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Studies on acetoacetate formation Caldwell, Ian Carl 1961

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STUDIES ON ACETOACETATE FORMATION by IAN C. CALDWELL A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department o f Pharmacology We accept t h i s t hes i s as conforming to the requ i red standard THE UNIVERSITY OF BRITISH COLUMBIA June, 1961 ~\. In presenting 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 permission 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 representatives. It 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 gain s h a l l not be allowed without my written 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 July 5, 1961 - i i -ABSTRACT In recent years, two mechanisms have been proposed for the enzymatic formation of acetoacetate by liver extracts. One of these, the "HMG-CoA cycle", involves the condensation of acetyl-CoA and acetoacety1-CoA to form ^-hydroxy- (3-methylglutaryl-CoA (HMG-CoA) via the action of the HMG-CoA condensing enzyme, with the release of free coenzyme A (CoASH) (reaction 1), Acetyl-CoA + acetoacetyI-CoA + H20 ^ ?: HMG-CoA + CoASH (1) followed by cleavage of the HMG-CoA to acetyl-CoA and free acetoacetate, via the action of the HMG-CoA cleavage enzyme (reaction 2). HMG-CoA acetoacetate + acetyl-CoA (2) The second mechanism which has been proposed involves a direct deacylation of acetoacety1 CoA through the action of a specific acetoacety1-CoA thio-esterase (reaction). Acetoacety 1-CoA + H20 >- acetoacetate + CoASH (3) Evidence is presented which indicates acetoacetate formation by a soluble enzyme system from bicarbonate extracts of whole beef liver proceeds largely, i f not exclusively, via HMG-CoA (reactions 1 and 2). Both the HMG-CoA condensing and cleavage enzymes have been partially purified from beef liver bicarbonate extracts, each free of contamination by the other, in good yields. The level of activity of these two enzymes is sufficiently high to account for a l l the acetoacetate formed by liver tissue. The possibility that the specific acetoacety1-CoA thioesterase may play a minor role in the enzymatic synthesis of acetoacetate is also discussed. The intracellular and tissue localization of the enzymes of aceto-acetate formation is also discussed. In liver homogenates, most, i f not a l l , of the acetoacetate-synthesizing activity appears to be associated with the mitochondrion. Evidence is also presented that the primary reason for the inability of extrahepatic tissue preparations to catalyze the accumulation of acetoacetate may be the lack of one of the enzymes involved, i.e., the HMG-CoA condensing enzyme, and not merely further metabolic degradation of acetoacetate, as has generally been assumed. An enzyme fraction in chicken liver extracts which inhibits the i n vi tro formation of acetoacetate by chicken liver homogenates has also been studied. Evidence is presented that this enzyme fraction exerts its effect through the inactivation of coenzyme A. Preliminary observations indicate that this enzyme may be a S'-nucleotidase, removing the 3*~ phosphate of coenzyme A, forming dephosphocoenzyme A. The occurrence of a highly active (2-hydroxybutyryl dehydrogenase in extracts of dry culture of C. kluyveri has been noted. This enzyme differs from the similar enzyme reported in mammalian tissues, in that i t is very specific for triphosphopyridine nucleotide, and is virtually inactive with diphosphopyridine nucleotide (DPN) (reaction ). Acetoacetyl-CoA + TPNH + H + ^ * ^ - hydroxybutyryl-CoA (*t) + TPN+ - iv -TABLE OF CONTENTS Page HISTORICAL BACKGROUND 1 MATERIALS AND METHODS 11 Miscellaneous 11 Purification of Acetoacetate Synthesizing System 12 Acetoacetate Synthesizing Assay System 16 Preparation and Assay of HMG-CoA Condensing Enzyme .. .. 18 Preparation and Assay of HMG-CoA Cleavage Enzyme 19 Preparation of Rat Tissue Homogenates 22 Resolution of Acetoacetate Synthesizing System . . . . 22 Purification of Chicken Liver "Inhi bi tor"tEnzyme Fraction .. 27 RESULTS 31 Purification of Acetoacetate Synthesizing System 31 Properties of Acetoacetate Synthesizing System 32 Resolution of Acetoacetate Synthesizing System k3 I. The Procedure of Bublitz , ^3 II. Ammonium Sulfate Fractionations h$ III. Attempted Resolution by Column Chromatography .. 49 IV. Studies on Heated Extracts 55 V. Fractionation with Ethanol in the Presence of Zinc 56 Localization and Distribution of Enzyme 65 The Chicken Liver "Inhibitor Enzyme". 67 £-Hydroxybutyryl Dihydrogenase from C. Kluyveri 73 DISCUSSION 79 BIBLIOGRAPHY 90 - v -LIST OF TABLES No. T i t l e Page I p u r i f i c a t i o n of HMG-CoA Cleavage Enzyme from Beef L i v e r Acetone Powder 2 1 II P u r i f i c a t i o n of Acetoacetate S y n t h e s i z i n g System from Beef L i v e r . E x t r a c t s .. 31 III P u r i f i c a t i o n of Acetoacetate S y n t h e s i z i n g System from Other L i v e r Preparations 3 3 IV Mg + + S t i m u l a t i o n and EDTA I n h i b i t i o n of Acetoacetate Synthesis 3 ^ V T h i o l I n t e r f e r e n c e w i t h the Determination of Aceto-acetate by the Walker Procedure 3 7 VI E f f e c t of Mg + +, GSH and EDTA on Acetoacetate Synthesis by Rat L i v e r Mitochondria 3 9 VII E f f e c t of EDTA on Acetoacetate Synthesis by Rat L i v e r Mitochondria 3 9 VI11 E f f e c t of Pretreatment of Enzyme w i t h I n h i b i t o r s .. .. k] IX Attempt to Reproduce the Re s o l u t i o n of the Acetoacetate S y n t h e s i z i n g System by the Procedure of B u b l i t z .. .. kS X Attempted R e s o l u t i o n of Acetoacetate S y n t h e s i z i n g System of Beef L i v e r Acetone Powder E x t r a c t s by S a l t F r a c t i o n a -t i o n k7 XI I n h i b i t i o n of Acetoacetate S y n t h e s i z i n g System by Ammonium S u l f a t e 8^ XII S i t e of I n h i b i t i o n of Acetoacetate Synthesis by Ammonium S u l f a t e kS XIII Chromatography of Acetoacetate S y n t h e s i z i n g System on Calcium Phosphate Gel 5 0 XIV P a r t i a l R e s o l u t i o n of the Acetoacetate S y n t h e s i z i n g System by Chromatography on Calcium Phosphate Gel .. .. 5 1 XV D i s t r i b u t i o n of HMG-CoA Condensing and Cleavage Enzymes i n Calcium Phosphate Gel Column Eluates 5 2 v i -LIST OF TABLES (cont'd.) No. T i t l e Page XVI Chromatography of Acetoacetate S y n t h e s i z i n g System on Calcium Phosphate Gel (Brushite) 53 XVII D i s t r i b u t i o n of HMG-CoA Condensing and Cleavage Enzymes i n Calcium Phosphate Gel Column Eluates Sk XVIII E f f e c t of P u r i f i e d HMG-CoA Condensing Enzyme on Aceto-acetate Synthesis by Heated L i v e r F r a c t i o n s 57 XIX HMG-CoA Condensing Enzyme from L i v e r F r a c t i o n s .. .. 59 XX Equivalence of HMG-CoA Condensing Enzyme Pre p a r a t i o n from Yeast and L i v e r 61 XXI I d e n t i f i c a t i o n of Th i o e s t e r Formed by Beef L i v e r Zn-ethanol F r a c t i o n .. 62 XXII Fate of HMG-CoA Condensing and Cleavage Enzymes During P u r i f i c a t i o n of the Acetoacetate S y n t h e s i z i n g System .. 6k XXIII D i s t r i b u t i o n of HMG-CoA Cleavage Enzyme i n Rat Tissue 66 XXIV P u r i f i c a t i o n of the Chicken L i v e r " I n h i b i t o r Enzyme" 68 XXV E f f e c t of Pre i n c u b a t i n g the Assay System Components w i t h the " I n h i b i t o r Enzyme" 70 XXVI I n a c t i v a t i o n of Coenzyme A by Chicken L i v e r " I n h i b i t o r Enzyme" 72 XXVII E f f e c t of TPNH and HMG-CoA Reductase on Acetoacetate by Beef L i v e r Enzymes 75 XXVIII Chromatography of Products of the § -Hydroxy bu t y r y l Dehydrogenase of C^ K l u y v e r i 78 — v i i — LIST OF FIGURES No. T i t l e Page 1 E f f e c t of D i v a l e n t Cation Concentration on the Rate of Acetoacetate Synthesis 35 2 E f f e c t of GSH Concentration on the Rate of Acetoacetate Synthesis 36 3 I n h i b i t i o n of the Acetoacetate S y n t h e s i z i n g System by Treatment w i t h lodoacetamide .. .. kl k F r a c t i o n a t i o n Procedure of Acetoacetate S y n t h e s i z i n g System kk 5 Interference by the Chicken L i v e r " I n h i b i t o r Enzyme" w i t h Acetoacetate Formation by Beef L i v e r Enzymes . . . . 69 6 E f f e c t of Coenzyme A Concentration on Depression of Acetoacetate Formation by Beef L i v e r Enzymes 71 7 P y r i d i n e Nucleotide S p e c i f i c i t y of 0-Hydroxybutyry1 Dihydrogenase of C. Kluyver? 77 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 used: a c e t y l - P , a c e t y l phosphate; acyl-CoA, a c y l t h i o e s t e r of coenzyme A; acyl-GSH, a c y l t h i o e s t e r of g l u t a t h i o n e ; ATP, adenosine triphosphate; BAL, 2 , 3 - d i m e r -captopropanol; CoASH, reduced coenzyme A; DEAE-cellulose, d i e t h y l -aminoethyl c e l l u l o s e ; DPN and DPNH, the o x i d i z e d and reduced forms, r e s p e c t i v e l y , of diphosphopyridine n u c l e o t i d e ; DTO, 6 , 8 - d i t h i o l o c t a n o i c ( d i h y d r o l i p o i c ) a c i d ; EOTA, ethylenediamine t e t r a a c e t i c a c i d ; GSH, reduced g l u t a t h i o n e ; HMG, ^-hydroxy- §-methy 1glutaric a c i d ; IAA, iodoacetamide; P i , orthophosphate; P P i , inorganic pyrophosphate; TPN and TPNH, the o x i d i z e d and reduced forms, r e s p e c t i v e l y of triphosphi p y r i d i n e n u c l e o t i d e ; T r i s , tris-(hydroxymethyl)-aminomethane. 1 . The p h y s i o l o g i c a l and p a t h o l o g i c a l aspects o f k e t o s i s and ke to -genesis have a t t r a c t e d the a t t e n t i o n o f many workers f o r more than a c e n t u r y , sparked by the accumulat ion o f the th ree ketone bod ies , acetone, ace toace ta te and @-hydroxybuty r a t e , as metabo l i c end-products i n the k e t o s i s assoc ia ted w i t h d iabetes m e l l i t u s . Over the y e a r s , t h i s r e l a t i o n s h i p between k e t o s i s and d iabetes mel1i tus has led to a g rea t deal o f i n t e r e s t i n the b iochemis t ry o f ke togenes is . Al though i t was e a r l y recognized t h a t f a t t y ac ids served as the d i e t a r y source o f the ketone bod ies , and t h a t the c h i e f s i t e o f ketogenesis was the l i v e r , i t is on ly i n recent years t h a t s i g n i f i c a n t progress has been made in the c l a r i f i c a t i o n on the p r e c i s e biochemical pathways through which t h e i r f o rma t i on proceeds. W i t h i n the l a s t decade, var ious enzymatic pathways f o r the fo rma t ion o f ace toace ta te , the pr imary ketone body, have been proposed, and abundant but i nconc lus i ve evidence presented f o r each p r o p o s a l . Dur ing the l a s t th ree y e a r s , i n p a r t i c u l a r , there has been cons iderab le con t roversy concern ing t h i s problem. This t hes i s represents the r e s u l t s o f a concer ted e f f o r t to e s t a b l i s h c o n c l u s i v e l y the u l t i m a t e pathway(s) through which ace toace ta te , and thus the o t h e r ketone bod ies , are formed. HISTORICAL BACKGROUND The s t o r y o f k e t o s i s began i n 1857, when Pe t te rs (1) repor ted the presence o f acetone in d i a b e t i c u r i n e . E igh t years l a t e r , Gerhardt (2) de tec ted ace toace ta te in d i a b e t i c u r i n e , a l though he i n c o r r e c t l y i d e n t i f i e d the compound as e t h y l ace toace ta te , To l lens (3) and Diechmul ler (k) l a t e r c o r r e c t l y i d e n t i f i e d Gerhard t ' s compound as the f r e e a c i d . The r e l a t i o n s h i p ' between these two ketones was po in ted o u t by Arno ld ( 5 ) , who recognized t h a t 2. ace toace ta te was the immediate p recursor o f acetone. In 1898, Geelmuyden (6) recognized t h a t f a t t y ac ids gave r i s e t o the ketone bod ies . This led to the p e r f u s i o n s tud ies o f Embden e t a_[ (7 ,8) w i t h i s o l a t e d dog l i v e r . These workers found t h a t ace toace ta te was formed when l i v e r s were per fused w i t h media c o n t a i n i n g f a t t y ac ids having an even number o f carbon atoms. Per fus ing the l i v e r s w i t h f a t t y ac ids having an odd number o f carbon atoms d i d not g ive r i s e to s i g n i f i c a n t amounts o f ace to -a c e t a t e . * These observa t ions led to the development o f the theory o f ^ - o x i d a t i o n , i n which f a t t y ac ids are degraded by a s tepwise removal o f two-carbon u n i t s through o x i d a t i o n a t the ^ - c a r b o n , to y i e l d a te rmina l f ou r -ca rbon fragment which could g ive r i s e to a c e t o a c e t a t e . These workers (9 ,10) and Friedman (11) a l so found t h a t ace toace ta te was formed when excised l i v e r s were per fused w i t h a c e t a t e . This was exp la ined as a ( 3 - o x i d a t i o n -condensat ion process; the two-carbon u n i t s formed du r ing @-ox ida t ion cou ld condense to form a fou r - ca rbon compound, which could then g i ve r i s e to ace toace ta te . Embden and Loeb (8) a l so found t h a t ace toace ta te was formed, when l i v e r s were per fused w i t h the aromat ic amino a c i d s , pheny la lan ine and t y r o s i n e . A l though these e a r l y observa t ions prov ided a f i r m founda t ion f o r f u r t h e r s tud ies o f ke togenes is , very l i t t l e progress was made f o r a lmost 30 y e a r s . In the l a t e 1930*s, the next step i n the t r a n s i t i o n f rom whole animal observa t ions to the study o f the i n d i v i d u a l enzymes was taken , namely the study o f ketogenesis by l i v e r s l i c e s . Jowett and Quastel (12) and L e l o i r and Munoz (13) showed t h a t when l i v e r s l i c e s were incubated w i t h even-numbered f a t t y a c i d s , ace toaceta te accumulated i n the medium. Var ious o t h e r groups found t h a t l i v e r s l i c e s could form acetoaceta te f rom a wide v a r i e t y o f compounds, i n c l u d i n g ace ta te ( 1 4 ) , c ro tona te ( 1 5 ) , v i n y l a c e t a t e ( 1 6 ) , 3. sorbate (14) and leuc ine (17). Quastel e t al_ (18) showed t h a t the o x i d a t i o n o f f a t t y ac ids by t i s s u e s l i c e s prepared f rom sp leen , t e s t i s and kidney a lso r e s u l t e d i n the accumulat ion o f ace toace ta te ; however, the ace toace ta te formed by these t i ssues amounted to on ly 1/40 t o 1/10 t h a t formed by an e q u i v a l e n t 1 i ve r p r e p a r a t i o n . The next advance, the fo rma t ion o f ace toaceta te by c e l l - f r e e p repara -t i o n s , was accomplished by Munoz and L e l o i r (19) i n 19^3. They found t h a t the " e a s i l y sedimentable f r a c t i o n " o f l i v e r homogenates cou ld c a r r y ou t the o x i d a t i o n o f f a t t y a c i d s , i n the presence o f M g + + , adeny l i c a c i d , cytochrome c and another o x i d i z a b l e s u b s t r a t e . Ketones were the major end p roduc t . T h e i r p r e p a r a t i o n , however, was q u i t e l a b i l e , and would not o x i d i z e f a t t y ac ids w i t h cha in lengths o f more than s i x carbons. Lehninger (20) repor ted the p r e p a r a t i o n o f a s i m i l a r " e a s i l y sedimentable f r a c t i o n " f rom r a t l i v e r which could o x i d i z e f a t t y ac ids w i t h cha in lengths o f up to 18 carbons, and which was r e l a t i v e l y s t a b l e . In the presence o f ATP and Mg 4 4 " , and w i t h the a d d i t i o n o f malonate to b lock te rmina l o x i d a t i o n v i a the t r i c a r b o x y l i c a c i d c y c l e , f a t t y ac ids were q u a n t i t a t i v e l y converted to a c e t o a c e t a t e . In a d d i t i o n , i n the absence o f a source o f o x a l a c e t a t e , and in the presence o f malonate, pyruvate was q u a n t i t a t i v e l y conver ted to ace toace ta te . Lehninger demonstated t h a t ace toace ta te was not f u r t h e r metabol ized in these l i v e r p r e p a r a t i o n s . S h o r t l y a f t e r t h i s , Kennedy and Lehninger (21,22) showed t h a t the mi tochond-r i o n was the ac tua l s i t e o f f a t t y a c i d o x i d a t i o n and ace toace ta te f o r m a t i o n . Lehninger (23) a l so prepared a s i m i l a r enzyme p r e p a r a t i o n f rom h e a r t , which could o x i d i z e f a t t y ac ids under the same c o n d i t i o n s as d i d the l i v e r p repara -t i o n . However, there was no accumulat ion o f ace toace ta te ; i n i t s p l a c e , Q L - k e t o g l u t a r a t e and succ ina te accumulated, i n d i c a t i n g t h a t a c e t o a c e t a t e , i f formed, was f u r t h e r metabol ized by e x t r a h e p a t i c t i s s u e s . Graff1 i n and Green 4. ( 2 4 ) , work ing w i t h what they termed the "cyc lophorase" system f rom l i v e r and k idney , demonstrated the fo rma t ion o f ace toace ta te f rom severa l unsatura ted s h o r t - c h a i n a c i d s . I t was l a t e r shown t h a t the "cyc lophorase" system was, to a l l i n t e n t s and purposes, s imply a m i tochondr ia l p r e p a r a t i o n . An impor tant breakthrough i n the study o f the p r e c i s e mechanism o f ketogenesis occur red w i t h the demonstrat ion o f ace toace ta te f o rma t i on by s o l u b l e enzymes. Soodak and Lipmann (25) repor ted t h a t s o l u b l e e x t r a c t s o f pigeon l i v e r acetone powders could c a t a l y z e the fo rma t i on o f ace toace ta te f rom a c e t a t e , i f supplemented w i t h ATP, M g + + , and coenzyme A. In 1951, Stadtman e t al_ (26) found t h a t pigeon l i v e r e x t r a c t s , supplemented w i t h b a c t e r i a l phosphotransacety lase and coenzyme A, cou ld form ace toace ta te f rom a c e t y l phosphate. Using t h i s coupled enzyme system and C ' ^ - l a b e l l e d compounds, these workers showed t h a t both two-carbon u n i t s condensing to form ace toace ta te came from " a c t i v e a c e t a t e " , which Lynen (27) i d e n t i f i e d as the coenzyme A t h i o e s t e r o f a c e t i c a c i d . The r e a c t i o n sequence is represented by r e a c t i o n 1 ( ca ta l yzed by phosphotransacety lase) and the ove r -a l l r e a c t i o n 2 is ca ta lyzed by the l i v e r e x t r a c t . 