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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 Brit ish 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 this thesis as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA October, ]96k In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of • B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study, I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n . s h a l l not be allowed without my w r i t t e n permission* Department of Pharmacology  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada Date October 9, 1964  i i ABSTRACT The p r e s e n c e 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 r e p o r t e d more tha n a c e n t u r y ago. S i n c e t h a t t i m e c o n s i d e r a b l e e f f o r t has been d e v o t e d t o s t u d i e s on k e t o s i s and k e t o g e n e s i s . 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 r e v e a l e d t h e p r e c i s e e n z y m a t i c mechanism f o r 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 , t h e p r i m a r y k e t o n e body. 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 l i v e r e x t r a c t s o b t a i n e d from v a r i o u s s p e c i e s such as b e e f , dog, r a b b i t , p i g e o n and c h i c k e n , as w e l l as omasum and rumen e p i t h e l i u m o b t a i n e d f r o m sheep. A s t u d y o f a c e t o a c e t a t e f o r m a t i o n i n e x t r a c t s o f c h i c k e n l i v e r i s c o m p l i c a t e d by t h e p r e s e n c e o f a f a c t o r w h i c h i n h i b i t s : i n v i t r o a c e t o a c e t a t e f o r m a t i o n i n t h e a s s a y u s e d . T h i s " i n h i b i t o r " a l s o d e p r e s s e s a c e t o a c e t a t e f o r m a t i o n by beef l i v e r e x t r a c t s . A s t u d y o f t h e n a t u r e o f t h i s i n h i b i t o r a c t i o n was begun i n t h i s l a b o r a t o r y i n t h e hope t h a t an u n d e r s t a n d i n g o f i t s a c t i o n might shed some l i g h t on 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 . The mechanism o f a c e t o a c e t a t e f o r m a t i o n was s u b s e q u e n t l y worked out by independ-ent means. I t was s t i l l o f i n t e r e s t , however, t o c l a r i f y t h e n a t u r e o f t h e i n h i b i t o r f a c t o r . T h i s t h e s i s c o n s t i t u t e s such a s t u d y . The " i n h i b i t o r " p r o t e i n has been p u r i f i e d 3 0 0 - f o l d from e x t r a c t s o f f r e s h c h i c k e n l i v e r . The 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 i n v i t r o a c e t o a c e t a t e - f o r m i n g s y s t e m have been examined. P r e l i m i n a r y s t u d i e s r e v e a l -ed t h a t t h e f a c t o r does not a c t on any o f t h e e n z y m a t i c components o f a c e t o -a c e t a t e f o r m a t i o n . E v i d e n c e i s p r e s e n t e d t h a t i t i n h i b i t s a c e t o a c e t a t e f o r -m a t i o n t h r o u g h t h e i n a c t i v a t i o n o f b o t h t h e f r e e and e s t e r i f i e d forms o f c o -enzyme A. The p r o d u c t o f i t s a c t i o n on coenzyme A has been i s o l a t e d and c h a r a c t e r i z e d by p a p e r and ion-exchange chromatography and c h e m i c a l a n a l y s i s i i i as 3 1-dephosphocoenzyme A. The i n h i b i t o r p r o t e i n can t h u s be more p r o p e r l y d e s i g n a t e d as a "coenzyme A 3 ' - p h o s p h o h y d r o l a s e . " In r e t r o s p e c t , i t i s now c l e a r t h a t t h i s enzyme c o u l d have a f f o r d e d no knowledge c o n c e r n i n g t h e en-z y m a t i c mechanism o f a c e t o a c e t a t e f o r m a t i o n . The enzyme e x h i b i t s maximal a c t i v i t y at pH 3.6, w i t h h a l f - m a x i m a l a c t i v i t y a t a " p l a t e a u " between pH 5.0 and pH 6.0, and a t some p o i n t below pH 2.5. The 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 p h o s p h o h y d r o l a s e i s n e i t h e r enhanced nor d e p r e s s e d by d i v a l e n t c a t i o n o r m e t a l - b i n d i n g a g e n t s . A l t h o u g h t h e most 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 phospho-h y d r o l a s e a c t i v i t y a g a i n s t a w i d e v a r i e t y o f p h o s p h a t e e s t e r s , e v i d e n c e i s p r e s e n t e d t o i n d i c a t e t h a t t h e coenzyme A 3 1 - p h o s p h o h y d r o l a s e a c t i v i t y may w e l l be a s p e c i f i c and u n i q u e enzyme. i v TABLE OF CONTENTS Page INTRODUCTION 1 EXPERIMENTAL SECTION 5 M a t e r i a l s 5 Methods 5 I. M i s c e l l a n e o u s 5 I I . P r e p a r a t i o n and A s s a y o f Enzymes 6 A. A u x i l i a r y Enzymes 6 B. C h i c k e n L i v e r Enzymes 9 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. 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 17 RESULTS I. E x i s t e n c e o f t h e I n h i b i t o r 21 I I . Locus o f t h e I n h i b i t o r E f f e c t 2k I I I . 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 IV. The 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 LIST OF FIGURES No. T i t l e Page 1 F o r m a t i o n 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 F o r m a t i o n o f a c e t o a c e t a t e by c h i c k e n l i v e r f r a c t i o n s 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 beef l i v e r enzyme i n t h e p r e s e n c e o f c h i c k e n l i v e r f r a c t i o n s 25 4 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 beef l i v e r enzyme i n t h e p r e s e n c e and absence 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 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 upon c o -enzyme A 37 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 by paper chrom-a t o g r a p h y 39 7 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 by ion-exchange chromatography 42 8 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 t i m e 45 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 46 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 on DEAE-c e l l u l o s e 48 11 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 pH 54 12 " 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 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 55 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 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 chromatography on DEAE-c e l l u l o s e 59a, v i L I ST OF TABLES No. T i t l e Page I 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 a s s a y components w i t h t h e " i n h i b i t o r enzyme" 27 II 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 o -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 28 I I I 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 beef l i v e r enzyme 29 IV 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 " i n h i b i t o r enzyme" 32 V C o r r e l a t i o n between " i n h i b i t o r enzyme" and coenzyme A i n -a c t i v a t i o n a c t i v i t i e s 34 VI E v i d e n c e f o r i n a c t i v a t i o n o f e s t e r i f i e d coenzyme A by t h e " i n h i b i t o r enzyme" 35 V I I C o m p o s i t i o n o f t h e p r o d u c t o f t h e r e a c t i o n 43 V I M 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 49 IX 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 upon a c t i v -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 52 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 t h e coenzyme A phosphohydro-l a s e 56 XI D i s t r i b u t i o n o f a c i d 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 i n c h i c k e n 1 i v e r f r a c t ions 58 X I I 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" 60 v i i ABBREVIATIONS The f o l l o w i n g a b b r e v i a t i o n s have been u s e d : acetoacety 1-CoA, t h e a c e t o a c e t y l t h i o e s t e r o f coenzyme A; a c e t y l - C o A and a c e t y l - G , t h e a c e t y l 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 , r e s p e c t i v e l y ; 2 ' - , 3 ' " and 5 ' -AMP, t h e 2 ' - , 3 ' - 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 a c e t o a c e t a t e - s y n t h e s i z i n g enzyme system; 3 '(2')-CMP, t h e mixed 2 1 - and 3 1 -p h o s p h a t e e s t e r s o f c y t i d i n e ; CoASH, reduced coenzyme A; D E A E - c e l l u l o s e , d i e t h y l a m i n o e t h y l c e l l u l o s e ; E C T E O L A - c e l l u l o s e , a c e l l u l o s e ion-exchange m a t e r i a l formed by r e a c t i n g c e l l u l o s e w i t h 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 -amine; 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 mixed 2 ' -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, reduced g l u t a t h i o n e ; 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 coenzyme 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 ; 3 , ( 2 1 ) " UMP, t h e mixed 2 ' - and 3'-phosphate e s t e r s o f u r i d i n e . v i i i ACKNOWLEDGEMENTS The a u t h o r would 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 p e r s o n s : Dr. George i . Drummond, Department o f Pharmacology, f o r a d v i c e and a s s i s t a n c e d u r i n g t h e c o u r s e o f t h i s p r o j e c t ; Dr. James G. F o u l k s , Head, Department o f Pharmacology, f o r making 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; The C a n d i d a t e ' s Committee - Dr. Drummond; Dr. F o u l k s ; Dr. J . J . R. C a m p b e l l , Department o f D a i r y i n g ; and Dr. G. M. T e n e r , Department o f B i o -c h e m i s t r y - f o r a d v i c e and encouragement; Dr. George M. L i n g , Department o f Pharmacology, f o r p e r s o n a l en-couragement; 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 o n a l s u p p o r t and r e s e a r c h s u p p o r t ; Mr. E l l i s , 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 c h i c k e n l i v e r ; Mr. Frampton, Canada P a c k e r s L t d . , 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 beef 1 i v e r ; M r s . D. W. ( L o v e r n e ) Duncan, Department o f Pharmacology, f o r l e n d i n g a h e l p i n g hand when needed; Many o t h e r s , t o o numerous t o mention i n d i v i d u a l l y , who have a s s i s t e d i n t h e c o m p l e t i o n o f t h i s p r o j e c t . 1 INTRODUCTION The p r e s e n c e 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 r e p o r t e d more t h a n a c e n t u r y ago (1). Much e f f o r t has been expended on t h e s t u d y o f k e t o s i s and k e t o g e n e s i s i n t h e e n s u i n g y e a r s , but i t i s o n l y w i t h i n t h e l a s t decade t h a t t h e p r e c i s e b i o c h e m i c a l pathways i n v o l v e d i n 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 , t h e p r i m a r y k e t o n e body, have been e l u c i d a t e d i n t h i s l a b o r a t o r y (2-4) and e l s e w h e r e (5"7). In I960, S t e r n and c o - w o r k e r s at Western R e s e r v e U n i v e r s i t y i n C l e v e -l a n d d e s c r i b e d 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 f r o m a c e t y l - C o A 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 (8). In most 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-? t i o n was 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 added. One n o t a b l e e x c e p t i o n was found i n t h e c a s e o f e x t r a c t s o f c h i c k e n l i v e r a c e t o n e powder. As t h e c o n c e n t r a t i o n o f c h i c k e n l i v e r p r o t e i n i n t h e i r r e a c t i o n m i x t u r e 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 formed under t h e c o n d i t i o n s o f t h e a s s a y r o s e and t h e n d e c l i n e d . 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 no a c e t o a c e t a t e was f o r m e d . The above w o r k e r s 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 t o t h e p r e s e n c e i n c h i c k e n l i v e r e x t r a c t s o f a s e c o n d a r y enzyme s y s t e m w h i c h i n -t e r f e r e d i n some manner w i t h t h e enzyme(s) i n v o l v e d i n a c e t o a c e t a t e f o r m a t i o n . When t h i s " i n h i b i t o r " was 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 f o r m a t i o n was not y e t u n d e r s t o o d . A s t u d y o f t h i s s e c o n d a r y enzyme s y s t e m was begun i n t h i s l a b o r a t o r y i n t h e hope t h a t i t might p r o v i d e some i n s i g h t i n t o t h e i n t i m -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 . A l t h o u g h i t became app a r e n t q u i t e e a r l y In t h e c o u r s e o f t h i s s t u d y t h a t s u c h would not be t h e c a s e , i t was s t i l l f e l t t h a t t h i s o b s e r v a t i o n was w o r t h y o f f u r t h e r s t u d y . It i s t h e p u r -2 p o s e 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 s e c o n d a r y enzyme sy s t e m and t o p r o v i d e an e x p l a n a t i o n f o r t h e above mentioned phenomenon. It was pr o p o s e d by Lynen and co - w o r k e r s ( 5 ) , and e s t a b l i s h e d by t h e a u t h o r (2-4) and o t h e r s (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 by t i s s u e e x t r a c t s i n v o l v e s two enzymes. These two enzymes a r e t h e HMG-CoA c o n d e n s i n g enzyme ( 3 " h y d r o x y - 3 - m e t h y l g l u t a r y l - C o A 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 as HMG-CoA s y n t h a s e ) d e s c r i b e d by Rudney and c o - w o r k e r s (9-13) , and t h e HMG-CoA c l e a v a g e enzyme ( 3-hydroxy - 3 _ m e t h y l g 1 u t a r y l-CoA a c e t o a c e t a t e -l y a s e , EC 4 . 1 . 3 . 4 ; a l s o known as HMG-CoA l y a s e ) d e s c r i b e d by Coon and c o -w o r k e r s . The f i r s t o f t h e s e enzymes, t h e HMG-CoA c o n d e n s i n g enzyme, c a t a l y z e s t h e c o n d e n s a t i o n o f one mole each o f a c e t y l - C o A and a c e t o a c e t y l - C o A t o fo r m one mole o f HMG-CoA and l i b e r a t e s one mole o f f r e e coenzyme A ( R e a c t i o n 1 ) . The second o f t h e s e enzymes, t h e HMG-CoA c l e a v a g e 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 one mole o f f r e e a c e t o a c e t a t e and one mole o f a c e t y l - C o A ( R e a c t i o n 2 ) . CH3-C-SC0A + CH 3-C-CH 2-C-SCoA + H 20 a c e t y l - C o A a c e t o a c e t y l - C o A 9H fl *• CH 3-C-CH 2-C-SCoA + CoASH (1) CH 2-C00" HMG-CoA OH ^0 CH 3-C-CH 2-C-SCoA 0 0 1 »- CH 3-C-CH 2-C00" + CH 3-C-SCoA (2) CH2-COO:* HMG-CoA a c e t o a c e t a t e a c e t y l - C o A 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 two enzymes i s t h e h y d r o -l y s i s o f one mole o f a c e t o a c e t y l - C o A , p r o d u c i n g one mole each o f a c e t o a c e t a t e and coenzyme A. In t h e a s s a y s y s t e m used 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 and a c e t o -a c e t y l - C o A were g e n e r a t e d f r o m a c e t y l p h o s p h a t e and c a t a l y t i c c o n c e n t r a t i o n s o f coenzyme A i n t h e p r e s e n c e 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 b a c t e r i a l e x t r a c t s e r v e d 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 : o r t h o p h o s p h a t e 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) and p - k e t o t h i o l a s e ( a c e t y l - C o A : a c e t y l - C o A 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 k). 