 2. F o r m a t i o n o f a c e t o a c e t a t e by T h e s t a n d a r d c o n d i t i o n s o f t h e A.S.E.S. • • , i n i t i a l extract; o • t i o n ammonium s u l f a t e f r a c t i o n ; * * u r a t i o n ammonium s u l f a t e f r a c t i o n .  chicken liver fractions. assay were employed. , 30 t o 4 0 p e r c e n t s a t u r a , hO t o 80 p e r c e n t s a t -  2k  t h e enzyme w h i c h was known. perimental  Methods.  acetate formation protein  added o v e r  (In t h i s  a r e given under Ex-  As F i g u r e 3 s h o w s , t h e e x t e n t o f i n h i b i t i o n o f a c e t o -  i s d i r e c t l y p r o p o r t i o n a l t o t h e amount o f c h i c k e n a f a i r l y wide range,  A.S.E.S. a c t i v i t y  under t h e s p e c i f i e d c o n d i t i o n s .  residual  A.S.E.S. a c t i v i t y .  experimental  Locus o f t h e I n h i b i t o r  The p r e s e n c e o f  results.)  Effect  Various s t u d i e s were c a r r i e d out t o determine i n h i b i t o r produced  its effects.  Stern et a K  t h a t t h e i n h i b i t o r was n e i t h e r a n a c e t y l acetyl-CoA  treat-  i n t h e i n h i b i t o r f r a c t i o n s w o u l d h a v e made i t v i r t u a l l y  impossible t o interpret  could  liver  e a r l y w o r k , t h e A S - I 4 0 - 8 0 f r a c t i o n was g i v e n a b r i e f h e a t  ment a t a c i d pH t o d e s t r o y  11.  The d e t a i l s o f t h e assay  hydrolase.  p r e c i s e l y how t h e  (8) had p r e v i o u s l y e s t a b l i s h e d  p h o s p h a t e p h o s p h o h y d r o l a s e n o r an  One p o s s i b l e e x p l a n a t i o n w a s t h a t t h e i n h i b i t o r  effect  be d u e t o t h e combined a c t i o n o f a " m i x e d t h i o l a s e " enzyme, s i m i l a r t o  t h a t d e s c r i b e d b y Drummond a n d S t e r n glutathione hydrolase  (36) ( R e a c t i o n 6 ) . T h i s would r e s u l t  Acetoacetyl-CoA  Acetyl-G  +  of acetoacetyl-CoA A.S.E.S. a s s a y  (35.43) ( R e a c t i o n 5 ) , and an S - a c e t y l -  +  GSH  acetyl-CoA  H 0  v  2  a s i t was f o r m e d .  acetate  system used f o r t h e i n h i b i t o r assay  spectrophotometric  assay,  b o t h o f t h e s e enzymes w e r e p r e s e n t  +  acetyl-G  (5)  GSH  (6)  In v i e w o f t h e f a c t t h a t t h e s t a n d a r d  h i g h c o n c e n t r a t i o n o f g l u t a t h i o n e ( 1 0 mM), t h i s By d i r e c t  +  i n t h e removal  contained  a  relatively  idea appeared very  t h e a u t h o r was a b l e t o d e t e r m i n e in a l l chicken  attractive. that  l i v e r f r a c t i o n s which ex-  25  F i g u r e 3. I n h i b i t i o n by c h i c k e n l i v e r f r a c t i o n s o f a c e t o a c e t a t e f o r m a t i o n by b e e f l i v e r e n z y m e . The s t a n d a r d c o n d i t i o n s o f t h e " i n h i b i t o r enzyme" a s s a y w e r e employed. ° o , heated c h i c k e n l i v e r e x t r a c t ; * * , second ammonium s u l f a t e f r a c t i o n ; • • , 2k t o kS p e r c e n t ethanol f r a c t i o n . The h e a t e d e x t r a c t was p r e p a r e d by h e a t i n g an i n i t i a l e x t r a c t o f c h i c k e n l i v e r f o r f o u r m i n u t e s a t pH 5.5 and 50 ; t h e o t h e r f r a c t i o n s w e r e p r e p a r e d f r o m t h i s h e a t e d e x t r a c t as d e s c r i b e d u n d e r E x p e r i m e n t a l M e t h o d s .  26  hibited  Inhibitor  activity.  t i o n was n o t v a l i d . A.S.E.S. f r a c t i o n , (8),  which  H o w e v e r , f u r t h e r s t u d y showed t h a t t h i s  I t was f o u n d t h a t t h e most h i g h l y p u r i f i e d t h e 2 0 t o 35 p e r c e n t e t h a n o l f r a c t i o n  exhibited  no s i g n w h a t s o e v e r  beef  explanaliver  of Stern et a l .  of inhibitor a c t i v i t y , contained  e v e n m o r e o f t h e s e t w o enzymes t h a n d i d t h e most p o t e n t  inhibitor  fraction.  Thus t h i s p o s s i b i l i t y c o u l d be e l i m i n a t e d . A t t e n t i o n was t h e n t u r n e d t o a n y p o s s i b l e e f f e c t on t h e e n z y m a t i c c o m p o n e n t s o f t h e A . S . E . S . a s s a y s y s t e m . enzyme was p r e i n c u b a t e d w i t h p h o s p h a t e enzyme s y s t e m .  t r a n s a c e t y l a s e and t h e beef  i n t h e u s u a l manner.  i n h i b i t i o n was v i r t u a l l y t h e same a s w o u l d been no p r e i n c u b a t i o n p e r i o d that the inhibitor  have been t h e c a s e  i f t h e r e had  of the inhibitor  enzyme upon r e -  c o n s i s t i n g o f a l i m i t i n g amount o f y e a s t  enzyme p l u s e x c e s s  HMG-  l i v e r HMG-CoA c l e a v a g e enzyme a n d o f a  amount o f l i v e r HMG-CoA c l e a v a g e enzyme p l u s e x c e s s y e a s t HMG-CoA  condensing  enzyme.  As T a b l e  l y t h e same i n e a c h did  In b o t h c a s e s , t h e d e g r e e o f  a c t e d d i r e c t l y on t h e s e enzymes.  c o n s t i t u t e d A.S.E.S. s y s t e m s ,  limiting  liver  (Table I ) . This v i r t u a l l y eliminated t h e poss-  In o t h e r e x p e r i m e n t s , t h e e f f e c t  CoA c o n d e n s i n g  The i n h i b i t o r  The o t h e r components o f t h e a s s a y s y s t e m were t h e n added and  t h e a s s a y was c o m p l e t e d  ibility  of the inhibitor  not interfere  case.  I I s h o w s , t h e d e g r e e o f i n h i b i t i o n was v i r t u a l -  This  indicated that t h e inhibitor  w i t h t h e f u n c t i o n o f e i t h e r o f t h e s e enzymes.  In v i e w o f t h e s e o b s e r v a t i o n s , i t became a p p a r e n t enzyme must a f f e c t  enzyme p r o b a b l y  that the inhibitor  one o f t h e nonenzymic components o f t h e a s s a y .  To d e t e r -  m i n e i f t h i s was t r u e , t h e r e a c t i o n m i x t u r e was i n c u b a t e d w i t h t h e i n h i b i t o r fraction marized  b e f o r e a d d i t i o n o f t h e beef i n Table  l i v e r enzyme.  T h i s experiment  I I I . When no c h i c k e n l i v e r f r a c t i o n  i s sum-  was a d d e d ( l a a n d 2 a ) ,  TABLE I Effect  o f p r e i n c u b a t i n g t h e enzymic a s s a y components w i t h i n h i b i t o r enzyme.  Experiment No.  I n h i b i t o r enzyme added mg  Acetoacetate formed  protein  1  -  2  0.70  3  0.70  umole  the  Inhibition  per  cent  0.36  v  0.25  31  0.24  33  The A . S . E . S . p r e p a r a t i o n u s e d was a 20 t o 35 p e r c e n t e t h a n o l f r a c t i o n o f b e e f l i v e r , 0.61 mg p r o t e i n . The inhibito r enzyme p r e p a r a t i o n was a c h i c k e n l i v e r g e l s u p e r n a t e f r a c t i o n , 0.70 mg p r o t e i n . The p r e i n c u b a t i o n p e r i o d was kS m i n utes in a l l cases. In e x p e r i m e n t 1, t h e b e e f l i v e r f r a c t i o n and t h e C. k l u y v e r ? e x t r a c t w e r e p r e i n c u b a t e d t o g e t h e r and t h e n added t o t h e o t h e r a s s a y components. In e x p e r i m e n t 2, t h e i n h i b i t o r enzyme f r a c t i o n was a l s o a d d e d a t t h e end o f t h e p r e incubation period. In e x p e r i m e n t 3, t h e i n h i b i t o r enzyme f r a c t i o n was a d d e d t o t h e o t h e r two enzyme f r a c t i o n s a t t h e s t a r t of the preincubation period. The g l u t a t h i o n e r e q u i r e d f o r t h e a s s a y was p r e s e n t d u r i n g t h e p r e i n c u b a t i o n p e r i o d , t o p r e v e n t i n a c t i v a t i o n o f t h e A.S.E.S. F o l l o w i n g t h e end o f t h e p r e i n c u b a t i o n p e r i o d , t h e u s u a l p r o c e d u r e was f o l l o w e d .  28  TABLE I I E f f e c t o f i n h i b i t o r enzyme o n f o r m a t i o n o f a c e t o a c e t a t e b y r e c o n s t i t u t e d A.S.E.S. p r e p a r a t i o n s .  Experiment No. ' 1  1  Inhibitor enzyme mg p r o t e i n  la lb  Beef l i v e r " "  fraction "  2a 2b  Reconstituted »  3a 3b  Reconstituted system " "  system "  I  II  Acetoacetate ' synthesized umole  Inhibition 1  1  p e r cent  0.14  0.60 0.38  0.14  0.44  0.29  34  0.14  0.61 0.37  39  36  T h e b e e f l i v e r f r a c t i o n u s e d was a 2 0 t o 35 p e r c e n t e t h a n o l f r a c t i o n ( 8 ) , 0.98 mg p r o t e i n . R e c o n s t i t u e d s y s t e m I c o n s i s t e d o f HMG-CoA c o n d e n s i n g enzyme f r o m y e a s t , 0.41 mg p r o t e i n , p l u s HMG-CoA c l e a v a g e enzyme f r o m b e e f l i v e r a c e t o n e p o w d e r , 1.1 mg p r o t e i n ( a f o u r - f o l d e x c e s s ) . Reconstitu t e d s y s t e m I I c o n s i s t e d o f t h e l i v e r HMG-CoA c l e a v a g e e n z y m e , 0.28 mg p r o t e i n , p l u s y e a s t HMG-CoA c o n d e n s i n g e n z y m e , 1.65 mg p r o t e i n ( a f o u r fold excess). T h e s t a n d a r d c o n d i t i o n s o f t h e i n h i b i t o r enzyme a s s a y w e r e employed.  TABLE  III  Effect of preincubation of the nonenzyme components of the assay system with the " i n h i b i t o r enzyme" before addition of the beef l i v e r enzyme.  