2 a c e t y l s + 2 CoASH 1 2 acety l -CoA + 2 P; (1) 2 acety l -CoA ^ a c e t o a c e t a t e + 2 CoASH (2) Net React ion: 2 a c e t y l - P » acetoaceta te + 2 Pj The d iscovery o f acetoacety l -CoA (28,29,30) and the demonstra t ion o f i t s f o rma t i on as an in te rmed ia te i n f a t t y a c i d metabol ism (31»32) p rov ided an a t t r a c t i v e candidate f o r the r o l e o f the immediate p recursor o f enzym-a t i c a l l y formed ace toace ta te . Acetoacety1-CoA could be formed from f a t t y acids v i a g -ox ida t i on , and a lso f rom the condensat ion o f two molecules o f acety l -CoA v i a the a c t i o n o f £ - k e t o t h i o l a s e ( r e a c t i o n 3 ) . 2 acety l -CoA • acetoacety1-CoA + CoASH (3) A t t h i s same t ime , Stern e t aj[ b r i e f l y repor ted (29) the p a r t i a l p u r i f i c a -t i o n f rom beef l i v e r e x t r a c t s o f an enzyme system which was capable o f c a t a l y z i n g the f o rma t i on o f ace toace ta te f rom added acety l -CoA, o r f rom acetyl -CoA generated i n s i t u w i t h phosphot ransacety lase, a c e t y l phosphate and c a t a l y t i c concen t ra t ions o f coenzyme A. They pos tu la ted t h a t ace to -acetyl -CoA formed by (2>-ketothiolase ( r e a c t i o n 3) was hydro lyzed by a s p e c i f i c deacylase to l i b e r a t e f r e e ace toace ta te ( r e a c t i o n k): Acetoacety 1-CoA + H 2 0 *- ace toace ta te + CoASH (4) U n f o r t u n a t e l y , the unequivocal demonstrat ion o f the presence o f t h i s s p e c i f i c deacylase o r t h i o e s t e r a s e in l i v e r e x t r a c t s has proven ext remely d i f f i c u l t . Another mechanism f o r the f o rma t i on o f ace toace ta te by t i s s u e e x t r a c t s was demonstrated by Stern e t aj. (30) and a lso by Green e t aj_ ( 3 1 ) . In the presence o f s u c c i n a t e , var ious t i s s u e e x t r a c t s can c a t a l y z e the fo rma t ion o f ace toace ta te f rom acetoacety1-CoA through the a c t i o n o f s u c c i n y l - @ - k e t o a c y 1 coenzyme th iophorase (CoA t r a n s f e r a s e , th iophorase) ^ r e a c t i o n 5 ) : Acetoacety 1-CoA + succ ina te « ace toace ta te + succinyl -CoA (5) This enzyme i s not found i n mammalian l i v e r ; i t s p h y s i o l o g i c a l r o l e appears to i n v o l v e the e x t r a h e p a t i c a c t i v a t i o n o f ace toaceta te p r i o r to i t s cleavage 6. to acety l -CoA and i t s subsequent o x i d a t i o n v i a the t r i c a r b o x y l i c a c i d c y c l e in these t i s s u e s . A f u r t h e r mechanism which could be considered was reversa l o f the ace toace ta te a c t i v a t i o n r e a c t i o n ( r e a c t i o n 6) which had been repor ted i n yeast (29) and pigeon l i v e r ( 3 6 ) . Acetoaceta te + ATP + CoASH acetoacety 1-CoA + AMP + PP j (6) However, l i k e the CoA t rans fe rase r e a c t i o n , t h i s r e a c t i o n does not occur i n mammalian l i v e r , and appears to be invo lved o n l y in e x t r a h e p a t i c a c t i v a t i o n o f ace toace ta te . Based on the f i n d i n g s t h a t (a) beef l i v e r e x t r a c t s hydro lyzed ace to -acetyl -CoA on ly on the a d d i t i o n o f g l u t a t h i o n e , (b) ace toace ta te syn thes is f rom a c e t y l phosphate and coenzyme A i n the coupled phosphot ransacety lase-l i v e r e x t r a c t system requ i red the a d d i t i o n o f t h i o l f o r maximal s y n t h e s i s , and (c) beef l i v e r e x t r a c t s conta ined a very a c t i v e a c e t o 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 , S te rn and Drummond (3*0 pos tu la ted a mechanism f o r ace toace ta te fo rmat ion i n v o l v i n g the t r a n s f e r o f the ace toace ty l moiety f rom coenzyme A to g l u t a t h i o n e by a t r ans fe rase ( r e a c t i o n 7) f o l l o w e d by h y d r o l y s i s o f the g l u t a t h i o n e t h i o e s t e r to l i b e r a t e f r e e ace toace ta te ( r e a c t i o n 8 ) . Acetoacety l -CoA + GSH »- acetoacety 1-GSH + CoASH (7) Acetoacetyl-;GSH + H 20 *• ace toaceta te + GSH (8) U n f o r t u n a t e l y , a l though r e a c t i o n 7 occurs very r a p i d l y over a cons iderab le range o f exper imenta l c o n d i t i o n s , and e s p e c i a l l y a t a l k a l i n e pH, the t r a n s f e r 7. Is non-enzymat ic , and lacks t h i o l and t h i o e s t e r s p e c i f i c i t y . The ex i s tence o f an enzyme c a t a l y z i n g t h i s r e a c t i o n has never been d e f i n i t e l y e s t a b l i s h e d . Al though the coup l i ng o f reac t ions 7 and 8 c o n s t i t u t e s a mechanism f o r the d e a c y l a t i o n o f acetoacety1-CoA, there is no evidence t h a t i t has any p h y s i o l o g i c a l s i g n i f i c a n c e i n ace toace ta te f o r m a t i o n . Lynen e t a^ (35) l a t e r repor ted t h a t the p u r i f i c a t i o n o f the ace toace ta te s y n t h e s i z i n g enzyme system d i d not p a r a l l e l the p u r i f i c a t i o n o f the acetoacetyl-GSH t h i o e s t e r a s e . In 1954, Bachhawat e t a l (36) repor ted the f o rma t i on o f ace toace ta te f rom HMG-CoA, a degradat ion product o f the amino ac id l e u c i n e , by the HMG-CoA cleavage enzyme, which is found in a wide v a r i e t y o f t i ssues ( r e a c t i o n 9). HMG-CoA *- ace toaceta te + acety l -CoA (9) These authors p a r t i a l l y p u r i f i e d the enzyme (37), and demonstrated abso lu te requirements f o r t h i o l and d i v a l e n t c a t i o n . The ace toace ta te s y n t h e s i z i n g system from beef l i v e r a l so showed these same requ i rements . In 1956, Rudney (38) b r i e f l y repor ted the fo rma t ion o f r a d i o a c t i v e HMG f o l l o w i n g the incuba-t i o n o f l i v e r enzyme p repara t ions w i t h ATP, coenzyme A, and aceta te-C . In a subsequent r e p o r t (39) , the same author demonstrated t h a t the product o f the r e a c t i o n was an HMG-CoA t h i o e s t e r , and was formed by the condensat ion o f acetoacety l -CoA and acety l -CoA. The enzyme was g iven the name "HMG-CoA condens-ing enzyme". Using a p u r i f i e d enzyme f r a c t i o n f rom y e a s t , which proved to be a source o f a more s t a b l e enzyme than d i d l i v e r , Rudney and Ferguson (40) were ab le to demonstrate t h a t the product o f the condensat ion r e a c t i o n was the HMG-CoA monoth ioes te r , and t h a t the condensat ion t h e r e f o r e proceeded accord ing to r e a c t i o n 10. To determine which t h i o e s t e r bond was hydro lyzed d u r i n g the condensat ion r e a c t i o n , acety1-1-c '^-CoA and un label led a c e t o a c e t y l -8. Acetoacety1-CoA + acety l -CoA + h^O * - HMG-CoA + CoASH (10) CoA were incubated w i t h p u r i f i e d , t h i o l a s e - f r e e HMG-CoA condensing enzyme, and the r a d i o a c t i v e HMG-CoA i s o l a t e d and incubated w i t h the HMG-CoA cleavage enzyme o f Bachhawat e t aj_ (37). The products o f the cleavage r e a c t i o n were i s o l a t e d and analyzed f o r r a d i o a c t i v i t y . The c'** was found e x c l u s i v e l y i n the carboxy1 group o f the ace toace ta te formed; t h u s , i t must have been the t h i o e s t e r bond o f acety l -CoA which was hydro lyzed du r ing the condensat ion , and the r e a c t i o n must have proceeded as shown by reac t ions 11 and 12. A more complete r e p o r t on the p u r i f i c a t i o n and p r o p e r t i e s o f the HMG-CoA condensing enzyme from baker ' s yeas t has s ince been pub l ished (41,42). In 1956, Lynen b r i e f l y repor ted (43) t h a t B u b l i t z , work ing i n the Munich l a b o r a t o r y , had separated an ace toace ta te s y n t h e s i z i n g enzyme system f rom beef l i v e r acetone powder i n t o two f r a c t i o n s , enzymes "A" and " B " , both o f which were requ i red f o r the f o rma t i on o f ace toace ta te f rom a c e t y l phosphate in the coupled t ransace ty lase system descr ibed above. L a t e r , Lynen e t aj[ (35) pub l i shed a longer repor t on these two enzymes, i d e n t i f y i n g enzyme " B " as the HMG-CoA condensing enzyme, and enzyme "A" as the HMG-CoA cleavage enzyme. Since t h e i r enzyme " B " p r e p a r a t i o n f rom l i v e r proved to be ? H 9 CH3-C-CH2-C-SCoA + CoASH CH2-C00H (10 (HMG-CoA) OH 0 CH3-C-CH2-C-SCoA CH2-COOH 9. u n s t a b l e , they prepared a p u r i f i e d HMG-CoA condensing enzyme from baker ' s y e a s t . Using t h i s p u r i f i e d yeast enzyme and a p u r i f i e d enzyme "A" p repara -t i o n f rom beef l i v e r , they c a r r i e d out a s e r i e s o f exper iments , e s s e n t i a l l y a d u p l i c a t i o n and ex tens ion o f the experiment o f Rudney and Ferguson as o u t l i n e d above, which demonstrated t h a t i f these two p u r i f i e d enzymes were coupled in an a p p r o p r i a t e assay system, ace toace ta te was formed. From these exper iments , they concluded t h a t ace toace ta te f o rma t i on i n l i v e r e x t r a c t s , w i t h the except ion o f t h a t formed from the aromat ic amino a c i d s , f o r which a d i f f e r e n t pathway is a l ready known (44), proceeds e x c l u s i v e l y v i a HMG-CoA. I t should be po in ted o u t , however, t h a t t h e i r evidence is de r i ved almost e n t i r e l y from experiments i n which yeast p repara t ions were used as the source o f the HMG-CoA condensing enzyme. When t h i s f a c t i s taken i n t o c o n s i d e r a t i o n , i t would appear t h a t the s ta tement , "Wi r konnen daher annehmen, dass d i e A c e t a c e t a t b i l d u n g i n der Leber, b is auf den Abbau der aromatischem Aminosauren, uber ^~ hydroxy-(3 -methy lg lu tary l -CoA f u h r t . " ' i s somewhat premature. In I960, S tern and co-workers presented evidence t h a t ace toace ta te f o r m a t i o n by r a t l i v e r m i tochondr ia l p repara t i ons (45) and p a r t i a l l y p u r i f i e d beef l i v e r e x t r a c t s (29,46,47) f rom acetoacety1-CoA need not proceed v i a HMG-CoA. I t was shown t h a t ace toace ta te could be formed from s u b s t r a t e amounts o f acetoacety l -CoA by l i v e r p repara t ions in which ( ^ -ke to th io lase and the HMG-CoA condensing and cleavage enzymes had been apparen t l y t o t a l l y i n a c t i v a t e d by prev ious t reatment w i t h iodoacetamide. S i m i l a r f i n d i n g s w i t h an un t rea ted r a t l i v e r m i tochondr ia l p r e p a r a t i o n have been independent ly ' "We can t h e r e f o r e assume t h a t ace toace ta te f o rma t i on in l i v e r , except t h a t formed i n the degradat ion o f the aromat ic amino a c i d s , proceeds v i a © - h y d r o x y - ^ - m e t h y l g l u t a r y l CoA." ( c f . re fe rence 35) • 10. repor ted by Segal and Menon ( 4 8 ) ; however, the v a l i d i t y o f the l a t t e r r e p o r t i s very much open to q u e s t i o n , as w i l l be more f u l l y d iscussed l a t e r . More r e c e n t l y , H i rd and Symons ( 4 9 ) have repor ted on the f o rma t i on o f ace toace ta te f rom C ^ - l a b e l led subs t ra tes by sheep omasum and rumen e p i t h e l i u m , which Pennington (50,51,52) has shown t o be f u l l y as a c t i v e as l i v e r t i s s u e i n c a t a l y z i n g the 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 i r r e s u l t s , ob ta ined w i t h whole c e l l p r e p a r a t i o n s , i n d i c a t e almost beyond d i s p u t e t h a t a t l e a s t 75%. and probably more, o f the ace toace ta te formed i n t h e i r e x p e r i -ments could not p o s s i b l y have been formed by d i r e c t d e a c y l a t i o n o f ace to -acety 1 -CoA; the HMG-CoA pathway is the on ly known mechanism which can e x p l a i n t h e i r f i n d i n g s . This w i l l be discussed i n more d e t a i l l a t e r . C l e a r l y , the p r e c i s e enzymatic mechanism f o r the fo rma t ion o f ace to -ace ta te i s c o n t r o v e r s i a l and u n s e t t l e d . This t hes i s represents an a t tempt to c l a r i f y the mechanism. Three main approaches to the problem have been employed. The f i r s t cons i s t s o f a t tempts to p u r i f y the ace toace ta te fo rming system i n l i v e r to a g r e a t e r degree than t h a t p r e v i o u s l y ach ieved . The second approach cons is ted o f e f f o r t s to reso lve the system i n t o two o r more enzymatic e n t i t i e s . I f indeed HMG-CoA is an i n t e r m e d i a t e , i t should be p o s s i b l e to separate the two enzymes i n v o l v e d , namely, the HMG-CoA condensing enzyme and the HMG-CoA cleavage enzyme. Synthesis o f ace toace ta te cou ld then proceed on ly upon recombinat ion o f the two enzymatic e n t i t i e s . F i n a l l y , the t h i r d approach has been to study the mechanism by which a p r o t e i n f a c t o r ob ta ined f rom chicken l i v e r i n h i b i t s ace toace ta te f o rma t i on i n the standard assay system. The f o l l o w i n g experiments desc r ibe our r e s u l t s to date and s t r o n g l y i n d i c a t e t h a t HMG-CoA is an i n t e r m e d i a t e . Two enzymes have been o b t a i n e d , each l a r g e l y f r e e o f the o t h e r , and ace toace ta te syn thes is proceeds on ly upon t h e i r recombinat ion . A b r i e f p r e l i m i n a r y account o f some o f our 1 1 . work has appeared (53). In a d d i t i o n , the reduc t ion o f acetoacety1-CoA by a TPNH-speci f ic dehydrogenase i n C. k l u y v e r i e x t r a c t s is desc r i bed . MATERIALS AND METHODS MISCELLANEOUS ATP (d ipo tass ium s a l t ) , coenzyme A ("75% p u r e " ) , g lucose 6-phosphate (bar ium s a l t ) , L -cys te ine HC1, g l u t a t h i o n e ( reduced) , TPN, TPNH, DPN, DPNH, HMG, glucose 6-phosphate dehydrogenase and 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 commercial p roduc ts . For some exper iments , a n a l y t i c a l l y pure coenzyme A was prepared f rom the commercial product by chromatography on DEAE c e l l u l o s e (Se lec tace l ) accord ing to M o f f a t t and Khorana (54). Glucose 6-phosphate was conver ted to the d ipotass ium s a l t through exhaus t ive t r e a t -ment w i t h A m b e r l i t e IR-120, potassium fo rm, before use. Ace ty l phosphate was prepared as the d i l i t h i u m s a l t f rom a c e t i c anhydr ide and or thophosphate accord ing to Avison (55) and p u r i f i e d ( f i n a l p u r i t y : 95%) by f r a c t i o n a l p r e c i p i t a t i o n from aqueous s o l u t i o n w i t h ethanol (56). Acetyl-CoA was prepared f rom a c e t i c anhydr ide and CoASH accord ing to Simon and Shemin (57), acetoacety l -CoA f rom d ike tene and CoASH accord ing to Lynen e t al_ (35), and HMG-CoA f rom HMG anhydr ide and CoASH accord ing to H i l z e t aj_ (58). These t h i o e s t e r s were used w i t h o u t f u r t h e r p u r i f i c a t i o n . Coenzyme A was measured by the c a t a l y t i c phosphotransacety lase assay (59). P r o t e i n was est imated by the spect rophometr ic method o f Warburg and C h r i s t i a n (60) o r by the mod i f i ed b i u r e t r e a c t i o n o f Gorna l l e t aj_ ( 6 1 ) . Nuc le ic ac id was est imated s p e c t r o p h o t o m e t r i c a l l y (60) o r by the t u r b i d i -m e t r i c method o f Jones and Lipmann ( 6 2 ) . 12. 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 source o f phospho-t ransace ty lase and as a supplementary source o f { 3 - k e t o t h i o l a s e , were p r e -pared e s s e n t i a l l y accord ing to Stadtman and Barker ( 6 3 ) . Vacuum-dried c e l l s (1 g) were ground to a smooth paste w i t h 10 ml o f 0.01 M potassium phosphate b u f f e r , pH 8 . 1 , and incubated w i t h f requen t s t i r r i n g for 4J hours a t 38° . The suspension was c e n t r i f u g e d f o r 15 minutes a t 20,000 x cj, 0 ° . The p r e c i p i t a t e was thoroughly e x t r a c t e d w i t h 5 ml o f co ld g l a s s - d i s t i l l e d w a te r , and the r e s u l t i n g suspension c e n t r i f u g e d as b e f o r e , The t w o - c e l l -f r e e supernatant s o l u t i o n s were combined; 13 to 14 ml o f a b r i g h t y e l l o w e x t r a c t were o b t a i n e d . This e x t r a c t can be shown to c o n t a i n phosphotrans-ace ty lase (64) and ^ - k e t o t h i o l a s e (65) by a p p r o p r i a t e assay procedures. An i n t e r e s t i n g s i d e - l i g h t i s t h a t the ^ - k e t o t h i o l a s e o f t h i s b a c t e r i a l e x t r a c t appears to be a b s o l u t e l y s p e c i f i c f o r CoASH and acetoacety1-CoA; u n l i k e the mammalian enzyme, i t cannot c a t a l y z e a t h i o l y s i s o f a c e t o a c e t y 1 -Pn by pan te the ine . PURIFICATION OF THE ACETOACETATE SYNTHESIZING ENZYME SYSTEM I . From Beef l i v e r Homogenates The acetoaceta te s y n t h e s i z i n g enzyme system o f beef l i v e r was p u r i f i e d accord ing to S te rn e t a]_ (46) f rom b icarbonate e x t r a c t s through 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 , adsorp t ion o f i n a c t i v e p r o t e i n on ca lc ium phosphate g e l , and p r e c i p i t a t i o n w i t h e t h a n o l . Data f rom a t y p i c a l p u r i f i c a t i o n procedure are presented i n Table I I . The p u r i f i c a t i o n procedure is r e a d i l y r e p r o d u c i b l e , and r e g u l a r l y r e s u l t s i n a f i v e - to seven - fo ld p u r i f i c a t i o n o f the system. I t should be noted t h a t i f the l i v e r used has an abnormal ly h igh l i p i d c o n t e n t , the ca lc ium phosphate gel s tep may not be as successfu l as is repor ted here; i n f a c t , i t may r e s u l t i n a decrease, ra the r than inc rease , 13. i n s p e c i f i c a c t i v i t y . 