2 CH 3-C-0-P-0" + 2 CoASH =^=^= 2 ChyC-SCoA + 2 P. (3) a c e t y l - C o A a c e t y l p h o s p h a t e 0 0 0 2 CH 3-C-SCoA CH 3-C^CH 2-d'-SCoA + CoASH (h) a c e t y l - C o A a c e t o a c e t y l - C o A An e x a m i n a t i o n o f t h e sequence o f r e a c t i o n s i n v o l v e d i n t h e a s s a y s y s t e m shows t h a t t h e " i n h i b i t o r " enzyme p r e s e n t i n c h i c k e n l i v e r e x t r a c t s might p r e v e n 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 i n a number o f ways. I t c o u l d be a p r o t e o l y t i c enzyme r e l e a s e d when t h e c e l l s a r e br o k e n d u r i n g p r e p a r a -t i o n o f t h e e x t r a c t s . S i m i l a r l y , 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 complex, p a r a l l e l i n g t h e s i t u a t i o n seen w i t h t r y p s i n 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 i n h i b i t o r s ( 1 6 ) . I n -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 would r e s u l t i n t h e o b s e r v e d I n h i b i t o r e f f e c t . On t h e o t h e r hand, t h e " i n h i b i t o r enzyme" c o u l d p r e v e n t 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 one o r more o f t h e e s s e n t i a l nonenzymic a s s a y components o r one o f t h e i n t e r m e d i a t e s formed i n t h e p r o c e s s . One o b v i o u s p o s s i b i l i t y would be t h a t i t might be i d e n t i c a l w i t h t h e HMG-CoA h y d r o l a s e (EC 3.1.2.5) o f c h i c k e n l i v e r d e s c r i b e d by Coon and c o - w o r k e r s 07). I f t h i s were t h e c a s e , t h e f i n a l s t e p i n a c e t o a c e t a t e f o r m a t i o n , t h e c l e a v a g e o f HMG-CoA ( R e a c t i o n 2 ) , would be p r e v e n t e d . I t c o u l d a l s o be an a c e t y l 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 (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 by Lipmann i n e x t r a c t s o f h o r s e l i v e r ( 1 8 ) . T h i s p o s s i b i l i t y i s r a t h e r remote, however, s i n c e a c e t y l p h o s p h a t e i s p r e s e n t i n t h e as s a y i n l a r g e e x c e s s . The 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 h y d r o l a s e (EC 3.1*2.1). It c o u l d be a c t i n g upon coenzyme A i n some manner - f o r example, i t c o u l d be i d e n t i c a l w i t h t h e p e p t i d a s e d e s c r i b e d i n a v i a n l i v e r e x t r a c t s by Novel 1 i and c o - w o r k e r s (19), 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 and p-mercaptoethylamine m o i e t i e s o f coenzyme A. S i n c e coenzyme A f u n c t i o n s c a t a l y t i c a l l y i n t h e a s s a y method u s e d , any removal o f coenzyme A would n a t u r a l l y r e s u l t i n d e p r e s s e d a c e t o a c e t a t e f o r m a t i o n . The i n h i b i t o r c o u l d a l s o p r o d u c e i t s e f f e c t i n some o t h e r way. Many o f t h e s e p o s s i b l e e x p l a n a t i o n s f o r t h e i n h i b i t o r e f f e c t w i l l be d i s c u s s e d l a t e r i n more d e t a i l . In t h i s t h e s i s , e v i d e n c e w i l l be p r e s e n t e d t h a t t h e 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 i n v i t r o by c h i c k e n l i v e r e x t r a c t s i s a d i r e c t r e s u l t o f t h e p r e s e n c e i n t h e e x t r a c t s o f a n o v e l a c i d p h o s p h o h y d r o l a s e . I t w i l l be shown t h a t t h e i n h i b i t o r e f f e c t i s due t o t h e h y d r o l y s i s o f t h e 3'-phospho* monoester l i n k a g e o f coenzyme A, r e s u l t i n g i n t h e f o r m a t i o n o f t h e e n z y m i c -a l l y - ? n a c t i v e 3'-dephosphocoenzyme A. 5 EXPERIMENTAL SECTION M a t e r i a l s Coenzyme A ( nA 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 " 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 p h o s p h a t e ("A gr a d e " ) were o b t a i n e d f r o m t h e 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 R e s e a r c h , Los A n g e l e s , C a l i f o r n i a . D r i e d 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 f r o m W o r t h i n g t o n B i o -c h e m i c a l Co., F r e e h o l d , N.J. Ion-exchange e e l l u l o s e s ( " S e l e c t a c e l s " ) were o b t a i n e d f r o m C a r l S c h l e i c h e r and S c h u e l l Co., Keene, N.H. A u t h e n t i c 3 ' -dephosphocoenzyme A was t h e g i f t o f Dr. J.G. M o f f a t t , S y n t e x I n s t i t u t e f o r M o l e c u l a r B i o l o g y , P a l o A l t o , C a l i f o r n i a . C a l c i u m p h o s p h a t e g e l was p r e -p a r e d by t h e method o f K e i l i n and H a r t r e e ( 2 0 ) . A c e t y l p h o s p h a t e was p r e -p a r e d 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 aqueous p y r i d i n e as d e s c r i b e d by Stadtman ( 2 1 ) . A c e t y l t h i o e s t e r s were p r e p a r e d by t h e method o f Simon and Shemin ( 2 2 ) , i n w h i c h t h e reduced t h i o l i s r e a c t e d w i t h a 40 t o 50 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 s i m i l a r manner, a c e t o a c e t y l t h i o e s t e r s were p r e p a r e d f r o m reduced t h i o l and d i k e t e n e as d e s c r i b e d by Lynen e t a l _ . ( 5 ) , and HMG t h i o e s t e r s f r o m reduced t h i o l and HMG a n h y d r i d e as d e s c r i b e d by H i l z e t a1_. (23). 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 were 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 s o u r c e s . Methods I. M i s c e l l a n e o u s Methods A c e t y l p h o s p h a t e was d e t e r m i n e d by t h e hydroxamic a c i d method o f Lipmann and T u t t l e ( 2 4 ) . Coenzyme A s o l u t i o n s were f r e s h l y p r e p a r e d as r e -q u i r e d and 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 p h o s p h a t e 6 t r a n s a c e t y l a s e method o f Stadtman (25). P r o t e i n c o n c e n t r a t i o n s o f enzyme f r a c t i o n s were d e t e r m i n e d by t h r e e methods. F o r t u r b i d f r a c t i o n s , o r f r a c -t i o n s w i t h 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 , t h e c o l o r i m e t r i c m o d i f i e d b i u r e t method o f Gornal 1 et_ a l _ . (26) was u s e d . The p r o t e i n c o n c e n t r a t i o n s o f o p t i c a l l y c l e a r f r a c t i o n s were d e t e r m i n e d by t h e s p e c t r o p h o t o m e t r i c method o f Warburg and C h r i s t i a n (27), w h i c h i s based upon t h e a b s o r p t i o n o f u l t r a v i o l e t r a d i a t i o n by a r o m a t i c amino a c y l r e s i d u e s i n t h e p r o t e i n s a t 280 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 f r o m t h e o t p i c a l d e n s i t y a t 260 mu. 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 i l u t e s o l u t i o n s - f o r ex-ample, i n e f f l u e n t s f r o m c h r o m a t o g r a p h i c columns - were d e t e r m i n e d f r o m t h e o p t i c a l d e n s i t i e s a t 215 mu and 225 mu as d e s c r i b e d by Murphy and K i e s ( 2 8 ) . B o v i n e serum a l b u m i n was used as a p r o t e i n s t a n d a r d . 11. P r e p a r a t i o n and Assay o f Enzymes Enzyme a c t i v i t i e s , e x c e p t where n o t e d , a r e e x p r e s s e d i n terms o f t h e enzyme u n i t recommended by t h e Commission on Enzymes o f t h e I n t e r n a t i o n a l Union o f B i o c h e m i s t r y (29). T h i s u n i t i s d e f i n e d as t h a t amount o f any en-zyme w h i c h w i l l 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 m i n u t e at 30* under s p e c i f i e d c o n d i t i o n s . Where t h i s would r e q u i r e t h e use o f i n c o n v e n i e n t numbers, a c t i v i t i e s a r e e x p r e s s e d i n m i l l i u n i t s (mU). Spec-i f i c a c t i v i t i e s a r e e x p r e s s e d as 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 . A. Aux ? 1i a r y Enzymes Crude 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 p r e p a r e d 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 i n p h o s p h a t e 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 (2). Phosphate t r a n s a c e t y l a s e was measured by a m o d i f i c a t i o n o f t h e method o f Stadtman (30), u s i n g 10 mumoles, r a t h e r t h a n 5 u n i t s , o f coenzyme A. 7 The a c e t o a c e t a t e - s y n t h e s i z i n g enzyme sy s t e m (A.S.E.S.) f r a c t i o n s were p r e p a r e d f r o m beef l i v e r as d e s c r i b e d by S t e r n ert a l _ . (8). The A.S.E.S. a c t i v i t y was measured by a m o d i f i c a t i o n o f t h e method o f t h e C l e v e l a n d group. (For an e x p l a n a t i o n o f t h e b a s i s o f t h i s a s s a y method, s e e e q u a t i o n s (1) t o (4), i n t h e I n t r o d u c t i o n . ) The a s s a y s y s t e m c o n s i s t e d o f T r i s h y d r o c h l o r i d e b u f f e r , pH 8 .1 , 50 umoles; p o t a s s i u m c h l o r i d e , 2.5 umoles; reduced g l u t a -t h i o n e , n e u t r a l i z e d w i t h p o t a s s i u m b i c a r b o n a t e i m m e d i a t e l y b e f o r e u s e , 5 umoles; magnesium c h l o r i d e , 1 umole; a t h i o e s t e r - g e n e r a t i n g s y s t e m , c o n s i s t -i n g o f d i l i t h i u m a c e t y l p h o s p h a t e , 10 umoles, coenzyme A, 0.0125 umoles, and C l o s t r i d i u m k l u y v e r ? e x t r a c t , 0.03 ml ( c o n t a i n i n g 2 u n i t s 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 ) ; and enzyme f r a c t i o n i n a f i n a l volume o f 0.5 m l . The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f t h e l i v e r f r a c t i o n . A f t e r 60 m i n u t e s at 38°, t h e t u b e s were c h i l l e d i n i c e and 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 0.5 ml o f 12 p e r c e n t t r i c h l o r o a c e t i c a c i d . Denatured p r o t e i n was removed by c e n t r i f u g i n g i n t h e c o l d . S u i t a b l e a l i q u o t s o f t h e s u p e r n a t a n t s o l u t i o n were t h e n removed f o r d e t e r m i n a t i o n o f a c e t o a c e t a t e by a m o d i f i c a t i o n o f t h e method o f W a l k e r (31). T h i s m o d i f i e d p r o c e d u r e , w h i c h i s e x t r e m e l y r e p r o d -u c i b l e , i s as f o l l o w s : A s u i t a b l e a l i q u o t o f t h e d e p r o t e i n i z e d s o l u t i o n was d i l u t e d t o 0.50 ml w i t h 6 p e r c e n t t r i c h l o r o a c e t i c a c i d i n a g l a s s - s t o p p e r e d t e s t t u b e a t 0°. To t h i s d i l u t e d a l i q u o t were added, i n r a p i d s u c c e s s i o n , 3.5 ml o f M sodium a c e t a t e b u f f e r , pH 5 .1 , and 3.0 ml o f W a l k e r ' s d i a z o t i z e d p - n i t r o a n i l i n e r e a g e n t . The t u b e s were t h e n p l a c e d i n a w a t e r b a t h at 30°. A f t e r 20 m i n u t e s , t h e t u b e s were c h i l l e d i n i c e f o r 3 t o 5 m i n u t e s . The s o l u t i o n s were t h e n a c i d i f i e d by t h e a d d i t i o n o f 6.0 ml o f 5 N h y d r o c h l o r i c a c i d , and kept a t 0° f o r 8 t o 10 m i n u t e s . E t h y l a c e t a t e (4.0 ml) was t h e n added, and 8 t h e 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 e x t r a c t e d i n t o t h e o r g a n i c l a y e r by v i g o r o u s s h a k i n g . The 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 measured a t 450 mu. Under t h e above c o n d i t i o n s , 0.1 umole o f a c e t o a c e t a t e w i l l p r o d -uce an o p t i c a l d e n s i t y o f O.36 (d = 0.5 cm). U s i n g t h i s p r o c e d u r e , t h e 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 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 range 0.05 t o 0.55 umole. The HMG-CoA c o n d e n s i n g was p u r i f i e d f r o m y e a s t a c c o r d i n g t o Rudney and Ferguson ( 1 2 ) . The 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 s u l f -a t e f r a c t i o n a t i o n , 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 and p r e c i p i t a t i o n w i t h ammonium s u l f a t e . The f i n a l two s t e p s - t r e a t m e n t w i t h i o d o a c e t a m i d e and a second t r e a t m e n t w i t h c a l c i u m p h o s p h a t e g e l - w h i c h t h e s e a u t h o r s employed t o remove p-keto-t h i o l a s e were o m i t t e d , s i n c e t h e y were u n n e c e s s a r y f o r o u r p u r p o s e s , and r e s u l t e d i n v e r y low r e c o v e r i e s o f enzyme. The above a u t h o r s r e p o r t e d t h a t e t h i s enzyme was v e r y u n s t a b l e i f s t o r e d as a s o l u t i o n , even a t -20 , and t h a t t h e enzyme was o n l y s l i g h t l y more s t a b l e i f s t o r e d as a f r o z e n p a s t 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 . In c o n t r a s t t o t h i s r e p o r t , we have found t h a t i f w a t e r d i s t i l l e d f r o m an a l l - g l a s s a p p a r a t u s i s used at a l l s t a g e s 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 , t h e f i n a l p r e p a r a t i o n i s s t a b l e f o r s e v e r a l months a t -20°. The HMG-CoA c o n d e n s i n g enzyme was ass a y e d by i t s a b i l i t y 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. a s s a y s y s t e m 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 enzyme f r o m beef l i v e r . The HMG-CoA c l e a v a g e enzyme was p r e p a r e d from beef l i v e r a c e t o n e powder by t h e method o f Lynen et_ a K ( 5 ) , o m i t t i n g t h e a c e t o n e f r a c t i o n a t i o n , w h i c h a f f o r d s c o n s i d e r a b l e p u r i f i c a t i o n but low 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 by i t s a b i l i t y 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. a s s a y s y s t e m i n t h e p r e s e n c e o f e x c e s s HMG-CoA c o n d e n s i n g enzyme f r o m y e a s t . B. The C h i c k e n L i v e r Enzymes 1. Enzyme a s s a y s The " i n h i b i t o r enzyme" a s s a y . " I n h i b i t o r enzyme" a c t i v i t y was e s t i m -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 1 i v e r f r a c t i o n s were a b l e t o i n h i b i t f o r m a t i o n o f a c e t o a c e t a t e by beef l i v e r A.S.E.S. p r e p a r a t i o n s . The c o n d i t i o n s o f t h e s t a n d a r d A.S.E.S. a s s a y were employed, w i t h s u f f i c i e n t beef l i v e r f r a c t i o n 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 t o 0.55 umole of a c e t o a c e t -a t e . The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f t h e beef l i v e r f r a c t i o n , f o l -lowed w i t h i n 15 seconds by t h e c h i c k e n l i v e r f r a c t i o n . A f t e r 60 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 and a c e t o a c e t a t e d e t e r m i n e d i n t h e u s u a l manner. One " i n h i b i t o r u n i t " was d e f i n e d as t h a t amount o f enzyme w h i c h under 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 r e d u c t i o n i n t h e r a t e o f a c e t o -a c e t a t e f o r m a t i o n . Coenzyme A p h o s p h o h y d r o l a s e . 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 p o t a s s i u m s u c c i n a t e b u f f e r , pH 6.0, kO umoles; c y s t e i n e , 10 umoles; reduced coenzyme A, 50 mumoles; and enzyme, 0.5 t o 1.5 m i l l i u n i t s , i n a f i n a l volume o f 0.5 m l . Where n e c e s s a r y , t h e enzyme f r a c t i o n s were a p p r o p r i a t e l y d i l u t e d w i t h 10 mM p o t a s s i u m s u c c i n a t e b u f f e r , pH 6.0. A l l components exc e p t t h e enzyme were e q u i l i b r a t e d t o 30° and t h e r e a c t i o n was i n i t i a t e d by a d d i t i o n o f t h e enzyme. A f t e r 15 m i n u t e s t h e tube s were p l a c e d i n a b o i l i n g w a t e r b a t h f o r 5 m i n u t e s and t h e n c h i l l e d 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 a p p r o x i m a t e l y 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 b i c a r b o n a t e , and 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 i f n e c e s s a r y . D u p l i c a t e 10 s t a n d a r d s (minus enzyme) and d u p l i c a t e c o n t r o l s (minus coenzyme A and enzyme) were c a r r i e d t h r o u g h t h e p r o c e d u r e s i m u l t a n e o u s l y . R e s i d u a l coenzyme A was t h e n determined i n s u i t a b l e a l i q u o t s o f the s u p e r n a t a n t s o l u t i o n by a m o d i f i c a t i o n o f t h e method d e s c r i b e d by Novel 1 i (32). T h i s method i s based upon t h e a b i l i t y o f coenzyme A, but not i t s de-g r a d a t i o n p r o d u c t s , t o c a t a l y z e t h e a r s e n o l y s i s o f a c e t y l p h o s p h a t e i n t h e p r e s e n 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 and a r s e n a t e . 