Experiment No.  '  Preincubation period  '  Acetoacetate formed  minutes  umoles  la lb lc  30 30 30  0.56 0.31 0.13  2a 2b 2c  60 60 60  0.56 0.31 0.00  A l l tubes contained a l l the nonenzymic components of the standard A.S.E.S. assay system. Acetoacetate-synthesizing system (beef l i v e r 20 to 35 per cent ethanol f r a c t i o n , 0 . 9 8 mg protein) was routinely added to a l l tubes at the end of the preincubation period. In experiments ^a and 2 a , no " i n h i b i t o r enzyme" was added. In experiments J_b and 2 b , " i n h i b i t o r enzyme" (chicken l i v e r ethanol f r a c t i o n , 0.13 mg protein) was added immediately following the beef l i v e r fraction. In experiments Jl_c and 2c_, the same chicken l i v e r f r a c t i o n was added at the start of the preincubation p e r i o d . The preincubation period was 30 or 60 minutes, as noted, at 38 . Following addition of the beef l i v e r f r a c t i o n , the usual procedure was followed.  30  0.56  u m o l e o f a c e t o a c e t a t e was  a d d e d a t t h e end of t h e beef  liver  when t h e c h i c k e n 2c),  enzyme ( l b and  liver  2 b ) , 0.3'  f r a c t i o n was  i n h i b i t i o n was  60 m i n u t e p r e i n c u b a t i o n . a c t i n g upon o n e From an  liver  fraction  u m o l e o f a c e t o a c e t a t e was  present  I t was  now  formed,  However,  during the pre-incubation period  much m o r e m a r k e d - 77 p e r c e n t 100  per cent  q u i t e apparent  inhibition  inhibition that the  fol-  following  of the composition  appeared t h a t t h e  i n h i b i t o r m u s t be p r o d u c i n g  u p o n c o e n z y m e A.  T h i s was  confirmed  by two  a  inhibitor  o r more o f t h e nonenzymic components o f t h e assay  examination  was  after the a d d i t i o n  inhibition of acetoacetate formation.  l o w i n g a 30 m i n u t e p r e i n c u b a t i o n , and  was  When t h e c h i c k e n  of the p r e i n c u b a t i o n p e r i o d , immediately  r e p r e s e n t i n g 45 p e r c e n t  ( l c and  formed.  enzyme system.  of the reaction mixture, i t i t s e f f e c t s through  simple experiments.  some a c t i o n Figure  4  shows t h e e f f e c t o f c o e n z y m e A c o n c e n t r a t i o n upon a c e t o a c e t a t e f o r m a t i o n a beef l i v e r The  fraction  in the presence  g r a p h shows t h a t a t  and  absence of chicken  l o w c o n c e n t r a t i o n s o f c o e n z y m e A,  liver  enzyme.  the chicken On  h a n d , when s u f f i c i e n t  rates of  acetate formation, the chicken periment,  liver  added t o p r o d u c e maximal e n z y m e had  the a b i l i t y  of the  inhibitor  enzyme t o  demonstrated d i r e c t l y  (Table  IV).  data  does  indeed  d e s t r o y coenzyme  I t had capable of formation by  The  other aceto-  In a n o t h e r  ex-  i n a c t i v a t e coenzyme A  was  inhibitor  enzyme  A.  t h e r e f o r e been e s t a b l i s h e d t h a t c h i c k e n  i n a c t i v a t i n g c o e n z y m e A,  and  that the  i n v i t r o p r o d u c e d by t h e c h i c k e n  i t c o u l d be f i n a l l y c o n c l u d e d  identical  effect.  indicate that the  liver  i n c r e a s i n g t h e c o n c e n t r a t i o n o f coenzyme A  fore  no  the  liver  enzyme c a u s e s a m a r k e d d e p r e s s i o n o f a c e t o a c e t a t e f o r m a t i o n . c o e n z y m e A was  by  that the  liver  extracts are  inhibition of  acetoacetate  e x t r a c t s c o u l d be  prevented  in the reaction mixture.  i n h i b i t o r e n z y m e was  w i t h t h e enzyme d e s t r o y i n g c o e n z y m e A,  i t was  necessary  Be-  indeed t o show a  31  •  0-2  *  COENZYME  •  0-4 A  •  1  0-6  (mM)  F i g u r e k. E f f e c t o f coenzyme A c o n c e n t r a t i o n on a c e t o a c e t a t e f o r m a t i o n by b e e f l i v e r enzyme i n t h e p r e s e n c e a n d a b s e n c e o f c h i c k e n l i v e r " i n h i b i t o r e n z y m e . " T h e s t a n d a r d c o n d i t i o n s o f t h e A.S.E.S. a s s a y w e r e e m p l o y e d , e x c e p t t h a t t h e c o e n z y m e A c o n c e n t r a t i o n was v a r i e d a s n o t e d . • • , b e e f l i v e r ammonium s u l f a t e f r a c t i o n ( 8 ) ( 1 . 1 0 mg p r o t e i n ) ; * * , a s a b o v e , w i t h t h e a d d i t i o n o f c h i c k e n l i v e r 2k t o kS p e r c e n t e t h a n o l f r a c t i o n ( 0 , 1 8 mg p r o t e i n ) .  TABLE IV I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n " i n h i b i t o r enzyme."  Experiment No.  Enzyme  mq  protein _  , Coenzyme A added  (  liver  Coenzyme A recovered  mumoles  mumoles  la lb  0.19  10 10  10 10  2a 2b  0.19  20 20  20 0  -  (  The r e a c t i o n m i x t u r e c o n t a i n e d T r i s h y d r o c h l o r i d e buffer, pH 8 . 1 , 100 u m o l e s ; M g C l , 1 u m o l e ; K C l , k u m o l e s ; c y s t e i n e , k u m o l e s ; and c o e n z y m e A and " i n h i b i t o r e n z y m e " ( c h i c k e n l i v e r e t h a n o l f r a c t i o n ) , as n o t e d ; f i n a l v o l u m e , 0.3 m l . The r e a c t i o n was s t a r t e d by a d d i t i o n o f enzyme. A f t e r kS m i n u t e s a t 38 , t h e r e a c t i o n was s t o p p e d by a d d i t i o n o f 5 N h y d r o c h l o r i c t o b r i n g t h e s o l u t i o n t o pH k f o l l o w e d by h e a t i n g a t 100 f o r two m i n u t e s . C o e n z y m e A i n t h e s o l u t i o n was t h e n d e termined enzymatically (32). 2  t  33 correlation T a b l e V.  b e t w e e n t h e two  Over a t e n - f o l d  enzyme A  inactivation:  a h i g h o f 5.2. this,  activities.  Such a c o r r e l a t i o n  range o f s p e c i f i c  inhibitor  enzyme a c t i v i t y  A l t h o u g h one might  in view of the v a r i a b i l i t y  One  observed  f r o m a l o w o f 3.3  in the i n h i b i t o r inactivation  assay, the  In v i e w o f t h e f a c t t h a t t h e phosphate  i t also  enzyme  inactivate ester ified  co-  i n h i b i t o r enzyme a s s a y s y s t e m  t r a n s a c e t y l a s e t o e s t e r i f y more t h a n  100  inhibitor coenzyme. was tion  low.  It would  enzyme w o u l d This  a s s a y , and  method.  It can  enzyme. depressed  in Table VI.  inhibitor  residual be s e e n  a t a p p r o x i m a t e l y 55  that t o produce  a c e t y l - C o A was that the  m e a s u r e d by t h e u s u a l  i n h i b i t o r enzyme d o e s  t h i s d e p r e s s i o n o f enzyme a c t i v i t y to the presence inhibits the  in the acetyl  inactiva-  acetyl-CoA,  i t inactivates the free phosphate  It i s not c l e a r  i s due t o t h e a c e t y l ; phosphate  phosphate  of orthophosphate, which  i n a c t i v a t i o n o f c o e n z y m e A by t h e c h i c k e n l i v e r  the  arsenolysis  inactivate  that the addition of acetyl  t h e r a t e o f d i s a p p e a r a n c e o f c o e n z y m e A.  would  acetyl-CoA  i n t h e coenzyme A  per cent of the r a t e at which  I t s h o u l d a l s o be n o t e d  time  esterified  Enzymatically-synthesized  enzyme f r a c t i o n  formed,  its effect,  o f n e c e s s i t y be e x p e c t e d t o a c t upon t h e  i s shown  i n c u b a t e d w i t h an  t h e r e f o r e appear  con-  umoles  e x p e c t t h a t t h e amount o f f r e e c o e n z y m e A p r e s e n t a t any  be e x t r e m e l y  and,  correla-  o f coenzyme A p e r h o u r , w h i l e l e s s t h a n one umole o f a c e t o a c e t a t e i s one would  to than  enzyme a s s a y  o t h e r p o i n t a l s o comes t o m i n d : d o e s t h e c h i c k e n l i v e r  tains sufficient  co-  satisfactory.  a c t o n l y upon f r e e c o e n z y m e A, o r d o e s enzyme A?  ranged  the r a t i o of  p r e f e r t o have a c l o s e r c o r r e l a t i o n  t o a much l e s s e r e x t e n t , i n t h e c o e n z y m e A t i o n c a n be c o n s i d e r e d  activities,  is presented in  alone  whether  itself markedly  enzyme.  co-  or  34  TABLE V Correlation  b e t w e e n i n h i b i t o r enzyme and c o e n z y m e A inactivation activities.  Inhibitor enzyme  Heated  (0  units/mg protein  mU/mg protein  0.9  4.1  4.5  supernate  2.1  7.2  3.4  fraction  2.1  7.0  3.3  0.5  2.5  5.0  extract  Second g e l Second s a l t  E t h a n o l , 0 t o 31 p e r E t h a n o l , 31  Ratio  Coenzyme A inact ivat ion (2)  Fract ion  cent  (2)/(D  t o 37 p e r  cent  5.8  30  5.2  E t h a n o l , 37 t o 45 p e r  cent  8.6  33  3.8  E t h a n o l , 45  cent  0  t o 56 p e r  0  T h e h e a t e d e x t r a c t was p r e p a r e d by h e a t i n g t h e f i r s t s a l t f r a c t i o n f o r 4 m i n u t e s a t pH 5.5 and 50 . T h e o t h e r f r a c t i o n s w e r e p r e p a r e d f r o m t h i s h e a t e d e x t r a c t as d e s c r i b e d u n d e r E x p e r i m e n t a l M e t h o d s .  