1 1 . From Beef L i v e r Acetone Powder Beef l i v e r was ob ta ined immediately a f t e r s laugh te r and packed in i ce f o r t r a n s p o r t to the l a b o r a t o r y . I f the t i s s u e was not to be used a t once, i t was f rozen w i t h i n two hours a f t e r s l a u g h t e r . To prepare the acetone powder, p a r t i a l l y thawed o r f r e s h t i s s u e was cu t i n t o small p ieces and homogenized w i t h 10 volumes o f co ld (-20°) acetone in a Waring Blendor o p e r a t i n g a t f u l l v e l o c i t y f o r 3 m inu tes . The suspension was then placed in a deep f reeze f o r 15 to 20 m inutes . The supernatant s o l u t i o n was decanted and d i sca rded , and the res idue f i l t e r e d by s u c t i o n on a la rge (25 cm) Buchner f u n n e l , us ing coarse (Whatman No. 5) f i l t e r paper. The damp "cake" which was ob ta ined was b r i e f l y homogenized in the b lendor w i t h a f u r t h e r 5 volumes (based on the we igh t o f the o r i g i n a l t i s sue ) o f co ld acetone, and again f i l t e r e d . The "cake" was washed on the f i l t e r w i t h co ld acetone, and d r i e d as thoroughly as p o s s i b l e by s u c t i o n . The damp mass was then crumbled by hand and d r i e d \n vacuo a t 0°. The d r i e d mass was rubbed to a f i n e powder and s to red over anhydrous ca lc ium s u l f a t e ( " D r i e r i t e " ) a t -20°. Using the above procedure, 20 to 25 g o f a f l u f f y tan -co loured powder can be ob ta ined f rom 100 g o f l i v e r t i s s u e . P repa ra t i on o f e x t r a c t . A p o r t i o n o f the acetone powder was weighed and ground to a smooth paste i n a p r e - c h i l l e d mor tar w i t h 10 volumes o f 0.2 M potassium b icarbonate s o l u t i o n , c o n t a i n i n g 0.005 M c y s t e i n e and ad jus ted to pH 8.2 to 8.3. This suspension was a l lowed to stand 30 to k$ minutes a t 0°, w i t h occas iona l s t i r r i n g , and c e n t r i f u g e d f o r 20 minutes a t 20,000 x c[, 0°. The p r e c i p i t a t e was then thorough ly e x t r a c t e d w i t h a f u r t h e r 2 volumes o f the same b u f f e r , and the suspension was c e n t r i f u g e d . The two supernatant s o l u t i o n s were combined. From 10 g o f acetone powder, 95 to 100 ml o f 14. e x t r a c t were o b t a i n e d . Ammonium s u l f a t e f r a c t i o n a t i o n . The b icarbonate e x t r a c t was d i l u t e d w i t h co ld g l a s s - d i s t i l l e d wa te r , i f necessary, to a d j u s t the p r o t e i n concen t ra -t i o n to 30 mg per m l . S o l i d ammonium s u l f a t e (21.2 g f o r each 100 ml o f d i l u t e d e x t r a c t ) was added g r a d u a l l y over a pe r iod o f 20 minutes , w i t h mechanical s t i r r i n g , to b r i n g the s o l u t i o n to 30% s a t u r a t i o n . A f t e r s tand -ing 15 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x 3, 0°. The p r e c i p i t a t e was d i sca rded . The super-natant s o l u t i o n was brought to 60% s a t u r a t i o n by a d d i t i o n o f another 21.2 g o f s o l i d ammonium s u l f a t e f o r each 100 ml o f o r i g i n a l d i l u t e d e x t r a c t , i n the manner i n d i c a t e d . The mix tu re 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 , and the supernatant f l u i d was d i sca rded . The p r e c i p i t a t e was d i sso l ved in a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e , 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 the same b u f f e r . The r e s u l t s are summarized in Table I I I . I I I . From Beef L i v e r M i tochondr ia l Acetone Powder The la rge sca le i s o l a t i o n o f m i tochondr ia from beef l i v e r and p repara -t i o n o f the acetone powder were c a r r i e d ou t accord ing to the procedure o f Mahler e t a_]_ (66). Prepara t i on o f e x t r a c t . B icarbonate e x t r a c t s o f the m i tochond r ia l acetone powder were prepared by the general procedure o u t l i n e d above f o r beef l i v e r acetone powder. Ammonium s u l f a t e f r a c t i o n a t i o n . The b icarbonate e x t r a c t was d i l u t e d w i t h g l a s s - d i s t i l l e d wa te r , i f necessary, to a d j u s t the p r o t e i n c o n c e n t r a t i o n to 30 mg per m l , and s t i r r e d a t 0°. S o l i d ammonium s u l f a t e (24.7 g f o r each 100 ml o f d i l u t e d e x t r a c t ) was added g r a d u a l l y over 20 m inutes , to b r i n g the s o l u t i o n to 35% s a t u r a t i o n . A f t e r s tand ing 15 minutes w i t h s t i r r i n g , 15. the suspension was c e n t r i f u g e d f o r 20 minutes a t 20>Q00 x 3, 0 ° . The p r e c i p i t a t e was d i sca rded . The supernatant f l u i d was brought to 55% s a t u r a t i o n by the a d d i t i o n o f another 14.1 g o f s o l i d ammonium s u l f a t e f o r each 100 ml o f the o r i g i n a l d i l u t e d e x t r a c t , i n the manner i n d i c a t e d . The suspension 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 , and the supernatant s o l u t i o n was d i sca rded . The p r e c i p i t a t e was d i s s o l v e d in a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7 .5 , c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y z e d o v e r n i g h t vs 4 l i t e r s o f the same b u f f e r . The r e s u l t s are summarized in Table I I I . IV. From Pigeon L i v e r Homoqenates P repa ra t i on o f e x t r a c t . Pigeons were stunned w i t h a blow on the head, d e c a p i t a t e d , and b l e d . The l i v e r s were r a p i d l y removed and immediately f r o z e n . For p r e p a r a t i o n o f the crude e x t r a c t , 28 g o f p a r t i a l l y thawed l i v e r , cu t i n t o small p i e c e s , were placed in a Waring Blendor and 140 ml o f 0.2 M potassium b ica rbona te , c o n t a i n i n g 0 . 0 0 5 M c y s t e i n e and ad jus ted to pH 8.2 to 8 . 4 , were added. The suspension was homogenized f o r 5 minutes a t f u l l v e l o c i t y , c h i l l e d i n ice f o r 10 minutes , and homogenized 5 minutes f u r t h e r a t f u l l v e l o c i t y . The homogenate was s t r a i n e d through f o u r layers o f cheeseclo th to remove f a t p a r t i c l e s and c e n t r i f u g e d f o r 3 0 minutes a t 30 ,000 x ^ , 0 ° . The p r e c i p i t a t e was d i sca rded . Ammonium s u l f a t e p r e c i p i t a t i o n . The crude e x t r a c t was d i l u t e d w i t h an equal volume o f co ld g l a s s - d i s t i l l e d wa te r , and s t i r r e d i n an i ce b a t h . S o l i d ammonium s u l f a t e (28.4 g f o r each 100 ml o f d i l u t e d e x t r a c t ) was added s lowly over a pe r iod o f 20 minutes , to b r i n g the s o l u t i o n to 40% s a t u r a t i o n . A f t e r s tand ing 15 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x 5, 0 ° , and the supernatant f l u i d was d i sca rded . The p r e c i p i t a t e was d i sso lved i n a minimal volume o f 0.02 M 16. potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y z e d o v e r n i g h t vs 4 l i t e r s o f the same b u f f e r . The s l i g h t l y t u r b i d d i a l y z e d e x t r a c t was c e n t r i f u g e d f o r 30 minutes a t 30,000 x a , 0 ° . The r e s u l t s are summarized i n Table 111. V. From Pigeon L i v e r Acetone Powder Pigeon l i v e r acetone powder was prepared by the general procedure o u t l i n e d above f o r beef l i v e r acetone powders. Ex t rac t s were prepared as descr ibed above, us ing e i t h e r the b icarbonate b u f f e r a l ready descr ibed o r 0.05 M potassium phosphate b u f f e r , pH 7.5. Ammonium s u l f a t e p r e c i p i t a t i o n . The crude e x t r a c t was d i l u t e d w i t h co ld g l a s s - d i s t i l l e d water to a d j u s t the p r o t e i n c o n c e n t r a t i o n to 30 mg per m l , and s t i r r e d a t 0 ° . S o l i d ammonium s u l f a t e (24.7 g f o r each 100 ml o f d i l u t e d e x t r a c t ) was added g r a d u a l l y over a pe r i od o f 20 minutes , to b r i n g the s o l u t i o n to 35% s a t u r a t i o n . A f t e r s tand ing 15 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x 0 ° . The supernatant s o l u t i o n was d i sca rded . The p r e c i p i t a t e was d i sso l ved i n a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y z e d o v e r n i g h t vs 4 l i t e r s o f the same b u f f e r . The r e s u l t s a re summarized in Table I I I . ACETOACETATE SYNTHESIZING ASSAY SYSTEM The ace toace ta te s y n t h e s i z i n g a c t i v i t y o f t i s s u e e x t r a c t s was measured as t h e i r a b i l i t y to c a t a l y z e the fo rma t ion o f ace toace ta te f rom a c e t y l phosphate in the presence o f phosphotransacety lase and c a t a l y t i c concen t ra -t i o n s o f CoASH. Two assay systems have been used in t h i s work, both based on the method o f S te rn e t aj[ (46) . Assay system I , used in e a r l i e r exper iments , cons is ted o f Tr is -HCl 17. b u f f e r , pH 8.15, 200 / jmoles; MgCl2, 2.5/umoles; KCl , 10/umoles; g l u t a t h i o n e , 20 / jmo les ; di 1 i th ium a c e t y l phosphate, 45^imoles; coenzyme A, 0.05A""o1es; C l o s t r i d i u m k l u y v e r i e x t r a c t , 0.02 to 0.10 m l , depending on the phosphotrans-ace ty lase a c t i v i t y ; enzyme to be assayed; g l a s s - d i s t i l l e d water to a f i n a l volume o f 2.0 m l . The g l u t a t h i o n e was n e u t r a l i z e d to pH 8 w i t h K0H immediately before use. A l l components except the enzyme f r a c t i o n to be ...... assayed were added to tubes a t 0 ° , and t h i s m i x t u r e was incubated a t 38° f o r 5 m inu tes . 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 incuba t ion was cont inued a t 38°. A f t e r 60 minu tes , the tubes were removed to an ice bath and 1.0 ml o f 12% (w/v) t r i c h l o r o a c e t i c a c i d added. Denatured p r o t e i n was removed by c e n t r i f u g a t i o n , and s u i t a b l e a l i q u o t s o f the super-natant ^ s o l u t i o n were taken f o r de te rm ina t i on o f ace toace ta te by the pheny l -hydrazone method o f Greenberg and Lester (67) as mod i f i ed by B a r k u l i s and Lehninger ( 6 8 ) . For Assay system I I , a l l components o f the above system were reduced to o n e - f o u r t h t h e s t a t e d amounts, w i t h a f i n a l volume o f 0.5 m l . Ace to -ace ta te i n the d e p r o t e i n i z e d s o l u t i o n was measured by a m o d i f i c a t i o n o f the method o f Walker (69) s i m i l a r to t h a t repor ted by K a l n i t s k y and Tapley ( 7 0 ) . The procedure which gave the most rep roduc ib le r e s u l t s , and which has been adopted f o r routine use, 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 the 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 to 0.05 ml w i t h k% (w/v) t r i c h l o r o a c e t i c a c i d i n a g lass-s toppered t e s t tube 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 rap id success ion, 3.5 ml o f M sodium ace ta te b u f f e r , pH 5.1, and 3.0 ml o f the d i a z o t i z e d p - n i t r o a n i1 i n e reagent descr ibed by Walker ( 6 9 ) . The tubes were then placed in a water bath a t 38°. A f t e r 30 m inu tes , the tubes were placed in ice f o r 3 to 5 m inu tes . The s o l u t i o n s were then a c i d i f i e d by the a d d i t i o n o f 6.0 ml o f 5N HCl, and 18. the tubes were kept i n ice f o r 8 to 10 m inu tes . Ethy l ace ta te (4.0 ml) was then added, and the formazan d e r i v a t i v e 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 the o rgan ic layer by shaking the tubes by hand 35 t imes . The absorbance o f the o rgan ic layer was then measured i n a Beckman DU spectrophotometer a t X = 450 itjfi. (d=0.5 cm). Under the above c o n d i t i o n s , 0.1/iimole o f ace to -ace ta te w i l l produce an absorbance o f 0.470. Using t h i s procedure, the co lou r developed was p r o p o r t i o n a l to the amount o f ace toace ta te present over the range o f 0.05 to 0.55yumo1es. One u n i t o f ace toace ta te s y n t h e s i z i n g a c t i v i t y i s de f ined as the amount o f enzyme wh ich , under the c o n d i t i o n s o f e i t h e r o f the above assay systems, ca ta lyzes the fo rma t i on o f 1.0/imole o f ace toace ta te per hour . S p e c i f i c a c t i v i t y is expressed as u n i t s per mg o f p r o t e i n . PREPARATION AND ASSAY OF HMG-CoA CONDENSING ENZYME The HMG-CoA condensing enzyme was p u r i f i e d f rom baker ' s yeast accord ing to the procedure o f Ferguson and Rudney ( 4 1 ) . The p u r i f i c a t i o n was c a r r i e d as f a r as the ammonium s u l f a t e p r e c i p i t a t i o n s tep f o l l o w i n g protamine t rea tmen t . The above authors found t h a t the enzyme a t t h i s stage o f the p u r i f i c a t i o n procedure was very uns tab le i f s to red as a s o l u t i o n , even when f r o z e n . I t was, however, somewhat more s t a b l e i f s to red as a p r e c i p i t a 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 . Even under these c o n d i t i o n s , they repor ted t h a t the enzyme l o s t 25 per cent o f i t s a c t i v i t y i n one week. In c o n t r a s t to t h i s , we found t h a t i f the enzyme s o l u t i o n was thoroughly d i a l y z e d vs d i l u t e b u f f e r (0.01 M Tr is -HCl b u f f e r , pH 7.0) prepared f rom g l a s s - d i s t i l l e d de ion ized w a t e r , i t re ta ined f u l l a c t i v i t y a f t e r 5 months. In l a t e r p repara t ions o f t h i s enzyme, the a u t o l y s i s procedure o f Lynen e t aj_ (35). r a t h e r than t h a t o f Ferguson and Rudney, was used. The HMG-CoA condensing enzyme p repara t ions were assayed by measuring the amount o f ace toace ta te formed in the c a t a l y t i c assay system supp le-mented w i t h an excess o f p a r t i a l l y p u r i f i e d HMG-CoA cleavage enzyme. In the absence o f HMG-CoA cleavage enzyme, the yeas t enzyme was unable to c a t a l y z e the f o rma t i on o f a c e t o a c e t a t e . For our purposes, one u n i t o f HMG-CoA condensing enzyme was de f ined as the amount o f enzyme wh ich , under the c o n d i t i o n s o f the assay, ca ta lyzed the f o rma t i on o f 1.0/umole o f ace to -a c e t a t e per hour . One o f these u n i t s is approx imate ly e q u i v a l e n t to one u n i t o f enzyme " B " a c t i v i t y as de f ined by Lynen e t aj^ (35), who used a s i m i l a r assay method, and is approx imate ly e q u i v a l e n t to th ree u n i t s o f HMG-CoA condensing a c t i v i t y as measured i n the spec t ropho tomet r i c assay o f Ferguson and Rudney ( 4 1 ) . The i r assay i s based on the disappearance o f acetoacety 1-CoA eno la te ion abso rp t i on a t X = 310 rati. The spec t ropho to -m e t r y assay procedure is ra the r u n r e l i a b l e f rom a q u a n t i t a t i v e s t a n d p o i n t , s ince most o f the enzyme f r a c t i o n s are cons iderab ly contaminated w i t h § - k e t o t h i o l a s e , which w i l l i n t e r f e r e w i t h the assay. PREPARATION AND ASSAY OF HMG-CoA CLEAVAGE ENZYME The HMG-CoA cleavage enzyme was p a r t i a l l y p u r i f i e d f rom beef- l i v e r acetone powder e x t r a c t s by a m o d i f i c a t i o n o f the procedure o f Lynen e t al_ (36). The procedure f i n a l l y adopted f o r r o u t i n e use is g iven below. Heat t rea tment . A phosphate b u f f e r e x t r a c t o f beef l i v e r acetone powder, prepared by the general procedure descr ibed p r e v i o u s l y , was heated i n a 55° water bath u n t i l i t s temperature reached 50°. I t was then t r a n s f e r r e d to a 50° bath and kept there f o r 10 m inu tes . The suspension was immediately 20. placed in an i ce bath to c o o l . 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 f o r 45 minutes a t 30,000 x a , 0°. Zinc p r e c i p i t a t i o n . The h e a t - t r e a t e d supernatant s o l u t i o n was d i l u t e d w i t h 0.05 M Tr is -HCl b u f f e r , pH 7.5, to a d j u s t the p r o t e i n c o n c e n t r a t i o n to 15 mg per m l , and s t i r r e d a t 0°. To t h i s d i l u t e d e x t r a c t , 0.25 volume o f 0.1 M z i n c ace ta te s o l u t i o n was added dropwise. A f t e r s tand ing 15 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r TO minutes a t 20,000 x c[, 0°, and the supernatant s o l u t i o n d i sca rded . 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 0.2 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n -ing 0.003 M g l u t a t h i o n e , w i t h the a i d o f a P o t t e r Elvjehm homogenizer. The suspension was c e n t r i f u g e d f o r 10 minutes a t 20,000 x _g, 0°. The res idue was washed w i t h a small volume o f the same b u f f e r and r e c e n t r i f u g e d . The two supernatant s o l u t i o n s were combined. Ammonium s u l f a t e p r e c i p i t a t i o n . The red isso lved z i n c p r e c i p i t a t e was mixed w i t h an equal volume o f a n e u t r a l sa tu ra ted (0°) ammonium s u l f a t e , to p r o -duce 50% s a t u r a t i o n . A f t e r s tand ing 20 to 30 minutes w i t h mechanical s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x a , 0°. The p r e c i p i t a t e was d i sso lved i n a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.002 M c y s t e i n e , and d i a l y z e d o v e r n i g h t vs 5 l i t e r s o f the same b u f f e r . The d i a l y z e d e x t r a c t was then c e n t r i f u g e d f o r 30 minutes a t 30,000 x 5, 0°. The HMG-CoA cleavage enzyme p repara t ions were assayed by measuring the amount o f ace toaceta te formed in the standard acetoaceta te syn thes is assay system supplemented w i t h an excess o f HMG-CoA condensing enzyme from y e a s t . In the absence o f the "condens ing" enzyme, the HMG-CoA cleavage enzyme f r a c t i o n s were unable to c a t a l y z e the fo rma t ion o f ace toace ta te . For our purposes, one u n i t o f HMG-CoA cleavage enzyme was de f ined as the 2 1 . amount o f enzyme w h i c h , under the c o n d i t i o n s o f the assay, ca ta lyzed the f o rma t i on o f l .O / jmo le o f ace toaceta te per hour . One o f these u n i t s is approx imate ly e q u i v a l e n t to one u n i t o f enzyme "A" a c t i v i t y as de f ined by Lynen e t a]_ ( 3 5 ) , who used a s i m i l a r assay procedure, and is approx imate ly equ iva len t to 1.5 to 2 . 