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 T r i s h y d r o c h l o r i d e b u f f e r , pH 8.1, 20 umoles; c y s t e i n e , 10 umoles; a c e t y l p h o s p h a t e , 6 umoles; 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 ; and t h e c o -enzyme A s o l u t i o n t o be a s s a y e d i n a f i n a l volume o f 0.7 m l . A f t e r t h e a s s a y O m i x t u r e was e q u i l i b r a t e d t o 30 , t h e a r s e n o l y s i s r e a c t i o n was i n i t i a t e d by t h e a d d i t i o n o f 0.3 ml o f O.I67 M p o t a s s i u m a r s e n a t e , pH 8.1, p r e v i o u s l y e q u i l i b r a t e d t o 30°. A f t e r 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 and r e s i d u a l a c e t y l p h o s p h a t e d e t e r m i n e d by t h e hydroxamic a c i d method o f Lipmann and T u t t l e ( 2 4 ) . Enzyme a c t i v i t y was d e f i n e d i n terms o f t h e s t a n d a r d u n i t s (see a b o v e ) . p - N i t r o p h e n y l 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 . The a b i l i t y o f c h i c k e n l i v e r f r a c t i o n s t o h y d r o l y z e t h e phosphomonoester bond o f p - n i t r o p h e n y l p h o s p h a t e was e s t i m a t e d i n a s y s t e m c o n s i s t i n g 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 , pH 6.0, 40 umoles; p - n i t r o p h e n y l p h o s p h a t e , 5 umoles; and enzyme f r a c t i o n i n a f i n a l volume o f 0.5 m l . The enzyme f r a c t i o n s were d i l u t e d when n e c e s s a r y w i t h 10 mM p o t a s s i u m s u c c i n a t e b u f f e r , pH 6.0. The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f t h e enzyme f r a c t i o n . A f t e r t e n m i n u t e s at 30*, 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 2.0 ml o f 0.05 N sodium h y d r o x i d e . C o n t r o l s c o n s i s t i n g o f s u b s t r a t e w i t h o u t enzyme and o f enzyme w i t h o u t s u b s t r a t e were c a r r i e d t h r o u g h t h e p r o c e d u r e c o n c u r r e n t l y . The amount o f p - n i t r o p h e n y l 11 p h o s p h a t e h y d r o l y z e d was c a l c u l a t e d f r o m t h e a b s o r b a n c e o f t h e r e s u l t a n t p - n i t r o p h e n y l a t e i o n a t 405 mu i n a Beckman DU s p e c t r o p h o t o m e t e r . Under t h e s e c o n d i t i o n s , t h e r a t e o f t h e r e a c t i o n i s p r o p o r t i o n a l 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 i s 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 B y r n e (33), t h e a u t h o r found t h a t p - n i t r o p h e n y l 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 n e i t h e r enhanced by magnesium i o n n o r i n h i b i t e d by EDTA. In f a c t , t h e p r e s -ence o f magnesium i o n i n t h e r e a c t i o n m i x t u r e i n t e r f e r e d w i t h t h e a s s a y , due t o t h e g r a d u a l appearance o f t u r b i d i t y a t a l k a l i n e pH, p r o b a b l y due t o t h e p r e c i p i t a t i o n o f i n s o l u b l e magnesium h y d r o x i d e o f c a r b o n a t e . " M i x e d t h i o l a s e " a s s a y . The "mixed t h i o l a s e " a c t i v i t y o f l i v e r f r a c -t i o n s ( s e e R e a c t i o n (5)) was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y by m e a s u r i n g 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 mu (34) i n t h e p r e s e n c e o f g l u t a t h i o n e . The c o m p l e t e a s s a y s y s t e m c o n -s i s t e d o f 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, 100 umoles; a c e t o a c e t y l - C o A , 0.4 umole; reduced g l u t a t h i o n e , 1.5 umoles; enzyme; and w a t e r t o a f i n a l volume o f 1.5 m l . C u v e t t e 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 -t a i n e d a l l components e x c e p t g l u t a t h i o n e ; and t h e r e f e r e n c e c u v e t t e c o n t a i n -ed a l l components e x c e p t a c e t o a c e t y l - C o A . C u v e t t e II s e r v e d as a c o n t r o l f o r t h e spontaneous d e a c y l a t i o n o f a c e t o a c e t y l - C o A , w h i c h o c c u r s a t a p p r e c -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 a s s a y (35). The r e a c t i o n was i n i t -i a t e d by a d d i t i o n o f g l u t a t h i o n e , and t h e d i s a p p e a r a n c e o f e n o l a t e i o n ab-s o r p t i o n a t 310 mu was r e c o r d e d a t 30 second i n t e r v a l s , r e a d i n g c u v e t t e s I and II a l t e r n a t e l y . The d i f f e r e n c e between t h e r a t e s o f change o f o p t i c a l d e n s i t y i n c u v e t t e s I and II was t a k e n as a measure o f t h e "mixed t h i o l a s e " r e a c t i o n . T h i s a s s a y method was o n l y a p p r o x i m a t e , due t o t h e o c c u r r e n c e o f 12 c o m p e t i n g r e a c t i o n s . T h i s has been more f u l l y d i s c u s s e d e l s e w h e r e ( 3 5 ) . S - A c e t y l g l u t a t h i o n e t h i o e s t e r a s e . T h i s enzymic a c t i v i t y was measur-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 method o f Drummond and S t e r n ( 3 6 ) . T h i s 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 bond a b s o r p t i o n a t 2k0 mu i n a Beckman model DU s p e c t r o p h o t o m e t e r i n q u a r t z c u v e t t e s (d = 0.5 cm). The c o m p l e t e a s s a y s y s t e m c o n s i s t e d o f T r i s h y d r o -c h l o r i d e b u f f e r , pH 7.5,100 umoles; S - a c e t y l g l u t a t h i o n e , 0.5 umole; enzyme; and w a t e r t o a f i n a l volume o f 1.5 m 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 components e x c e p t s u b s t r a t e . The r e a c t i o n was i n i t i a t e d by a d d i t i o n o f enzyme, and t h e change i n o p t i c a l 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 -ond i n t e r v a l s . N o n - s p e c i f i c a c i d p h o s p h o h y d r o l a s e . 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 a c t i v i t y 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 f r o m t h e a b i l i t y o f t h e f r a c t i o n s t o h y d r o l y z e a number o f phosphomonoesters. The a s s a y s y s t e m c o n -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 pH, 100 umoles; sub-s t r a t e , 10 umoles; enzyme; and w a t e r t o a f i n a l volume o f 1.0 m l . The r e -a c t i o n was i n i t i a t e d by a d d i t i o n o f t h e enzyme f r a c t i o n . A f t e r 15 m i n u t e s a t 30*, 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 20 p e r c e n t t r i c h l o r o a c e t i c a c i d . The t u b e s were c h i l l e d i n i c e f o r 10 m i n u t e s , and 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 i n a 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 the n d e t e r m i n e d by t h e method o f D r y e r e t a l . . ( 3 7 ) . 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" A p r o c e d u r e fcr t h e p a r t i a l 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" fr o m e x t r a c t s o f c h i c k e n l i v e r had been d e v i s e d p r e v i o u s l y i n t h i s l a b o r a -t o r y by Dr. G.I. Drummond. T h i s p r o c e d u r e i n v o l v e d s a l t f r a c t i o n a t i o n , heat 13 t r e a t m e n t at a c i d pH, removal o f i n e r t p r o t e i n w i t h c a l c i u m p h o s p h a t e g e l , p r e c i p i t a t i o n w i t h ammonium s u l f a t e and f r a c t i o n a t i o n w i t h e t h a n o l i n t h e p r e s e n c e o f z i n c i o n s . T h i s p r o c e d u r e gave a s i x - t o t e n - f o l d p u r i f i c a t i o n , w i t h v a r i a b l e r e c o v e r y o f t o t a l a c t i v i t y . A f t e r t h e n a t u r e o f t h e enzyme had 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 , i t was found t h a t some o f t h e s t e p s i n t h e e a r l i e r f r a c t i o n a t i o n p r o c e d u r e were u n s a t i s f a c t o r y w i t h r e s p e c t t o r e c o v e r y o f a c t i v i t y . The f o l l o w i n g p r o c e d u r e i s t h e one w h i c h was f i n a l l y adopted f o r t h e p u r i f i c a t i o n o f t h e enzyme. C h i c k e n l i v e r s were o b t a i n e d as soon as p o s s i b l e a f t e r s l a u g h t e r and packed i n i c e f o r t r a n s p o r t t o t h e l a b o r a t o r y . A l l subsequent o p e r a t i o n s were p e r f o r m e d a t o r near 0 , e x c e p t where n o t e d o t h e r w i s e . L i v e r s were ob-t a i n e d f r o m b r o i l i n g o r f r y i n g c h i c k e n s whenever p o s s i b l e , s i n c e t h e l i p i d c o n t e n t o f l i v e r s f r o m l a y i n g hens made p r o c e s s i n g o f t h e e x t r a c t s r a t h e r d i f f i c u l t . 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 one hour a f t e r removal f r o m t h e c h i c k e n s . When t h i s was not f e a s i b l e , t h e l i v e r s were f r o z e n u n t i l r e q u i r e d . 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 at -20* f o r s e v e r a l months w i t h o u t l o s s o f enzyme. E x t r a c t i o n . 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 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 d i v i d e d i n t o 130 g p o r t i o n s . Each p o r t i o n was homogenized w i t h 200 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 b u f f e r , pH 8.2, c o n t a i n i n g 5 mjl c y s t e i n e f o r a p p r o x i m a t e l y one m i n u t e i n a W a r i n g b l e n d o r . The c o a r s e homogenate was t h e n homogenized f o r f i v e m i n u t e s more i n a S e r v a l l Omni-m i x e r w i t h t h e s t a i n l e s s s t e e l chamber immersed i n a b a t h o f i c e w a t e r . A f u r t h e r 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 b u f f e r was added and homogeniz-in g was c o n t i n u e d f o r f i v e m i n u t e s . The homogenate was s t r a i n e d t h r o u g h two l a y e r s o f c h e e s e c l o t h and c e n t r i f u g e d f o r 60 m i n u t e s a t 13,000 x £ i n a 14 S e r v a l l a n g l e c e n t r i f u g e . The p r e c i p i t a t e was d i s c a r d e d . The s u p e r n a t a n t s o l u t i o n - " I n i t i a l e x t r a c t " - was s t r a i n e d t h r o u g h a pad o f c h e e s e c l o t h t o remove l i p i d . A p p r o x i m a t e l y t w e l v e t o f o u r t e e n l i v e r s (400 g o f t i s s u e ) c o u l d be p r o c e s s e d c o n v e n i e n t l y a t one t i m e . F i r s t s a l t f r a c t i o n a t i o n . The e n t i r e i n i t i a l e x t r a c t was d i l u t e d w i t h c o l d g l a s s - d i s t i l l e d w a t e r t o a p r o t e i n c o n c e n t r a t i o n o f 30 mg/ml and s t i r r e d m e c h a n i c a l l y . The o p a l e s c e n t e x t r a c t was brought t o 30 p e r c e n t s a t u r a t i o n w i t h 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 o f 45 t o 50 m i n u t e s , o f s o l i d ammonium s u l f a t e (16.8 g f o r each 100 ml o f d i l u t e d e x t r a c t ; c a l c u l a t e d f o r 0° a c c o r d i n g t o Noltmann e t aj_. ( 3 8 ) ) . The e x t r a c t was main-t a i n e d a t pH 7.5 t o 7.8 by o c c a s i o n a l a d d i t i o n o f s o l i d p o t a s s i u m b i c a r b o n a t e . The s u s p e n s i o n was s t i r r e d f o r 60 m i n u t e s a f t e r a d d i t i o n o f s a l t was com-p l e t e d and c e n t r i f u g e d f o r 45- m i n u t e s at 10,000 x £. The p r e c i p i t a t e was t h o r o u g h l y e x t r a c t e d w i t h a 30 p e r c e n t s a t u r a t e d s o l u t i o n o f ammonium s u l f -a t e ( a p p r o x i m a t e l y 0.1 volume, based on t h e Volume o f t h e d i l u t e d e x t r a c t ) . A f t e r s t a n d i n g f o r 30 m i n u t e s t h i s s u s p e n s i o n was c e n t r i f u g e d f o r 20 m i n u t e s a t 20,000 x £. The p r e c i p i t a t e was d i s c a r d e d . 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 volume n o t e d . T h i s combined s u p e r n a t a n t s o l u t i o n was brought t o 40 p e r c e n t s a t u r a -t i o n w i t h ammonium s u l f a t e by a d d i t i o n o f t h e s o l i d s a l t (5.8 g f o r each 100 ml o f s o l u t i o n ) as d e s c r i b e d above. The s u s p e n s i o n was s t i r r e d and c e n t r i -f u g ed as b e f o r e . The p r e c i p i t a t e was e x t r a c t e d w i t h 0.1 volume o f 40 p e r c e n t 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 and c e n t r i f u g e d . The p r e c i p i t a t e was d e s i g n a t e d as f r a c t i o n AS-I 30-40. 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 volume n o t e d . The c l e a r r e d s o l u t i o n was brought t o 80 p e r c e n t s a t u r a t i o n by a d d i -15 t i o n o f s o l i d ammonium s u l f a t e (26.3 g f o r each 100 ml o f s o l u t i o n ) i n t h e manner i n d i c a t e d . The s u s p e n s i o n was s t i r r e d and c e n t r i f u g e d as b e f o r e . The s u p e r n a t a n t s o l u t i o n was d i s c a r d e d . The p r e c i p i t a t e was d e s i g n a t e d as f r a c t i o n AS-I 40-80. The two p r e c i p i t a t e s (AS-1 30-40 and AS-I 40-80) were d i s s o l v e d i n m i n i m a l volumes 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. Each f r a c t i o n was d i a l y z e d v s 6 l i t e r s o f t h e same b u f f e r f o r 12 t o 16 h o u r s . I n s o l u b l e m a t e r i a l was t h e n removed by c e n t r i f u g a t i o n . At t h i s s t a g e 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 d i v i d e d i n t o two p o r t i o n s . I f t h i s was not done, t h e volumes i n t h e n e x t s t e p became unmanageable. One p o r t i o n was p r o c e s s e d as d e s c r i b e d below; t h e e o t h e r was s t o r e d a t -20 u n t i l needed. Gel t r e a t m e n t . The f r a c t i o n AS-I 40-80 was d i l u t e d w i t h c o l d g l a s s -d i s t i l l e d w a t e r t o a p r o t e i n c o n c e n t r a t i o n o f 10 mg p e r ml and brought t o pH 5.5 (0°, 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 d r o p w i s e a d d i t i o n o f 2 N a c e t i c a c i d . The c l o u d y s u s p e n s i o n was s t i r r e d a t 0° and s u f f i c i e n t c a l c i u m p h o s -p h a t e g e l (20 mg s o l i d s p e r ml) was added i n a t h i n s t r e a m t o p r o d u c e a g e l / p r o t e i n r a t i o o f 0.35. The s u s p e n s i o n was a l l o w e d t o s t a n d f o r 15 m i n u t e s and c e n t r i f u g e d b r i e f l y a t 5,000 x £. The p r e c i p i t a t e was washed w i t h c o l d g l a s s - d i s t i l l e d w a t e r and t h e wash s o l u t i o n added t o t h e s u p e r n a t a n t s o l u -t i o n . The p r e c i p i t a t e was t h e n d i s c a r d e d . The s u p e r n a t a n t s o l u t i o n was s t i r r e d and c a l c i u m p h o s p h a t e g e l was a g a i n added (60 mg g e l f o r each 100 mg o f p r o t e i n i n t h e i n i t i a l AS-I 40-80 f r a c t i o n ) . The s u s p e n s i o n was c e n -t r i f u g e d and t h e p r e c i p i t a t e washed as b e f o r e . The p r e c i p i t a t e was t h e n d i s -c a r d e d . The two s u p e r n a t a n t s o l u t i o n s were combined, b r o u g h t t o pH 7.5 by t h e c a r e f u l 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 b u f f e r e d by t h e a d d i t i o n 16 o f 0.03 volume 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 i s s o l u t i o n was t h e n brought t o 90 p e r c e n t s a t u r a t i o n w i t h ammonium s u l f a t e by a d d i t i o n o f t h e s o l i d s a l t (61.5 g f o r each 100 ml o f s o l u t i o n ) o v e r a p e r i o d o f 60 m i n -u t e s . The s u s p e n s i o n was s t i r r e d f o r 60 t o 90 m i n u t e s and c e n t r i f u g e d f o r 60 m i n u t e s a t 10,000 x £. The p r e c i p i t a t e was d i s s o l v e d i n a m i n i m a l volume o f 10 mM p o t a s s i u m s u c 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 1 mM p o t a s s i u m EDTA, and d i a l y z e d f o r 16 hours vs_ 6 l i t e r s o f t h e same b u f f e r . The d i a l y z e d s o l u -t i o n was d e s i g n a t e d as f r a c t i o n A S - 1 I . E t h a n o l f r a c t i o n s . S u f f i c i e n t M magnesium c h l o r i d e and M p o t a s s i u m s u c c i n a t e b u f f e r , pH 6.0, were added t o t h e AS-11 f r a c t i o n t o p r o d u c e f i n a l c o n c e n t r a t i o n s o f 0.10 and 0.05 M, r e s p e c t i v e l y . T h i s s o l u t i o n was s t i r r e d m e c h a n i c a l l y i n a w a t e r - e t h a n o l - d r y i c e b a t h a t -6*, and s u f f i c i e n t c h i l l e d e t h a n o l (-20°) added t o g i v e a f i n a l c o n c e n t r a t i o n o f 15 p e r c e n t e t h a n o l ( v / v ) . The 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 , and more c h i l l e d e t h a n o l added u n t i l t h e e t h a n o l c o n c e n t r a t i o n was 2k p e r c e n t ( v / v ) . The s u s p e n s i o n was s t i r r e d a t -12° f o r 15 m i n u t e s , and c e n t r i f u g e d f o r 15 m i n u t e s a t 13,000 x £ and -10°. In a s i m i l a r manner, f r a c t i o n s were c o l l e c t e d at 35, k3, 55 and 6k o o o o p e r c e n t e t h a n o l ( v / v ) a t -15 , "15 , "20 and -20 , r e s p e c t i v e l y . Each p r e c i p i t a t e was d i s s o l v e d a t 0° i n a mi n i m a l volume o f 10 mM p o t a s s i u m s u c -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 1 mM p o t a s s i u m EDTA, and d i a l y z e d vs_ 3 l i t e r s o f t h e same b u f f e r . The 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 h o u r l y , f o r f o u r c hanges. At 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 by 7 l i t e r s o f f r e s h b u f f e r and 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 . I n s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n . The 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 t h e f r a c t i o n p r e c i p i t a t i n g between 35 and k$ p e r c e n t e t h a n o l . 