TABLE VI Evidence f o r i n a c t i v a t i o n o f ester i f i e d i n h i b i t o r enzyme  Additions  coenzyme A by t h e  Rate o f i n a c t i v a t i o n mumo1es/mg/m ? n  None Acetyl  84 phosphate  Acetyl phosphate plus phosphate t r a n s a c e t y l a s e  42  23  S t a n d a r d a s s a y c o n d i t i o n s w e r e e m p l o y e d , w i t h 50 mum o l e s o f c o e n z y m e A, a n d t h e a d d i t i o n o f a c e t y l p h o s p h a t e , 0.6 u m o l e , a n d p h o s p h a t e t r a n s a c e t y l a s e , 0.3 u n i t , w h e r e n o t e d . T h e p h o s p h o h y d r o l a s e u s e d was a c h i c k e n liver ethanol f r a c t i o n .  36 III.  Precise Action o f the Inhibitor A survey o f t h epertinent  bonds by  l i t e r a t u r e showed t h a t  i n t h e coenzyme A m o l e c u l e t h a t  enzymes f r o m v a r i o u s Several  (44),  sources.  Enzyme there  are several  a r e known t o b e s u s c e p t i b l e t o a t t a c k  This  i s summarized  5.  i n Figure  enzymes, i n c l u d i n g p r o s t a t i c a c i d p h o s p h o h y d r o l a s e  t h e a l k a l i n e phosphohydrolase  (EC 3 . 1 . 3 . 1 ) o f E s c h e r i c h i a c o l i  the 3 ' - n u c l e o t i d e phosphohydrolase o f plant known t o h y d r o l y z e  t h e 3'-phosphomonoester  f a c t , been employed f o r t h e p r e p a r a t i o n  tissues  (45) a n d  l i n k a g e o f coenzyme A ( p o i n t a ) , T h e s e enzymes h a v e , i n  o f dephosphocoenzyme A  (47,48).  (EC 3 . 6 . 1 . 9 ) f r o m s n a k e venom (47,49), p l a n t  Pyrophosphatases  (19)  3.1.3.2)  (46) a r e  t i s s u e (EC 3.1.3.6)  f o r m i n g 3'-dephosphocoenzyme A and orthophosphate.  (50) a n d a n i m a l  (EC  a r e known t o h y d r o l y z e  sources  t h e p y r o p h o s p h a t e bond o f  c o e n z y m e A ( p o i n t c_), f o r m i n g 4 - p h o s p h o p a n t e t h e i n e a n d 3' , 5 ' - d i p h o s p h o a d e n o sine  (19,47,51). Purified  intestinal  g r a d e c o e n z y m e A.  This  phosphatase  enzyme h y d r o l y z e s  a l s o t h e p h o s p h o m o n o e s t e r bonds dj,  (52)  i s a l s o known t o e x t e n s i v e l y d e -  t h e p y r o p h o s p h a t e bond  i n t h e r e s u l t i n g fragments  ( p o i n t c ) and  ( p o i n t s a, b a n d  f o r m i n g a d e n o s i n e , p a n t e t h e i n e and t h r e e moles o f o r t h o p h o s p h a t e In a d d i t i o n , N o v e l 1 i  p i g e o n and c h i c k e n the pantothenic  and co-workers  (19) d e s c r i b e d  l i v e r e x t r a c t s which hydrolyzed  a c i d and p-mercaptoethylamine  (53,54).  a peptidase i n  t h e amide l i n k a g e  (cysteamine) moieties  between of co-  enzyme A ( p o i n t e ) , f o r m i n g p - m e r c a p t o e t h y l a m i n e a n d p ' - 3 ' - p h o s p h o a d e n o s i n e -  2 5',  P -pantothenic  acid-4'  pyrophosphate.  In v i e w o f t h e s e v e r a l p o s s i b l e p o i n t s i t was i m p e r a t i v e by  a t w h i c h t h e enzyme c o u l d  t o c h a r a c t e r i z e t h eproduct o f t h e r e a c t i o n .  chromatography on paper and on E C T E O L A - c e l l u l o s e .  act,  T h i s was d o n e  In o n e e x p e r i m e n t , t r i -  NH  37 2  -N  (b)  (c) (d)  CH  I  HCCH -0-P-9-P-0-CH 00. C-(CH ) 2  '\? ?A Vc  7H  H  i  i  HO 0 (a) i _ . I 0—P—0  2  3  2  H-C-OH l  /\  0  NH-CH  0  /  2  NH-CH -CH -SH 2  2  ^ P a n t o t h e n i c i — p - m e r c a p t o e t h y l amine-*] acid H ( c y s t earn i n e ) i Pantetheine• 3 , 1  5*-diphosphoadenosine  4-phosphopantetheine-  F i g u r e 5. P o s s i b l e s i t e s o f a c t i o n o f t h e i n h i b i t o r enzyme o n c o e n z y m e A. For e x p l a n a t i o n s e e t e x t .  38 e t h y l ammonium a c e t a t e b u f f e r , pH 6 . 0 , chicken  liver  f r a c t i o n , 2 mg p r o t e i n ,  ethanol  w e r e i n c u b a t e d a t 30°. a d d i t i o n o f 0.5  4 0 0 u m o l e s ; c o e n z y m e A, 4 u m o l e s ; and  After  in a total  ml  15 m i n u t e s , t h e r e a c t i o n was s t o p p e d by t h e  ml o f 10 p e r c e n t p e r c h l o r i c a c i d .  removed by c e n t r i f u g a t i o n .  v o l u m e o f 1.5  Denatured p r o t e i n  T h e s u p e r n a t a n t s o l u t i o n was  by a d d i t i o n o f 5 N, p o t a s s i u m h y d r o x i d e .  was 7.2  a d j u s t e d t o pH  A f t e r 30 m i n u t e s a t 0°,  t h e suspen-  s i o n was c e n t r i f u g e d t o r e m o v e t h e i n s o l u b l e p o t a s s i u m p e r c h l o r a t e and t h e s u p e r n a t a n t s o l u t i o n was  lyophilized.  T h e d r y r e s i d u e was d i s s o l v e d  s m a l l v o l u m e o f w a t e r and a g a i n l y o p h i l i z e d . o u g h l y e x t r a c t e d w i t h 0.5 centrifugation. the perchloric  A control acid  ;  ml o f w a t e r and  t h e e x p e r i m e n t a l and c o n t r o l  ed o f e t h a n o l , 2.  150;  Solvent system  i d e , 8:  w a t e r , 78;  i n two s o l v e n t s y s t e m s .  1 M ammonium a c e t a t e , pH 7.5,  II c o n s i s t e d o f i s o b u t y r i c a c i d , and 2 - m e r c a p t o e t h a n o l , 2.  In s o l v e n t s y s t e m  ethanol,  The chromatograms were d e v e l o p e d The  results  6.  I , t h e r e was g o o d s e p a r a t i o n o f t h e s t a r t i n g  ( r e d u c e d c o e n z y m e A)  (R  p  0.08)  and t h e p r o d u c t o f t h e r e a c t i o n  (Rp  T h e p r o d u c t moved a t t h e same r a t e a s a u t h e n t i c d e p h o s p h o c o e n z y m e A.  However, t h i s  s o l v e n t s y s t e m d o e s n o t s e p a r a t e r e d u c e d c o e n z y m e A and  5'-diphosphoadenosine,  another p o s s i b l e product  (55).  gave o n l y a s l i g h t s e p a r a t i o n o f reduced coenzyme A (R_  I consist-  N_ ammonium h y d r o x -  h o u r s a t room t e m p e r a t u r e .  in Figure  filter  and 2-mercapto 114; 15  after  Aliquots of  Solvent system 60;  thor-  removed by  e x t r a c t s w e r e a p p l i e d t o Whatman No. 1  are reproduced d i a g r a m a t i c a l l y  0.31).  i n s o l u b l e m a t t e r was  i n e x a c t l y t h e same m a n n e r .  by t h e d e s c e n d i n g t e c h n i q u e f o r 13  material  T h e r e s u l t i n g p o w d e r was  t u b e i n w h i c h t h e enzyme f r a c t i o n was a d d e d  was t r e a t e d  p a p e r and c h r o m a t o g r a p h e d  in a  3',  S o l v e n t s y s t e m II (Rp 0.55)  0 . 6 3 ) , w h i c h a g a i n moved a t t h e same r a t e as a u t h e n t i c  and t h e p r o d u c t  3'-dephospho-  39  1 2 0  0  0  3  4  0  °  1 2  3  4  Origin  0  Solvent front  F i g u r e 6. C h a r a c t e r i z a t i o n o f t h e r e a c t i o n p r o d u c t b y p a p e r c h r o m a t o g r a p h y . S a m p l e 1, e x p e r i m e n t a l ; s a m p l e 2, c o n t r o l ; s a m p l e 3, r e d u c e d c o enzyme A; s a m p l e k, r e d u c e d d e p h o s p h o c o e n z y m e A. Chromatogram A r e p r e s e n t s t h e r e s u l t s o b t a i n e d w i t h s o l v e n t S y s t e m I ; c h r o m a t o g r a m B, t h o s e o b t a i n e d w i t h s o l v e n t System I I . F o r d e t a i l s s e e text.  40  However, 3 ' , 5 ' ~ d i p h o s p h o a d e n o s i n e ,  c o e n z y m e A. at  i f p r e s e n t , would  a p p r o x i m a t e l y 70 p e r c e n t o f t h e r a t e a t w h i c h  ultraviolet tected. lowed  absorbing material  h a v e moved  c o e n z y m e A moved.  corresponding t o diphosphoadenosine  U n f o r t u n a t e l y , n e i t h e r o f t h e s e two s o l v e n t systems  would  No was d e have a l -  t h e u n e q u i v o c a l d e m o n s t r a t i o n o f t h e p r e s e n c e o r a b s e n c e o f 5'-AMP,  s i n c e c o e n z y m e A a n d 5'"AMP move t o g e t h e r i n m o s t s o l v e n t s y s t e m s .  (The  p o s s i b l e p r e s e n c e o f s m a l l amounts o f 5'-AMP i n b o t h t h e c o e n z y m e A a n d d e phosphocoenzyme A might in s o l v e n t system  account  f o r the f a i n t slowly-moving spots  observed  I f o r t h e e x p e r i m e n t a l a l i q u o t and a u t h e n t i c dephospho-  c o e n z y m e A.) The  i d e n t i t y o f t h e r e a c t i o n p r o d u c t was c o n f i r m e d by  chromatography.  Triethylammonium  ion-exchange  a c e t a t e b u f f e r , pH 6 . 0 , 200 u m o l e s ; c o -  enzyme A, 10 u m o l e s ; a n d c h i c k e n l i v e r  e t h a n o l f r a c t i o n , 2 mg p r o t e i n ,  in a  O  total was  v o l u m e o f 2.8 ml w e r e i n c u b a t e d a t 30 .  b y a d d i t i o n o f 0.2 ml o f 70 p e r c e n t p e r c h l o r i c  stopped  p r o t e i n was removed by c e n t r i f u g a t i o n . ed t o pH 7.2 30 m i n u t e s  A f t e r 30 m i n u t e s , acid.  Denatured  T h e s u p e r n a t a n t s o l u t i o n was a d j u s t -  by a d d i t i o n o f s a t u r a t e d p o t a s s i u m b i c a r b o n a t e s o l u t i o n .  a t 0 * , t h e s u s p e n s i o n was c e n t r i f u g e d t o r e m o v e t h e  potassium perchlorate.  