0 u n i t s o f HMG-CoA cleavage enzyme a c t i v i t y as de f ined by Bachhawat e t aj_ ( 3 6 ) , who measured the ace toace ta te formed from s t o i c h i o m e t r i c concen t ra t ions o f HMG-CoA. The p u r i f i c a t i o n procedure o u t l i n e d above is e s s e n t i a l l y t h a t o f Lynen e_t aT ( 3 5 ) , out w i t h the omiss ion o f the acetone s t e p . We found t h a t the acetone s t e p , w h i l e ach iev ing a cons iderab le p u r i f i c a t i o n , r e s u l t e d in a cons iderab le loss o f t o t a l a c t i v i t y , due to " s p r e a d i n g " o f the enzyme over a wide range o f acetone concen t ra t ions and to loss o f p r o t e i n through d e n a t u r a t i o n . Thus, t h i s procedure r e s u l t s i n on ly a f i v e - to s e v e n - f o l d p u r i f i c a t i o n , compared to the 3 0 - f o l d p u r i f i c a t i o n repor ted by the above a u t h o r s . However, the t o t a l enzyme recovery was c o n s i s t e n t l y in the reg ion o f 75%, compared to 10%. In any event , the p u r i f i c a t i o n achieved through t h i s mod i f i ed procedure was adequate f o r our purposes. A summary o f a t y p i c a l p u r i f i c a t i o n is g iven in Table I . TABLE I . P u r i f i c a t i o n o f HMG-CoA cleavage enzyme from beef l i v e r acetone powder F r a c t i o n P r o t e i n S p e c i f i c To ta l A c t i v i t y A c t i v i t y Y i e l d mg uni ts/mq uni ts % Phosphate e x t r a c t Heated e x t r a c t Z inc p r e c i p i t a t e (NHi f ) 2 S0^ p r e c i p i t a t e 2100 1404 541 281 0.90 1890 1.28 1790 2.96 1600 4.76 1340 "100" 95 84 71 22. PREPARATION OF RAT TISSUE HOMOGENATES Female ra ts (150 g , f a s t e d 48 hours) were stunned by a blow on the head, decap i ta ted and b l e d . The h e a r t , l i v e r and kidney were r a p i d l y per fused w i t h a small volume o f co ld s a l i n e . The var ious organs were q u i c k l y exc ised and placed i n co ld aerated 0.25 M sucrose s o l u t i o n s . The t i ssues were cu t i n t o small p ieces , b l o t t e d w i t h f i l t e r paper and weighed. Homogenates ( b r a i n , 20%; a l l o t h e r t i s s u e s , 10%) were prepared i n 0.25 M sucrose s o l u t i o n in a Pot te r -E lveh jem homogenizer equipped w i t h a T e f l o n p e s t l e . The homogenates were s to red f rozen f o r 5 days before assay; t h i s has been shown by Bucher e t aj_ (71) and Segal e t a_l_ ( 7 2 ) , and conf i rmed in t h i s l a b o r a t o r y , to produce an " a c t i v a t i o n " o f the ace toace ta te s y n t h e s i z i n g enzyme system, poss ib l y by f r e e i n g the enzyme(s) from the p a r t i c l e - b o u n d fo rm. For some exper iments , m i tochondr ia were i s o l a t e d f rom r a t l i v e r homogenates 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 (73). The mi tochondr ia were a l so " a c t i v a t e d " by f rozen s torage f o r 5 days. RESOLUTION OF THE ACETOACETATE SYNTHESIZING ENZYME SYSTEM I . Procedure o f Bubl i t z (43) To 110 ml o f a phosphate e x t r a c t o f beef l i v e r acetone powder, a t 0 ° , 6.1 ml o f N a c e t i c ac id was added, b r i n g i n g the s o l u t i o n to pH 5.4 (g lass e l e c t r o d e ) . A f t e r s tand ing 10 minutes w i t h g e n t l e s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 30,000 x a , 0 ° . The p r e c i p i t a t e was d i s -carded. The supernatant s o l u t i o n was read jus ted to pH 7.5 by the cau t ious a d d i t i o n o f 1.2 ml 5N K0H. To 120 ml o f ac id supernatant f r a c t i o n , i n a -5° b a t h , 30 ml o f co ld 23. ( -10°) ethanol was added dropwise, w i t h e f f i c i e n t s t i r r i n g , t o produce a f i n a l ethanol c o n c e n t r a t i o n o f 20% by volume. A f t e r s tand ing 10 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d 40 minutes a t 13,000 x - 5 ° . The supernatant s o l u t i o n (142 ml) was brought to a f i n a l ethanol c o n c e n t r a t i o n o f 40% by volume by the dropwise a d d i t i o n o f 47 ml o f co ld ( -20°) e t h a n o l , du r i ng which the temperature was g r a d u a l l y lowered to - 1 5 ° . A f t e r s t i r r i n g f o r an a d d i t i o n a l 10 minutes , the suspension was c e n t r i f u g e d 90 minutes a t 13,000 x -15°. The supernatant s o l u t i o n was d i sca rded . The two p r e c i p i t a t e s were d i sso l ved in minimal volumes o f 0.02 M potassium phosphate b u f f e r , pH 7 .5 , c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y z e d ove rn igh t vs 5 l i t e r s o f the same b u f f e r . The d i a l y z e d 0 to 20% ethanol f r a c t i o n (38 ml) was brought to 32% s a t u r a t i o n by the a d d i t i o n o f 8.6 g o f s o l i d ammonium s u l f a t e g r a d u a l l y over 20 minutes , w i t h mechanical s t i r r i n g . A f t e r s tand ing 20 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x cj, 0 ° . The supernatant s o l u t i o n was brought to 90% s a t u r a t i o n by the a d d i t i o n o f a f u r t h e r 15.6 g o f s o l i d ammonium s u l f a t e , i n the manner i n d i c -a t e d . A f t e r s tand ing 20 minutes , the suspension was c e n t r i f u g e d f o r 20 minutes a t 20,000 x 3, 0 ° . The supernatant s o l u t i o n was d i s c a r d e d . The d i a l y z e d 20 to 40% ethanol f r a c t i o n (25 ml) was brought to 63% s a t u r a t i o n by the a d d i t i o n o f 11.1 g o f s o l i d ammonium s u l f a t e i n the manner i n d i c a t e d . A f t e r s t i r r i n g f o r 20 minutes , the suspension was c e n t r i f u g e d as above. The supernatant s o l u t i o n was brought to 80% s a t u r a t i o n by the a d d i t i o n o f a f u r t h e r 3 g o f s o l i d ammonium s u l f a t e . The suspension was s t i r r e d and c e n t r i f u g e d as descr ibed above. The supernatant s o l u t i o n was d i searded. The f o u r ammonium s u l f a t e p r e c i p i t a t e s were d i sso lved i n minimal 24. volumes o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y z e d vs 3 l i t e r s o f the same b u f f e r . A f t e r 3 hours , the b u f f e r was changed and d i a l y s i s cont inued f o r 5 hours . The d i a l y z e d f r a c t i o n s were c l a r i f i e d by c e n t r i f u g a t i o n f o r 30 minutes a t 30,000 x 3, 0°. I I . Ammonium S u l f a t e F r a c t i o n a t i o n s Ten ml o f a phosphate b u f f e r e x t r a c t o f beef l i v e r acetone powder, prepared by the general procedure o u t l i n e d above, was brought to 50% s a t u r a t i o n by the dropwise a d d i t i o n o f 10 ml o f a n e u t r a l sa tu ra ted (0°) s o l u t i o n o f ammonium s u l f a t e , w i t h e f f i c i e n t s t i r r i n g . A f t e r s t i r r i n g f o r 15 minutes , the suspension was c e n t r i f u g e d 15 minutes a t 20,000 x 3, 0°. The supernatant s o l u t i o n was brought to 85% s a t u r a t i o n by the a d d i t i o n o f 4.9 g o f s o l i d ammonium s u l f a t e over a pe r iod o f 20 m inutes , w i t h mechanical s t i r r i n g . A f t e r 15 minutes the suspension was c e n t r i f u g e d f o r 30 minutes a t 20,000 x a , 0°. Both p r e c i p i t a t e s were d i sso lved i n minimal volumes o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e . An a l i q u o t o f the 50 to 85% sa tu ra ted f r a c t i o n was immediately f r o z e n . The balance o f the 50 to 85% s a t u r a t i o n f r a c t i o n and the 0 t o 50% s a t u r a t i o n f r a c t i o n were d i a l y z e d vs 3 l i t e r s o f the same b u f f e r . A f t e r 2 hours , the b u f f e r was replaced and d i a l y s i s cont inued f o r 2 hours . The b u f f e r was again changed, and d i a l y s i s cont inued f o r a f u r t h e r 2 hours . The d i a l y z e d f r a c t i o n s were c e n t r i f u g e d f o r 30 minutes a t 30,000 x a , 0°. Immediately before assay, a second se t o f f r a c t i o n s was prepared by the same procedure, but w? thout d i a l y s i s . These f r a c t i o n s were assayed as r a p i d l y as poss ib le a f t e r p r e p a r a t i o n . I I I . Chromatography on Calcium Phosphate Gel Experiment 1. Calcium phosphate gel ( " b r u s h i t e " fo rm, 2CaO.P2O5.5H2O) was prepared accord ing to T i s e l i u s (74) and thoroughly washed w i t h 0.002 M 25. potassium phosphate b u f f e r , pH 7.0. A column o f the g e l , 2 cm x 18 cm, was prepared i n a j a c k e t e d chromatography tube. Water, c h i l l e d by passage through Tygon tub ing c o i l e d in a s a l t bath a t -2 to -4°, was c i r c u l a t e d through the j a c k e t . This served to keep the e f f l u e n t a t 2 to k°. The column was e q u i l i b r a t e d f o r temperature in the co ld room f o r k hours . Beef l i v e r ammonium s u l f a t e f r a c t i o n ( t o t a l o f 325 mg p r o t e i n ) , p r e v i o u s l y d i a l y z e d thoroughly vs 0.002 M potassium phosphate b u f f e r , pH 7.0, was placed on top o f the column and thoroughly washed in w i t h the same b u f f e r . The p r o t e i n was e l u t e d w i t h a l i n e a r g r a d i e n t . The mix ing f l a s k i n i t i a l l y conta ined 1 l i t e r o f 0.002 M potassium phosphate b u f f e r , pH 7.0, and the r e s e r v o i r f l a s k 1 l i t e r o f 0.2 M potassium phosphate b u f f e r , pH 7.0. The f l ow ra te was main ta ined a t 1 ml per minute by a p p l i c a t i o n o f s l i g h t pressure to the r e s e r v o i r f l a s k f rom a n i t r o g e n tank . F rac t i ons o f 5 ml were c o l l e c t e d i n t o tubes c o n t a i n i n g 0.05 ml o f 0.1 M neu t ra l c y s t e i n e , to g i ve a f i n a l t h i o l c o n c e n t r a t i o n o f 0.001 M. P r o t e i n c o n c e n t r a t i o n o f the e f f l u e n t was fo l l owed by l i g h t a b s o r p t i o n a t >v= 280 irju. A f t e r 160 tubes had been c o l l e c t e d , p r o t e i n remaining on the column was e l u t e d w i t h 0.2 M potassium phosphate b u f f e r , pH 7.0. (As l a t e r experiments showed, a cons iderab le amount o f p r o t e i n is not e l u t e d w i t h 0.2 M b u f f e r . ) The e lua tes were pooled in l o t s o f approx imate ly 20 tubes (100 ml) and the p r o t e i n p r e c i p i t a t e d by a d d i t i o n o f s o l i d ammonium s u l f a t e (60 g f o r each 100 ml o f s o l u t i o n ) to produce 85% s a t u r a t i o n . The p r e c i p i t a t e s were c o l l e c t e d by c e n t r i f u g a t i o n , d i sso lved i n minimal volumes o f 0.02 M potassium phosphate b u f f e r , pH 7-5, and d i a l y z e d o v e r n i g h t vs 6 l i t e r s o f the same b u f f e r . Experiment 2 . A column o f ca lc ium phosphate gel ( " b r u s h i t e " ) was prepared by the procedure descr ibed above, and e q u i l i b r a t e d w i t h 0.05 M potassium phosphate b u f f e r , pH 7.0. Beef l i v e r ammonium s u l f a t e f r a c t i o n ( t o t a l o f 26. 365 mg o f p r o t e i n ) was adsorbed onto the gel as descr ibed above. P r o t e i n was removed f rom the gel by s tepwise e l u t i o n w i t h phosphate b u f f e r , pH 7.0, o f g radua l l y i nc reas ing i o n i c s t r e n g t h . The peaks (as determined by l i g h t abso rp t i on a t >v= 280 mju) were pooled and l y o p h i l i z e d . The l yophy l i zed powders were d i sso l ved in minimal volumes o f g l a s s - d i s t i l l e d w a t e r , and d ia l yzed vs 7 l i t e r s o f 0.005 M neu t ra l c y s t e i n e f o r 8 hours . The ex te rna l f l u i d was then replaced w i t h 7 l i t e r s o f 0.005 M potassium phosphate b u f f e r pH 7.5, c o n t a i n i n g 0.001 M c y s t e i n e , and d i a l y s i s cont inued f o r 6 hours . IV. Z inc -e thano l F r a c t i o n a t i o n . Ten ml o f beef l i v e r 20 to 35% ethanol f r a c t i o n were d i l u t e d w i t h 10 ml o f g l a s s - d i s t i l l e d wa te r , to produce a p r o t e i n c o n c e n t r a t i o n o f 15 mg per m l . The s o l u t i o n was ad jus ted to pH 6.0 w i t h 2 ml o f potassium succ ina te b u f f e r , pH 6.0. Whi le the s o l u t i o n was e f f i c i e n t l y s t i r r e d , 8 ml o f 0.1 M z i n c ace ta te s o l u t i o n were added dropwise. A f t e r s tand ing f o r 10 minutes w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 10 minutes a t 13,000 x a , 0°. To t h e . c l e a r supernatant s o l u t i o n , 2.0 ml o f co ld (0°) e thanol was added, dropwise, to produce a f i n a l ethanol c o n c e n t r a t i o n o f 6.2% by volume. The suspension was s t i r r e d f o r 10 m inu tes , and c e n t r i f u g e d f o r 15 minutes a t 13,000 x a , 0°. The r e s u l t i n g supernatant s o l u t i o n was c h i l l e d to -5° i n a dry i c e -ethanol ba th , and 2.0 ml o f co ld (-5°) e thanol added dropwise, w i t h s t i r r i n g , to produce a f i n a l ethanol c o n c e n t r a t i o n o f 11.8% by volume. A f t e r s tand ing 10 minutes a t -5° w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 15 minutes a t 13,000 x 5, -5°. The supernatant s o l u t i o n ob ta ined was c h i l l e d to -5°, and 2.0 ml o f -co ld ethanol was added dropwise, w i t h s t i r r i n g , to produce a c o n c e n t r a t i o n 27. o f 16.6% by volume. The suspension was s t i r r e d 15 minutes f u r t h e r and c e n t r i f u g e d f o r 15 minutes a t 13,000 x 3, -5°. This f i n a l supernatant s o l u t i o n was c h i l l e d to - 1 5 ° , and 15 ml o f co ld (-15°) ethanol was added dropwise, w i t h s t i r r i n g , to produce an ethanol c o n c e n t r a t i o n o f 30% by volume. A f t e r s tand ing 15 minutes a t -15° w i t h cont inued s t i r r i n g , the suspension was c e n t r i f u g e d f o r 10 minutes a t 13,000 x - 1 6 ° . A l l p r e c i p i t a t e s were immediately taken up i n minimal volumes o f 0.02 M Tr is -HCl b u f f e r , pH 7-5, c o n t a i n i n g 0.01 M EDTA and 0.1% g l u t a t h i o n e , and d i a l y z e d w i t h o u t c e n t r i f u g i n g f o r 10 hours vs 6 l i t e r s o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M EDTA and 0.001 M c y s t e i n e . The b u f f e r was then replaced w i t h a s i m i l a r b u f f e r , but w i t h o u t EDTA, and d i a l y s i s was cont inued f o r 3 hours . The b u f f e r was replaced by 3 l i t e r s o f f r e s h b u f f e r , a l so w i t h o u t EDTA, and d i a l y s i s was cont inued f o r a f u r t h e r 3 hours . A l l p repara t ions were c e n t r i f u g e d to remove t u r b i d i t y . (The z i n c p r e c i p i t a t e f r a c t i o n conta ined a cons iderab le amount o f i n s o l u b l e p r o t e i n . ) PURIFICATION OF CHICKEN LIVER "INHIBITOR" ENZYME FRACTION Prepara t ion o f e x t r a c t . L i ve rs o f young (8 week) chickens were ob ta ined immediately a f t e r s l a u g h t e r and packed i n i c e . Por t ions o f c h i l l e d l i v e r (150 g) were placed in a Waring Blendor , and 200 ml o f 0.2 M potassium b icarbonate s o l u t i o n , c o n t a i n i n g 0.005 M c y s t e i n e and ad jus ted to pH 8.2 -8.4, were added. The suspension was homogenized a t f u l l v e l o c i t y f o r 5 minutes , and cooled in an ice bath f o r 10 to 15 minutes . Another 100 ml o f the same b u f f e r was added, and homogenization cont inued f o r 5 m inu tes . Homogenates f rom several p o r t i o n s o f l i v e r were poo led , s t r a i n e d through two layers o f cheesec lo th , and c e n t r i f u g e d f o r 60 minutes a t 13,000 x j j , 0°. 28. F i r s t ammonium s u l f a t e f r a c t i o n a t i o n . The b icarbonate e x t r a c t was d i l u t e d w i t h 0.6 volumes o f co ld g l a s s - d i s t i l l e d w a t e r , and s t i r r e d in an i c e - b a t h . S o l i d ammonium s u l f a t e (28.2 g f o r each 100 ml o f d i l u t e d e x t r a c t ) was added g radua l l y over 20 minutes , to b r i n g the s o l u t i o n to 40% s a t u r a t i o n . A f t e r s t i r r i n g f o r 20 minutes , the suspension was c e n t r i f u g e d f o r 60 minutes a t 13>000 x 5, 0 ° . The p r e c i p i t a t e was d i sca rded . The supernatant s o l u t i o n was brought to 70% s a t u r a t i o n by adding another 21.2 g o f s o l i d ammonium s u l f a t e f o r each 100 ml o f o r i g i n a l d i l u t e d e x t r a c t , i n the manner i n d i c a t e d . The mix tu re was s t i r r e d and c e n t r i f u g e d , and the p r e c i p i t a t e d i sso l ved in a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.005 M c y s t e i n e . The s o l u t i o n was d i a l y z e d vs 4 l i t e r s o f the same b u f f e r f o r 3 hours . The b u f f e r was then replaced w i t h 4 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 cont inued f o r 4 hours . Heat t reatment a t a c i d pH. The d i a l y z e d s o l u t i o n was s low ly warmed to room temperature, and ad jus ted to pH 5.5 (g lass e lec t rode ) by the caut ious a d d i t i o n o f 2 N a c e t i c a c i d . The p r e p a r a t i o n was placed i n a bath a t 55° u n t i l the temperature reached 50°, and was then t r a n s f e r r e d t o a bath a t 50°. A f t e r 5 minutes a t t h i s temperature the e x t r a c t was c h i l l e d in an i ce ba th . The pH o f the e x t r a c t was ad jus ted to 7.0 - 7.5, and 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 . This heat t reatment serves to remove any remain-ing a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y , and the heated f r a c t i o n is considered as the s t a r t i n g m a t e r i a l f o r p u r i f i c a t i o n o f the " i n h i b i t o r " f r a c t i o n . Treatment w i t h ca lc ium phosphate g e l . The heated f r a c t i o n was d i l u t e d w i t h an equal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, and the s o l u -t i o n was ad jus ted t o pH 5.5 (g lass e lec t rode ) by the caut ious a d d i t i o n o f 2N a c e t i c a c i d . S u f f i c i e n t ca lc ium phosphate gel (dry w e i g h t , 20 mg per ml) was added in a t h i n s t ream, w i t h e f f i c i e n t s t i r r i n g , to p rov ide a g e l / p r o t e i n 29. r a t i o o f 35/100. S t i r r i n g was cont inued f o r 10 minutes , and the suspension was c e n t r i f u g e d a t 13,000 x c[, 0 ° , f o r 10 minutes . The p r e c i p i t a t e was d i s -carded. To the superna tan t , f u r t h e r ca lc ium phosphate gel (1 ml f o r each 100 mg o f p r o t e i n in the o r i g i n a l heated supernatant) was added in the manner i n d i c a t e d . The suspension was s t i r r e d f o r 10 minutes 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 i sca rded . The supernatant s o l u t i o n was ad jus ted to pH 7.5 by the caut ious a d d i t i o n o f 5N K0H. Second ammonium s u l f a t e f r a c t i o n a t i o n . S o l i d ammonium s u l f a t e (28.2 g f o r each 100 ml o f gel supernatant ) was added g r a d u a l l y over 20 minutes , w i t h mechanical s t i r r i n g , to b r i n g the s o l u t i o n to 40% s a t u r a t i o n . I f a s i g n i f -i c a n t p r e c i p i t a t e formed, i t was removed by c e n t r i f u g a t i o n and d i sca rded . The supernatant s o l u t i o n was brought to 70% s a t u r a t i o n by the a d d i t i o n o f a f u r t h e r 21.2 g o f s o l i d ammonium s u l f a t e f o r each 100 ml o f o r i g i n a l gel superna tan t , i n the manner i n d i c a t e d . A f t e r 20 minutes , the suspension was c e n t r i f u g e d a t 20,000 x c[, 0 ° . The p r e c i p i t a t e was d i sso l ved i n a minimal volume o f 0.02 M potassium phosphate b u f f e r , pH 7.5, c o n t a i n i n g 0.001 M L - c y s t e i n e , and d i a l y z e d o v e r n i g h t vs 6 l i t e r s o f the same b u f f e r . Z inc -e thano l f r a c t i o n a t i o n . Ten ml o f the d i a l y z e d ammonium s u l f a t e f r a c t i o n was d i l u t e d w i t h an equal volume o f co ld g l a s s - d i s t i l l e d wa te r , and s t i r r e d i n an i c e - b a t h . The s o l u t i o n was ad jus ted to pH 6 by a d d i t i o n o f 1.0 ml o f M potassium succ ina te b u f f e r , pH 6 . 