17 Chromatography. D E A E - e e l l u l o s e was p r e p a r e d f o r u s e , e q u i l i b r a t e d w i t h 5 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 7.2, c o n t a i n i n g 1 mM EDTA and packed i n t o j a c k e t e d g l a s s columns under hand-bulb p r e s s u r e as d e s c r i b e d by P e t e r -son and Sober (39). The columns were e q u i l i b r a t e d t o 0 i n a r e f r i g e r a t e d 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 u s e . The e t h a n o l f r a c t i o n was p r e p a r e d f o r chromatography by o v e r n i g h t d i a l y s i s vs_ 6 l i t e r s o f t h e same b u f f e r used t o p r e p a r e t h e column. The enzyme f r a c t i o n was a l l o w e d t o run i n t o t h e column a t a r a t e o f a p p r o x i m a t e l y 1 ml p e r m i n u t e . The column was t h e n e l u t e d w i t h t h e above b u f f e r . The f r a c t i o n s c o n t a i n i n g t h e enzyme were p o o l e d and t h e s o l u t i o n was c o n c e n t r a t e d by l y o p h i l i z a t i o n . The c o n c e n t r a t e d s o l u t i o n was t h e n a d j u s t e d t o pH 6.0 and 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 o f 10 mM p o t a s s i u m s u c 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 1 mM EDTA. T h i s f i n a l f r a c t i o n r e p r e s e n t s a 200- t o 3 0 0-fold p u r i f i c a t i o n o v e r t h e i n i t i a l e x t r a c t . The f i n a l f r a c t i o n i s s t a b l e f o r a t l e a s t t h r e e months when s t o r e d a t -20 . Repeated f r e e z i n g and t h a w i n g appears t o have l i t t l e o r no e f f e c t upon t h e enzyme a t any s t 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 . 3. 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 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 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 o f homogenates p r e p a r e d i n s u c r o s e s o l u t i o n s . The p r o c e d u r e g i v e n below i s a compromise between t h e method o f S c h n e i d e r and Hogeboom (40) f o r t h e i s o -l a t i o n o f t h e u s u a l s u b c e l l u l a r f r a c t i o n s ( n u c l e i , m i t o c h o n d r i a , microsomes and t h e s o l u b l e phase) and t h e method o f Sawant e t a l _ . (41) f o r t h e i s o l a -t i o n o f l y s o s o m e s . The l i v e r was o b t a i n e d f r o m a b r o i l i n g c h i c k e n w i t h i n f i v e m i n u t e s o f s l a u g h t e r and i m m e d i a t e l y immersed i n c o l d 0.25 M s u c r o s e f o r t r a n s p o r t t o t h e l a b o r a t o r y . A l l subsequent o p e r a t i o n s were c a r r i e d out a t o r near 18 0°. Workup o f t h e l i v e r was begun w i t h i n 20 m i n u t e s o f d e a t h . 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 homogenized i n p o r t i o n s w i t h f o u r volumes o f 0 .25 M s u c r o s e c o n t a i n i n g 2 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, i n a 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 . The homogenate was d i l u t e d w i t h 0 . 2 5 M s u c r o s e t o g i v e a 10 p e r c e n t (w/v) homogenate and s t r a i n e d t h r o u g h two l a y e r s o f c h e e s e c l o t h . " N u c l e a r " f r a c t i o n . A c r u d e " n u c l e a r " f r a c t i o n ( i n c l u d i n g b l o o d c e l l s , unbroken l i v e r c e l l s 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 c e n t r i f u g -i n g f o r 10 m i n u t e s a t 750 x £. The s u p e r n a t a n t s o l u t i o n - Sj - was s e t a s i d e . The p r e c i p i t a t e was washed t w i c e by s u s p e n d i n g i n 50 ml o f 0 . 2 5 M s u c r o s e each t i m e w i t h t h e a i d o f t h e homogenizer and c e n t r i f u g i n g as b e f o r e . The twic e - w a s h e d p r e c i p i t a t e was suspended i n 0 .25 M s u c r o s e - F r a c t i o n N w 2 M i t o c h o n d r i a l f r a c t i o n . The s u p e r n a t a n t s o l u t i o n S] and t h e two n u c l e a r washes were combined and c e n t r i f u g e d f o r 10 m i n u t e s at 5,000 x £. The o p a l e s c e n t s u p e r n a t a n t s o l u t i o n ( i n c l u d i n g t h e b u l k o f t h e " f l u f f y l a y e r " ( 4 2 ) ) was removed and s e t a s i d e - S2 . The p r e c i p i t a t e was washed t w i c e by r e s u s p e n d i n g i n 50 ml 0 . 2 5 M s u c r o s e each t i m e and r e c e n t r i f u g i n g as b e f o r e . The twic e - w a s h e d p r e c i p i t a t e was suspended i n 0 .25 M s u c r o s e - F r a c t i o n M w 2 " L y s o s o m e - r i c h f r a c t i o n . " The s u p e r n a t a n t s o l u t i o n S 2 and t h e two m i t o c h o n d r i a l washes were combined and c e n t r i f u g e d f o r 20 m i n u t e s at 16,300 x £. The s u p e r n a t a n t s o l u t i o n - - was s e t a s i d e . The p r e c i p i t a t e was suspended 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 a t 6,000 o x £ and 0 . The p e l l e t was d i s c a r d e d . The r e m a i n i n g s u s p e n s i o n was t h e n c e n t r i f u g e d f o r 30 m i n u t e s a t 14,000 x £ and 0°, and t h e s u p e r n a t a n t s o l u -t i o n d i s c a r d e d . The p e l l e t was washed t w i c e by r e s u s p e n d i n g i n 40 ml o f 19 0.7 M s u c r o s e each t i m e and s e d i m e n t i n g as b e f o r e . The p e l l e t was suspended i n 0.7 M s u c r o s e - F r a c t i o n LRF. M i c r o s o m a l f r a c t i o n . The s u p e r n a t a n t s o l u t i o n was c e n t r i f u g e d f o r 90 m i n u t e s a t 66,000 x £ and 0 . The t i g h t l y - p a c k e d p e l l e t was r e s u s -pended i n 0.25 M s u c r o s e - F r a c t i o n P. S o l u b l e f r a c t i o n . The s u p e r n a t a n t s o l u t i o n r e m a i n i n g a f t e r s e d i m e n t a -t i o n o f t h e m i c r o s o m a l f r a c t i o n r e p r e s e n t s t h e s o l u b l e p o r t i o n o f t h e c e l l -F r a c t i o n S/^ A summary o f t h e f r a c t i o n a t i o n i s g i v e n i n t h e " f l o w s h e e t " form i n F i g u r e 12. A l l 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 were f r o z e n and s t o r e d a t -20* o v e r -n i g h t . To p r e p a r e t h e f r a c t i o n s f o r enzyme a s s a y s , t h e thawed s u s p e n s i o n s were mixed w i t h s u f f i c i e n t T r i t o n X-100 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 , pH 6 . 0 , t o p r o d u c e f i n a l c o n c e n t r a t i o n s o f 0.2 p e r c e n t and 0.02 M, r e s p e c t i v e l y . The 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 were a l l o w e d t o s t a n d a t 0* f o r 30 m i n u t e s b e f o r e t h e v a r i o u s e n z y m a t i c a c t i v i t i e s were d e t e r m i n e d . Received without pape(s) 0 Filmed as r e c e i v e d . U n i v e r s i t y M i c r o f i l m s , Inc. 21 RESULTS I. Existence of the Inhibitor The formation of acetoacetate in v itro by extracts of l iver from most species is proportional to the amount of tissue protein added. From Figure 1 it can be seen that the rate of acetoacetate formation by pigeon l iver enzymes exhibits direct dependence upon protein concentration under the conditions of the assay. The same is true in the case of beef l i ver . This is not true, however, in the case of chicken l i ver . As Figure 1 shows, the rate of acetoacetate formation by an extract of chicken l iver is not proportional to protein concentration when relatively large amounts of protein are added. This indicates the presence of an inhibitory factor in extracts of chicken l i ver . When a crude extract of chicken l iver is sub-jected to salt fractionation, this becomes even more apparent. Figure 2 shows that the enzyme(s) responsible for acetoacetate formation can be separ-ated from the inhibitory factor. The fraction precipitating between the limits of 30 and kO per cent saturation with ammonium sulfate contains the A.S.E.S., relat ively free of the inhibitory factor. The fraction precip i tat -ing between the limits of kO and 70 per cent saturation with ammonium sulfate also exhibits some A'.S.E.S. act iv i ty , but is relat ively rich in the inhibit -ory factor. Preliminary studies of the inhibitor in this laboratory, begun by Or. G.I. Drummond and continued by the author, were based on the observation that this inhibitor was able to prevent the formation of acetoacetate by beef l iver enzyme fractions. An assay system was devised on the basis of this observation, for the simple reason that this was the only property of 22 2 0 4-0 PROTEIN (mg) Figure 1. Formation of acetoacetate by extracts of various tissues. The standard conditions of the A.S.E.S. assay were employed. -• *r beef l iver extract; *• *, pigeon l iver extract; * x, chicken l iver extract. The extracts were prepared in bicarbonate-cysteine buffer as described for chicken l iver in the Experimental Section. 23 2-0 4 0 PROTEIN (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 c h i c k e n l i v e r f r a c t i o n s . The 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 were employed. • • , i n i t i a l e x t r a c t ; o • , 30 t o 40 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 ; * * , hO 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 . 2k t h e enzyme w h i c h was known. The d e t a i l s o f t h e a s s a y a r e g i v e n under Ex-p e r i m e n t a l Methods. As F i g u r e 3 shows, 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 -a c e t a t e f o r m a t i o n 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 l i v e r p r o t e i n added o v e r a f a i r l y w i d e r a n g e , under t h e s p e c i f i e d c o n d i t i o n s . ( I n t h i s e a r l y work, t h e AS-I 40-80 f r a c t i o n was g i v e n a b r i e f heat t r e a t -ment a t a c i d pH t o d e s t r o y r e s i d u a l A.S.E.S. a c t i v i t y . The p r e s e n c e o f A.S.E.S. a c t i v i t y i n t h e i n h i b i t o r f r a c t i o n s would have made i t v i r t u a l l y i m p o s s i b l e t o i n t e r p r e t e x p e r i m e n t a l r e s u l t s . ) 11. Locus o f t h e I n h i b i t o r E f f e c t V a r i o u s s t u d i e s were c a r r i e d o ut t o d e t e r m i n e p r e c i s e l y how t h e i n h i b i t o r p r o d uced i t s e f f e c t s . S t e r n e t a K (8) had p r e v i o u s l y e s t a b l i s h e d t h a t t h e i n h i b i t o r was n e i t h e r an a c e t y l 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 . One p o s s i b l e e x p l a n a t i o n was t h a t t h e i n h i b i t o r e f f e c t c o u l d be due t o t h e combined a c t i o n o f a "mixed 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 by Drummond and S t e r n (35.43) ( R e a c t i o n 5 ) , and an S - a c e t y l -g l u t a t h i o n e h y d r o l a s e (36) ( R e a c t i o n 6 ) . T h i s would r e s u l t i n t h e removal A c e t o a c e t y l - C o A + GSH a c e t y l - C o A + a c e t y l - G (5) A c e t y l - G + H 20 v a c e t a t e + GSH (6) o f a c e t o a c e t y l - C o A as i t was formed. 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 A.S.E.S. a s s a y s y s t e m used f o r t h e i n h i b i t o r a s s a y c o n t a i n e d a r e l a t i v e l y 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 (10 mM), t h i s i d e a appeared v e r y a t t r a c t i v e . By d i r e c t s p e c t r o p h o t o m e t r i c a s s a y , 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 t h a t b o t h o f t h e s e enzymes were p r e s e n t i n a l l c h i c k e n l i v e r f r a c t i o n s w h i c h 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 beef l i v e r enzyme. 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 were employed. ° o , h e a t e d c h i c k e n l i v e r e x t r a c t ; * * , s e c -ond 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 e t h a n o l f r a c t i o n . The heated 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 were 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 under E x p e r i m e n t a l Methods. 26 h i b i t e d I n h i b i t o r a c t i v i t y . However, f u r t h e r s t u d y showed t h a t t h i s e x p l a n a -t i o n was not v a l i d . I t was found t h a t t h e most h i g h l y p u r i f i e d beef l i v e r A.S.E.S. f r a c t i o n , t h e 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 S t e r n e t a l . (8), w h i c h e x h i b i t e d no s i g n w h a t s o e v e r o f i n h i b i t o r a c t i v i t y , c o n t a i n e d even more o f t h e s e two enzymes th a n d i d t h e most p o t e n t i n h i b i t o r f r a c t i o n . 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 any p o s s i b l e e f f e c t o f t h e i n h i b i t o r on t h e e n z y m a t i c components o f t h e A.S.E.S. a s s a y s y s t e m . The i n h i b i t o r 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 t r a n s a c e t y l a s e and t h e beef l i v e r enzyme s y s t e m . The o t h e r components o f t h e assay s y s t e m were t h e n added and t h e a s s a y was co m p l e t e d i n t h e u s u a l manner. In bo t h c a s e 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 l y t h e same as would have been t h e c a s e i f t h e r e had been no p r e i n c u b a t i o n p e r i o d ( T a b l e I ) . T h i s v i r t u a l l y e l i m i n a t e d t h e p o s s -i b i l i t y t h a t t h e i n h i b i t o r a c t e d d i r e c t l y on t h e s e enzymes. 