a d d i t i o n o f 2 H h y d r o c h l o r i c a c i d , mixed  ethanol  and l e f t  a t room t e m p e r a t u r e  reported t o effect  After  insoluble  T h e s u p e r n a t a n t s o l u t i o n was a d j u s t e d t o pH 6.0 by  careful  a complete  w i t h 3 ml o f 2 - m e r c a p t o -  f o r k hours.  T h i s t r e a t m e n t has been  r e d u c t i o n o f any d i s u l f i d e s p r e s e n t  s o l u t i o n was t h e n d i l u t e d t o 100 ml w i t h d i s t i l l e d 2.0 cm by 15 cm c o l u m n o f E C T E O L A - c e l l u l o s e umn  the reaction  was w a s h e d w i t h 150 ml o f d i s t i l l e d  c h l o r i d e a n d 150 ml o f d i s t i l l e d  water.  water  (55).  The  and a p p l i e d t o a  i n the c h l o r i d e form.  The c o l -  w a t e r , 250 ml o f 0.02 M ammonium E l u t i o n was t h e n s t a r t e d w i t h a  41  linear chloride gradient.  mixing f l a s k  initially  c o n t a i n e d one  liter  of  0.003 N. h y d r o c h l o r i c a c i d ; t h e r e s e r v o i r  initially  c o n t a i n e d one  liter  of  0.15  M  lithium chloride  each were c o l l e c t e d is presented this  carried  The  The  dashed-1ine  out  and  1.46  x  determined  hydryl  i n 0.01  The  N hydrochloric acid  determined  standard.  The  The 1.82:0.92. expect  results  are presented  f o r d e p h o s p h o c o e n z y m e A.  a r e somewhat l o w , h o w e v e r . small  amount o f 5'-AMP.  mediately  b e f o r e reduced  experiment  in Table  (55).)  The  These r a t i o s a r e almost reduced  (Peak  II).  es-  (57).  was  Total  et_ a]_. ( 3 7 ) .  Sulfwas  g l u t a t h i o n e as  a  VM.  quite closely  The  enzyme  Ribose content  reaction  ( 5 8 ) , u s i n g reduced  I were  to those which  values f o r total  1.00:1.04: one  p h o s p h a t e and  T h i s c o u l d p o s s i b l y be due  would thiol  to the presence  ( 5 A M P w o u l d h a v e been e l u t e d f r o m  of  a  t h e column  im-  3'-dephosphocoenzyme A under t h e c o n d i t i o n s o f  the  , -  corresponding  r a t i o s f o r Peak  r e d u c t i o n , w e r e 1 . 0 0 : 1 . 0 2 : 2 . 0 3 : 0 . 0 0 and  for  (56).  r a t i o s a d e n i n e : r i b o s e : p h o s p h a t e : t h i o l f o r Peak These values correspond  control  coefficient  r e d u c t i o n w i t h sodium borohydride,  m e a s u r e d by t h e n i t r o p r u s s i d e r e a c t i o n  of  A d e n i n e c o n t e n t was  by t h e m e t h o d o f D r y e r  after  ml  f r a c t i o n s c o m p r i s i n g each peak were  by t h e D i s c h e m o d i f i c a t i o n o f t h e o r c i n o l  c o n t e n t , b e f o r e and  10  diagram  that the  a b s o r p t i o n , assuming a molar e x t i n c t i o n  mu  p h o s p h a t e c o n t e n t was  elution  t h e same m a n n e r , e x c e p t  c o n c e n t r a t e d t o a s m a l l volume i n vacuo.  10** a t 260  An  p r o f i l e represents the r e s u l t s of a  in p r e c i s e l y  t i m a t e d by u l t r a v i o l e t  F r a c t i o n s of  s o l i d - l i n e p r o f i l e represents the results  added a f t e r t h e p e r c h l o r i c a c i d .  pooled  of  i n 0.003 N h y d r o c h l o r i c a c i d .  a t a f l o w r a t e o f 60 ml p e r h o u r .  i n F i g u r e 7.  experiment.  experiment was  The  identical  dephosphocoenzyme A Further evidence  1.00:1.02:2.03:1.05,  to those which  (Peak  i s found  I I , b e f o r e and  I) and  one  after  respectively.  would expect  to obtain  o x i d i z e d dephosphocoenzyme A  in the s a l t  concentrations required to  50  _  40  o CD  £1 301  (CoASH)  20  10  i i .i i i i  100 FRACTION F i g u r e 7. tion  Characterization  see text.  150  NUMBER  o f t h e r e a c t i o n p r o d u c t by i o n - e x c h a n g e c h r o m a t o g r a p h y .  For explana-  43  TABLE V I I Composition of the product of the r e a c t i o n .  Total Adenine * Ribose ' Phosphate  1  Sulfhydryl  umoles/ml  Peak A  1.00  1.04  1.83  0.92  0.95 -  0.97  1.93  0.0 1.05  Peak B (1) (2)  Before reduction After reduction  For d e t a i l s , see Experimental  Methods.  44  e l u t e t h e s e t w o p e a k s f r o m t h e c o l u m n - a p p r o x i m a t e l y 0.02 M f o r P e a k 0.04 M f o r P e a k p e a k s a t 2 6 0 mu,  i t (55).  From t h e c o m b i n e d o p t i c a l  density of the original  r e s u l t s o f these chromatographic  that t h e inhibitor apparent virtually  It  is  product.  studies provide strong evidence  enzyme i s a "coenzyme A 3 - p h o s p h o h y d r o l a s e . " 1  that the inhibitor  f o r 95 p e r  coenzyme A s o l u t i o n .  t h u s o b v i o u s t h a t t h e r e c o u l d be no o t h e r major The  d e n s i t i e s o f t h e two  i t was c a l c u l a t e d t h a t t h e t w o p e a k s a c c o u n t e d  cent o f t h e o p t i c a l  I and  enzyme p r e p a r a t i o n u s e d  It i s a l s o  f o rthese experiments i s  f r e e o f enzymes c a p a b l e o f d e g r a d i n g c o e n z y m e A i n a n y o t h e r man-  ner.  IV.  T h e Coenzyme A Assay.  direct  3'-Phosphohydrolase  When t h e s i t e o f t h e i n h i b i t o r  assay based  e f f e c t was e s t a b l i s h e d , a  upon t h e d i s a p p e a r a n c e o f c o e n z y m e A was d e v i s e d t o r e -  p l a c e t h e r a t h e r cumbersome and u n r e l i a b l e " i n h i b i t o r " a s s a y .  In t h i s  dir-  e c t a s s a y , t h e enzyme was i n c u b a t e d w i t h c o e n z y m e A u n d e r s p e c i f i e d c o n d i t i o n s , t h e enzyme was i n a c t i v a t e d  by h e a t  and r e s i d u a l  ured e n z y m a t i c a l l y . The d e t a i l e d p r o c e d u r e Methods.  The a s s a y method  i s f a r from  c o e n z y m e A was meas-  i s g i v e n under E x p e r i m e n t a l  ideal, since several  tions are involved.  However,  and  (One a l t e r n a t i v e a s s a y m e t h o d w o u l d  i s inexpensive.  ment o f o r t h o p h o s p h a t e  i t is satisfactory  r e l e a s e d f r o m c o e n z y m e A.  in that  i tisfairly  vated  t o both time  rapid  T h i s , however, would r e become p r o h i b i -  U n d e r t h e c o n d i t i o n s o f t h e a s s a y , t h e amount o f c o e n z y m e A i sproportional  addi-  i n v o l v e t h e measure-  q u i r e h i g h e r s u b s t r a t e c o n c e n t r a t i o n s , and t h e expense would tive.)  critical  inacti-  ( F i g . 8 ) and p r o t e i n c o n c e n t r a t i o n ( F i g .  9), p r o v i d i n g t h a t n o t m o r e t h a n 4 0 p e r c e n t o f t h e s u b s t r a t e i s u t i l i z e d . Purification  o f t h e enzyme.  A summary o f t h e c o u r s e o f t h e p u r i f i c a -  h5  10  20  30  40  TIME (minutes)  F i g u r e 8. I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n l i v e r enzyme a s a function of time. The s t a n d a r d c o n d i t i o n s o f t h e coenzyme A p h o s p h o h y d r o l a s e a s s a y w e r e e m p l o y e d . T h e e n z y m e f r a c t i o n u s e d was an i n i t i a l e x t r a c t o f c h i c k e n l i v e r (0.7 mg p r o t e i n ) .  46  0-2 PROTEIN  0-4  0-6  (mg)  F i g u r e 9. I n a c t i v a t i o n o f coenzyme A by c h i c k e n l i v e r enzyme as a f u n c t i o n o f p r o t e i n c o n c e n t r a t i o n . The standard c o n d i t i o n s o f t h e coenzyme A p h o s p h o h y d r o l a s e assay w e r e employed. • • , 40 t o 80 p e r c e n t s a t u r a t i o n ammonium s u l f a t e f r a c t i o n ; * x , second ammonium s u l f a t e f r a c t i o n ; o — — o , 35 t o 43 p e r c e n t e t h a n o l fraction. T h e enzyme f r a c t i o n s w e r e p r e p a r e d a s d e s c r i b e d u n d e r Experimental Methods.  47  t i o n procedure and  is given  regularly  in Table V I I I .  g i v e s a 250-  The  procedure  to 300-fold p u r i f i c a t i o n .  which problems o c c a s i o n a l l y arose were the ethanol c h r o m a t o g r a p h y on  DEAE-cellulose.  is quite reproducible, The  o n l y two  fractionation  steps  and  In b o t h o f t h e s e s t e p s , c o n t r o l  in  the  o f pH  is  cr it i c a l . I f t h e pH  i s a p p r e c i a b l y a b o v e 6.0  t h e enzyme, enzyme a c t i v i t y purification  is achieved.  i s found The  during ethanol p r e c i p i t a t i o n  i n almost  presence  a l l f r a c t i o n s , and  very  of d i v a l e n t c a t i o n i s a l s o  of little  important.  In t h e a b s e n c e o f a d d e d c a t i o n , h i g h e r c o n c e n t r a t i o n s o f e t h a n o l  are required  t o p r e c i p i t a t e t h e enzyme.  becomes a  p r o b l e m , and tion  Under t h e s e c i r c u m s t a n c e s ,  recoveries of a c t i v i t y  studies, zinc  found  r e p l a c e d by In t h e f i n a l  is extremely  In some o f t h e e a r l i e r step.  T h i s was  a problem.  purifica-  quite satisfactory  H o w e v e r , when t h e p r o c e d u r e  t h a t removal of t h e z i n c p r e s e n t e d  z i n c was  can  low.  i o n s w e r e added f o r t h i s  for small-scale fractionations. was  are  stability  was  s c a l e d up i t  For t h i s  reason,  the  magnesium. s t e p , c h r o m a t o g r a p h y on  important.  