0 , and 5.0 ml o f 0.1 M z i n c ace ta te s o l u t i o n added dropwise. A f t e r 5 m inu tes , the suspension was c e n t r i f u g e d a t 20,000 x 5 , 0 ° . The p r e c i p i t a t e was d i sca rded . The supernatant s o l u t i o n was placed in a bath a t -3°, and s u f f i c i e n t co ld ethanol was added dropwise, w i t h mechanical s t i r r i n g , to produce a f i n a l ethanol c o n c e n t r a t i o n o f 18% by volume. The suspension was s t i r r e d 15 minutes a t -3° and c e n t r i f u g e d a t 20,000 x a., -3°. In a s i m i l a r manner, 30. the r e s u l t i n g supernatant s o l u t i o n was brought to 27% ethanol by volume, w h i l e the temperature was g radua l l y reduced to - 6 ° . The suspension was s t i r r e d f o r 15 minutes a t -6° and c e n t r i f u g e d f o r 15 minutes a t 13,000 x al, - 6 ° . The supernatant f l u i d was then brought to 37.4% ethanol by volume i n a s i m i l a r manner, w h i l e the temperature was g radua l l y reduced to - 1 5 ° . The suspension was c e n t r i f u g e d a t 13,000 x - 1 5 ° , and the supernatant s o l u -t i o n d i sca rded . The three ethanol p r e c i p i t a t e s were d i sso l ved in minimal volumes o f 0.02 M potassium phosphate b u f f e r , pH 7 .5 , c o n t a i n i n g 0.001 M EDTA and 0 .1% g l u t a t h i o n e and d i a l y z e d wi thou t c e n t r i f u g i n g vs 2 l i t e r s o f 0.02 M potassium phosphate b u f f e r , pH 7 . 5 , c o n t a i n i n g 0.001 M EDTA and 0.005 M L - c y s t e i n e . A f t e r 2 hours , f r e s h b u f f e r was added and d i a l y s i s cont inued f o r 3 hours . At t h i s t ime, the ex te rna l f l u i d was replaced by a s i m i l a r b u f f e r , but wi thout EDTA, and d i a l y s i s was cont inued f o r a t o t a l o f 5 hours vs 3 changes o f 2 l i t e r s each o f t h i s b u f f e r . An assay system has been devised f o r the " i n h i b i t o r enzyme" on the basis o f i t s i n t e r f e r e n c e w i t h ace toace ta te fo rma t ion by beef l i v e r enzymes. One u n i t o f " i n h i b i t o r enzyme" has been de f ined as the amount o f enzyme which produces a 50% decrease in the ra te o f ace toaceta te fo rma t ion by 2.0 to 2.2 u n i t s o f 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 under the standard c o n d i t i o n s o f assay system I , o r 0.5 to 0.55 u n i t s under the standard cond i t i ons o f assay system I I . The decrease in the ra te o f ace toaceta te f o rma t i on is p ropor -t i o n a l to the amount o f " i n h i b i t o r enzyme" p r o t e i n added, p r o v i d i n g t h a t the ra te is not decreased by more than 60% ( c f . F igure 5 ) • 31. RESULTS PURIFICATION OF THE ACETOACETATE-SYNTHESIZING SYSTEM Our understanding o f the p r e c i s e mechanism o f ace toace ta te syn thes is has been hindered i n the past due t o f a i l u r e o f var ious i n v e s t i g a t o r s to achieve a h igh degree o f p u r i f i c a t i o n o f the enzyme system. Our f i r s t approach to c l a r i f y i n g the mechanism invo lved e f f o r t s to improve the degree o f p u r i t y beyond t h a t repor ted by Stern e t a j ( 4 6 ) . These at tempts us ing f r e s h homogenates o f beef l i v e r as s t a r t i n g m a t e r i a l have l a r g e l y met w i t h f a i l u r e . A t y p i c a l p u r i f i c a t i o n o f 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 system is summarized in Table I I . Attempts to achieve f u r t h e r p u r i f i c a t i o n by the use o f alumina Cy gel and o rgan ic so lven ts (acetone, methanol , t e r t . - b u t a n o l , n - b u t a n o l , n-propanol) were unsuccess fu l . These at tempts i n v a r i a b l y r e s u l t e d in f r a c t i o n s w i t h less a c t i v i t y than the s t a r t i n g 20-35% ethanol f r a c t i o n ; the o rgan ic s o l v e n t s , p a r t i c u l a r l y the h igher a l c o h o l s , u s u a l l y produced cons iderab le p r o t e i n d e n a t u r a t i o n and very cons iderab le loss o f a c t i v i t y . Recombinations o f var ious f r a c t i o n s ob ta ined by the above procedures prov ided no evidence tha t more than one enzymatic e n t i t y was requ i red f o r ace toace ta te f o r m a t i o n . TABLE I I . P u r i f i c a t i o n o f a c e t o a c e t a t e - s y n t h e s i z i n g system from beef l i v e r e x t r a c t s Enzyme A c t i v i ty F r a c t i o n Pro te i n S p e c i f i c To ta l A c t i v i t y A c t i v i t y Y i e l d mg uni ts/mg uni ts % Bicarbonate e x t r a c t 5780' 0.123 712 0.480 532 0.590 455 0.985 246 100"** (NH^) 2 S0 i f , 30-60% s a t . 1100 Gel supernatant 770 Ethano l , 20-35% 250 * 300 ml o f d i l u t e d e x t r a c t * * A r b i t r a r i l y taken as 100% 75 64 35 32. Because our at tempts to improve the p u r i f i c a t i o n from f r e s h beef l i v e r homogenates f a i l e d , we undertook p u r i f i c a t i o n s tud ies us ing beef l i v e r acetone powder and beef l i v e r m i tochondr ia l acetone powder as s t a r t i n g m a t e r i a l . I t might be emphasized t h a t Lynen e t a_l_ (35) used beef l i v e r acetone powder as s t a r t i n g ma te r i a l f o r t h e i r s t u d i e s . We have a lso at tempted p u r i f i c a t i o n us ing f r e s h pigeon l i v e r homogenates and pigeon l i v e r acetone powder. The data on the p a r t i a l p u r i f i c a t i o n o f the system from these sources are shown in Table 111. I t can be seen t h a t the crude e x t r a c t s o f the acetone powders o f whole beef l i v e r and o f beef l i v e r mi tochondr ia e x h i b i t a h igher s p e c i f i c a c t i v i t y than do b icarbonate e x t r a c t s o f beef l i v e r . Attempts to o b t a i n h i g h l y a c t i v e f r a c t i o n s f rom these e x t r a c t s have again been unsuccess fu l . Pigeon l i v e r p r e p a r a t i o n s , both homogenates and acetone powder e x t r a c t s , are much more a c t i v e than the corresponding beef l i v e r p r e p a r a t i o n s . This appeared promis ing a t f i r s t , but a l l a t tempts to achieve e i t h e r ex tens ive p u r i f i c a t i o n o f the pigeon l i v e r system o r r e s o l u t i o n o f i t i n t o two or more f r a c t i o n s r e s u l t e d i n f a i l u r e . A l l the procedures t r i e d (iise o f adsorbents , s a l t and o rgan ic so lven ts ) r e s u l t e d on ly i n loss of a c t i v i t y . Thus the at tempts to o b t a i n a h i g h l y p u r i f i e d a c e t o a c e t a t e - s y n t h e s i z i n g system have been unsuccessful and have t h e r e f o r e y i e l d e d no i n f o r m a t i o n regard ing the enzymatic mechan-s ims. PROPERTIES OF THE ACETOACETATE-SYNTHESIZING SYSTEM The p r o p e r t i e s o f 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 system have been s tud ied by Lynen e t aj . (35) and by Drummond and Stern (47). A r e i n v e s t i -g a t i o n o f some o f these mat ters is repor ted here . I t was f e l t t h a t a c l e a r knowledge o f the p r o p e r t i e s o f the system could shed l i g h t on the TABLE I I I . P u r i f i c a t i o n o f ace toace ta te -syn thes i z ing system f rom o t h e r l i v e r p repara t ions Source o f E x t r a c t F r a c t i o n Pro te i n mg S p e c i f i c A c t i v i ty uni ts/mg Tota l A c t i v i ty uni ts Recovery % Beef l i v e r acetone pdr . Bicarbonate e x t r a c t (NHz f) 2S0/ 4 30-60% s a t . 4860* 1575 0.253 0.540 1230 850 " 1 0 0 " * * 69 Beef 1i ver mi t o -chondr ia acetone pd r . B icarbonate e x t r a c t ( N H 4 ) 2 S 0 i f 35-55% s a t . 2800* 880 0.450 0.870 1260 766 " 1 0 0 " * * 61 Pigeon l i v e r homogenate Bicarbonate e x t r a c t (NH4) 2S0^ Q-45% s a t . 4080*** 1185 0.430 1.340 1755 1590 " 1 0 0 " * * 91 Pigeon l i v e r acetone pdr . B icarbonate e x t r a c t (NH/^SOk 0-35% s a t . 2190* 1030 0.845 1.300 1850 1340 II 100' ' * * 73 Pigeon l i v e r acetone pdr . Phosphate e x t r a c t (NHj^SOi,. 0-35% s a t . 2020* 775 2.55 4.50 5150 3490 " 1 0 0 " * * 68 * From 10 g o f acetone powder. * * A r b i t r a r i l y taken as 100%. * * * From 28 g o f pigeon l i v e r . 34. enzymatic mechanisms i n v o l v e d . Of p a r t i c u l a r i n t e r e s t is the repor ted t h i o l and magnesium requirement (35, 47). These two f a c t o r s are known to be requ i red f o r HMG-CoA cleavage enzyme a c t i v i t y and i f HMG-CoA were an in te rmed ia te i n ace toace ta te fo rma t ion the t h i o l and magnesium requirement o f the l a t t e r system could r e a d i l y be e x p l a i n e d . E f f e c t o f D i va len t Cat ions and T h i o l s . Enzymatic syn thes is o f ace toace ta te by p u r i f i e d beef l i v e r f r a c t i o n s e x h i b i t e d a marked requirement f o r d i v a l e n t c a t i o n . I f d i v a l e n t c a t i o n is om i t ted from the assay system, ace toace ta te syn thes is is depressed by 15 to 25% (Table I V ) . I f d i v a l e n t c a t i o n is o m i t t e d , and EDTA added to the assay system, ace toace ta te syn thes is is v i r t u a l l y complete ly b locked . M n + + was found to be the most e f f e c t i v e a c t i v a t o r , w i t h M g + + o n l y s l i g h t l y less e f f e c t i v e (F igure 1). Low concen-t r a t i o n s o f C o + + were found to produce a s l i g h t a c t i v a t i o n o f ace toace ta te TABLE IV. M g + + s t i m u l a t i o n and EDTA i n h i b i t i o n o f ace toace ta te syn thes is Cond i t ions Acetoaceta te synthes ized / imoles per hour Complete system 0.68 No Mg++ 0.52 No Mg++, p lus 10" 3 M EDTA 0.05 Standard assay c o n d i t i o n s were employed, except as i n d i c -a t e d . The 20-35% e thanol f r a c t i o n o f beef l i v e r (1.1 mg p r o t e i n ) was used. f o r m a t i o n . A l l o t h e r d i v a l e n t ca t ions tes ted ( F e + + , C d + + , S r + + , C a + + , N i + + and Cu"1"1") were i n h i b i t o r y . At h igh c o n c e n t r a t i o n s , a l l the d i v a l e n t ca t ions were i n h i b i t o r y . Acetoaceta te syn thes is by both crude and p u r i f i e d l i v e r f r a c t i o n s was a lso markedly s t i m u l a t e d by a d d i t i o n o f t h i o l s . In the absence o f added [ M * * ] (pmoles/ml) Figure 1. E f f e c t o f d i v a l e n t c a t i o n c o n c e n t r a t i o n on the ra te o f ace toaceta te s y n t h e s i s . Standard assay c o n d i t i o n s were employed, except as i n d i c a t e d . The 20-35% ethanol f r a c t i o n o f beef l i v e r (1 .1 mg p r o t e i n ) was used. 0-6 , i 1— Ui o E n_ 0-4 ITATE [TOACE 0-2 U J o < 0 ...i. .... L. • 0 10 20" 30 [GSH] (umoles/ml) Figure 2. E f f e c t o f GSH c o n c e n t r a t i o n on the ra te o f ace to-ace ta te s y n t h e s i s . Standard assay c o n d i t i o n s were employed, except as i n d i c a t e d . The 20-35% ethanol f r a c t i o n o f beef l i v e r (1.1 mg p r o t e i n ) was used. 37. t h i o l , p u r i f i e d f r a c t i o n s were v i r t u a l l y i n a c t i v e (F igure 2 ) . Drummond and Stern (47) repor ted t h a t several monothio ls (GSH, c y s t e i n e , mercapto-e t h a n o l , mereaptoethy1 amine, and 0 ( - t h i o l g l y c e r o l ) were approx imate ly equa l l y e f f e c t i v e in producing maximal a c t i v a t i o n a t 10"^ M c o n c e n t r a t i o n , w h i l e th ioma la te and t h i o g l y c o l l a t e were p a r t i a l l y e f f e c t i v e , and pante-the ine was i n h i b i t o r y . D i t h i o l compounds (DTO, BAL, and reduced l ipoamide) were e f f e c t i v e a t I0"3 M c o n c e n t r a t i o n . In the present s tudy , GSH was r o u t i n e l y used as the t h i o l a c t i v a t o r , s ince v i r t u a l l y a l l o t h e r t h i o l s i n t e r f e r e w i t h the e s t i m a t i o n o f ace toace ta te by the method o f Walker (69) by producing h i g h l y - c o l o u r e d b lanks . The co lou r produced by severa l t h i o l s is g iven in Table V. This obse rva t i on in f a c t ra ises the p o s s i b i l i t y o f TABLE V. Th io l i n t e r f e r e n c e w i t h the de te rm ina t ion o f ace toace ta te by the Walker procedure Th io l /,> .Absorbance ( A = 450 mu) G lu ta th ione 0.000 Cyste ine 0.040 BAL 0.109 DTO 0.319 2-Mercaptoethanol 0.098 T h i o g l y c o l l a t e 0.046 Ten/umoles o f each t h i o l in 0.5 ml o f 4% t r i -c h l o r o a c e t i c ac id was t rea ted as descr ibed under Ma te r ia l s and Methods f o r de te rm ina t ion o f ace toace ta te by the mod i f ied procedure o f Walker ( 6 9 ) . adapt ing the Walker procedure f o r use as an assay f o r t h i o l compounds. Al though the procedure is ra the r complex and t ime-consuming, i t has two marked advantages over the n i t r o p r u s s i d e method o f Grunert and P h i l l i p s (75): the co lou r produced is q u i t e s t a b l e , and d i t h i o l compounds r e a c t . 38. This l a t t e r p o i n t may make f u r t h e r i n v e s t i g a t i o n i n t o t h i s procedure w o r t h -w h i l e , s ince none o f the c u r r e n t methods a v a i l a b l e f o r de te rm ina t i on o f d i t h i o l compounds is very s a t i s f a c t o r y . Drummond and Stern (47) showed t h a t h igh concen t ra t ions o f d i t h i o l s depressed ace toace ta te by l i v e r enzymes. As F igure 2 shows, GSH is a l so i n h i b i t o r y a t h igh c o n c e n t r a t i o n s . The p r e c i s e mechanism by which t h i o l s a c t i v a t e the enzymatic symthesis o f ace toace ta te is not y e t d e f i n i t e l y e s t a b l i s h e d . Drummond and Stern (47) demonstrated t h a t maintenance o f coenzyme A i n the reduced form is not the mechanism, s ince reducing agents such as sodium s u l f i d e , potassium borohydr ide , and sodium cyan ide , which can reduce d i s u l f i d e s , produced on ly a s l i g h t a c t i v a t i o n . Al though phosphotransacety lase and ^ - k e t o t h i o l a s e are a c t i v a t e d by t h i o l s , t h e i r a c t i v i t i e s were a l ready excessive i n the absence o f added t h i o l . By p r e i n c u b a t i o n o f enzyme w i t h t h i o l , f o l l owed by e f f e c t i v e removal o f the t h i o l by r o t a r y d i a l y s i s o r d i l u t i o n and assay o f the enzyme w i t h o u t added t h i o l , these workers were ab le to achieve a p a r t i a l a c t i v a t i o n o f ace toace ta te s y n t h e s i s , i n d i c a t i n g the p o s s i b i l i t y t h a t reduc t i on o f an enzyme o r bound coenzyme may be i n v o l v e d . Consider ing the wide v a r i e t y o f t h i o l s which can a c t i v a t e the system, i t i s u n l i k e l y t h a t the t h i o l becomes bound as a coenzyme. In a b r i e f examinat ion o f ace toace ta te syn thes is by r a t l i v e r m i t o -chondr ia , i t was found t h a t t h i o l s a c t i v a t e d the system, as w i t h beef l i v e r enzymes, but t h a t a d d i t i o n o f Mg 4 4" caused a decrease, ra the r than an i n -crease, i n ace toace ta te f o rma t i on (Table V I ) . I t was a lso found t h a t 10"3 M EDTA, which almost t o t a l l y blocked ace toace ta te syn thes is by beef 1 i ve r enzymes, had very l i t t l e e f f e c t on ace toace ta te syn thes is by r a t l i v e r m i tochondr ia . Fur ther experiments showed t h a t h igher concen t ra t i ons o f EDTA d id produce a s i g n i f i c a n t i n h i b i t i o n o f ace toaceta te f o r m a t i o n by 39. TABLE V I . E f f e c t o f M g + + , GSH and EDTA on ace toace ta te syn thes is by r a t l i v e r mi tochondr ia System Acetoaceta te synthes ized Aimoles per hour Complete 0.731 No t h i o l 0 . 2 9 6 No M g + + 0.900 No M g - ^ , no t h i o l 0 . 4 2 6 No M g + + , no t h i o l , p lus 10-3 M EDTA 0 . 3 5 2 No M g + + , p lus 10-3 M EDTA 0 . 8 4 5 Standard c o n d i t i o n s o f assay system I I were used, except as i n d i c -a t e d . The enzyme used was a r a t l i v e r " a c t i v a t e d " m i tochondr ia l p r e p a r a t i o n (1.82 mg p r o t e i n ) . the m i tochondr ia l p r e p a r a t i o n (Table V I I ) . A poss ib le e x p l a n a t i o n o f these observa t ions could be the presence o f s u f f i c i e n t M g + + i n the mi tochondr ia l p r e p a r a t i o n to produce maximal s t i m u l a t i o n o f the enzyme system, so t h a t TABLE V I I . E f f e c t o f EDTA on acetoaceta te syn thes is by r a t l i v e r mi tochondr ia EDTA c o n c e n t r a t i o n Acetoaceta te synthes ized Aimoles per hour 1 x 1 0 - 3 M 0 . 4 4 9 2 x 1 0 - 3 M 0 . 4 2 1 1 x 1 0 - 2 M 0 . 3 2 3 2 x 1 0 - 2 M 0 . 2 4 4 Standard c o n d i t i o n s o f assay system I I were used, w i t h the omiss ion o f MgCl2, and the a d d i t i o n o f EDTA as i n d i c a t e d . The enzyme f r a c t i o n used was a r a t l i v e r " a c t i v a t e d " m i tochondr ia l p r e p a r a t i o n ( 0 . 9 mg o f p r o t e i n ) . 