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 o f t h e i n h i b i t o r enzyme upon r e -c o n s t i t u t e d A.S.E.S. s y s t e m s , 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 HMG-CoA c o n d e n s i n g enzyme p l u s e x c e s s l i v e r HMG-CoA c l e a v a g e enzyme and o f a l i m i t i n g 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 c o n d e n s i n g enzyme. As T a b l e II shows, 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 -l y t h e same i n each c a s e . T h i s i n d i c a t e d t h a t t h e i n h i b i t o r enzyme p r o b a b l y d i d not i n t e r f e r e 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 t h a t t h e i n h i b i t o r enzyme must a f f e c t one o f t h e nonenzymic components o f t h e a s s a y . To d e t e r -mine 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 f r a c t i o n b e f o r e a d d i t i o n o f t h e beef l i v e r enzyme. T h i s e x p e r i m e n t i s sum-m a r i z e d i n T a b l e 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 was added ( l a and 2 a ) , TABLE I 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 a s s a y components w i t h t h e i n h i b i t o r enzyme. E x p e r i m e n t I n h i b i t o r enzyme A c e t o a c e t a t e I n h i b i t i o n No. added formed mg p r o t e i n umole p e r c e n t 1 - 0.36 2 0.70 0.25 31 3 0.70 v 0.24 33 The A.S.E.S. p r e p a r a t i o n used 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 beef l i v e r , 0.61 mg p r o t e i n . The i n h i b i t -o 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 -u t e s i n a l l c a s e s . In e x p e r i m e n t 1, t h e beef 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 were 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 added at 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 . 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 added 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 o f t h e p r e i n c u b a t i o n p e r i o d . 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 II E f f e c t o f 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 o 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 . E x p e r i m e n t I n h i b i t o r A c e t o a c e t a t e I n h i b i t i o n 1 No. ' 1 enzyme ' s y n t h e s i z e d 1 1 mg p r o t e i n umole p e r c e n t l a Beef l i v e r f r a c t i o n - 0.60 l b " " " 0.14 0.38 36 2a R e c o n s t i t u t e d s y s t e m I - 0.44 2b » " 0.14 0 . 2 9 34 3a R e c o n s t i t u t e d s y s t e m II - 0.61 3b " " 0.14 0.37 39 The beef l i v e r f r a c t i o n used was a 20 t o 35 p e r ce 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 condens-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 beef l i v e r a c e t o n e powder, 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 ) . R e c o n s t i t -u t e d s y s t e m II 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 enzyme, 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 enzyme, 1.65 mg p r o t e i n (a f o u r -f o l d e x c e s s ) . 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 were employed. TABLE III Effect of preincubation of the nonenzyme components of the assay system with the " inhib i tor enzyme" before addition of the beef l iver enzyme. Experiment Preincubation Acetoacetate No. ' period ' formed minutes umoles la 30 0.56 lb 30 0.31 lc 30 0.13 2a 60 0.56 2b 60 0.31 2c 60 0 . 0 0 Al l tubes contained a l l the nonenzymic components of the standard A.S.E.S. assay system. Acetoacetate-synthes-izing system (beef l iver 20 to 35 per cent ethanol fract ion, 0.98 mg protein) was routinely added to a l l tubes at the end of the preincubation period. In experiments ^a and 2 a , no " inhib i tor enzyme" was added. In experiments J_b and 2b , " inhib i tor enzyme" (chicken l iver ethanol fract ion, 0.13 mg protein) was added immediately following the beef l iver fract ion. In experiments Jl_c and 2c_, the same chicken l iver fraction was added at the start of the preincubation period. The preincubation period was 30 or 60 minutes, as noted, at 38 . Following addition of the beef l iver fract ion, the us-ual procedure was followed. 30 0.56 umole o f a c e t o a c e t a t e was f o r m e d . When t h e c h i c k e n l i v e r f r a c t i o n was added a t 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 , i m m e d i a t e l y a f t e r t h e a d d i t i o n o f t h e beef l i v e r enzyme ( l b and 2 b ) , 0.3' umole o f a c e t o a c e t a t e was formed, r e p r e s e n t i n g 45 p e r c e n t 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 . However, when t h e c h i c k e n l i v e r f r a c t i o n 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 ( l c and 2 c ) , i n h i b i t i o n was much more marked - 77 p e r c e n t i n h i b i t i o n f o l -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 100 p e r c e n t i n h i b i t i o n f o l l o w i n g a 60 m i n u t e p r e i n c u b a t i o n . It was now q u i t e a p p a r e n t t h a t t h e i n h i b i t o r enzyme was a c t i n g upon one o r more o f t h e nonenzymic components o f t h e a s s a y s y s t e m . From an e x a m i n a t i o n o f t h e c o m p o s i t i o n o f t h e r e a c t i o n m i x t u r e , i t appeared t h a t t h e i n h i b i t o r must be p r o d u c i n g i t s e f f e c t s t h r o u g h some a c t i o n upon coenzyme A. T h i s was c o n f i r m e d by two s i m p l e e x p e r i m e n t s . F i g u r e 4 shows t h e e f f e c t o f coenzyme 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 by a beef l i v e r f r a c t i o n i n t h e p r e s e n c e and absence o f c h i c k e n l i v e r enzyme. The graph shows t h a t a t low c o n c e n t r a t i o n s o f coenzyme A, t h e c h i c k e n l i v e r enzyme c a u s e s a marked 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 . On t h e o t h e r hand, when s u f f i c i e n t coenzyme A was added t o p r o d u c e maximal r a t e s o f a c e t o -a c e t a t e f o r m a t i o n , t h e c h i c k e n l i v e r enzyme had no e f f e c t . In a n o t h e r ex-p e r i m e n t , t h e a b i l i t y o f t h e i n h i b i t o r enzyme t o i n a c t i v a t e coenzyme A was d e m o n s t r a t e d d i r e c t l y ( T a b l e I V ) . The d a t a i n d i c a t e t h a t t h e i n h i b i t o r enzyme does indeed d e s t r o y coenzyme A. I t had 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 l i v e r e x t r a c t s a r e c a p a b l e o f i n a c t i v a t i n g coenzyme A, and t h a t t h e 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 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 l i v e r e x t r a c t s c o u l d be p r e v e n t e d by 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 i n t h e r e a c t i o n m i x t u r e . Be-f o r e i t c o u l d be f i n a l l y c o n c l u d e d t h a t t h e i n h i b i t o r enzyme was indeed i d e n t i c a l w i t h t h e enzyme d e s t r o y i n g coenzyme A, i t was n e c e s s a r y t o show a 31 • * • • 1 0-2 0-4 0-6 COENZYME A (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 and absence o f c h i c k e n l i v e r " i n -h i b i t o r enzyme." The 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 were employed, e x c e p t t h a t t h e coenzyme A c o n c e n t r a t i o n was v a r i e d as n o t e d . • • , beef l i v e r ammonium s u l f a t e f r a c t i o n (8) (1.10 mg p r o t e i n ) ; * * , as above, 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 (0,18 mg p r o t e i n ) . TABLE IV 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 " i n h i b i t o r enzyme." Exp e r i m e n t Enzyme , Coenzyme A ( Coenzyme A ( No. added r e c o v e r e d mq p r o t e i n mumoles mumoles l a _ 10 10 l b 0.19 10 10 2a - 20 20 2b 0.19 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 b u f f e r , pH 8 . 1 , 100 umoles; M g C l 2 , 1 umole; K C l , k umoles; c y s t e i n e , k umoles; and coenzyme A and " i n h i b i t o r enzyme" ( 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 volume, 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 kt 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 . Coenzyme A i n t h e s o l u t i o n was t h e n de-t e r m i n e d e n z y m a t i c a l l y ( 3 2 ) . 33 c o r r e l a t i o n between t h e two a c t i v i t i e s . Such a c o r r e l a t i o n i s p r e s e n t e d i n T a b l e V. Over a t e n - f o l d range o f s p e c i f i c a c t i v i t i e s , t h e r a t i o o f c o -enzyme A i n a c t i v a t i o n : i n h i b i t o r enzyme a c t i v i t y ranged from a low o f 3.3 t o a h i g h o f 5 . 2 . A l t h o u g h one might 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 h a n t h i s , i n v i e w o f t h e v a r i a b i l i t y o b s e r v e d i n t h e i n h i b i t o r enzyme a s s a y and, t o a much l e s s e r e x t e n t , i n t h e coenzyme A i n a c t i v a t i o n a s s a y , t h e c o r r e l a -t i o n can be c o n s i d e r e d s a t i s f a c t o r y . One o t h e r p o i n t a l s o comes t o mind: does t h e c h i c k e n l i v e r enzyme a c t o n l y upon f r e e coenzyme A, o r does i t a l s o i n a c t i v a t e e s t e r i f i e d c o -enzyme A? In v i e w o f t h e f a c t t h a t t h e i n h i b i t o r enzyme a s s a y s y s t e m c o n -t a i n s s u f f i c i e n t p h o s p h a t e 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 umoles 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 formed, one would e x p e c t t h a t t h e amount o f f r e e coenzyme A p r e s e n t a t any t i m e would be e x t r e m e l y low. I t would t h e r e f o r e appear t h a t t o p r o d u c e i t s e f f e c t , t h e i n h i b i t o r enzyme would 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 e s t e r i f i e d coenzyme. T h i s i s shown i n T a b l e V I . E n z y m a t i c a l l y - s y n t h e s i z e d a c e t y l - C o A was i n c u b a t e d w i t h an i n h i b i t o r enzyme f r a c t i o n i n t h e coenzyme A i n a c t i v a -t i o n a s s a y , and r e s i d u a l a c e t y l - C o A was measured by t h e u s u a l a r s e n o l y s i s method. It can be seen t h a t t h e i n h i b i t o r enzyme does i n a c t i v a t e a c e t y l - C o A , a t a p p r o x i m a t e l y 55 p e r c e n t o f t h e r a t e a t w h i c h i t i n a c t i v a t e s t h e f r e e c o -enzyme. It s h o u l d a l s o be n o t e d t h a t 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 a l o n e d e p r e s s e d t h e r a t e o f d i s a p p e a r a n c e o f coenzyme A. I t i s not c l e a r whether 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 i s due t o t h e a c e t y l ; p hosphate i t s e l f o r t o t h e p r e s e n c e i n t h e a c e t y l p h o s p h a t e o f o r t h o p h o s p h a t e , w h i c h m a r k e d l y i n h i b i t s t h e 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 l i v e r enzyme. 34 TABLE V C o r r e l a t i o n between i n h i b i t o r enzyme and coenzyme A i n a c t i v a t i o n a c t i v i t i e s . F r a c t i o n I n h i b i t o r enzyme (0 Coenzyme A i n a c t i v a t i o n (2) R a t i o (2)/(D Heated e x t r a c t Second g e l s u p e r n a t e Second s a l t f r a c t i o n E t h a n o l , 0 t o 31 p e r c e n t E t h a n o l , 31 t o 37 p e r c e n t E t h a n o l , 37 t o 45 p e r c e n t E t h a n o l , 45 t o 56 p e r c e n t u n i t s / m g p r o t e i n 0.9 2.1 2.1 0.5 5.8 8.6 0 mU/mg p r o t e i n 4.1 7.2 7.0 2.5 30 33 0 4.5 3.4 3.3 5.0 5.2 3.8 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 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 . The o t h e r f r a c t i o n s were 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 under E x p e r i m e n t a l Methods. TABLE VI E v i d e n c e f o r i n a c t i v a t i o n o f e s t e r i f i e d coenzyme A by t h e i n h i b i t o r enzyme A d d i t i o n s R a t e o f i n a c t i v a t i o n mumo1es/mg/m ? n None 84 A c e t y l p h o s p h a t e 42 A c e t y l p h o s p h a t e p l u s p h o s p h a t e t r a n s a c e t y l a s e 23 S t a n d a r d a s s a y c o n d i t i o n s were employed, w i t h 50 mu-moles o f coenzyme A, and 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 umole, and 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 , where n o t e d . The p h o s p h o h y d r o l a s e used was a 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 . 36 I I I . 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 A s u r v e y o f t h e p e r t i n e n t l i t e r a t u r e showed t h a t t h e r e a r e s e v e r a l bonds i n t h e coenzyme A m o l e c u l e t h a t a r e known t o be s u s c e p t i b l e t o a t t a c k by enzymes f r o m v a r i o u s s o u r c e s . T h i s i s summarized i n F i g u r e 5. S e v e r a l 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 (EC 3.1.3.2) (44), t h e a l k a l i n e p h o s p h o h y d r o l a s e (EC 3.1.3.1) o f E s c h e r i c h i a c o l i (45) and t h e 3 ' - n u c l e o t i d e p h o s p h o h y d r o l a s e o f p l a n t t i s s u e (EC 3.1.3.6) (46) a r e known t o h y d r o l y z e t h e 3 '-phosphomonoester l i n k a g e o f coenzyme A ( p o i n t a ) , f o r m i n g 3 '-dephosphocoenzyme A and o r t h o p h o s p h a t e . These enzymes have, i n f a c t , been employed f o r t h e p r e p a r a t i o n o f dephosphocoenzyme A (47,48). P y r o p h o s p h a t a s e s (EC 3.6 .1.9) f r o m s n a k e venom (47,49), p l a n t s o u r c e s (50) and ani m a l t i s s u e s (19) a r e known t o h y d r o l y z e t h e p y r o p h o s p h a t e bond o f coenzyme 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 and 3' , 5 ' -diphosphoadeno-s i n e (19,47,51). P u r i f i e d i n t e s t i n a l p h o s p h a t a s e (52) i s a l s o known t o e x t e n s i v e l y de-gra d e coenzyme A. T h i s enzyme h y d r o l y z e s t h e p y r o p h o s p h a t e bond ( p o i n t c ) and a l s o t h e phosphomonoester bonds i n t h e r e s u l t i n g f r a g m e n t s ( p o i n t s a, b and d j , 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 (53,54). In a d d i t i o n , Novel 1i and co - w o r k e r s (19) d e s c r i b e d a p e p t i d a s e i n p i g e o n and c h i c k e n l i v e r e x t r a c t s w h i c h h y d r o l y z e d 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 and p-mercaptoethylamine ( c y s t e a m i n e ) m o i e t i e s o f c o -enzyme A ( p o i n t e ) , f o r m i n g p-mercaptoethylamine and p ' - 3 ' - p h o s p h o a d e n o s i n e -2 5 ' , P - p a n t o t h e n i c a c i d - 4 ' p y r o p h o s p h a t e . 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 a t w h i c h t h e enzyme c o u l d a c t , i t was i m p e r a t i v e t o c h a r a c t e r i z e t h e p r o d u c t o f t h e r e a c t i o n . T h i s was done by chromatography on pa p e r and on E C T E O L A - c e l l u l o s e . In one e x p e r i m e n t , t r i -NH 2 37 I HC--N CH '\? ?A H V c 7 H i i HO 0 (a) i _ . I 0 — P — 0 (b) (c) (d) CH 2 -0-P-9 -P-0-CH 2 0. 