A typical  DEAE-cellulose, control  chromatogram  i s shown  of  pH  i n F i g u r e 10.  It  be s e e n t h a t u n d e r t h e c o n d i t i o n s e m p l o y e d t h e b u l k o f t h e p r o t e i n i n  the ethanol effluent ficiently specific  fraction  i s not  r e t a i n e d i n t h e c o l u m n and  as a " b r e a k t h r o u g h "  peak.  The  phosphohydrolase,  r e t a r d e d by t h e e x c h a n g e r t o y i e l d activity.  ed o n l y s l i g h t l y  I f t h e pH  on p a s s i n g t h r o u g h  o v e r l a p o f t h e p r o t e i n and chromatography  i s lowered  t o 6.0  t o 6.5,  t h e c o l u m n , and  a t pH  7.3  t o 7.5,  t i o n o f t h e enzyme f r o m t h e b u l k o f t h e p r o t e i n .  there  column  increase in  t h e enzyme i s r e t a r d -  there On  in the  however, i s s u f -  a 15- t o 2 0 - f o l d  enzyme a c t i v i t y p e a k s .  i s c a r r i e d out  appears  is considerable  t h e o t h e r hand, i f is a very wide  separa-  However, under t h e s e  con-  FRACTION  NUMBER  F i g u r e 10. Chromatography o f t h e coenzyme A p h o s p h o h y d r o l a s e o n D E A E - c e l l u l o s e . protein concentration; , enzyme a c t i v i t y . For d e t a i l s , s e eExperimental  Methods.  49  TABLE V I I I Purification  o f t h e coenzyme A  phosphohydrolase.  Recovery Fract ion  Protein  Specific activity  mg.  mU/mq  Total activity  Overall  mU  per  From preceding step cent  6.5  171,500  12,500  9  111,500  6,600  16.5  110,000  Second g e l s u p ' t ,  3,300  30  99,000  58  90  AS-11  2,850  30  83,000  49  84  765  82  62,500  37  75  Initial AS-I First  extract  40-80 gel sup't.  EtOH p p t .  27,000  DEAE-cellulose eluate  From 200 g c h i c k e n  a  1500  b  a  850°  (100)  (100)  65  65 100  60  22  d  liver.  Estimated s p e c i f i c a c t i v i t y . The p r o t e i n c o n c e n t r a t i o n o f t h i s t i o n was t o o l o w t o a l l o w r e l i a b l e d e t e r m i n a t i o n . In t h i s p a r t i c u l a r e x p e r i m e n t , 17.5 mg o f e t h a n o l f r a c t i o n (1450 m i l l i u n i t s ) w e r e a p p l i e d t o a 1.0 x 25 cm c o l u m n .  frac-  protein  A c t u a l l y , e s s e n t i a l l y 100 p e r c e n t o f t h e enzyme was a c c o u n t e d f o r i n t h e e l u a t e . H o w e v e r , t h e l e a d i n g e d g e o f t h e enzyme p e a k o v e r l a p p e d t h e t r a i l i n g edge o f t h e " b r e a k t h r o u g h " p r o t e i n p e a k , s o t h a t t h e f i r s t f e w f r a c t i o n s c o n t a i n e d enzyme o f r e l a t i v e l y l o w s p e c i f i c a c t i v i t y . T h i s a c c o u n t e d f o r a p p r o x i m a t e l y 20 p e r c e n t o f t h e a c t i v i t y a p p l i e d t o t h e c o l u m n , a n d was d i s c a r d e d . A f u r t h e r 20 p e r c e n t o f t h e a c t i v i t y was f o u n d i n a v e r y l a r g e v o l u m e i n t h e " t a i l " o f t h e enzyme p e a k ; t h i s t o o was d i s c a r d e d .  50 d i t i o n s t h e enzyme i s e l u t e d f r o m t h e column l a r g e volume.  ( I n o n e s u c h e x p e r i m e n t , 2,800 m i l l i u n i t s  were chromatographed 7.3. was  The  on a 2.0  cm  by 25  cm c o l u m n  f r o m t h e column  from t h e column  in fractions  in fractions  15  120 t o 2 6 0 ,  in a very  (kO mg  t o 20. in a total  The  e n z y m e was  Two  peaks  However, t h i s  findings with  gradient peak  i s unnecessary.  instance.  acid phosphohydrolases  t h e enzyme a c t i v i t y  ml.)  protein  o f coenzyme A p h o s p h o h y d r o l a s e c a n be s e e n  T h i s has o c c u r r e d i n e v e r y  Effect of divalent cations.  in Figure  Other workers have r e p o r t e d (59).  The  i n t o two d i s t i n c t p e a k s  reason f o r the s p l i t t i n g  Although the a l k a l i n e  phosphohydrolases  f o r maximal  by m e t a l - b i n d i n g a g e n t s , t h e  tion with  inhibited  respect t o the acid phosphohydrolases  m o n o g r a p h s by S c h m i d t - f o r example, and  t o be  rat liver  ( 6 0 ) and  Stadtman  (61).)  is less clear. Some a c i d  situa-  phosphohydrolases (33)  ( 6 2 ) , and t h e ^ - g l y c e r o p h o s p h a t e p h o s p h o h y d r o l a s e o f y e a s t a d e f i n i t e magnesium r e q u i r e m e n t .  On  h a n d , some - n o t a b l y t h e a c i d p h o s p h o h y d r o l a s e s o f p r o s t a t e and - do n o t a p p e a r t o r e q u i r e m a g n e s i u m , n o r a r e t h e y  presence of metal-binding agents. t h e e f f e c t s o f magnesium phohydrolase  cation  (See t h e  the 0-phosphoserine phosphohydrolase of chicken l i v e r  (63) - appear t o e x h i b i t  cytes  of  i s unknown.  upon t h e p r e s e n c e o f d i v a l e n t  and  10.  similar  a r e g e n e r a l l y c o n s i d e r e d t o be d e p e n d e n t activity  mu,  eluted  v o l u m e o f 700  l o w e r i n g t h e pH o f t h e e l u a n t b u f f e r a f t e r t h e i n i t i a l  has been o b t a i n e d .  pH  d e n s i t y a t 280  l a t t e r p r o b l e m c o u l d p r o b a b l y be o v e r c o m e by u s e o f a s a l t  o r by  protein)  of DEAE-cellulose at  b u l k o f t h e p r o t e i n , as d e t e r m i n e d by o p t i c a l  eluted  This  v e r y s l o w l y and  activity.  I t was  therefore of  the other erythro-  inhibited  interest  in the  to determine  i o n s and o f c h e l a t i n g a g e n t s u p o n c o e n z y m e A  phos-  51  The  a d d i t i o n o f magnesium i o n s t o t h e r e a c t i o n m i x t u r e d i d n o t  c r e a s e t h e r a t e o f i n a c t i v a t i o n o f coenzyme A by t h e c h i c k e n In f a c t ,  i t was f o u n d t h a t  EDTA d i d n o t s i g n i f i c a n t l y these observations,  alter  o f enzyme a c t i v i t y .  the  bound m e t a l  EDTA.  sufficient  To t e s t t h i s p o s s i b i l i t y ,  N o l t m a n n et, a]_. ( 3 8 ) .  dialysis  reaction  rates  vessel  18 l i t e r s o f t h e same b u f f e r .  flushed with  ions  The  with  and s t o r e d  a t -20°.  fraction  had a p p a r e n t l y  dialysis  v s EDTA h a d r e s t o r e d  through  thoroughly  10 mM T r i s b u f f e r w i t h o u t  experiment a r e summarized dialysis  EDTA.  i n T a b l e IX.  had been p r e p a r e d  three  I t c a n be seen f r o m t h e t a b l e t h a t  l o s t 20 p e r c e n t o f i t s a c t i v i t y on s t o r a g e , t h e enzyme a c t i v i t y  view o f these f i n d i n g s , i t i s quite u n l i k e l y that cation.  t o flow  o f 4 8 h o u r s t o remove;. EDTA f r o m t h e  enzyme u s e d f o r t h e e x h a u s t i v e  weeks p r e v i o u s l y  8 0 0 ml o f  The r e s e r v o i r c o n -  T h e a p p a r a t u s was t h e n  D i a l y s i s was c a r r i e d o u t o v e r a p e r i o d The r e s u l t s o f t h i s  by  ( T h i s t r e a t m e n t has been r e p o r t e d t o  (64).)  w a t e r , a n d s e t up a g a i n  enzyme s o l u t i o n .  was a b e a k e r c o n t a i n i n g  T h e b u f f e r was a l l o w e d  o f 96 h o u r s .  remove enzyme-bound m e t a l  f r a c t i o n (33 mg  vs_ a n E D T A - c o n t a i n i n g s o l u t i o n .  b u f f e r , pH 7.0, a n d 1 mM EDTA.  system over a period  valent  that  a p p a r a t u s u s e d was b a s e d upon t h a t d e s c r i b e d  The d i a l y s i s  10 mM T r i s h y d r o c h l o r i d e  the  possible  t o produce maximal  an a l i q u o t o f t h e e t h a n o l  t o exhaustive  continuous flow d i a l y s i s  tained  dependent  a d d e d c a t i o n , a n d t h a t t h e b o u n d m e t a l was n o t r e a d i l y a c c e s s i b l e t o  p r o t e i n ) was s u b j e c t e d The  H o w e v e r , i t was s t i l l  1 mM  On t h e b a s i s  i t w o u l d a p p e a r t h a t t h e enzyme i s n e i t h e r ions.  without  Furthermore,  t h e a c t i v i t y o f t h e enzyme.  upon n o r a c t i v a t e d by m a g n e s i u m enzyme c o n t a i n e d  enzyme.  t h e a d d i t i o n o f 5 mM m a g n e s i u m c h l o r i d e t o t h e  assay produced 40 p e r cent depression  of  liver  in-  to its initial this  this  and t h a t  level.  In  enzyme r e q u i r e s d i -  TABLE IX Effect  '  o f e x h a u s t i v e d i a l y s i s ys_ EDTA s o l u t i o n upon o f t h e coenzyme A 3'-phosphohydrolase.  S t a t u s o f enzyme  {  Total  enzymatic a c t i v i t y mi 1 1 i u n i t s  Freshly  prepared  470  F r o z e n 21 d a y s  375  EDTA-dialyzed  460  For e x p l a n a t i o n see t e x t .  activity  '  53 E f f e c t o f pH. the chicken liver show a f a i r l y  T h e e f f e c t o f pH upon  enzyme i s shown  i n a c t i v a t i o n o f coenzyme A by  i n F i g u r e 11.  