4 0 . added c a t i o n would produce an i n h i b i t o r y c o n c e n t r a t i o n ( c f . F igure 1)• The f a c t t ha t EDTA is i n h i b i t o r y on ly a t very h igh concen t ra t ions might i n d i c a t e the presence o f bound;.metal. Attempts to unequ ivoca l l y demonstrate a M g i + requirement w i t h d ia l yzed mi tochondr ia and w i t h mi tochondr ia fragmented by b r i e f homogenization a t 14,000 rpm i n a Se rva l l Omni-mixer and subsequent ly d i a l y z e d were unsuccess fu l , as both procedures r e s u l t e d in almost t o t a l loss o f enzyme. Heat I n a c t i v a t i o n . The ou ts tand ing p roper t y o f the a c e t o a c e t a t e - s y n t h e s i z -ing sys temi i s i t s marked heat l a b i l i t y . The a b i l i t y o f l i v e r f r a c t i o n s to c a t a l y z e the f o rma t i on o f ace toace ta te is almost complete ly dest royed by hea t ing the e x t r a c t s f o r 5 minutes a t 50°, a t e i t h e r neu t ra l o r a c i d pH. I t has been shown by Lynen e t a l (35). and conf i rmed in t h i s l a b o r a t o r y , t h a t heated e x t r a c t s can be r e a c t i v a t e d by the a d d i t i o n o f p u r i f i e d HMG-CoA condensing enzyme. This w i l l be discussed in more d e t a i l in a l a t e r s e c t i o n . I n h i b i t o r s . Acetoaceta te synthes is by l i v e r f r a c t i o n s is not i n h i b i t e d by te t rae thy lpy rophospha te (5 x 10"4 M ) (47), o r by cyanide (10"^ M). Drummond and S te rn (47) showed t h a t concen t ra t ions o f a r s e n i t e g rea te r than 10"3 M produced s i g n i f i c a n t i n h i b i t i o n . Pret reatment o f enzyme f r a c t i o n s w i t h iodoacetamide, N-ethylmaleimide o r pe r ioda te produces s i g n i f i c a n t i n h i b i t i o n o f the enzyme (Table V I I I , F igure 3). TABLE VI1 I E f f e c t o f p re t rea tment o f enzyme w i t h i n h i b i t o r s I n h i b i t o r Concent ra t ion Acetoaceta te /U moles None 1.380 lodoacetamide 2.0 x 10"3 M 0.617 N-ethylmale imide 2.0 x 10 _ l f M 0.795 2.0 x 10-3 M ' 0.150 Potassium pe r ioda te 2.8 x \0"1* M 0.0 Beef l i v e r (NH^SOZ* 30 to 60% s a t . , thoroughly d i a l y z e d vs 0.02 M potassium phosphate b u f f e r , pH 7.5. to remove t h i o l , was incubated a t 0° w i t h the above i n h i b i t o r s a t 0°. A f t e r 30 m inutes , excess iodoacetamide and N - e t h y l -maleimide were d ischarged w i t h excess c y s t e i n e , and excess pe r i oda te was discharged w i t h excess Tr is -HCl b u f f e r , pH 7.5. A l i q u o t s o f each s o l u t i o n , rep resen t ing 1.5 mg p r o t e i n , were then taken f o r assay o f ace toace ta te syn-t h e s i z i n g a c t i v i t y under standard c o n d i t i o n s . Figure 3. I n h i b i t i o n o f 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 system by t reatment w i t h iodoacetamide. Beef l i v e r (NHi^SO^ 30-60% s a t u r a t i o n f r a c t i o n was d i l u t e d to 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 per m l , and incubated a t 0° i n the presence o f 1 x 10""3 M iodoacetamide. At i n t e r v a l s , as no ted , a l i q u o t s were removed and remaining iodoacetamide discharged w i t h excess c y s t e i n e . P r o t e i n was p r e c i p i t a t e d w i t h ammonium s u l f a t e (80% s a t u r a t i o n ) , c o l l e c t e d by c e n t r i f u g a t i o n , and d i sso l ved in 0.02 M potassium phosphate b u f f e r , pH 7 . 5 . The d i sso lved p r o t e i n was then d ia l yzed f o r 2 hours vs the same b u f f e r , c o n t a i n i n g 1 x 10"2 c y s t e i n e , and then 3 hours vs^  f r e s h b u f f e r c o n t a i n i n g 1 x 10*3 M c y s t e i n e . The enzyme f r a c t i o n s were then assayed i n the standard assay system. 43. RESOLUTION OF THE ACETOACETATE-SYNTHESIZING SYSTEM The second major approach to the problem o f c l a r i f y i n g the mechanism o f ace toace ta te fo rmat ion cons is ted o f e f f o r t s to reso lve the system i n t o two or more enzymatic e n t i t i e s . Complete r e s o l u t i o n would r e s u l t i n loss o f a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y . This a c t i v i t y would then be res to red a f t e r recombinat ion o f the requ i red enzymatic e n t i t i e s . S p e c i f i c a l l y , i f HMG-CoA were an in te rmed ia te i n ace toaceta te f o r m a t i o n , then i t should be p o s s i b l e to separate the two enzymes i n v o l v e d , i . e . , the HMG-CoA condensing and cleavage enzymes. Acetoaceta te fo rma t ion could proceed on ly upon t h e i r combinat ion . Our f i r s t experiments cons is ted o f e f f o r t s to con f i rm the presence and the r e s o l u t i o n o f these two enzymes by the methods repor ted by the Munich group. I . The Procedure o f B u b l i t z In 1956, Lynen b r i e f l y repor ted (43) t h a t B u b l i t z , work ing i n h i s (Lynen's) l a b o r a t o r y , had achieved the separa t ion o f the ace toace ta te -s y n t h e s i z i n g system o f beef l i v e r acetone powder e x t r a c t s i n t o two f r a c t i o n s , both o f which were requ i red f o r ace toace ta te f o r m a t i o n . No d e t a i l s o f the exact technique used f o r the separa t ion have ever been pub l i shed ; the on ly i n f o r m a t i o n a v a i l a b l e is a ra the r sketchy " f l o w s h e e t " , which is reproduced in F igure 4. Using t h i s " f l o w sheet" as a gu ide , we devised a d e t a i l e d procedure in an a t tempt to reproduce the r e s o l u t i o n o f the system i n t o two d i s t i n c t f r a c t i o n s . The r e s u l t s are summarized in Table IX. From the data in the t a b l e , i t is obvious t h a t a complete sepa ra t i on had not been ach ieved, s ince 43% o f the a c e t o a c e t a t e - s y n t h e s i z -ing a c t i v i t y present i n the s t a r t i n g m a t e r i a l was recovered in one o f the f i n a l f r a c t i o n s . A l l poss ib le combinat ions o f the two ethanol f r a c t i o n s 44. Beef l i v e r acetone powder e x t r a c t A c e t i c ac id to pH 5.4 Supernatant 0-20% ethanol P rec i p i ta te Ammonium s u l f a t e 0-32% s a t u r a t i o n (Enzyme A) Supernatant 20-40% ethanol P r e c i p i t a t e Ammonium s u l f a t e 0-63% s a t u r a t i o n P rec ip i t a t e Supernatant Ammonium s u l f a t e 63-80% s a t u r a t i o n (Enzyme B) F igure 4. O u t l i n e o f f r a c t i o n a t i o n procedure f o r separa t i on o f 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 system. This o u t l i n e is the o n l y i n f o r m a t i o n a v a i l a b l e on the procedure used by B u b l i t z (43). 45. TABLE IX. Attempt to reproduce the resolution of the acetoacetate-synthesizing system into two components by the procedure of Bublitz (4-3). ^Fraction Protein Specific Total Yield activity activity \". M mg uni ts/mg uni ts % 1; Phosphate buffer extract 4150 0.245 1018 "100" 2. Acid supernatant 3230 0.319 1000 98 3. a. Ethanol ppt., 0-20% 494 0.110 54 5 b. Ethanol ppt., 20-40% 849 0.568 483 48 4. Low ethanol: , a. (NH^ OSOL, 0-32% sat. 198 0.0 0 0 b. " 32-90% sat. 216 0.185 40 4 5. High ethanol: a. (NHi.)2S0it, 0-63% sat. 712 0.610 435 43 b. " 63-80% sat. 26 0.0 0 0 For d e t a i l s o f the procedure used, see under Ma te r ia l s and Methods. and the four ammonium sulfate fractions were tried. In every case, there was no evidence for any degree of resolution of the system into two com-ponents; the acetoacetate formed by a combination of any two fractions was always the same as or less than the sum of the acetoacetate formed by the fractions when assayed individually. It is quite possible that our detailed procedure may differ from the exact method used by Bublitz in one or more important details; this could account for his success and our failure. In any event, we were unable to resolve the system into two components by a combination of ethanol and ammonium sulfate fractionations. 11. Ammonium Sulfate Fractionations In 1958, Lynen e_t a_l_ (35) reported that a preparation of "enzyme B", which they equated with the HMG-CoA condensing enzyme, could be obtained by fractionation of a beef liver acetone powder extract with ammonium sulfate between the limits of 50 and 80% saturation. They also reported that this "enzyme B" preparation from liver extracts was very unstable. 46. I t could not be d i a l y z e d ; i t was uns tab le i f s to red a t 0 ° , l o s i n g 50% of i t s a c t i v i t y i n 8 hours and v i r t u a l l y a l l i t s a c t i v i t y i n 24 hours . Al though i t was s t a b l e f o r several months a t - 2 0 ° , i t r a p i d l y l o s t a c t i v i t y i f repeatedly f rozen and thawed. A se r ies o f experiments were c a r r i e d o u t w i t h e x t r a c t s o f beef l i v e r acetone powder f r a c t i o n a t e d a t 0-50% and 50-80% s a t u r a t i o n w i t h ammonium s u l f a t e , as descr ibed i n Experimental S e c t i o n . The r e s u l t s o f these experiments are summarized in Table X. From Experiment I , i t can be seen t h a t d i a l y z e d 0-50% sa tu ra ted and d i a l y z e d 50-80% sa tu ra ted f r a c t i o n s were approx imate ly equa l l y e f f e c t i v e c a t a l y z i n g the syn thes is o f ace toace ta te . When these two f r a c t i o n s were assayed i n combina t ion , the amount o f ace toace ta te formed ( 2 . 5 4 / jmo les ) was almost exac t l y the sum o f the ace toace ta te formed by the two f r a c t i o n s when assayed separa te l y (2 .52 /Umoles) . This ha rd ly i nd i ca tes r e s o l u t i o n o f the enzyme: systern i n t o two components. I t was a lso found t h a t f r e s h l y prepared f r a c t i o n s and f r a c t i o n s prepared the prev ious day and s to red o v e r n i g h t a t - 1 8 ° , w i t h o u t d i a l y s i s , had i d e n t i c a l a c t i v i t y . When the und ia lyzed 50-80% s a t u r a t i o n f r a c t i o n s were assayed in combinat ion w i t h the d ia l yzed 0-50% s a t u r a t i o n f r a c t i o n , the ace toace ta te formed was con-s i d e r a b l y less than the sum o f the ace toace ta te formed by the i n d i v i d u a l f r a c t i o n s , i n d i c a t i n g the presence o f some i n h i b i t o r y f a c t o r i n the un-d i a l y z e d f r a c t i o n . In Experiment M , f r e s h l y prepared ammonium s u l f a t e f r a c t i o n s were assayed a lone , t o g e t h e r , and w i t h p u r i f i e d HMG-CoA condensing and cleavage enzymes. Aga in , the acetoaceta te formed when the two f r a c t i o n s are com-bined was cons iderab ly less than the sum o f the acetoaceta te formed by the i n d i v i d u a l f r a c t i o n s (0.52 /Umoles vs 0.84/umoles). However, i t can be seen t h a t ace toace ta te f o rma t i on by the 0-50% s a t u r a t i o n f r a c t i o n was 47 . TABLE X. Attempted r e s o l u t i o n o f a c e t o a c e t a t e - s y n t h e s i z i n g system o f beef l i v e r acetone powder e x t r a c t s by s a l t f r a c t i o n a t i o n Expt . Ammonium s u l f a t e f r a c t i o n s Acetoaceta te >umo 1 es (1) 0-50% s a t . , d i a l y z e d 1.27 (6 .1 mg p r o t e i n ) (2) 50-80% s a t . , d i a l y z e d 1.25 (7 .8 mg p r o t e i n ) (3) 50-80% s a t . , u n d i a l y z e d , f r ozen and thawed 0.41 ( 6 . 8 mg p r o t e i n ) (4) 50-80% s a t . , u n d i a l y z e d , f r e s h l y prepared 0.38 (6 .2 mg p r o t e i n ) (1) p lus (2) 2.54 (1) p lus (3) 0.75 (1) p lus (4) 0.68 (1) 0-50% s a t . , und ia l yzed , f r e s h l y prepared 0.65 (6 .5 mg p r o t e i n ) (2) 50-80% s a t . , u n d i a l y z e d , f r e s h l y prepared 0.19 (6 .1 mg p r o t e i n ) (1) p lus (2) 0.52 (1) p lus HMG-CoA condensing enzyme (4 u n i t s ) 2.38 (2) p lus HMG-CoA cleavage enzyme (4 u n i t s ) 0.39 The ammonium s u l f a t e f r a c t i o n s were prepared as descr ibed under M a t e r i a l s and Methods. HMG-CoA condensing and cleavage enzymes were prepared and assayed as descr ibed e a r l i e r . Standard assay c o n d i t i o n s (assay system I) were employed. increased almost f o u r - f o l d on a d d i t i o n o f p u r i f i e d HMG-CoA condensing enzyme, and t h a t ace toace ta te fo rma t ion by the 50-80% s a t u r a t i o n f r a c t i o n was doubled on a d d i t i o n o f p u r i f i e d HMG-CoA cleavage enzyme. This was a t l e a s t a t e n t a t i v e i n d i c a t i o n t h a t HMG-CoA might be an i n t e r m e d i a t e . I t was a l s o a t e n t a t i v e i n d i c a t i o n t h a t perhaps the two enzymes had been p a r t i a l l y separa ted . However, no f i r m conc lus ions could be drawn and, moreover, the recovery o f a c t i v i t y was not s a t i s f a c t o r y . We have thus been unable to reproduce the r e s u l t s repor ted by the German workers . The r e s u l t s o f the above experiments i n d i c a t e a p o s s i b l e i n h i b i t i o n o f 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 system by ammonium s u l f a t e . To s e t t l e t h i s q u e s t i o n , a p p u r i f i e d beef l i v e r enzyme and a crude e x t r a c t o f beef l i v e r acetone powder were assayed in the presence and absence o f ammonium s u l f a t e (Table X I ) . From the r e s u l t s , i t is obvious t h a t the ace toace ta te -s y n t h e s i z i n g system is i n h i b i t e d e i t h e r by ammonium s u l f a t e o r by some i m p u r i t y present i n commercial ammonium s u l f a t e . This obse rva t i on p o i n t s up the necess i ty f o r adequate d i a l y s i s o f a l l enzyme f r a c t i o n s used in s tud ies on ace toace ta te s y n t h e s i s . TABLE X I . I n h i b i t i o n o f the ace toaceta te s y n t h e s i z i n g system by ammonium s u l f a t e > (NHi t) 2SO/ f F r a c t i o n P r o t e i n concen- Acetoaceta te t r a t i o n mg M yumoles Beef l i v e r 20-35% ethanol 3.80 - 2.24 3.80 0.2 0.72 Beef l i v e r acetone powder e x t r a c t 6.25 - 1.17 " 6.25 0.2 0.31 Standard assay c o n d i t i o n s (assay system I) were employed, except as i n d i c a t e d . The ammonium s u l f a t e c o n c e n t r a t i o n r e f e r s to the f i n a l c o n c e n t r a t i o n i n the assay system, and corresponds to 5% s a t u r a t i o n . A p r e l i m i n a r y a t tempt has been made to determine the s i t e o f i n h i b i -t i o n by ammonium s u l f a t e . HMG-CoA condensing enzyme, HMG-CoA cleavage enzyme and excess C. k l u y v e r i e x t r a c t were added to p u r i f i e d 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 in the presence o f s u f f i c i e n t ammonium s u l f a t e to depress ace toace ta te fo rma t ion 45% (Table X I l ) . Acetoaceta te syn thes is 4 9 . was res to red on ly by the p u r i f i e d HMG-CoA condensing enzyme, i n d i c a t i n g tha t t h i s may be the s i t e o f i n h i b i t i o n . However, t h i s is not i n accord w i t h the statement by Rudney and Ferguson (41) t h a t the HMG-CoA condensing enzyme is not i n h i b i t e d by the presence o f ammonium s u l f a t e . Lack o f t ime has prevented f u r t h e r i n v e s t i g a t i o n o f t h i s aspect o f the problem. TABLE X I I . S i t e o f i n h i b i t i o n o f ace toace ta te syn thes is by ammonium s u l f a t e System Acetoaceta te Aimoles Enzyme 0 .52 Enzyme + ammonium s u l f a t e 0 .29 Enzyme + ammonium s u l f a t e + HMG-CoA condensing enzyme 0.51 Enzyme + ammonium s u l f a t e + HMG-CoA cleavage enzyme 0 .30 Enzyme + ammonium s u l f a t e + excess C. k l u y v e r i e x t r a c t 0.31 Standard assay c o n d i t i o n s (assay system I I ) were employed, except as i n d i c a t e d . The enzyme used was a 20-35% ethanol f r a c t i o n o f beef l i v e r (1.1 mg p r o t e i n ) . Where ammonium s u l f a t e was added, the f i n a l c o n c e n t r a t i o n in the assay system was 0 .05 M. Where no ted , 5 u n i t s each o f HMG-CoA condensing and cleavage enzymes were added. "Excess" C_. k l u y v e r i e x t r a c t was produced by adding th ree times the normal amount o f the e x t r a c t . I I I . Attempted Reso lu t ion by Column Chromatography The use o f column chromatography f o r the p u r i f i c a t i o n and separa t i on o f enzymes is now we l l e s t a b l i s h e d . Since prev ious e f f o r t s to reso lve the system by c l a s s i c a l procedures had met w i t h l i t t l e success, we exp lored the use o f chromatography. A s i n g l e a t tempt a t chromatography on DEAE-c e l l u l o s e as descr ibed by Peterson and Sober (76) was s i n g u l a r l y unsuccess-f u l . The on ly r e s u l t was almost t o t a l d e s t r u c t i o n o f the system. Since the standard p u r i f i c a t i o n procedure o f 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 system, as o u t l i n e d above, invo lves a ca lc ium phosphate gel s t e p , chromatography 50. o f the system on ca lc ium phosphate gel ( " b r u s h i t e " f o r m ) , as descr ibed by T i s e l i u s ( 7 4 ) , was a t tempted. Al though a more s t a b l e form o f ca lc ium phosphate gel ( " h y d r o x y l a p a t i t e " ) f o r enzyme chromatography has been des-c r i b e d ( 7 7 ) , the somewhat less s t a b l e " b r u s h i t e " form was se lec ted s ince i t permi ts more rap id f l o w r a t e s . Rapid f l o w rates seemed d e s i r a b l e , due to the repor ted i n s t a b i l i t y o f l i v e r HMG-CoA condensing enzyme (35.39). Experiment 1 . In the f i r s t exper iment , the enzyme was adsorbed on a column o f " b r u s h i t e " and e l u t e d by a l i n e a r g r a d i e n t o f phosphate b u f f e r , w i t h i nc reas ing c o n c e n t r a t i o n as the parameter. The recovery o f 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 system, as summarized in Table X I I I , was very low (12.6% o f the a c t i v i t y placed on the gel column was recovered) . To determine whether t h i s low recovery was due s imply to d e s t r u c t i o n o f TABLE X I I I . Chromatography o f 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 system on ca lc ium phosphate gel ( " B r u s h i t e " ) . Gradient e l u t i o n . F r a c t i o n Tubes P r o t e i n Speci f i c Tota l Y i e l Pooled A c t i v i ty A c t i v i ty uni ts/mq uni ts 1 Beef l i v e r 325.0 0.740 240.0 (NHL ) 2S0U 30-60% s a t . 1 26-45 15.4 0 0 11 46-65 15.8 0 0 111 66-85 23.0 0 0 IV 86-105 39.2 0.172 6.7 2.8 V 106-125 26.1 0.473 12.4 5.2 VI 126-144 11.5 0.397 4.6 2.0 VI1 145-170 6.1 0.497 3.0 1.3 VI 1 1 170-196 58.2 0.050 3.0 1.3 Tota ls 199.3 29.7 12.6 d For d e t a i l s , see under M a t e r i a l s and Methods. 51. enzyme o r to separa t i on o f the enzyme system i n t o more than one component, var ious recombinat ions were a t tempted . One such recombinat ion experiment is summarized i n Table XIV. That a t l e a s t a p a r t i a l r e s o l u t i o n had occurred was obv ious ; two f r a c t i o n s which when assayed i n d i v i d u a l l y ca ta lyzed the f o r m a t i o n o f a t o t a l o f 0 .14/ jmoles o f ace toace ta te ca ta lyzed the fo rmat ion o f 0.70/timoles o f ace toaceta te when combined, a f i v e - f o l d inc rease . A l l the f r a c t i o n s e l u t e d f rom the gel column were then assayed f o r HMG-CoA condensing and cleavage enzyme a c t i v i t i e s ; the r e s u l t s are summarized in Table XV. The r e s u l t s showed t h a t the HMG-CoA condensing enzyme was e l u t e d from the gel a t r e l a t i v e l y low b u f f e r c o n c e n t r a t i o n s , and tha t the bu lk o f the HMG-CoA cleavage enzyme was e l u t e d a t h igher b u f f e r c o n e n t r a t i o n s , a l though some HMG-CoA cleavage enzyme a c t i v i t y was found in TABLE XIV. P a r t i a l r e s o l u t i o n o f 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 system by chromatography on ca lc ium phosphate gel Experiment Enzyme Acetoacetate Ai moles F r a c t i o n V I , 0.280 mg p r o t e i n 0.10 2 F r a c t i o n V I I I , 0.570 mg p r o t e i n 0.04 3 F r a c t i o n V I , 0.280 mg p r o t e i n p lus F r a c t i o n V I I I , 0.570 mg p r o t e i n 0.70 4 F r a c t i o n V I , 0.280 mg p r o t e i n p lus F r a c t i o n V I I I , 1.140 mg p r o t e i n 0.78 Standard assay c o n d i t i o n s (assay system I I ) were employed. For a d e s c r i p t i o n o f the f r a c t i o n s , see Table X I I I . 