3 1 , 5 * - d i p h o s p h o a d e n o s i n e 0 -C-(CH 3 ) 2 H-C-OH l / \ 0 NH-CH 2 0 / NH-CH 2 -CH 2 -SH ^ P a n t o t h e n i c i — p-mercaptoethyl amine-*] a c i d H ( c y s t earn i n e ) i P a n t e t h e i n e • 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 on coenzyme 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 , 400 umoles; coenzyme A, 4 umoles; and 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 , i n a t o t a l volume o f 1.5 ml were i n c u b a t e d a t 30°. A f t e r 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 a d d i t i o n o f 0.5 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 . Denatured p r o t e i n was removed by c e n t r i f u g a t i o n . The 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 7.2 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 . A f t e r 30 m i n u t e s 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 remove 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 l y o p h i l i z e d . The d r y r e s i d u e was d i s s o l v e d i n a s m a l l volume o f w a t e r and a g a i n l y o p h i l i z e d . The r e s u l t i n g powder was t h o r -o u g h l y e x t r a c t e d w i t h 0.5 ml o f w a t e r and i n s o l u b l e m a t t e r was removed by c e n t r i f u g a t i o n . A c o n t r o l t u b e i n w h i c h t h e enzyme f r a c t i o n was added a f t e r t h e p e r c h l o r i c a c i d ; was t r e a t e d i n e x a c t l y t h e same manner. A l i q u o t s o f t h e e x p e r i m e n t a l and c o n t r o l e x t r a c t s were a p p l i e d t o Whatman No. 1 f i l t e r p a p e r and chromatographed i n two s o l v e n t s y s t e m s . S o l v e n t s y s t e m I c o n s i s t -ed o f e t h a n o l , 150; 1 M ammonium a c e t a t e , pH 7.5, 60; and 2-mercapto e t h a n o l , 2. S o l v e n t s y s t e m 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 , 114; 15 N_ ammonium h y d r o x -i d e , 8: w a t e r , 78; and 2 - m e r c a p t o e t h a n o l , 2. The chromatograms were d e v e l o p e d 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 hours at room t e m p e r a t u r e . The r e s u l t s a r e r e p r o d u c e d d i a g r a m a t i c a l l y i n F i g u r e 6. In s o l v e n t s y s t e m I , t h e r e was good s e p a r a t i o n o f t h e s t a r t i n g m a t e r i a l ( r e d u c e d coenzyme 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 0.31). The p r o d u c t moved a t t h e same r a t e as a u t h e n t i c dephosphocoenzyme A. However, t h i s s o l v e n t s y s t e m does not s e p a r a t e reduced coenzyme A and 3 ' , 5 ' - d i p h o s p h o a d e n o s i n e , a n o t h e r p o s s i b l e p r o d u c t (55). S o l v e n t s y s t e m II 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 (Rp 0.55) and t h e p r o d u c t (R_ 0 .63) , 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 3 '-dephospho-39 1 2 3 4 0 0 0 ° 1 2 3 4 0 0 Origin 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 by paper chromatography. Sample 1, e x p e r i -m e n t a l ; sample 2, c o n t r o l ; sample 3, reduced c o -enzyme A; sample k, reduced dephosphocoenzyme 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 System I; chromatogram B, t h o s e ob-t a i n e d w i t h s o l v e n t System I I . For d e t a i l s s e e t e x t . 40 coenzyme A. However, 3 ' , 5 ' ~ d i p h o s p h o a d e n o s i n e , i f p r e s e n t , would have moved at 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 coenzyme A moved. No u l t r a v i o l e t a b s o r b i n g m a t e r i a l c o r r e s p o n d i n g t o d i p h o s p h o a d e n o s i n e was de-t e c t e d . 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 have a l -lowed 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 absence o f 5'-AMP, s i n c e coenzyme A and 5'"AMP move t o g e t h e r i n most 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 coenzyme A and de-phosphocoenzyme A might account f o r t h e f a i n t s l o w l y - m o v i n g s p o t s o b s e r v e d i n s o l v e n t s y s t e m 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-coenzyme 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 ion-exchange chromatography. T r i e t h y l a m m o n i u m a c e t a t e b u f f e r , pH 6 .0 , 200 umoles; c o -enzyme A, 10 umoles; and 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 , i n a O t o t a l volume o f 2.8 ml were i n c u b a t e d a t 30 . A f t e r 30 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 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 a c i d . Denatured p r o t e i n was removed by c e n t r i f u g a t i o n . The 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 -ed t o pH 7.2 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 f t e r 30 m i n u t e s 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 remove 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 . The 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 c a r e f u l 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 w i t h 3 ml o f 2-mercapto-e t h a n o l and l e f t a t room t e m p e r a t u r e f o r k h o u r s . 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 e f f e c t a c o m p l e t e 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 (55). The 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 w a t e r and a p p l i e d t o a 2.0 cm by 15 cm column o f E C T E O L A - c e l l u l o s e i n t h e c h l o r i d e f o r m . The c o l -umn was washed w i t h 150 ml o f d i s t i l l e d w a t e r , 250 ml o f 0.02 M ammonium c h l o r i d e and 150 ml o f d i s t i l l e d w a t e r . 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 l i n e a r c h l o r i d e g r a d i e n t . The m i x i n g f l a s k i n i t i a l l y c o n t a i n e d one l i t e r o f 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 i n i t i a l l y c o n t a i n e d one l i t e r o f 0.15 M l i t h i u m c h l o r i d e i n 0.003 N h y d r o c h l o r i c a c i d . F r a c t i o n s o f 10 ml each were c o l l e c t e d at a f l o w r a t e o f 60 ml p e r h o u r . An e l u t i o n d i a g r a m i s p r e s e n t e d i n F i g u r e 7. The s o l i d - l i n e p r o f i l e r e p r e s e n t s t h e r e s u l t s o f t h i s e x p e r i m e n t . The d a s h e d - 1 i n e p r o f i l e r e p r e s e n t s t h e r e s u l t s o f a c o n t r o l e x p e r i m e n t c a r r i e d out i n p r e c i s e l y t h e same manner, e x c e p t t h a t t h e enzyme was added a f t e r t h e p e r c h l o r i c a c i d . The f r a c t i o n s c o m p r i s i n g each peak were p o o l e d and c o n c e n t r a t e d t o a s m a l l volume i n vacuo. A d e n i n e c o n t e n t was e s -t i m a t e d by u l t r a v i o l e t a b s o r p t i o n , assuming a m o l a r e x t i n c t i o n c o e f f i c i e n t o f 1.46 x 10** a t 260 mu i n 0.01 N h y d r o c h l o r i c a c i d ( 5 6 ) . R i b o s e c o n t e n t was d e t e r m i n e d 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 r e a c t i o n ( 5 7 ) . T o t a l p h o s p h a t e c o n t e n t was d e t e r m i n e d by t h e method o f D r y e r et_ a]_. ( 3 7 ) . S u l f -h y d r y l c o n t e n t , b e f o r e and a f t e r r e d u c t i o n w i t h sodium b o r o h y d r i d e , was measured 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 ( 5 8 ) , u s i n g reduced g l u t a t h i o n e as a s t a n d a r d . The 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 V M . The 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 I were 1.00:1.04: 1.82:0 . 9 2 . These v a l u e s c o r r e s p o n d q u i t e c l o s e l y t o t h o s e w h i c h one would exp e c t f o r dephosphocoenzyme A. The v a l u e s f o r t o t a l p h o s p h a t e and t h i o l a r e somewhat low, however. T h i s c o u l d p o s s i b l y be due t o t h e p r e s e n c e o f a s m a l l amount o f 5'-AMP. (5 , -AMP would have been e l u t e d f r o m t h e column im-m e d i a t e l y b e f o r e reduced 3'-dephosphocoenzyme A under t h e c o n d i t i o n s o f t h e e x p e r i m e n t ( 5 5 ) . ) The c o r r e s p o n d i n g r a t i o s f o r Peak I I , b e f o r e and a f t e r r e d u c t i o n , were 1.00:1.02:2.03:0.00 and 1.00:1.02:2.03:1.05, r e s p e c t i v e l y . These r a t i o s a r e a l m o s t i d e n t i c a l t o t h o s e w h i c h one would e x p e c t t o o b t a i n f o r reduced dephosphocoenzyme A (Peak I) and o x i d i z e d dephosphocoenzyme A (Peak I I ) . F u r t h e r e v i d e n c e i s found i n t h e s a l t c o n c e n t r a t i o n s r e q u i r e d t o 5 0 _ 4 0 o CD £1 301 20 (CoASH) 10 i i .i i i i 100 150 FRACTION NUMBER F i g u r e 7. 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 by ion-exchange c h r o m a t o g r a p h y . F o r e x p l a n a -t i o n s e e t e x t . 43 TABLE V I I C o m p o s i t i o n o f t h e p r o d u c t o f t h e r e a c t i o n . T o t a l A d e n i n e * R i b o s e ' Phosphate 1 S u l f h y d r y l umoles/ml Peak A 1.00 1.04 1.83 0.92 Peak B (1) B e f o r e r e d u c t i o n 0.95 0.97 1.93 0.0 (2) A f t e r r e d u c t i o n - 1.05 F o r d e t a i l s , s e e E x p e r i m e n t a l Methods. 44 e l u t e t h e s e two peaks from t h e column - a p p r o x i m a t e l y 0.02 M f o r Peak I and 0.04 M f o r Peak i t (55). From t h e combined o p t i c a l d e n s i t i e s o f t h e two peaks a t 260 mu, i t was c a l c u l a t e d t h a t t h e two peaks a c c o u n t e d f o r 95 p e r c e n t o f t h e o p t i c a l d e n s i t y o f t h e o r i g i n a l coenzyme A s o l u t i o n . It i s 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 p r o d u c t . The r e s u l t s o f t h e s e c h r o m a t o g r a p h i c s t u d i e s p r o v i d e s t r o n g e v i d e n c e t h a t t h e i n h i b i t o r enzyme i s a "coenzyme A 3 1 - p h o s p h o h y d r o l a s e . " It i s a l s o a p p a r e n t t h a t t h e i n h i b i t o r enzyme p r e p a r a t i o n used f o r t h e s e e x p e r i m e n t s i s v i r t u a l l y 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 coenzyme A i n any o t h e r man-n e r . IV. The Coenzyme A 3 ' - P h o s p h o h y d r o l a s e A s s a y . When t h e s i t e o f t h e i n h i b i t o r e f f e c t was e s t a b l i s h e d , a d i r e c t a s s a y based upon t h e d i s a p p e a r a n c e o f coenzyme 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 d i r -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 coenzyme A under 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 heat and r e s i d u a l coenzyme A was meas-ur e d 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 i s g i v e n under E x p e r i m e n t a l Methods. The a s s a y method i s f a r f r o m i d e a l , s i n c e s e v e r a l c r i t i c a l a d d i -t i o n s a r e i n v o l v e d . However, i t i s s a t i s f a c t o r y i n t h a t i t i s f a i r l y r a p i d and i s i n e x p e n s i v e . (One a l t e r n a t i v e a s s a y method would i n v o l v e t h e measure-ment o f o r t h o p h o s p h a t e r e l e a s e d from coenzyme A. T h i s , however, would r e -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 become p r o h i b i -t i v e . ) Under 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 coenzyme A i n a c t i -v a t e d i s p r o p o r t i o n a l t o b o t h t i m e ( 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 not more t h a n 40 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 . P u r i f i c a t i o n 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 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 t i 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 coenzyme A phos-p h o h y d r o l a s e a s s a y were employed. The enzyme f r a c t i o n used 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 0-4 0-6 PROTEIN (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 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 were 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 f r a c t i o n . The enzyme f r a c t i o n s were p r e p a r e d as d e s c r i b e d under E x p e r i m e n t a l Methods. 47 t i o n p r o c e d u r e i s g i v e n i n T a b l e V I I I . The p r o c e d u r e i s q u i t e r e p r o d u c i b l e , and r e g u l a r l y g i v e s a 250- t o 3 0 0 - f o l d p u r i f i c a t i o n . The o n l y two s t e p s i n w h i c h p r o b l e m s o c c a s i o n a l l y a r o s e were t h e e t h a n o l f r a c t i o n a t i o n and t h e chromatography on D E A E - c e l l u l o s e . In b o t h o f t h e s e s t e p s , c o n t r o l o f pH i s c r i t i c a l . I f t h e pH i s a p p r e c i a b l y above 6.0 d u r i n g e t h a n o l p r e c i p i t a t i o n o f t h e enzyme, enzyme a c t i v i t y i s found i n almost a l l f r a c t i o n s , and v e r y l i t t l e p u r i f i c a t i o n i s a c h i e v e d . The p r e s e n c e o f d i v a l e n t c a t i o n i s a l s o i m p o r t a n t . In t h e absence o f added 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 a r e r e q u i r e d t o p r e c i p i t a t e t h e enzyme. Under t h e s e c i r c u m s t a n c e s , s t a b i l i t y becomes a p r o b l e m , and r e c o v e r i e s o f a c t i v i t y a r e low. In some o f t h e e a r l i e r p u r i f i c a -t i o n s t u d i e s , z i n c i o n s were added f o r t h i s s t e p . T h i s was q u i t e s a t i s f a c t o r y f o r s m a l l - s c a l e f r a c t i o n a t i o n s . However, when t h e p r o c e d u r e was s c a l e d up i t was found t h a t removal o f t h e z i n c p r e s e n t e d a p r o b l e m . For t h i s r e a s o n , t h e z i n c was r e p l a c e d by magnesium. In t h e f i n a l s t e p , chromatography on D E A E - c e l l u l o s e , c o n t r o l o f pH i s e x t r e m e l y i m p o r t a n t . A t y p i c a l chromatogram i s shown i n F i g u r e 10. I t can be seen t h a t under t h e c o n d i t i o n s employed t h e b u l k o f t h e p r o t e i n i n t h e e t h a n o l f r a c t i o n i s not r e t a i n e d i n t h e column and appears i n t h e column e f f l u e n t as a " b r e a k t h r o u g h " peak. The p h o s p h o h y d r o l a s e , however, i s s u f -f i c i e n t l y r e t a r d e d by t h e exchanger t o y i e l d a 15- t o 2 0 - f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y . I f t h e pH i s lowered t o 6.0 t o 6.5, t h e enzyme i s r e t a r d -ed o n l y s l i g h t l y on p a s s i n g t h r o u g h t h e column, and t h e r e i s c o n s i d e r a b l e o v e r l a p o f t h e p r o t e i n and enzyme a c t i v i t y p e a k s . On t h e o t h e r hand, i f chromatography i s c a r r i e d out a t pH 7.3 t o 7.5, t h e r e i s a v e r y w i d e s e p a r a -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 . However, under t h e s e c o n -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 on D E A E - c e l l u l o s e . p r o t e i n c o n c e n t r a t i o n ; , enzyme a c t i v i t y . F or d e t a i l s , s e e E x p e r i m e n t a l Methods. 49 TABLE V I I I P u r i f i c 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 . F r a c t i o n I n i t i a l e x t r a c t AS-I 40-80 F i r s t g e l s u p ' t . Second g e l s u p ' t , AS-11 EtOH p p t . D E A E - c e l l u l o s e e l u a t e S p e c i f i c T o t a l P r o t e i n a c t i v i t y a c t i v i t y R ecovery From O v e r a l l p r e c e d i n g s t e p mg. mU/mq mU p e r c e n t 27,000 a 6.5 171,500 a (100) (100) 12,500 9 111,500 65 65 6,600 16.5 110,000 100 3,300 30 99,000 58 90 2,850 30 83,000 49 84 765 82 62 ,500 37 75 1500b 850° 60 d 22 From 200 g c h i c k e n l i v e r . E s t i m a t e d 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 f r a c -t i o n was t o o low 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 p r o t e i n (1450 m i l l i u n i t s ) were a p p l i e d t o a 1.0 x 25 cm column. 