Most a c i d  phosphohydrolases  b r o a d o p t i m u m b e t w e e n pH 4 a n d pH 6, t h e p r e c i s e o p t i m u m d e -  p e n d i n g upon t h e p a r t i c u l a r c o m b i n a t i o n o f enzyme, s u b s t r a t e a n d b u f f e r employed. optimal  To t h e b e s t o f t h e a u t h o r ' s k n o w l e d g e , no p h o s p h o h y d r o l a s e activity  b e l o w pH 4 h a s been p r e v i o u s l y  reported  i n animal  with tissues,  although t h enon-specific phosphohydrolase o f yeast i s reported t o e x h i b i t optimal later  activity  a t pH 4 . 0 (60).  i n more d e t a i l .  o p t i m u m a t pH 3 . 6 , technical  This unique feature w i l l  be d i s c u s s e d  I t s h o u l d be n o t e d t h a t a l t h o u g h t h i s  enzyme h a s  its  t h e a s s a y h a v e been r o u t i n e l y c a r r i e d o u t a t pH 6.0 f o r  reasons.  Intracellular  localization.  A c i d p h o s p h o h y d r o l a s e s have been r e -  p o r t e d t o be a s s o c i a t e d w i t h s e v e r a l p r o s t a t i c acid phosphohydrolase  subcellular fractions.  i s largely  F o r example,  associated with mitochondria  (65),  p h o s p h a t i d i c a c i d and g l u c o s e 6-phosphate p h o s p h o h y d r o l a s e s w i t h m i c r o -  somes  (66,67) a n d t h e m a j o r p o r t i o n o f t h e n o n - s p e c i f i c a c i d  of rat l i v e r with are t i g h t l y the  i t a p p e a r s t h a t most components  (68).  phosphohydrolases In v i e w o f t h i s ,  l o c a l i z a t i o n o f t h e c o e n z y m e A p h o s p h o h y d r o l a s e was exam-  The v a r i o u s s u b c e l l u l a r f r a c t i o n s were  e n a t e by d i f f e r e n t i a l given  In f a c t ,  bound t o p a r t i c u l a t e c e l l u l a r  intracellular  ined.  lysosomes.  phosphohydrolase  centrifugation.  i n " f l o w sheet" form  particulate structures become a p p a r e n t u n t i l  isolated  f r o m a s u c r o s e homog-  A summary o f t h e p r o c e d u r e u s e d i s  i n F i g u r e 1 2 . S i n c e many enzymes  exhibit "latency" - that  i s ,their  these structures are disrupted  f r a c t i o n s were t r e a t e d w i t h T r i t o n oxyalkylene type) before assay  X-100  (69).  associated  with  a c t i v i t y does n o t  (41,69,70,71)  -t h e  (a n o n i o n i c detergent o f t h e p o l y -  T h e r e s u l t s a r e p r e s e n t e d i n T a b l e X.  54  •  i 3  i  •  •  4  . 5  •  ! • . 6  i 7  i 8  pH  F i g u r e 11. I n a c t i v a t i o n o f c o e n z y m e A by c h i c k e n l i v e r e n z y m e a s a f u n c t i o n o f pH. T h e enzyme f r a c t i o n e m p l o y e d was a l y o p h i l i z e d e l u a t e f r o m a D E A E - c e l l u l o s e c o l u m n . The s t a n d a r d c o n d i t i o n s o f t h e coenzyme A p h o s p h o h y d r o l a s e a s s a y w e r e e m p l o y e d , e x c e p t t h a t t h e pH was v a r i e d . as n o t e d , x *, g l y c y l g l y c i n e h y d r o c h l o r i d e b u f f e r ; • •, potassium succinate buffer; o o, T r i s h y d r o c h l o r i d e b u f f e r . C o m p a n i o n c o n t r o l s w i t h o u t enzyme w e r e c a r r i e d t h r o u g h t h e e n t i r e a s s a y p r o c e d u r e a t e a c h pH t o c o r r e c t f o r n o n e n z y m i c i n a c t i v a t i o n o f c o e n z y m e A.  10 p e r c e n t h o m o g e n a t e 750 x £ , 10 I Pellet 11  Nuclear"  minutes  Supernate - Sj  5,000 x £ , 10 m i n u t e s  fraction  Pellet  Supernate - S  Mitochondria  16,300 x £ , 20 m i n u t e s  I  Supernate - S^  Pellet I  Suspend  i n 0,7  2  66,000 x £ , 90 m i n u t e s  M sucrose  6,000 x £ , *30 m i n u t e s Pellet Supernate  Pellet  Microsomes  S u p e r n a t e - S4 Soluble  fraction  14,000 x £ , 30 m i n u t e s  I Pellet  Supernate  Lysosome-rich fraction  F i g u r e 12. "Flow sheet" f o r i s o l a t i o n of s u b c e l l u l a r see E x p e r i m e n t a l Methods.  fractions  by d i f f e r e n t i a l  centrifugation.  For d e t a i l s  56  TABLE X I n t r a c e l l u l a r d i s t r i b u t i o n o f coenzyme  Coenzyme A phosphohydrolase  3'-phosphohydrolase.  ,p-Nitrophenyl phosphate phosphohydrolase  (  Ribonuclease  Fract ion Specific act i v i t y mU/mg protein  Recovery  per  cent  Homogenate  1.2  100  Nuc1e i  1.3  Mitochondr i a  Spec i f i c act i v i t y mU/mg protein  Recovery  Spec i f i c act i v i t y mU/mg protein  a  per  cent  -  85  100  27  60  16  1.5  1.2  16  82  15  1.4  Lysosomes  4.7  14  170  8  2.4  Microsomes  2.4  14  200  18  0.6  "Soluble" f r a c t ion  Q.5  20  9  0.7  b  R i b o n u c l e a s e was m e a s u r e d i n a m o d i f i c a t i o n o f t h e a s s a y m e t h o d o f A n f i n s e n e t a j _ . (72), i n w h i c h t h e enzyme f r a c t i o n i s i n c u b a t e d w i t h dialyzed yeast ribonucleic acid. Undigested r i b o n u c l e i c acid i s prec i p i t a t e d w i t h uranium a c e t a t e i n p e r c h l o r i c a c i d , and t h e o p t i c a l d e n s i t y o f t h e d i g e s t e d p r o d u c t s i s d e t e r m i n e d a t 260 mu. T h e r i b o nuclease a c t i v i t i e s in t h i s t a b l e a r e expressed in a r b i t r a r y u n i t s . Arbitrarily  t a k e n a s 100 p e r c e n t .  57 The a c t i v i t i e s  of the fractions  against p-nitrophenyl  phosphate were  d e t e r m i n e d , as a m e a s u r e o f n o n - s p e c i f i c p h o s p h o h y d r o l a s e  levels.  also  It can  b e s e e n t h a t t h e h i g h e s t c o e n z y m e A p h o s p h o h y d r o l a s e a c t i v i t y was f o u n d i n t h e lysosome f r a c t i o n , fairly  high level  fraction, somes  of a c t i v i t y .  ribonuclease, which  (73),  i t might  and t h a t t h e m i c r o s o m a l  was  level  is reportedly  fractions  a  exclusively  in the lyso-  From t h e r i b o n u c l e a s e a s s a y s ,  f r a c t i o n , w h i c h h a s an  appreciable  i s almost devoid o f r i b o n u c l e a s e a c t i v i t y .  one s i m p l e and a d m i t t e d l y c r u d e e x p e r i m e n t , o f t h e coenzyme A p h o s p h o h y d r o l a s e  of the  c o u l d c o n c e i v a b l y be due t o l y s o s o m a l  However, t h e m i c r o s o m a l  o f t h e enzyme,  chondrial  located  t h a t t h e coenzyme A p h o s p h o h y d r o l a s e a c t i v i t i e s  n u c l e a r and m i t o c h o n d r i a l contamination.  also exhibited  To t e s t f o r l y s o s o m a l c o n t a m i n a t i o n o f e a c h  a l s o measured i n each f r a c t i o n .  be i n f e r r e d  fraction  i t would  appear  From  this  that the bulk  i s a s s o c i a t e d w i t h t h e l y s o s o m a l and m i t o -  fractions.  Substrate s p e c i f i c i t y .  In an a t t e m p t t o d e t e r m i n e w h e t h e r  thei n -  a c t i v a t i o n o f c o e n z y m e A was d u e t o t h e a c t i o n o f a s p e c i f i c c o e n z y m e A  phos-  phohydrolase o r merely t h e r e s u l t o f t h e a c t i o n o f a n o n - s p e c i f i c phosphoh y d r o l a s e , a study o f t h e h y d r o l y s i s o f a v a r i e t y o f phosphomonoesters carried poses  out.  The r e s u l t s o f t h i s  s t u d y a r e summarized  in Table XI.  o f c o m p a r i s o n , r e l a t i v e r a t e s o f h y d r o l y s i s p e r mg p r o t e i n  w i t h t h e r a t e o f i n a c t i v a t i o n o f coenzyme A t a k e n as 100. a c t i v i t y o f each f r a c t i o n parentheses  i n t h e coenzyme A  ( m i l l i u n i t s p e r mg p r o t e i n ) .  the phosphohydrolase a c t i v i t i e s carried  through the procedure  i t y varied over a 120-fold  inactivation  are given, specific  is given in  From t h e d a t a , i t c a n b e s e e n  against a l l of the substrates tested  in a parallel  range.  For pur-  The a c t u a l assay  was  that  were  manner, w h i l e t h e s p e c i f i c  activ-  I t i s i n t e r e s t i n g t o n o t e t h a t t h e most  TABLE X I .  D i s t r i b u t i o n o f acid phosphohydrolase a c t i v i t i e s  AS-I Substrate  Coenzyme A p-NPP  Relative act i v i t y  40-80 3  '  100 ( 2 . 5 )  , EtOH 4 5 - 5 5 p e r c e n t ,  3.6  PH  b  pH 6.0  c  -  -  Relat ive act i v i t y  '  3.6  PH  pH 6.0  350  -  100 (20)  i n chicken  liver fractions  EtOH 2 4 - 4 5 p e r c e n t Relat ive act i v i t y  •  3.6  PH  pH 6.0  DEAE e l u a t e Relat ive act i v i t y  pH 3.6 pH 6.0  100  2.00  360  -  330  0.85  100 (60)  (300)  2'-AMP  190  1.00  185  1.00  190  0.90  195  0.50  V "AMP  155  1.10  140  1.05  140  1.10  150  0.95  5'-AMP  80  0.85  55  0.90  60  0.95  65  0.85  3'(2')-CMP  160  0.85  145  0.85  135  0.90  145  0.85  3'(2')-UMP  100  1.00  100  0.95  95  1.05  100  0.85  3'(2')-GMP  110  1.05  95  1.00  95  1.15  95  1.10  Glucose 6-phosphate  110  1.00  115  1.15  105  1.20  115  1.05  p-glycerophosphate  195  0.90  165  1.10  180  170  0.95  Relative activities t a k e n a s 100.  a r egiven  T h e r a t i o , pH 3.6/pH 6 . 0 , two pH v a l u e s .  