52. TABLE XV.. Chromatography o f 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 system on ca lc ium phosphate gel ( " b r u s h i t e " ) . D i s t r i b u t i o n o f HMG-CoA condensing and cleavage enzymes in e l u a t e s . HMG-CoA condensing enzyme HMG-CoA cleavage enzyme F r a c t i o n S p e c i f i c To ta l Speci f i c To ta l A c t i v i ty A c t i v i ty Y i e l d A c t i v i ty A c t i v i ty Y i e l d uni ts/mq uni ts % uni ts/mg uni ts % Beef l i v e r (NHi f ) 2 S0 i f 0.740 240.0 1.23 400.0 30-60% s a t . 1 0 0 0 0 0 0 11 0 0 0 0 0 0 111 0 0 0 0.100 2.3 0.6 IV 0.324 12.7 5.3 0.172 6.7 1.7 V 2.780 56.3 23.4 0.473 12.4 3.1 VI 0.397 4.6 2.0 0.571 6.6 1.6 VI 1 0.497 3.0 1.3 1.310 8.0 2.0 VI1 1 0.050 3.0 1.3 1.510 82.4 20.6 To ta l s 79.6 33.3 118.4 29.6 For d e s c r i p t i o n o f the f r a c t i o n s , see Table X I I I . HMG-CoA condensing and cleavage enzymes were assayed as descr ibed under M a t e r i a l s and Methods. Y ie lds are c a l c u l a t e d on basis o f % o f the a c t i v i t y placed on the column which is recovered i n the f r a c t i o n . almost a l l f r a c t i o n s . Whi le r e s o l u t i o n was incomplete, the recover ies were low, there was a s t rong i n d i c a t i o n t h a t both the HMG-CoA condensing and cleavage enzymes were p resen t , and t h a t they had been p a r t i a l l y reso lved . Experiment 2 . With t h i s encouraging i n d i c a t i o n , another chromatographic separa t ion was a t tempted , us ing a s tep-w ise e l u t i o n technique r a t h e r than the g rad ien t used i n the f i r s t exper iment . The r e s u l t s are summarized i n Tables XVI and X V I I . The recovery o f a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y (Table XVI) was somewhat h igher than t h a t i n the f i r s t column experiment (34.3% vs 12.6%). However, as Table XVII shows, the recover ies o f HMG-CoA TABLE XVI . Chromatography o f a c e t o a c e t a t e - s y n t h e s i z i n g system on ca lc ium phosphate gel ( " B r u s h i t e " ) F r a c t i o n Beef l i v e r (NH i f ) 2 S04, 30-60% s a t . I 11 111 IV V VI Eluant concen t r a t i o n M 0.01 0.05 0.10 0.10 0.30 0.50 P r o t e i n S p e c i f i c Tota l a c t i v i t y a c t i v i t y mg 33.2 37.8 47.4 35.4 111.4 18.0 uni ts/mq uni ts 334.0 0.450 0 0 0.455 0.845 0 0 0 0 21 .6 29.9 0 0 Y i e l d % 150.0 "100.0" 0 0 14.4 19.9 0 0 To ta l s 273.2 51.5 34.3 * Eluant was potassium phosphate b u f f e r , pH 7 . 0 . For d e t a i l e d procedure, see M a t e r i a l s and Methods. * * A r b i t r a r i l y taken as 100%. 54. TABLE X V I I . Chromatography o f the ace toace ta te s y n t h e s i z i n g system on ca lc ium phosphate gel ( " b r u s h i t e " ) . D i s t r i b u t i o n o f o f HMG-CoA condensing and cleavage enzymes in the e l u a t e s . F r a c t i o n HMG-CoA condensing enzyme S p e c i f i c To ta l A c t i v i t y A c t i v i t y Y i e l d HMG-CoA cleavage enzyme S p e c i f i c To ta l A c t i v i t y A c t i v i t y Y i e l d uni ts/mg uni ts JL uni ts/mg uni ts % Beef l i v e r (NH/t)2S0if, 0.450 150 "100.0" 0.770 257.0 "100.0' 30-60% s a t . 1 0 0 0 0 0 0 1 1 0 0 0 0.270 10.2 4.0 1 1 1 2.010 95.4 63.6 0.455 21 .6 8.4 IV 1.070 37.9 25.3 1 .220 43.2 16.8 V 0 0 0 1.330 148.0 57.6 VI 0 0 0 0 0 0 Tota ls 133.3 88.9 223.0 86.8 For a d e s c r i p t i o n o f the f r a c t i o n s , see Table XVI . HMG-CoA condensing and cleavage enzyme a c t i v i t i e s were determined as descr ibed under M a t e r i a l s and Methods. Y ie lds are c a l c u l a t e d on the basis o f % o f a c t i v i t y p laced on the column which was recovered i n the f r a c t i o n . 55. condensing and cleavage enzymes were e s s e n t i a l l y q u a n t i t a t i v e , a cons ide r -ab le improvement over the f i r s t exper iment . I t can be seen f rom Table XVII t h a t f r a c t i o n I I I was e s s e n t i a l l y f r e e o f cleavage enzyme but 63.6% o f the condensing enzyme a c t i v i t y . F r a c t i o n V was complete ly devoid o f condens-ing enzyme, but conta ined 57.6% o f the cleavage enzyme a c t i v i t y . Thus, a very c l e a r r e s o l u t i o n o f the two enzymes had occurred in these two f r a c t i o n s w i t h e x c e l l e n t recovery o f the o r i g i n a l a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y . This was a .very s t rong i n d i c a t i o n t h a t HMG-CoA is indeed an i n te rmed ia te . A l l a t tempts to o b t a i n a more complete r e s o l u t i o n o f the system on " b r u s h i t e " columns have been unsuccess fu l . T i s e l i u s has p o s t u l a t e d t h a t the ca lc ium phosphate gels f u n c t i o n as weak ion-exchangers; i t would appear f rom our r e s u l t s t h a t the s p e c i f i c i t y o f the m a t e r i a l s w i t h respect to ion-exchange is i n s u f f i c i e n t to g ive a good r e s o l u t i o n o f the HMG-CoA condensing and cleavage enzymes. Another p o s s i b i l i t y i s t h a t the phys ica l c h a r a c t e r i s t i c s o f the two enzymes are very s i m i l a r ; t h i s would e x p l a i n the g rea t d i f f i c u l t y i n separa t i ng them by convent ional enzyme p u r i f i c a t i o n techn iques . TV. Studies on Heated Ex t rac ts I t has been mentioned p r e v i o u s l y t h a t an o u t s t a n d i n g f e a t u r e o f 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 system is i t s marked heat l a b i l i t y . I t was r e -por ted by Lynen e t a]_ ( 3 5 ) , and has been conf i rmed in t h i s l a b o r a t o r y , t h a t l i v e r e x t r a c t s l o s t t h e i r a b i l i t y to syn thes ize ace toace ta te i n the c a t a l y t i c assay system a f t e r b r i e f h e a t - t r e a t m e n t , and t h a t the a d d i t i o n o f p u r i f i e d HMG-CoA condensing enzyme to these heated e x t r a c t s res to red 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 a c t i v i t y to the i n i t i a l l e v e l s . The e f f e c t o f heat t reatment on acetoaceta te syn thes is by crude e x t r a c t s o f beef l i v e r 56. and o f beef and pigeon l i v e r acetone powders, and by p u r i f i e d f r a c t i o n s from beef l i v e r is shown in Table XVII I . As the t a b l e shows, a d d i t i o n o f p u r i f i e d HMG-CoA condensing enzyme to the crude e x t r a c t s (bu t not to the 20-35% ethanol f r a c t i o n ) produced a s t i m u l a t i o n o f a c e t o a c e t a t e - s y n t h e s i z -ing a c t i v i t y . S i m i l a r observa t ions have been repor ted by Wieland e t a l (78) and by Bucher e t aj_ (71) f o r r a t l i v e r e x t r a c t s . Assuming t h a t ace toaceta te fo rma t ion does proceed v i a HMG-CoA, t h i s would i n d i c a t e t h a t t h a t the HMG-CoA condensing enzyme is the r a t e - l i m i t i n g f a c t o r i n ace to -ace ta te syn thes is by crude e x t r a c t s . Heat ing such e x t r a c t s a t neu t ra l pH f o r 8 minutes destroyed complete ly t h e i r a b i l i t y to form ace toace ta te (Table X V I I l ) • When excess p u r i f i e d HMG-CoA condensing enzyme from yeas t was added to these heated e x t r a c t s , a c e t o a c e t a t e - s y n t h e s i z i n g a b i l i t y was complete ly r e s t o r e d . However, a d d i t i o n o f the yeas t enzyme to the heat -t r e a t e d 20-35% ethanol f r a c t i o n d i d not r e s t o r e the leve l o f ace toace ta te syn thes is to t h a t o f the i n i t i a l p r e p a r a t i o n . As the r e s u l t s o f the d? rec t HMG-CoA cleavage enzyme assays show, hea t ing had l i t t l e o r no e f f e c t on t h i s enzyme in the crude e x t r a c t s , but almost complete ly removed i t f rom the 20-35% e thanol f r a c t i o n . The r e s u l t s i n d i c a t e t h a t the heat l a b i l i t y o f 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 system may indeed be due to d e s t r u c t i o n o f the HMG-CoA condensing enzyme, and tha t i n the most p u r i f i e d ace to -a c e t a t e - s y n t h e s i z i n g f r a c t i o n (20-35% e thanol f r a c t i o n ) , the cleavage enzyme is a l so l a r g e l y removed by hea t . This is f u r t h e r s t rong evidence t h a t ace toace ta te syn thes is may proceed v i a HMG-CoA. V. F r a c t i o n a t i o n w i t h Ethanol ?n the Presence o f Z inc A major c r i t i c i s m o f the work o f Lynen e t a_[ (35) i s t h a t most o f the s tud ies were c a r r i e d ou t us ing a p u r i f i e d HMG-CoA condensing enzyme from y e a s t . Any study o f the HMG-CoA pathway as the mechanism f o r ace toace ta te TABLE XVII I. Effect of purified HMG-CoA condensing enzyme on acetoacetate synthesis by heated liver fractions Fraction Fresh beef 1 ?ver: Crude extract After heat-treatment Ethanol ppt., 20-35% After heat-treatment Beef liver acetone powder: Crude extract After heat-treatment Pigeon liver acetone powder Crude extract After heat-treatment Protein mq/ml 62.0 31.6 44.0 18.2 30.2 20.2 31.4 24.0 HMG-CoA cleavage enzyme units/ml 64.0 4.3 36.2 35.4 Aceto-; acetate synthesi s uni ts/ml 8.3 0.8 25.4 1.4 14.3 1.2 Acetoacetate syn-thesis with HMG-CoA condensing enzyme uni ts/ml 13.7 14.7 24.9 4.2 22.0 22.0 Recove ry % 107 16.6 100 48.7 5.8 100.0 96.1 96.1 . The fresh beef liver fractions were prepared as described by Stern et aj_ (46) (cf. Table II). The acetone powder extracts were prepared as described under Materials and Methods. Heat treat-ment was carried out by adjusting the fractions to pH 7.5 and heating for 8 minutes at 50°, followed by cooling and centrifugation. The HMG-CoA cleavage enzyme activities were determined by the direct assay of Bachhawat et |_[ (37), except that the Mg+* and thiol concentrations were reduced to produce maximal conditions, the other components were reduced by one-half, and acetoacetate was determined by a modification of the method of Walker (69), as already described. For measurement of acetoacetate-synthesizing activity, standard conditions (assay system II) were employed. 58. syn thes is should inc lude the i s o l a t i o n and i d e n t i f i c a t i o n o f HMG-CoA con-densing enzyme from 1 i v e r e x t r a c t s . Rudney (39) and Lynen e t aj_ (35) have repor ted t h a t the HMG-CoA condensing enzyme o f l i v e r e x t r a c t s is very un-s t a b l e , and c la imed t h a t t h i s l a b i l i t y prec luded at tempts to o b t a i n a p u r i f i e d enzyme from l i v e r e x t r a c t s . However, the . a c e t o a c e t a t e - s y n t h e s i z -ing system o f l i v e r e x t r a c t s is s t a b l e f o r r e l a t i v e l y long p e r i o d s . P u r i f i e d a c e t o a c e t a t e - s y n t h e s i z i n g e x t r a c t s r e t a i n f u l l a c t i v i t y f o r severa l weeks when f r o z e n , i f repeated ly thawed and r e f r o z e n , and one p u r i f i e d e x t r a c t , f rozen immediately a f t e r p r e p a r a t i o n and s to red con t inuous ly a t -18° w i t h o u t thawing, was f u l l y a c t i v e a f t e r more than one year . These two o b s e r v a t i o n s , the marked l a b i l i t y o f the HMG-CoA condensing enzyme and the r e l a t i v e s t a b i l i t y o f 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 system, d id not seem compat ib le w i t h the concept t h a t HMG-CoA was an o b l i g a t o r y i n t e r -mediate i n ace toaceta te f o r m a t i o n . In an at tempt to shed f u r t h e r l i g h t on the mechanism, we have sub jec ted the most p u r i f i e d ace toace ta te -s y n t h e s i z i n g p r e p a r a t i o n (20-35% ethanol f r a c t i o n ) to var ious f r a c t i o n a -t i o n procedures. By p r e c i p i t a t i o n w i t h z i n c ion fo l l owed by inc reas ing concen t ra t ions o f e t h a n o l , several f r a c t i o n s were ob ta ined in which a c e t o a c e t a t e - s y n t h e s i z i n g a b i l i t y was v i r t u a l l y absent (Table X I X ) . The f r a c t i o n s ob ta ined by ethanol p r e c i p i t a t i o n showed no increase in a c t i v i t y when assayed i n the presence o f excess yeast condensing enzyme. These f r a c t i o n s , however, showed a very great increase in a c t i v i t y when assayed in the presence o f excess HMG-CoA cleavage enzyme (Table X I X ) . The l a t t e r enzyme was shown by separate s p e c i f i c assay to be present on ly i n very small amounts i n two o f these f r a c t i o n s . This s t r o n g l y i n d i c a t e d t h a t these f r a c t i o n s conta ined the HMG-CoA condensing enzyme l a r g e l y f r e e o f cleavage enzyme a c t i v i t y . In f a c t , these f r a c t i o n s conta ined more than 90% TABLE XIX. HMG-CoA condensing enzyme from l i v e r f r a c t i o n s Acetoaceta te- HMG-CoA condensing enzyme syn thes iz ing system F r a c t i o n P r o t e i n Speci f i c A c t i v i ty Tota l A c t i v i ty Yi.ejd S p e c i f i c A c t i v i ty Total A c t i v i ty Y i e l d ma uni ts/mg uni ts 1 uni ts/mg uni ts % Ethanol p p t . , 20-35% 407.0 0.585 239.0 "100.0" 1.030 420.0 "100.0" Zinc p p t . 53.3 0.250 13.3 5.6 0.735 39.2 9.3 Ethanol p p t . , 0-6.2% 44.5 0.130 5.4 2.3 2.080 92.5 22.0 Ethanol p p t . , 6.2-11.8% 60.0 0.102 6.1 2.5 2.450 147.0 35.0 Ethanol p p t . , 11.8-16.6% 81.0 0.154 12.5 5.0 1.640 133.0 3K5 Ethanol p p t . , 16.6-30.0% 21.5 0.081 1.8 0.7 0.500 10.8 2.6 T o t a l s : 260.3 (64%) 39.1 16.1 422.5 100.5 For d e s c r i p t i o n o f the f r a c t i o n a t i o n procedure, see Mate r ia l s and Methods. Standard assay eond i t i ons f o r a c e t o a c e t a t e - s y n t h e s i z i n g system and HMG-CoA condensing enzyme a c t i v i t i e s were employed. 6 0 . of the o r i g i n a l HMG-CoA condensing enzyme a c t i v i t y o f the i n i t i a l 2 0 - 3 5 % ethanol f r a c t i o n , as determined by ace toace ta te fo rma t ion in the presence o f excess HMG-CoA cleavage enzyme, and less than 1 0 % o f the i n i t i a l ace to -ace ta te -syn thes i z i n g a c t i v i t y . The r a t i o o f HMG-CoA condensing enzyme a c t i v i t y to a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y i n one f r a c t i o n was as h igh as 2k. This procedure has been repeated several t imes, and appears to be r e a d i l y r e p r o d u c i b l e . The recovery o f HMG-CoA condensing enzyme in a l l cases has been e s s e n t i a l l y q u a n t i t a t i v e . The data are a very c l e a r i n d i c a t i o n f o r the almost complete and q u a n t i t a t i v e sepa ra t i on o f the HMG-CoA condensing enzyme from the cleavage enzyme. A f t e r several months a t - 1 8 ° , these " Z n + + - e t h a n o l " f r a c t i o n s have re ta ined t h e i r f u l l HMG-CoA condensing enzyme a c t i v i t y . However, they are now t o t a l l y unable to syn thes ize any ace toace ta te i n the absence o f supplementary HMG-CoA cleavage enzyme. This is an i n d i c a t i o n t h a t the small amount o f cleavage a c t i v i t y they o r i g i n a l l y conta ined has d e t e r i o r a t e d and we now have con-densing enzyme p repara t ions w i t h a b s o l u t e l y no cleavage enzyme contamina-t i o n . That the " Z n + + - e t h a n o l " f r a c t i o n s do indeed con ta in the HMG-CoA condensing enzyme has been f u r t h e r demonstrated i n several ways. The f i r s t p o i n t o f ev idence, o f course, i s the assay system used: syn thes is o f ace toace ta te i n the presence o f p u r i f i e d HMG-CoA cleavage enzyme. The f r a c t i o n s were a lso f u l l y ab le to replace yeast enzyme i n r e s t o r i n g ace to -ace ta te syn thes is i n heated l i v e r e x t r a c t s (Table XX) which have been shown p r e v i o u s l y to c o n t a i n the cleavage enzyme. In another exper iment , HMG-CoA was accumulated by i ncuba t ing a " Z n + + - e t h a n o l " f r a c t i o n in a mod i f ied assay system and i d e n t i f i e d as the hydroxamic ac id d e r i v a t i v e by paper chromatography (Table XXI) . A procedure f o r the i s o l a t i o n and TABLE XX Equivalence o f HMG-CoA condensing enzyme prepara t ions from yeas t and l i v e r System Acetoaceta te /imp les /hou r Heat t r e a t e d l i v e r f r a c t i o n 0 . 0 9 H e a t - t r e a t e d l i v e r f r a c t i o n p lus HMG-CoA condensing enzyme (yeast) 1 . 0 0 H e a t - t r e a t e d l i v e r f r a c t i o n plus l i v e r "In** - e t h a n o l " f r a c t i o n 0 . 9 9 The heated l i v e r f r a c t i o n was an ammonium s u l f a t e 3 0 - 6 0 % s a t . f r a c t i o n o f beef l i v e r e x t r a c t s , heated f o r 8 minutes a t 5 0 ° f o l l owed by c o o l i n g and c e n t r i -f u g a t i o n ( 1 . 4 mg p r o t e i n ) . Where no ted , 4 u n i t s o f p u r i f i e d yeast HMG-CoA condensing enzyme was added. The l i v e r " Z n + + - e t h a n o l " f r a c t i o n was the 1 1 . 8 - 1 6 . 6 % ethanol f r a c t i o n descr ibed in Table XIX ( 0 . 