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 . However, t h e l e a d i n g edge o f t h e enzyme peak 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 peak, so t h a t t h e f i r s t few 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 low 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 column, and 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 fo u n d i n a v e r y l a r g e volume i n t h e " t a i l " o f t h e enzyme peak; 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 v e r y s l o w l y and i n a v e r y l a r g e volume. ( I n one 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 (kO mg p r o t e i n ) were chromatographed on a 2.0 cm by 25 cm column o f D E A E - c e l l u l o s e a t pH 7.3. The 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 d e n s i t y a t 280 mu, was e l u t e d f r o m t h e column i n f r a c t i o n s 15 t o 20. The enzyme was e l u t e d f r o m t h e column i n f r a c t i o n s 120 t o 260, i n a t o t a l volume o f 700 ml.) T h i s 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 overcome by use o f a s a l t g r a d i e n t o r by 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 p r o t e i n peak has been o b t a i n e d . However, t h i s i s u n n e c e s s a r y . Two peaks o f coenzyme A p h o s p h o h y d r o l a s e can be seen i n F i g u r e 10. T h i s has o c c u r r e d i n e v e r y i n s t a n c e . Other w o r k e r s have r e p o r t e d s i m i l a r f i n d i n g s w i t h a c i d p h o s p h o h y d r o l a s e s (59). The r e a s o n f o r t h e s p l i t t i n g o f t h e enzyme a c t i v i t y i n t o two d i s t i n c t peaks i s unknown. E f f e c t o f d i v a l e n t c a t i o n s . A l t h o u g h t h e a l k a l i n e p h o s p h o h y d r o l a s e s 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 dependent upon t h e p r e s e n c e o f d i v a l e n t c a t i o n f o r maximal a c t i v i t y and t o be i n h i b i t e d by m e t a l - b i n d i n g a g e n t s , t h e s i t u a -t i o n w i t h r e s p e c t t o t h e a c i d p h o s p h o h y d r o l a s e s i s l e s s c l e a r . (See t h e monographs by Schmidt (60) and Stadtman ( 6 1 ) . ) Some a c i d p h o s p h o h y d r o l a s e s - f o r example, t h e 0 - p h o s p h o s e r i n e p h o s p h o h y d r o l a s e o f c h i c k e n l i v e r (33) and r a t l i v e r ( 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 (63) - appear t o e x h i b i t a d e f i n i t e magnesium r e q u i r e m e n t . On t h e o t h e r hand, 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 e r y t h r o -c y t e s - do not appear t o r e q u i r e magnesium, nor a r e t h e y i n h i b i t e d i n t h e p r e s e n c e o f m e t a l - b i n d i n g a g e n t s . It was t h e r e f o r e o f i n t e r e s t t o d e t e r m i n e t h e e f f e c t s o f magnesium io n s and o f c h e l a t i n g a g e n t s upon 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 . 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 not i n -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 l i v e r enzyme. In f a c t , i t was f o u n d t h a t t h e a d d i t i o n o f 5 mM magnesium c h l o r i d e t o t h e as s a y produced 40 p e r c e n t d e p r e s s i o n o f enzyme a c t i v i t y . F u r t h e r m o r e , 1 mM EDTA d i d not s i g n i f i c a n t l y a l t e r t h e a c t i v i t y o f t h e enzyme. On t h e b a s i s o f t h e s e o b s e r v a t i o n s , i t would appear t h a t t h e enzyme i s n e i t h e r dependent upon nor a c t i v a t e d by magnesium i o n s . However, i t was s t i l l p o s s i b l e t h a t t h e enzyme c o n t a i n e d s u f f i c i e n t bound metal t o p r o d u c e maximal r e a c t i o n r a t e s w i t h o u t added c a t i o n , and t h a t t h e bound m e t a l was not r e a d i l y a c c e s s i b l e t o EDTA. To t e s t t h i s p o s s i b i l i t y , an a l i q u o t o f t h e e t h a n o l f r a c t i o n (33 mg p r o t e i n ) was s u b j e c t e d t o e x h a u s t i v e d i a l y s i s vs_ an E D T A - c o n t a i n i n g s o l u t i o n . The c o n t i n u o u s f l o w d i a l y s i s a p p a r a t u s used was based upon t h a t d e s c r i b e d by Noltmann et, a]_. ( 3 8 ) . The d i a l y s i s v e s s e l was a beaker c o n t a i n i n g 800 ml 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 7.0, and 1 mM EDTA. The r e s e r v o i r c o n -t a i n e d 18 l i t e r s o f t h e same b u f f e r . The b u f f e r was a l l o w e d t o f l o w t h r o u g h t h e s y s t e m o v e r a p e r i o d o f 96 h o u r s . ( 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 remove enzyme-bound m e t a l ions ( 6 4 ) . ) The a p p a r a t u s was t h e n t h o r o u g h l y f l u s h e d w i t h w a t e r , and s e t up a g a i n w i t h 10 mM T r i s b u f f e r w i t h o u t EDTA. 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 o f 48 hours t o remove;. EDTA fr o m t h e enzyme s o l u t i o n . The r e s u l t s o f t h i s e x p e r i m e n t a r e summarized i n T a b l e IX. The enzyme used f o r t h e e x h a u s t i v e d i a l y s i s had been p r e p a r e d t h r e e weeks p r e v i o u s l y and s t o r e d a t -20°. I t can be seen f r o m t h e t a b l e t h a t t h i s f r a c t i o n had a p p a r e n t l y 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 , and t h a t d i a l y s i s vs EDTA had r e s t o r e d t h e enzyme a c t i v i t y t o i t s i n i t i a l l e v e l . In v i e w o f t h e s e f i n d i n g s , i t i s q u i t e u n l i k e l y t h a t t h i s enzyme r e q u i r e s d i -v a l e n t c a t i o n . TABLE IX 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 ys_ EDTA s o l u t i o n upon a c t i v i t y o f t h e coenzyme A 3'-phosphohydrolase. ' S t a t u s o f enzyme { T o t a l e n z y m a t i c a c t i v i t y ' mi 11iun i t s F r e s h l y p r e p a r e d 470 F r o z e n 21 days 375 E D T A - d i a l y z e d 460 Fo r e x p l a n a t i o n s e e t e x t . 53 E f f e c t o f pH. The e f f e c t o f pH upon 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 l i v e r enzyme i s shown i n F i g u r e 11. Most a c i d p h o s p h o h y d r o l a s e s show a f a i r l y b r oad optimum between pH 4 and pH 6, t h e p r e c i s e optimum de-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 and b u f f e r em-p l o y e d . To t h e b e s t o f t h e a u t h o r ' s knowledge, no p h o s p h o h y d r o l a s e w i t h o p t i m a l a c t i v i t y below pH 4 has been p r e v i o u s l y r e p o r t e d i n an i m a l t i s s u e s , a l t h o u g h t h e 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 o f y e a s t i s r e p o r t e d t o e x h i b i t o p t i m a l a c t i v i t y a t pH 4 . 0 (60). T h i s u n i q u e f e a t u r e w i l l be d i s c u s s e d l a t e r i n more d e t a i l . It 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 has i t s optimum a t pH 3 .6 , t h e a s s a y have 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 t e c h n i c a l r e a s o n s . I n t r a c e l l u l a r l o c a l i z a t i o n . 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 s u b c e l l u l a r f r a c t i o n s . F or example, 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 i s l a r g e l y a s s o c i a t e d w i t h m i t o c h o n d r i a (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) and t h e major 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 p h o s p h o h y d r o l a s e o f r a t l i v e r w i t h l y s o s o m e s . In f a c t , i t appears t h a t most p h o s p h o h y d r o l a s e s a r e t i g h t l y bound t o p a r t i c u l a t e c e l l u l a r components (68). In v i e w o f t h i s , t h e i n t r a c e l l u l a r l o c a l i z 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 was exam-i n e d . 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 i s o l a t e d f r o m a s u c r o s e homog-e n a t e 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 . A summary o f t h e p r o c e d u r e used i s g i v e n i n " f l o w s h e e t " f o r m i n F i g u r e 12. S i n c e many enzymes a s s o c i a t e d w i t h p a r t i c u l a t e s t r u c t u r e s e x h i b i t " l a t e n c y " - t h a t i s , t h e i r a c t i v i t y does not become ap p a r e n t u n t i l t h e s e s t r u c t u r e s a r e d i s r u p t e d (41 ,69,70,71) - t h e 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 X-100 (a n o n i o n i c d e t e r g e n t o f t h e p o l y -o x y a l k y l e n e t y p e ) b e f o r e a s s a y (69). The 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 i • • . • ! • . i i 3 4 5 6 7 8 pH F i g u r e 11. 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 pH. The enzyme f r a c t i o n employed 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 column. 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 were employed, 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 ; • • , 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 o, T r i s h y d r o c h l o r i d e b u f f e r . Companion c o n t r o l s w i t h o u t enzyme were 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 each pH t o c o r r e c t f o r nonenzymic i n a c t i v a t i o n o f coenzyme A. 10 p e r c e n t homogenate 750 x £, 10 m i n u t e s I P e l l e t 1 1 N u c l e a r " f r a c t i o n S u p e r n a t e - Sj 5,000 x £, 10 m i n u t e s P e l l e t P e l l e t M i t o c h o n d r i a I P e l l e t I Suspend i n 0,7 M s u c r o s e 6,000 x £, *30 minutes S u p e r n a t e 14,000 x £, 30 minutes P e l l e t L y s o s o m e - r i c h  f r a c t i o n I S u p e r n a t e S u p e r n a t e - S 2 16,300 x £, 20 m i n u t e s S u p e r n a t e - S^ 66,000 x £, 90 m i n u t e s P e l l e t Microsomes S u p e r n a t e - S4 S o l u b l e f r a c t i o n F i g u r e 12. " 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 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 . F or d e t a i l s s e e E x p e r i m e n t a l Methods. 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 3 ' - p h o s p h o h y d r o l a s e . Coenzyme A , p - N i t r o p h e n y l 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 p h o s p h o h y d r o l a s e R i b o n u c l e a s e F r a c t i o n S p e c i f i c a c t i v i t y R e c overy Spec i f i c a c t i v i t y R e c overy Spec i f i c a c t i v i t y mU/mg p r o t e i n p e r c e n t mU/mg p r o t e i n p e r c e n t mU/mg a p r o t e i n Homogenate 1.2 10 0 b 85 100 -Nuc1e i 1.3 27 60 16 1.5 M i t o c h o n d r i a 1.2 16 82 15 1.4 Lysosomes 4.7 14 170 8 2.4 Microsomes 2.4 14 200 18 0.6 " S o l u b l e " f r a c t i o n Q.5 20 9 0.7 R i b o n u c l e a s e was measured 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 method o f A n f i n s e n e t aj_. (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 d i a l y z e d y e a s t r i b o n u c l e i c a c i d . U n d i g e s t e d r i b o n u c l e i c a c i d i s p r e -c i p i t a t e d w i t h u r a n i u m 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. The r i b o -n u c l e a s e a c t i v i t i e s i n t h i s t a b l e a r e e x p r e s s e d i n a r b i t r a r y u n i t s . A r b i t r a r i l y t a k e n as 100 p e r c e n t . 57 The a c t i v i t i e s o f t h e f r a c t i o n s a g a i n s t p - n i t r o p h e n y l p h o s p h a t e were a l s o d e t e r m i n e d , as a measure 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 l e v e l s . I t can be seen t h a t t h e h i g h e s t 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 was found i n t h e lysosome f r a c t i o n , and t h a t t h e m i c r o s o m a l f r a c t i o n a l s o e x h i b i t e d a f a i r l y h i g h l e v e l o f a c t i v i t y . 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 each f r a c t i o n , r i b o n u c l e a s e , w h i c h i s r e p o r t e d l y l o c a t e d e x c l u s i v e l y i n t h e l y s o -somes (73), was a l s o measured i n each f r a c t i o n . From t h e r i b o n u c l e a s e a s s a y s , i t might be i n f e r r e d 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 o f t h e n u c l e a r and m i t o c h o n d r i a l f r a c t i o n s 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 c o n t a m i n a t i o n . However, t h e m i c r o s o m a l f r a c t i o n , w h i c h has an a p p r e c i a b l e l e v e l o f t h e enzyme, i s alm o s t d e v o i d o f r i b o n u c l e a s e a c t i v i t y . From t h i s 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 , i t would appear t h a t t h e b u l k o f t h e coenzyme A p h o s p h o h y d r o l a s e 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 -c h o n d r i a l f r a c t i o n s . S u b s t r a t e s p e c i f i c i t y . In an attempt t o d e t e r m i n e whether t h e i n -a c t i v a t i o n o f coenzyme A was due t o t h e a c t i o n o f a s p e c i f i c coenzyme A phos-p h o h y d r o l a s e o r m e r e l y 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 phospho-h y d r o l a s e , a s t u d y 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 was c a r r i e d o u t . The r e s u l t s o f t h i s s t u d y a r e summarized i n T a b l e X I . For p u r -poses 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 a r e g i v e 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. The a c t u a l s p e c i f i c a c t i v i t y o f each f r a c t i o n i n t h e coenzyme A i n a c t i v a t i o n a s s a y i s g i v e n i n p a r e n t h e s e s ( m i l l i u n i t s p e r mg p r o t e i n ) . From t h e d a t a , i t can be seen t h a t t h e 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 a g a i n 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 were c a r r i e d t h r o u g h t h e p r o c e d u r e i n a p a r a l l e l manner, w h i l e t h e s p e c i f i c a c t i v -i t y v a r i e d o v e r a 1 2 0 - f o l d range. 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 a c i d 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 i n c h i c k e n l i v e r f r a c t i o n s AS-I 40-80 , EtOH 45-55 p e r c e n t , EtOH 24-45 p e r c e n t DEAE e l u a t e S u b s t r a t e R e l a t i v e 3 a c t i v i t y ' PH 3 . 6 b pH 6.0 R e l a t i v e a c t i v i t y ' PH 3.6 pH 6.0 R e l a t i v e a c t i v i t y • PH 3.6 pH 6.0 R e l a t i v e a c t i v i t y pH 3.6 pH 6.0 Coenzyme A 100 ( 2 . 5 ) c - 100 (20) - 100 (60) - 100 2.00 (300) d p-NPP - - 350 - 360 - 330 0.85 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 G l u c o s e 6-phosphate 110 1.00 115 1.15 105 1.20 115 1.05 p-glycero-p h o s p h a t e 195 0.90 165 1.10 180 a. 170 0.95 R e l a t i v e a c t i v i t i e s a r e g i v e n i n comparison t o t h e a c t i v i t y a g a i n s t coenzyme A, w h i c h was a r b i t r a r i l y t a k e n as 100. The r a t i o , pH 3.6/pH 6 .0 , r e f e r s t o t h e r a t i o o f t h e r a t e s o f h y d r o l y s i s o f t h e s u b s t r a t e a t t h e s e two pH v a l u e s . 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 i n t h e coenzyme A i n a c t i v a -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 . T h i s v a l u e i s a p p r o x i m a t e o n l y . T h i s f r a c t i o n was v e r y d i l u t e , and t h e p r o t e i n c o n c e n t r a t i o n c o u l d not be a c c u r a t e l y d e t e r m i n e d . 59 p u r i f i e d 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-p h o s p h a t e a t a r e l a t i v e l y r a p i d r a t e - a p p r o x i m a t e l y t w o - t h i r d s o f t h e r a t e at w h i c h i t h y d r o l y z e d p-glycerophosphate. T h i s i s i n marked c o n t r a s t t o f i n d i n g s w i t h o t h e r p h o s p h o h y d r o l a s e s . For example, t h e p u r i f i e d p r o s t a t i c 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-phosphate p h o s p h o h y d r o l a s e a c t i v i t y ; 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 enzymes ( 6 0 , 7 4 ) . In t h e t a b l e , t h e r a t i o s o f t h e h y d r o l y t i c a c t i v i t i e s a t pH 3.6 and pH 6.0 a r e a l s o g i v e n . 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 not a p p r e c i a b l y d i f f e r e n t f r o m u n i t y . T h i s i s q u i t e c h a r a c t e r i s t i c o f a c i d p h o s p h o h y d r o l a s e s ( 6 0 ) . The s h a r p optimum a t pH 3.6 appears t o be u n i q u e t o 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 . A n o t h e r approach 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 t h e m o n i t o r i n g o f a l l f r a c t i o n s o b t a i n e d d u r i n g f r a c t i o n a t i o n s f o r a c t i v i t y a g a i n s t p - n i t r o p h e n y l p h o s p h a t e , t a k e n as a measure o f n o n - s p e c i f i c a c i d p h o s p h o h y d r o l a s e a c t i v i t y , as w e l l as f o r a c t i v i t y a g a i n s t coenzyme A. In a l m o s t e v e r y f r a c t i o n a t i o n r u n , t h e r a t i o 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 -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 remained c o n s t a n t a t 2.8 t o 3.2 o v e r t h e e n t i r e 3 0 0-fold range o f s p e c i f i c a c t i v i t i e s . T h i s was t r u e even o f t h e i n d i v i d u a l f r a c t i o n s o b t a i n e d d u r i n g chromatography on D E A E - c e l l u l o s e ( F i g . 13 ) . 