i n comparison t o t h e a c t i v i t y  a.  a g a i n s t c o e n z y m e A, w h i c h was a r b i t r a r i l y  refers t o t h e r a t i o of t h e rates o f hydrolysis o f t h esubstrate at these  The v a l u e s i n p a r e n t h e s e s r e f e r t o t h e s p e c i f i c a c t i v i t y o f t h e f r a c t i o n t i o n a s s a y ; m i l l i u n i t s p e r mg p r o t e i n . This value i s approximate only. not be a c c u r a t e l y d e t e r m i n e d .  This  f r a c t i o n was v e r y  dilute,  i n t h e coenzyme A  inactiva-  and t h e p r o t e i n c o n c e n t r a t i o n  could  d  59 purified  f r a c t i o n , t h e e l u a t e f r o m D E A E - c e l l u l o s e , h y d r o l y z e d g l u c o s e 6-  phosphate at which  at a r e l a t i v e l y  rapid  rate - approximately two-thirds of the  i t h y d r o l y z e d p-glycerophosphate.  findings with other phosphohydrolases.  T h i s i s i n marked c o n t r a s t  For example,  the purified  enzyme e x h i b i t s n e g l i g i b l e g l u c o s e 6 - p h o s p h a t e p h o s p h o h y d r o l a s e t h e same i s t r u e o f t h e e r y t h r o c y t e and s e m i n a l p l a s m a the table, the ratios of the hydrolytic a c t i v i t i e s also given.  enzymes  a t pH  3.6  phosphohydrolases  from u n i t y .  (60).  activity; (60,74).  t h e coenzyme A p h o s p h o h y d r o l a s e  3.6  In  and pH 6.0  This is quite c h a r a c t e r i s t i c of  T h e s h a r p o p t i m u m a t pH  to  prostatic  W i t h a l m o s t a l l o f t h e s u b s t r a t e s t e s t e d , t h e r a t i o was  appreciably different  rate  are  not  acid  a p p e a r s t o be u n i q u e t o  activity.  A n o t h e r a p p r o a c h t o t h e p r o b l e m o f t h e s p e c i f i c i t y o f t h e enzyme  was  the monitoring of a l l fractions obtained during fractionations f o r a c t i v i t y against p-nitrophenyl  phosphate,  t a k e n as a m e a s u r e o f n o n - s p e c i f i c as f o r a c t i v i t y  acid  phosphohydrolase a c t i v i t y ,  as w e l l  almost every f r a c t i o n a t i o n  run, the r a t i o of p-nitrophenyl phosphate  enzyme A p h o s p h o h y d r o l a s e a c t i v i t i e s t h e e n t i r e 300-fold individual 13).  a g a i n s t c o e n z y m e A.  r e m a i n e d c o n s t a n t a t 2.8  range o f s p e c i f i c  activities.  T h i s was  f r a c t i o n s o b t a i n e d d u r i n g chromatography  However, i n one p u r i f i c a t i o n  r u n , w h i c h was  t o 3.2  In  and  co-  over  t r u e even o f t h e  on D E A E - c e l l u l o s e ( F i g .  otherwise relatively  unsuc-  c e s s f u l , a s e p a r a t i o n o f t h e coenzyme A p h o s p h o h y d r o l a s e a c t i v i t y  from the  bulk of the p-nitrophenyl  achieved.  This experiment i e s w e r e 35 the f i r s t  p h o s p h a t e p h o s p h o h y d r o l a s e a c t i v i t y was  i s summarized  in Table X l l .  in the undialyzed extract,  salt  fraction  ammonium s u l f a t e ) , 4 . 5  19.5  The  r a t i o s o f t h e two  in the dialyzed  activit-  extract,  10 i n  ( c o l l e c t e d b e t w e e n 4 0 and 80 p e r c e n t s a t u r a t i o n in t h e second s a l t  f r a c t i o n , and 3.5  i n each o f  with  the  80  >-  >  40  40  60  FRACTION  80  NUMBER  F i g u r e 13. D i s t r i b u t i o n o f p - n i t r o p h e n y l phosphate and coenzyme A p h o s p h o h y d r o l a s e a c t i v i t i e s d u r i n g chromatography on D E A E - c e l l u l o s e . These data a r e taken from t h e column d e p i c t e d i n F i g u r e 10. , p-nitrophenyl phosphate phosphohydrolase; , coenzyme A phospho hydrolase.  60  TABLE X l l D i s t r i b u t i o n o f p - n i t r o p h e n y l p h o s p h a t e and coenzyme A p h o s p h o h y d r o l a s e s d u r i n g p u r i f i c a t i o n o f t h e " i n h i b i t o r enzyme."  Coenzyme A phosphohydrolase Fract ion  Spec i f i c act i v i t y mU/mg protein  Initial  extract  Initial extract (after d i a l y s i s ) AS-I  30-40  1.7  b  c  ASf  11  per  Specific act i v i t y  cent  100  a  mU/mg protein  60  Recovery  per  cent  100  1.7  33  0.3  40  14  60  26  18  11  23  49  3  2.5  AS-I 40-80  Recovery  , p-Nitrophenyl phosphate phosphohydrolase  E t h a n o l , 2 4 t o 35 p e r  cent  18  3  66  1  E t h a n o l , 35 t o 43 p e r  cent  60  12  205  1  E t h a n o l , 43 t o 55 p e r  cent  20  8  70  1  Arbitrarily  taken  a s 100 p e r  a  cent.  An a l i q u o t o f t h e i n i t i a l e x t r a c t was d i a l y z e d f o r 16 h o u r s v s 3 0 0 v o l u m e s o f 10 mM T r i s h y d r o c h l o r i d e b u f f e r , pH 7.5, a n d c e n t r i f u g e d b r i e f l y t o r e move i n s o l u b l e p r o t e i n . For  an e x p l a n a t i o n o f t h e f r a c t i o n s ,  see Experimental  Methods.  61  three ethanol "AS-I  fractions.  30-40" f r a c t i o n ,  phenyl  It should  exhibited considerable activity  p h o s p h a t e , b u t was e s s e n t i a l l y On t h e b a s i s o f t h e s e  phosphohydrolase a c t i v i t y it  a l s o be n o t e d t h a t o n e f r a c t i o n , t h e  i s at least  a g a i n s t c o e n z y m e A.  data, t h e author  feels that  i f t h e coenzyme A  i s associated with a non-specific phosphohydrolase  not associated w i t h al1 o f t h e n o n - s p e c i f i c phosphohydrolase  a c t i v i t y of chicken problem,  inert  against p - n i t r o -  liver  extracts.  i t would be necessary  To p r o v i d e  a d e f i n i t e answer t o t h i s  t o o b t a i n a t l e a s t o n e f r a c t i o n w h i c h was  a c t i v e a g a i n s t coenzyme A b u t n o t a g a i n s t t h e o t h e r phosphomonoesters. has  n o t y e t been Stability  This  achieved. o f t h e enzyme.  As was m e n t i o n e d e a r l i e r ,  t h e coenzyme A O  phosphohydrolase a c t i v i t y stages ing. ed  appeared t o be s t a b l e t o s t o r a g e  o f t h e p u r i f i c a t i o n p r o c e d u r e , even w i t h One e t h a n o l  repeated  a t -20  f r e e z i n g and thaw-  f r a c t i o n , s t o r e d f r o z e n f o r more t h a n two y e a r s ,  a c o n s i d e r a b l e amount o f d e n a t u r e d p r o t e i n when t h a w e d .  assayed f o r " i n h i b i t o r  increase  in specific  enzyme" a c t i v i t y ,  activity  ject,  (75"77).  During  h a s f o u n d no e v i d e n c e t h a t s u c h  enzyme A p h o s p h o h y d r o l a s e , a l t h o u g h been s p e c i f i c a l l y  examined.  t h e course  o f phosphoof this  pro-  i s t h e case with t h e co-  i t must be a d m i t t e d  that this  has n o t  Column e f f l u e n t f r a c t i o n s , w i t h p r o t e i n c o n -  c e n t r a t i o n s o f l e s s t h a n f i v e ug p r o t e i n p e r m l , l o s t  o  solution  had been e f f e c t e d .  t o surface denaturation  t h e author  in-  i t was f o u n d t h a t a t w o - f o l d  Many r e p o r t s h a v e a p p e a r e d o n t h e m a r k e d s u s c e p t i b i l i t y hydrolases  contain-  When t h i s  s o l u b l e m a t e r i a l was removed b y c e n t r i f u g a t i o n a n d t h e s u p e r n a t a n t was  ata l l  no a c t i v i t y  i n seven  days at 0 , d e s p i t e c o n s i d e r a b l e m a n i p u l a t i o n d u r i n g t h i s p e r i o d . The e n z y m e i s m o d e r a t e l y s t a b l e t o m i l d h e a t t r e a t m e n t . When a n  62  ammonium s u l f a t e f r a c t i o n  ("AS-I 4 0 - 8 0 ; " p r o t e i n c o n c e n t r a t i o n , 30 mg p e r  m l ) was h e a t e d a t pH 5.5 a n d 50° f o r h m i n u t e s , 25 enzyme A p h o s p h o h y d r o l a s e a c t i v i t y was l o s t .  t o 35 p e r c e n t o f t h e c o -  T h e a d d i t i o n o f 20 t o 5 0 mM  m a g n e s i u m c h l o r i d e t o t h e f r a c t i o n d i d n o t p r e v e n t l o s s o f t h e enzyme o n heating.  This differs  f r o m t h e f i n d i n g s o f some w o r k e r s w i t h o t h e r p h o s p h o -  hydrol ases ( 6 2 ) . Inhibitors.  As was p r e v i o u s l y m e n t i o n e d ,  a d d i t i o n o f magnesium  c h l o r i d e t o t h e r e a c t i o n m i x t u r e d e p r e s s e d coenzyme A p h o s p h o h y d r o l a s e i t y , w h i l e t h e a d d i t i o n o f EDTA h a d n o e f f e c t does  inhibit  t h e enzyme, w i t h 50 p e r c e n t  t r a t i o n o f 1.2 mM. phosphohydrolases  upon t h e e n z y m e .  activ-  Orthophosphate  i n h i b i t i o n o c c u r r i n g at a concen-  V a r i o u s compounds w h i c h h a v e b e e n r e p o r t e d t o i n h i b i t - f o r example,  other  p - c h l o r o m e r c u r i b e n z o a t e (75) and c u p r i c i o n  (78) - c o u l d n o t be t e s t e d , s i n c e t h e p r e s e n c e o f h i g h c o n c e n t r a t i o n s o f t h i o l in t h e assay system would (79) .  