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 o t h e r w i s e r e l a t i v e l y 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 f r o m t h e b u l k o f t h e p - n i t r o p h e n y l 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 a c h i e v e d . T h i s e x p e r i m e n t i s summarized i n T a b l e X l l . The r a t i o s o f t h e two a c t i v i t -i e s were 35 i n t h e u n d i a l y z e d e x t r a c t , 19.5 i n t h e d i a l y z e d e x t r a c t , 10 i n t h e f i r s t s a l t f r a c t i o n ( c o l l e c t e d between 40 and 80 p e r c e n t s a t u r a t i o n w i t h ammonium s u l f a t e ) , 4 . 5 i n t h e second s a l t f r a c t i o n , and 3.5 i n each o f t h e 8 0 >-> 4 0 4 0 6 0 8 0 FRACTION 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 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 chromato-graphy on D E A E - c e l l u l o s e . These d a t a a r e t a k e n f r o m t h e column d e p i c t e d i n F i g u r e 10. , p - n i t r o p h e n y l 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 ; , coenzyme A phospho h y d r o l a s e . 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 p h o s p h o h y d r o l a s e , p - N i t r o p h e n y l 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 F r a c t i on I n i t i a l e x t r a c t I n i t i a l e x t r a c t ( a f t e r d i a l y s i s ) b AS-I 3 0 - 4 0 c AS-I 40-80 ASf 11 E t h a n o l , 24 t o 35 p e r c e n t E t h a n o l , 35 t o 43 p e r c e n t E t h a n o l , 43 t o 55 p e r c e n t Spec i f i c a c t i v i t y mU/mg p r o t e i n 1.7 1.7 0.3 2.5 11 18 60 20 Recovery p e r c e n t 1 0 0 a 60 23 3 12 8 S p e c i f i c a c t i v i t y mU/mg p r o t e i n 60 33 40 26 49 66 205 70 Recovery p e r c e n t 1 0 0 a 14 18 3 1 1 1 A r b i t r a r i l y t a k e n as 100 p e r c e n t . 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 hours vs 300 volumes 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, and 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 . F o r 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 , s e e E x p e r i m e n t a l Methods. 61 t h r e e e t h a n o l f r a c t i o n s . It s h o u l d a l s o be n o t e d t h a t one f r a c t i o n , t h e "AS-I 30-40" f r a c t i o n , e x h i b i t e d c o n s i d e r a b l e a c t i v i t y a g a i n s t p - n i t r o -p h e n y l p h o s p h a t e , but was e s s e n t i a l l y i n e r t a g a i n s t coenzyme A. On t h e b a s i s o f t h e s e d a t a , t h e a u t h o r f e e l s t h a t i 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 i s a s s o c i a t e d w i t h a 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 i t i s a t l e a s t not a s s o c i a t e d w i t h a l 1 o f t h e 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 a c t i v i t y o f c h i c k e n l i v e r e x t r a c t s . To p r o v i d e a d e f i n i t e answer t o t h i s p r o b l e m , i t would be n e c e s s a r y t o o b t a i n at l e a s t one 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 but not a g a i n s t t h e o t h e r phosphomonoesters. T h i s has not y e t been a c h i e v e d . S t a b i l i t y o f t h e enzyme. As was mentioned e a r l i e r , t h e coenzyme A O p h o s p h o h y d r o l a s e a c t i v i t y appeared t o be s t a b l e t o s t o r a g e at -20 a t a l l s t a g e s 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 r e p e a t e d f r e e z i n g and thaw-i n g . One e t h a n o l 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 , c o n t a i n -ed 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 thawed. When t h i s i n -s o l u b l e m a t e r i a l was removed by c e n t r i f u g a t i o n and t h e s u p e r n a t a n t s o l u t i o n was a s s a y e d f o r " i n h i b i t o r enzyme" a c t i v i t y , i t was found t h a t a t w o - f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y had been e f f e c t e d . Many r e p o r t s have appeared on t h e marked s u s c e p t i b i l i t y o f phospho-h y d r o l a s e s t o s u r f a c e d e n a t u r a t i o n (75"77). D u r i n g t h e c o u r s e o f t h i s p r o -j e c t , t h e a u t h o r has found no e v i d e n c e t h a t s u c h i s t h e c a s e w i t h t h e c o -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 i t must be a d m i t t e d t h a t t h i s has not been s p e c i f i c a l l y examined. 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 no a c t i v i t y i n seven o 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 enzyme i s m o d e r a t e l y s t a b l e t o m i l d heat t r e a t m e n t . When an 62 ammonium s u l f a t e f r a c t i o n ("AS-I 40-80;" 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 ml) was heated at pH 5.5 and 50° f o r h m i n u t e s , 25 t o 35 p e r c e n t o f t h e c o -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 . The a d d i t i o n o f 20 t o 50 mM magnesium c h l o r i d e t o t h e f r a c t i o n d i d not p r e v e n t l o s s o f t h e enzyme on h e a t i n g . T h i s d i f f e r s 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 phospho-h y d r o l ases ( 6 2 ) . I n h i b i t o r s . 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 a c t i v -i t y , w h i l e t h e a d d i t i o n o f EDTA had no e f f e c t upon t h e enzyme. O r t h o p h o s p h a t e does i n h i b i t t h e enzyme, w i t h 50 p e r c e n t i n h i b i t i o n o c c u r r i n g a t a c o n c e n -t r a t i o n o f 1.2 mM. V a r i o u s compounds w h i c h have been r e p o r t e d t o i n h i b i t o t h e r p h o s p h o h y d r o l a s e s - f o r example, 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 not 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 i n t h e a s s a y s y s t e m would r e v e r s e any e f f e c t o f t h e s e compounds v e r y r e a d i l y (79) . 63 DISCUSSION The e v i d e n c e p r e s e n t e d i n t h i s t h e s i s has been c o n c e r n e d w i t h f o u r main o b j e c t i v e s . The f i r s t o f t h e s e was t h e d e m o n s t r a t i o n t h a t a f a c t o r c a p a b l e o f i n h i b i t i n g t h e e n z y m a t i c s y n t h e s i s o f a c e t o a c e t a t e f r o m a c e t y l -CoA i s indeed p r e s e n t i n e x t r a c t s o f c h i c k e n l i v e r . The second i n v o l v e d t h e d e t e r m i n a t i o n o f t h e s i t e o f a c t i o n o f t h i s i n h i b i t o r y f a c t o r . The t h i r d o b j e c t i v e - t h e d e m o n s t r a t i o n o f p r e c i s e l y how t h e i n h i b i t o r y f a c t o r p r o duced i t s 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 t h e second; i n f a c t , one might c o n s i d e r t h a t t h e t h i r d p r o b l e m 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 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 t h e i s o l a t i o n and c h a r a c t e r i z a t i o n o f t h e i n h i b i t o r . That t h e i n h i b i t o r i s indeed p r e s e n t i n e x t r a c t s o f c h i c k e n l i v e r has been w e l l e s t a b l i s h e d . The o r i g i n a l o b s e r v a t i o n - t h a t o f a l l t h e l i v e r ex-t r a c t s s t u d i e d , o n l y t h o s e p r e p a r e d f r o m c h i c k e n l i v e r f a i l e d t o show p r o -p o r t i o n a l i t y between a c e t o a c e t a t e f o r m a t i o n and p r o t e i n c o n c e n t r a t i o n ( F i g . 1) - was r e i n f o r c e d by t h e o b s e r v a t i o n t h a t p a r t i a l l y - p u r i f i e d c h i c k e n l i v e r p r e p a r a t i o n s m a r k e d l y d e p r e s s e d 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 by beef l i v e r enzymes ( F i g . 3). The f a c t s t h a t t h e i n h i b i t o r y f a c t o r was n o n - d i a l y z a b l e , was d e s t r o y e d by b o i l i n g , and c o u l d be p u r i f i e d by t h e c l a s s i c a l t e c h n i q u e s o f p r o t e i n f r a c t i o n - p r e c i p i t a t i o n by s a l t and s o l v e n t s and a d s o r p t i o n on g e l s - l e d t o t h e a s s u m p t i o n t h a t t h e i n h i b i t o r was p r o t e i n i n n a t u r e , and was p r e sumably an enzyme. The second o b j e c t i v e - l o c a l i z i n g t h e i n h i b i t o r e f f e c t - was more complex, due t o t h e m u l t i p l i c i t y o f p o s s i b l e s i t e s o f a c t i o n . S t e r n and h i s c o - w o r k e r s had a l r e a d y e s t a b l i s h e d t h a t t h e i n h i b i t o r was 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 ( 8 ) . The a u t h o r was a b l e t o d e m o n s t r a t e t h a t t h e i n h i b i t o r enzyme n e i t h e r a c t e d upon any o f t h e enzymes i n v o l v e d i n a c e t o a c e t a t e f o r m a t i o n p e r s e ( T a b l e I) nor p r e v e n t e d t h e i r o p e r a t i o n ( T a b l e I I ) . 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 f i n a l l y came t o t h e r e a l i z a t i o n t h a t t h e i n h i b i t o r enzyme must be a c t i n g upon c o -enzyme A. T h i s was e s t a b l i s h e d by a s e r i e s o f d i r e c t and i n d i r e c t e x p e r i -ments ( T a b l e s I I I t o V, F i g . 4 ) . T h i s b r ought up t h e t h i r d o b j e c t i v e - s p e c i f i c a l l y , t h e p r e c i s e man-ner i n w h i c h t h e i n h i b i t o r i n a c t i v a t e d coenzyme A. The a u t h o r has dem o n s t r a t e d by p a p e r ( F i g . 6) and ion-exchange ( F i g . 7, T a b l e V I I ) chromatography t e c h -n i q u e s t h a t t h e a c t i o n o f t h e i n h i b i t o r enzyme upon coenzyme A r e s u l t s i n t h e q u a n t i t a t i v e f o r m a t i o n o f a compound i n d i s t i n g u i s h a b l e f r o m 3'-dephospho-coenzyme A. On t h e b a s i s o f t h i s e v i d e n c e , i t has been c o n c l u d e d t h a t t h e i n h i b i t o r enzyme a c t i v i t y i s due t o t h e p r e s e n c e i n t h e e x t r a c t s o f a " c o -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 , one comes t o t h e f o u r t h o b j e c t i v e - t h e i s o l 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 and an 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 . An i s o l a -t i o n p r o c e d u r e has been d e v i s e d w h i c h r e g u l a r l y r e s u l t s i n a 200- t o 3 0 0 - f o l d p u r i f i c a t i o n o f t h e enzyme, w i t h r e c o v e r y o f 20 t o 30 p e r c e n t o f t h e i n i t i a l e n z y m a t i c a c t i v i t y i n t h e most a c t i v e f r a c t i o n . When one examines t h e su b -s t r a t e s p e c i f i c i t y o f t h i s enzyme, however, one f i n d s t h a t even t h e most h i g h l y - p u r i f i e d f r a c t i o n e x h i b i t s p h o s p h o h y d r o l a s e a c t i v i t y a g a i n s t a w i d e v a r i e t y o f p h o s p h a t e e s t e r s ( T a b l e X I ) . F u r t h e r m o r e , t h e r e l a t i v e a c t i v i t y a g a i n 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 remains r e l a t i v e l y c o n s t a n t t h r o u g h o u t a 1 2 0 - f o l d change i n s p e c i f i c a c t i v i t y . Two p o s s i b l e e x p l a n a t i o n s f o r t h i s come t o mind. One p o s s i b i l i t y i s t h a t t h e c h i c k e n l i v e r e x t r a c t s c o n t a i n 65 o n l y one p h o s p h o h y d r o l a s e , and t h a t t h i s p h o s p h o h y d r o l a s e i s c a p a b l e o f 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 . The a l t e r n a t i v e e x p l a n a t i o n 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 o r more p h o s p h o h y d r o l a s e s , so s i m i l a r i n n a t u r e t h a t t h e y f r a c t i o n a t e as a s i n g l e e n t i t y under t h e c o n d i -t i o n s employed. A d e f i n i t i v e s o l u t i o n t o t h i s q u e s t i o n w i l l be a t t a i n e d o n l y t h r o u g h a s e p a r a t i o n o f t h e v a r i o u s 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 . How-e v e r , t h e a u t h o r c o n s i d e r s t h a t t h e r e a r e at l e a s t some i n d i c a t i o n s 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 y i s not s i m p l y a m a n i f e s t a t i o n o f a non-s p e c i f i c enzyme. One p o i n t o f e v i d e n c e i n d i c a t i n g t h e e x i s t e n c e o f more t h a n one p h o s p h o h y d r o l a s e i n t h e e x t r a c t s i s seen i n T a b l e X l l . T h i s t a b l e shows t h a t i n a t l e a s t one p u r i f i c a t i o n r u n , a c o n s i d e r a b l e s e p a r a t i o n o f t h e coenzyme A and p - n i t r o p h e n y l 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 i e s was a c h i e v e d . A s i m i l a r 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 two a c t i v i t i e s a l s o o c c u r r e d upon t h e 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 o f a s u c r o s e homogenate o f c h i c k e n l i v e r ( T a b l e X ) . One might a l s o c o n s i d e r t h e e f f e c t o f pH upon e n z y m a t i c a c t i v i t y . The 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 e x h i b i t e d a f a i r l y s h a r p optimum a t pH 3.6, w i t h a marked " s h o u l d e r " i n t h e range o f pH 5.0 t o 6.0, and t h e r a t i o o f t h e s p e c i f i c a c t i v i t y at pH 3.6 t o t h e s p e c i f i c a c t i v i t y a t pH 6.0 was 2.0. On t h e o t h e r hand, t h e c o r r e s p o n d i n g r a t i o s w i t h t h e o t h e r p h o s p h a t e e s t e r s u b s t r a t e s were near u n i t y . From t h e s e r a t i o s , one might be tempted t o a s -sume t h a t t h e h y d r o l y s i s o f t h e phosphomonoester g r o u p i n g o f coenzyme A i s c a t a l y z e d by a s p e c i f i c u n i q u e enzyme. However, i t must be kept i n mind t h a t t h e e f f e c t s o f pH upon e n z y m a t i c a c t i v i t y a r e complex, i n v o l v i n g changes i n i o n i z a t i o n i n b o t h enzyme and s u b s t r a t e , and must be i n t e r p r e t e d w i t h c a u t i o n . In s u m m a r i z i n g t h e above p o i n t s , one might say t h a t a l t h o u g h t h e r e 66 may be some j u s t i f i c a t i o n f o r p o s t u l a t i n g t h e e x i s t e n c e o f a s p e c i f i c and u n i q u e coenzyme A p h o s p h o h y d r o l a s e , more work must be done b e f o r e a d e f i n i t e c o n c l u s i o n can be r e a c h e d . 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 r o l e o f t h e p h o s p h o h y d r o l a s e a l s o a r i s e s . Obvious p h y s i o l o g i c a l f u n c t i o n s can be a t t r i b u t e d t o many o f t h e known p h o s p h o h y d r o l a s e s . 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 i s t h e f i n a l s t e p i n t h e f o r m a t i o n o f b l o o d s u g a r t h r o u g h g l y c o g e n o l y s i s (79). S i m i l a r l y , t h e h y d r o l y s i s o f O - p h o s p h o s e r i n e has been p r o p o s e d as t h e f i n a l 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). A n o t h e r example would be t h e r o l e o f p h o s p h a t i d i c a c i d p h o s p h o h y d r o l a s e i n t h e b i o s y n t h e s i s o f t r i g l y c e r i d e s (81). I t has been 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 a c t t o e n s u r e a s u p p l y o f 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 (68). A p h y s i o l o g i c a l 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 be more d i f f i c u l t t o v i s u a l i z e , e x c e p t p o s s i b l y i n a s t r a i g h t f o r w a r d d e g r a d a t i v e r o l e . 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 d e v e l o p m e n t s . 67 BIBLIOGRAPHY 1. P e t t e r s , W., P r a g e r V i e r t e l j a h r s c h r i f t f U r d i e p r a k t f s c h e HeMkunde, 5J5_, 81 (1857); c i t e d by H. J . Deuel i n "The L i p i d s , " v o l . I l l , p. 121. I n t e r s c i e n c e P u b l i s h e r s I n c . , New Y o r k , 1957. 2. 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