r e v e r s e a n y e f f e c t o f t h e s e compounds v e r y  readily  63  DISCUSSION  The  evidence presented  main o b j e c t i v e s . capable CoA  of  The  first  o f t h e s e was  i s indeed  present  of the s i t e of a c t i o n of t h i s  that the t h i r d problem  from  second  the  inhibitory  acetyl-  involved The  the  third  f a c t o r produced  i n f a c t , one  might  i s more p r o p e r l y a p a r t o f t h e s e c o n d . the  i s o l a t i o n and  The  c h a r a c t e r i z a t i o n of  inhibitor. That t h e  inhibitor  been w e l l e s t a b l i s h e d . tracts  - was  r e i n f o r c e d by  present  original prepared  between a c e t o a c e t a t e  preparations enzymes  i s indeed  The  studied, only those  portionality  ( F i g . 3).  destroyed  observation from chicken  formation  the observation  by  The  f a c t s that the  boiling,  and  could  p r e s u m a b l y an  c o m p l e x , due  and  - precipitation  - that of a l l the liver  failed  of acetoacetate  inhibitory  f a c t o r was by  by  s o l v e n t s and  and  i n h i b i t o r was  (Fig.  chicken by  beef  liver liver  non-dialyzable,  the c l a s s i c a l  protein  ex-  techniques  adsorption in nature,  on and  enzyme.  second o b j e c t i v e - l o c a l i z i n g the  inhibitor effect  to the m u l t i p l i c i t y of p o s s i b l e s i t e s of a c t i o n .  c o - w o r k e r s had  liver  has  t o show p r o -  that p a r t i a l l y - p u r i f i e d  salt  liver  protein concentration  be p u r i f i e d  gels - led to the assumption that the  The  in e x t r a c t s of chicken  markedly depressed the formation  of p r o t e i n f r a c t i o n  was  The  four  that a factor  inhibitory factor.  o f p r e c i s e l y how  f o u r t h o b j e c t i v e , q u i t e n a t u r a l l y , was  was  liver.  e f f e c t s - f o l l o w e d q u i t e n a t u r a l l y from the second;  consider  1)  the demonstration  in extracts of chicken  o b j e c t i v e - the demonstration  the  been c o n c e r n e d w i t h  i n h i b i t i n g the enzymatic synthesis of acetoacetate  determination  its  i n t h i s t h e s i s has  already  established that the  i n h i b i t o r was  - was  more  Stern  and  his  n e i t h e r an a c e t y l  64  p h o s p h a t e p h o s p h o h y d r o l a s e n o r an a c e t y l - C o A h y d r o l a s e able  t o demonstrate that  enzymes  involved  their operation  the inhibitor  in acetoacetate formation (Table  II).  ments  This  (Tables This  ner  the  I) n o r prevented finally  enzyme must b e a c t i n g upon c o -  I I I t o V, F i g . 4 ) . b r o u g h t up t h e t h i r d o b j e c t i v e  q u a n t i t a t i v e formation  The a u t h o r has demonstrated  ( F i g . 7, T a b l e V I I ) c h r o m a t o g r a p h y  tech-  enzyme upon c o e n z y m e A r e s u l t s i n  o f a compound  On t h e b a s i s o f t h i s  i n h i b i t o r enzyme a c t i v i t y  - s p e c i f i c a l l y , t h e p r e c i s e man-  i n a c t i v a t e d c o e n z y m e A.  the action of the inhibitor  c o e n z y m e A.  upon a n y o f t h e  by a s e r i e s o f d i r e c t a n d i n d i r e c t e x p e r i -  ( F i g . 6) and i o n - e x c h a n g e  niques that  p e r se (Table  the inhibitor  was e s t a b l i s h e d  i n which t h e i n h i b i t o r  by p a p e r  acted  By a p r o c e s s o f e l i m i n a t i o n , t h e a u t h o r  came t o t h e r e a l i z a t i o n t h a t enzyme A.  enzyme n e i t h e r  ( 8 ) . T h e a u t h o r was  i n d i s t i n g u i s h a b l e from 3'-dephospho-  evidence,  i t has been c o n c l u d e d t h a t t h e  i s due t o t h e presence  in the extracts of a"co-  enzyme A 3 ' - p h o s p h o h y d r o l a s e . " F i n a l l y , o n e comes t o t h e f o u r t h o b j e c t i v e  - the isolation ofthe  c o e n z y m e A p h o s p h o h y d r o l a s e and a n e x a m i n a t i o n o f i t s p r o p e r t i e s . t i o n p r o c e d u r e has been d e v i s e d  which r e g u l a r l y r e s u l t s  p u r i f i c a t i o n o f t h e enzyme, w i t h  i n t h e most a c t i v e f r a c t i o n .  strate specificity  of this  against  (Table X I ) . tested  One p o s s i b i l i t y  e v e n t h e most against  i s that  a wide  Furthermore, t h e r e l a t i v e a c t i v i t y  remains r e l a t i v e l y constant  change i n s p e c i f i c a c t i v i t y .  come t o m i n d .  When o n e e x a m i n e s t h e s u b -  f r a c t i o n e x h i b i t s phosphohydrolase a c t i v i t y  a l l of the substrates  a 120-fold  i n a 200- t o 300-fold  enzyme, h o w e v e r , o n e f i n d s t h a t  v a r i e t y o f phosphate esters  isola-  r e c o v e r y o f 2 0 t o 30 p e r c e n t o f t h e i n i t i a l  enzymatic a c t i v i t y  highly-purified  An  Two p o s s i b l e e x p l a n a t i o n s t h e chicken  liver  extracts  throughout for this contain  65 o n l y one  p h o s p h o h y d r o l a s e , and  that t h i s phosphohydrolase  is capable  h y d r o l y z i n g a l a r g e number o f d i s s i m i l a r p h o s p h a t e e s t e r s . explanation similar  i s t h a t t h e e x t r a c t s c o n t a i n two  in nature  t i o n s employed.  that they  f r a c t i o n a t e as  of  The a l t e r n a t i v e  o r more p h o s p h o h y d r o l a s e s ,  a s i n g l e e n t i t y under the  A definitive solution to this  question w i l l  be  condi-  attained  only through a separation of the various phosphohydrolase a c t i v i t i e s . ever,  the author  considers  t h a t t h e r e a r e at  coenzyme A p h o s p h o h y d r o l a s e a c t i v i t y specific  enzyme.  t h a n one  phosphohydrolase  shows t h a t  One  in at  point of  A similar  activities  also occurred  homogenate o f c h i c k e n  The pH  simply  i s seen  in Table X l l .  liver  activity  (Table  a t pH  c e n t r i f u g a t i o n of a  t h e e f f e c t o f pH  3.6  in the  upon e n z y m a t i c  exhibited a f a i r l y r a n g e o f pH  to the s p e c i f i c  5.0  table of was  a specific  sucrose  activity  activity.  and  a t pH  the  6.0  ratio  was  2.0.  r a t i o s with the other phosphate e s t e r  From t h e s e  u n i q u e enzyme.  r a t i o s , one  However,  upon e n z y m a t i c a c t i v i t y  i n b o t h enzyme and  two  sharp optimum at  t o 6.0,  m i g h t be t e m p t e d t o  sume t h a t t h e h y d r o l y s i s o f t h e p h o s p h o m o n o e s t e r g r o u p i n g  ionization  This  X).  hand, t h e c o r r e s p o n d i n g  t h e e f f e c t s o f pH  more  run, a considerable separation  upon t h e d i f f e r e n t i a l  s u b s t r a t e s were near u n i t y .  c a t a l y z e d by  the  a m a n i f e s t a t i o n of a non-  phosphate phosphohydrolase a c t i v i t i e s  w i t h a marked " s h o u l d e r "  the other  l e a s t some i n d i c a t i o n s t h a t  indicating the existence of  coenzyme A p h o s p h o h y d r o l a s e a c t i v i t y 3.6,  How-  though q u a n t i t a t i v e l y l e s s marked s e p a r a t i o n o f t h e  might a l s o c o n s i d e r  of the s p e c i f i c On  purification  p-nitrophenyl  achieved.  One  evidence  in the extracts  l e a s t one  t h e c o e n z y m e A and  i s not  so  i t m u s t be  are complex,  s u b s t r a t e , and  In s u m m a r i z i n g t h e a b o v e p o i n t s , o n e  o f coenzyme A  must be  kept  i n mind  i n v o l v i n g changes  interpreted with  might say  that although  asis that in  caution. there  66 may  b e some j u s t i f i c a t i o n  f o r postulating the existence of a s p e c i f i c  u n i q u e c o e n z y m e A p h o s p h o h y d r o l a s e , m o r e w o r k must be d o n e b e f o r e a c o n c l u s i o n c a n be  Obvious p h y s i o l o g i c a l  known p h o s p h o h y d r o l a s e s . is the f i n a l (79). final  definite  reached.  The q u e s t i o n o f t h e p h y s i o l o g i c a l arises.  and  r o l e of the phosphohydrolase  f u n c t i o n s c a n be a t t r i b u t e d  also  t o many o f t h e  For example, t h e h y d r o l y s i s o f g l u c o s e 6-phosphate  step in the formation of blood sugar through  S i m i l a r l y , t h e h y d r o l y s i s of O-phosphoserine  glycogenolysis  h a s b e e n p r o p o s e d as  the  s t e p i n a b i o s y n t h e t i c pathway f o r t h e f o r m a t i o n o f s e r i n e from c a r b o -  h y d r a t e p r e c u r s o r s (80). acid phosphohydrolase  Another example would  be t h e r o l e o f p h o s p h a t i d i c  in the biosynthesis of t r i g l y c e r i d e s  s u g g e s t e d t h a t n o n - s p e c i f i c p h o s p h o h y d r o l a s e s may  (81).  difficult  (68).  r o l e f o r a coenzyme A p h o s p h o h y d r o l a s e , however, would  to visualize,  except p o s s i b l y  in a straightforward  T h i s a s p e c t o f t h e p r o b l e m must a l s o a w a i t f u r t h e r  been  act to ensure a supply of  o r t h o p h o s p h a t e f o r m e t a b o l i c , e x c r e t o r y and s e c r e t o r y p u r p o s e s physiological  I t has  A be m o r e  degradative role.  developments.  67  BIBLIOGRAPHY  1.  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