7 5 mg p r o -t e i n ) . Standard assay c o n d i t i o n s were employed. TABLE XXI . I d e n t i f i c a t i o n o f t h i o e s t e r formed by beef l i v e r " Z n + + - e t h a n o l " f r a c t i o n Experiment System Rf values o f No. hydroxamic ac ids 1 No enzyme 0.64 2 Yeast condensing enzyme 0.65, 0.37 3 Beef l i v e r enzyme 0.65, 0.37 Known HMG hydroxamic a c i d 0.35 Known acethydroxamic a c i d 0.63 A l l tubes conta ined Tr is -HCl b u f f e r , pH 8.15, 100/umoles; KCl , 5/Cimoles; MgCl2, 1 / imole ; g l u t a t h i o n e , 10 / jmoles; d i l i t h i u m ace ty l phosphate, 45/umoles; coenzyme A, 1 / imole ; C_. k l u y v e r i e x t r a c t , 0.05 m l . Experiment 1 was a c o n t r o l , w i t h no enzyme added. The yeast HMG-CoA condensing enzyme was 10 u n i t s , ass-ayed as descr ibed under M a t e r i a l s and Methods. The beef l i v e r enzyme was the 11.8-16.6% e thanol f r a c t i o n descr ibed i n Table  XIX (4,9 mg p r o t e i n ) . F ina l volume, 1.0 m l . The tubes were incubated f o r 60 minutes a t 38°. The r e a c t i o n was stopped by a c i d i f i c a t i o n to pH 4 w i t h 2N a c e t i c a c i d and hea t ing a t 100° f o r th ree minutes ; t h i s a l so serves to dest roy res idua l a c e t y l phosphate. Th ioes te rs present were then converted to t h e i r hydroxamic d e r i v a t i v e s and prepared f o r chromatography accord-ing to Hele e t a [ (79), and separated by ascending paper chromatography on Whatman 3MM paper accord ing to Lynen e t a l (35). i d e n t i f i c a t i o n o f the HMG-CoA per se is p r e s e n t l y being developed, but t ime l i m i t a t i o n s have precluded complet ion o f the s t u d y . The above-descr ibed "Zri** - e t h a n o l " f r a c t i o n a t i o n s were performed on a 20-35% ethanol f r a c t i o n prepared w i t h 0.02 M phosphate as b u f f e r . When a s i m i l a r l i v e r p r e p a r a t i o n , w i t h 0.02 M Tr is -HCl as b u f f e r , was 63. sub jec ted to the same t rea tment , the f r a c t i o n a t i o n was unsuccess fu l . This i nd i ca tes the p o s s i b i l i t y t h a t the HMG-CoA cleavage enzyme is bound to and/or destroyed by the p r e c i p i t a t e o f z i n c phosphate which forms when the z i n c ace ta te s o l u t i o n is added to the phosphate-buf fered enzyme p repara -t i o n s . A l though HMG-CoA cleavage enzyme can be recovered f o l l o w i n g p r e c i p i t a t i o n w i t h Z n + + f rom less p u r i f i e d l i v e r f r a c t i o n s by e x t r a c t i o n w i t h h igh concen t ra t ions (0 .2 M o r g rea te r ) o f phosphate, no more than 15 to 20% can be recovered when the 20-35% e thanol f r a c t i o n s are t r e a t e d by the procedure used here . This might i n d i c a t e t ha t the HMG-CoA cleavage enzyme in t h i s f r a c t i o n is dest royed on a d d i t i o n o f Z n + + . However, i t is poss ib le t h a t the enzyme is very s t r o n g l y adsorbed to the z i n c phosphate p r e c i p i t a t e , and i t might s t i l l be poss ib le to recover i t by s u i t a b l e i t rea tmen t . Our experiments have s t r o n g l y i m p l i c a t e d HMG-CoA as an in te rmed ia te in ace toace ta te s y n t h e s i s . I t thus became o f g rea t i n t e r e s t to examine the d i s t r i b u t i o n o f the two enzymes concerned du r ing the course o f the usual procedure f o r p u r i f i c a t i o n o f 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 system. Thus the var ious f r a c t i o n s ob ta ined du r ing t h e p p u r i f i c a t i o n procedure were assayed f o r t h e i r a b i l i t y to syn thes ize ace toace ta te , a lone , i n the presence o f excess condensing enzyme, and i n the ipresence o f excess cleavage enzyme. The r e s u l t s are shown in Table X X I I . The data c l e a r l y show t h a t p u r i f i c a -t i o n o f 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 system proceeds a t the expense o f HMG-CoA cleavage enzyme, s ince in the f i n a l f r a c t i o n on ly 10% o f t h i s enzyme remained. The procedure a c t u a l l y r e s u l t s i n a p u r i f i c a t i o n o f the condensing enzyme, the cleavage enzyme being d iscarded so tha t i n the end i t becomes r a t e - l i m i t i n g . On the basis o f these f i g u r e s and those o f Table XIX, i t can be c a l c u l a t e d t h a t the HMG-CoA condensing enzyme o f TABLE XXI I . Fate o f HMG-CoA condensing and cleavage enzymes du r ing p u r i f i c a t i o n o f the ace toace ta te -syn thes i z ing system Ace toace ta te -Synthes is HMG-CoA condens i ng enzyme HMG-CoA cleavage enzyme F r a c t i o n To ta l A c t i v i ty S p e c i f i c A c t i v i ty Tota l A c t i v i ty Y i e l d S p e c i f i c A c t i v i ty Tota l A c t i v i ty Y i e l d uni ts uni ts/mg uni ts .1 uni ts/mg uni ts % Crude e x t r a c t 712 0.123 712 "100" 0.440 2550 "100" (NH i t) 2S0/ f, 30-60% s a t . 532 0.480 532 75 0.785 864 34 Gel supernatant 455 0.590 455 64 0.800 616 24 E t h a n o l , 20-35% 246 1.190 297 42 0.985 246 10 HMG-CoA condensing and cleavage enzyme a c t i v i t i e s were determined as descr ibed under M a t e r i a l s  and Methods. Standard assay c o n d i t i o n s (assay system l l ) were employed. 6 5 . l i v e r has been p u r i f i e d 15 to 2 0 - f o l d o r more, w i t h recover ies approaching 40%. I t might be o f i n t e r e s t to record t h a t the data in Table XXII c a r r y an i r o n i c a l i m p l i c a t i o n . In 1957, Rudney was a t temp t ing to c l a r i f y the mechanism o f HMG-CoA fo rma t ion from acety l -CoA by yeast e x t r a c t s and to p u r i f y the enzyme which l a t e r became known as the HMG-CoA condensing enzyme. Stern and co-workers , work ing d i r e c t l y downstai rs from Rudney's l a b o r a t o r y , had p u r i f i e d 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 system and w h i l e do ing so had, i n r e a l i t y , succeeded in p u r i f y i n g the HMG-CoA condensing enzyme from beef l i v e r . I t was not poss ib le a t t h a t t ime to know t h a t HMG-CoA might be invo lved in ace toace ta te f o r m a t i o n . LOCALIZATION AND DISTRIBUTION OF THE ENZYME A study on the 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 the ace toace ta te -s y n t h e s i z i n g system and the HMG-CoA condensing and cleavage enzymes in r a t l i v e r homogenates i n d i c a t e d t h a t these enzyme a c t i v i t i e s are s i t u a t e d v i r t u a l l y e x c l u s i v e l y i n the m i tochondr ion . This has a l ready been repor ted by Bucher e t aj_ (71). However, as these authors po in ted o u t , HMG-CoA condensing enzyme is known to be present i n microsomes, where i t is thought to f u n c t i o n i n s t e r o l s y n t h e s i s . The leve l o f t h i s enzyme in microsomal p repara t ions is much lower than t h a t in m i tochondr ia l p r e p a r a t i o n s , and would not be detec ted by our assay method. Rudney (39) has demonstrated the presence o f the HMG-CoA condensing enzyme in r a t l i v e r microsomes by the use o f i s o t o p i c assay procedures, which are much more s e n s i t i v e than our assay system. The d i s t r i b u t i o n o f these enzyme a c t i v i t i e s in r a t t i s s u e homogenates was a lso s t u d i e d . A c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y and HMG-CoA condensing 66. enzyme a c t i v i t y were de tec tab le on ly in l i v e r homogenates; the r e s u l t s w i t h kidney homogenates were somewhat e q u i v o c a l , due to a h igh t i s s u e blank i n the absence o f s u b s t r a t e . This is somewhat anomalous, s ince many workers have shown t h a t some e x t r a h e p a t i c t i ssues can form ace toace ta te ( c f . H i s t o r i c a l Background). However, as was po in ted ou t i n the prev ious paragraph, these enzymes could be present a t l eve l s too low to be detec ted i n our assay system. The HMG-CoA cleavage enzyme, on the o t h e r hand, was detected in almost a l l t i ssues examined (Table XXI11) . Bachhawat e t a l (37) a l so found t h a t the HMG-CoA cleavage enzyme i s found in a wide v a r i e t y o f t i s s u e s . Lynen e t aj_ (35) repor ted r e s u l t s s i m i l a r to our f i n d i n g s . TABLE XXII I . D i s t r i b u t i o n o f HMG-CoA cleavage enzyme i n r a t t i ssues Tissue S p e c i f i c A c t i v i t y uni ts/mg p r o t e i n L i v e r 0.122 Heart 0.188 Kidney 0.086 Bra in 0.021 Uterus 0 Ovary 0.239 Adrenal 0.224 The homogenates were prepared as descr ibed under M a t e r i a l s and Methods. Standard assay c o n d i t i o n s were employed, except t h a t 0.01 M potassium cyanide was added to the system, to b lock o x i d a t i v e meta-bo l i sm. P r e l i m i n a r y experiments w i t h the p u r i f i e d enzyme i n d i c a t e d t h a t cyanide d i d not i n t e r f e r e w i t h the assay. 67. THE CHICKEN LIVER "INHIBITOR ENZYME" Stern e t aj_ (46) repor ted t h a t the ra te o f ace toaceta te fo rma t ion by crude e x t r a c t s o f ch icken l i v e r in the c a t a l y t i c assay system was not p r o -p o r t i o n a l to p r o t e i n c o n c e n t r a t i o n , and a t t r i b u t e d t h i s to the presence o f a secondary enzyme system(s) which competed w i t h the ace toace ta te -s y n t h e s i z i n g system f o r the s u b s t r a t e o r one o f the i n t e r m e d i a t e s . The i n h i b i t o r y f a c t o r could be l a r g e l y removed f rom the ace toace ta te -s y n t h e s i z i n g system in t h i s t i s s u e by f r a c t i o n a t i o n o f the homogenates w i t h ammonium s u l f a t e . In the present i n v e s t i g a t i o n i t was f e l t t h a t an under-s tand ing o f how t h i s f a c t o r exer ted i t s i n h i b i t o r y a c t i v i t y might shed impor tant l i g h t on the mechanism o f ace toace ta te f o r m a t i o n . To t h i s end, the " i n h i b i t o r enzyme" has been p u r i f i e d from chicken l i v e r homogenates by a se r ies o f steps i n v o l v i n g s a l t p r e c i p i t a t i o n , d e s t r u c t i o n o f res idua l ace toace ta te s y n t h e s i z i n g a c t i v i t y by heat t rea tmen t , a d s o r p t i o n o f i n a c t i v e p r o t e i n on ca lc ium phosphate g e l , r e p r e c i p i t a t i o n w i t h s a l t , and p r e c i p i t a -t i o n w i t h ethanol i n the presence o f z i n c i ons . The data f rom a t y p i c a l p u r i f i c a t i o n are shown in Table XXIV. The procedure is r e a d i l y r e p r o d u c i b l e , and a s i x - to t e n - f o l d p u r i f i c a t i o n is r e g u l a r l y ach ieved. For convenience, t h i s enzyme i s t empora r i l y being designated as the " i n h i b i t o r enzyme". When the p r e c i s e na ture o f i t s a c t i o n is d e f i n i t e l y e s t a b l i s h e d , a more a c c u r a t e l y d e s c r i p t i v e name w i l l be proposed. As descr ibed under M a t e r i a l s and Methods, the assay system f o r the " i n h i b i t o r enzyme" was based on i t s i n t e r f e r e n c e w i t h ace toace ta te forma-t i o n by beef l i v e r enzymes, f o r the s imple reason tha t t h i s was the on ly p roper ty o f the enzyme which was known a t t h a t t ime. As F igure 5 shows, the degree o f i n h i b i t i o n o f ace toaceta te fo rma t ion is d i r e c t l y p r o p o r t i o n a l to the c o n c e n t r a t i o n o f ch icken l i v e r p r o t e i n added over a f a i r l y wide 6 8 . TABLE XXIV. P u r i f i c a t i o n o f the ch icken l i v e r " i n h i b i t o r enzyme" S p e c i f i c To ta l F r a c t i o n P r o t e i n A c t i v i ty A c t i v i ty Recovery mg uni ts/mq uni ts % 1. Heated supernatant 3710 0.31 1160 "100" 2. Gel supernatant - - - -3. (NHOoSOj., 40-70% s a t . 642 0 .91 584 51 4 . a . Ethanol p p t . , 18-•27% 102 1.05 107 9 b. Ethanol p p t . , 27-•37% 147 2.50 368 32 c. Ethanol p p t . , 37-•50% 44 1.80 80 7 For d e t a i l s o f the p u r i f i c a t i o n procedure, assay method and d e f i n i t i o n o f the u n i t , see M a t e r i a l s and Methods. range, under the s p e c i f i e d c o n d i t i o n s . P r e l i m i n a r y a t tempts to determine the exact means by which the " i n h i b i t o r enzyme" i n t e r f e r e s w i t h ace toaceta te fo rmat ion showed t h a t the enzyme was not an a c e t y l phosphatase o r an acety l -CoA deacy lase, nor was i t the HMG-CoA deacylase descr ibed by Dekker e t aj_ ( 8 0 ) . I t was not a p r o t e o l y t i c enzyme h y d r o l y z i n g any o f the enzymatic components o f the c a t a l y t i c assay system. In a d d i t i o n , the " i n h i b i t o r enzyme" d i d not c a t a l y z e the f u r t h e r metabol ism o f any ace toace ta te formed. Two observa-t i ons prov ided the f i r s t d e f i n i t e c lue to i t s mechanism o f a c t i o n . The " i n h i b i t o r enzyme" was incubated w i t h the non-enzymatic components o f the assay system, in the presence and absence o f the £ . k l u y v e r i e x t r a c t , before a d d i t i o n o f 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 (Table XXV)'.. When no " i n h i b i t o r enzyme" was added (Exp ts . l a and 2 a ) , 2 .27/J m o1es o f ace to -ace ta te accumulated. When the " i n h i b i t o r f r a c t i o n " was added s i m u l t a n -eously w i t h the beef l i v e r enzyme, ace toace ta te syn thes is was depressed approx imate ly 45%. However, when the " i n h i b i t o r enzyme" was incubated w i t h 69. Figure 5. I n t e r f e r e n c e by the chicken l i v e r " i n h i b i t o r enzyme" w i t h ace toace ta te f o rma t i on by beef l i v e r enzymes. The beef l i v e r enzyme was a 20-35% ethanol f r a c t i o n (2.5 mg p r o t e i n ) . The chicken l i v e r " i n h i b i t o r enzyme" was a 27-37.4% ethanol f r a c t i o n . Standard assay c o n d i t i o n s were employed. 70. TABLE XXV. E f f e c t o f p r e - i n c u b a t i n g the assay system components w i t h the " i n h i b i t o r enzyme" before a d d i t i o n o f the ace toace ta te -s y n t h e s i z i n g f r a c t i o n Experiment No. P re incuba t ion Time Phosphotransacety lase added: S t a r t o f p r e i n c u b a t i o n End o f p r e i n c u b a t i o n mi nutes la l b 1c 30 30 30 2.27 1.27 0 . 0 2.27 1.28 0.53 2a 2b 2c 60 60 60 2.27 1.23 0 . 0 2.27 1.23 0.12 A l l tubes conta ined a l l the nonenzymatic components o f assay system I f o r ace toace ta te f o r m a t i o n . 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 (beef l i v e r 20-35% ethanol f r a c t i o n , 3 .9 mg p r o t e i n ) was r i rout ine ly- 'added to a l l tubes a t the end o f the p r e i n c u b a t i o n per-i o d . In experiments la and 2 a , no " i n h i b i t o r enzyme" f r a c t i o n was added. In experiments l b and 2b, " i n h i b i t o r enzyme" (ch icken l i v e r 27-37.4% e thanol f r a c t i o n , 0 .49 mg p ro te in ) was added a t the end o f the p r e i n c u b a t i o n p e r i o d , s imul taneous ly w i t h the beef l i v e r enzyme f r a c t i o n . In exper iments:- lc and 2 c , the same " i n h i b i t o r enzyme" f r a c t i o n was added a t the s t a r t o f the p r e -incuba t ion p e r i o d . C. k l u y v e r i e x t r a c t (0 .05 m l , 0 . 8 mg p r o t e i n ) was added as no ted . The p r e i n c u b a t i o n was f o r 30 o r 60 minu tes , as no ted , a t 3 8 ° . Fo l l ow ing a d d i t i o n o f the beef l i v e r f r a c t i o n , the usual procedure was f o l l o w e d . the assay components be fore a d d i t i o n o f 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 f r a c t i o n , i n h i b i t i o n o f ace toace ta te syn thes is was complete (1c and 2 c , f i r s t co lumn). The s i g n i f i c a n t obse rva t i on in these experiments was t h a t the ex ten t o f i n h i b i t i o n was decreased when the C. k l u y v e r i e x t r a c t was present d u r i n g the p r e i n c u b a t i o n p e r i o d , i n d i c a t i n g t h a t the b a c t e r i a l e x t r a c t was " p r o t e c t i n g " the s e n s i t i v e component o f the assay system i n some manner. 71. The o t h e r impor tant obse rva t i on was tha t the i n h i b i t i o n o f ace to -ace ta te syn thes is observed when the " i n h i b i t o r enzyme" was added to the assay system could be overcome by inc reas ing the c o n c e n t r a t i o n o f coenzyme A in the assay system. When the coenzyme A c o n c e n t r a t i o n was increased to s i x times i t s usual l e v e l , the e f f e c t o f the " i n h i b i t o r enzyme" on ace toace ta te f o rma t i on by beef l i v e r enzymes was complete ly blocked (F igure 6). CoASH (umoles) Figure 6. E f f e c t o f coenzyme A c o n c e n t r a t i o n on depres-s ion o f ace toace ta te fo rma t ion by beef l i v e r enzymes in presence o f the ch icken l i v e r " i n h i b i t o r enzyme". Curve I i s the ra te o f ace toaceta te fo rma t ion by beef l i v e r enzymes (20-35% ethanol f r a c t i o n , 1.1 mg p r o t e i n ) , i n the absence o f " i n h i b i t o r enzyme". Curve I I is the same, in the presence o f " i n h i b i t o r enzyme" (ch icken l i v e r ammonium s u l f a t e 40-70% s a t . f r a c t i o n , 1.4 mg p r o t e i n ) . 72. These two observa t ions i n d i c a t e d t h a t the " i n h i b i t o r enzyme" might be depressing ace toace ta te fo rma t ion by i n a c t i v a t i n g the coenzyme A present i n the assay system. To t e s t t h i s p o s s i b i l i t y , coenzyme A was incubated w i t h the " i n h i b i t o r enzyme", and the dep ro te in i zed s o l u t i o n was assayed f o r coenzyme A by the phosphotransacety lase method (59) (Table XXVI) . As the data in the t a b l e show, the " i n h i b i t o r enzyme" does indeed appear to be i n a c t i v a t i n g coenzyme A in some way. TABLE XXVI. I n a c t i v a t i o n o f coenzyme A by ch icken l i v e r " i n h i b i t o r enzyme" Experiment _ Coenzyme A Coenzyme A No. y added recovered mg Aimoles /jmoles la - 0.01 0.01 l b 0.19 0.01 0 2a - 0.02 0.02 2b 0.19 0.02 0 The r e a c t i o n m ix tu re conta ined Tr is -HCl b u f f e r , pH 8.15, 100/timoles; MgCl2, 1 /timole; KCl , 4 yumoles; c y s t e i n e , k /Umoles; and coenzyme A and " i n h i b i t o r enzyme" (ch icken l i v e r , 27-37% ethanol f r a c t i o n ) , as no ted ; 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, and incuba t ion was a t 38°. A f t e r kS minutes, the reac-t i o n was stopped by a c i d i f i c a t i o n to pH k w i t h 5N HC1 and hea t ing in a b o i l i n g water bath f o r 1 m inu te , f o l l owed by c o o l i n g . Coenzyme A i n the r e a c t i o n m ix tu re was then determined by the phosphotransacety lase method (59). A p r e l i m i n a r y a t tempt has been made to determine p r e c i s e l y what a c t i o n the " i n h i b i t o r enzyme has on coenzyme A. Coenzyme A was incubated w i t h the enzyme, and the dep ro te in i zed supernatant s o l u t i o n sub jec t to paper chromatography. The r e s u l t s (which w i l l not be repor ted here in 73. d e t a i l , s ince they are q u i t e i nconc lus i ve and incomplete) i n d i c a t e the fo rma t ion o f a new compound, which e x h i b i t e d u l t r a v i o l e t l i g h t abso rp t i on and which reacted w i t h the Toennies and Kolb reagent (81) f o r f r e e t h i o l compounds. One obvious p o s s i b l e i n t e r p r e t a t i o n o f t h i s obse rva t i on is t h a t the " i n h i b i t o r enzyme" is a phosphatase o r 3 ' - n u c l e o t i d a s e , removing the 3*-phosphate o f coenzyme A to form dephosphocoenzyme A. The occur -rence o f t h i s type o f nuc leo t idase i n p l a n t t i s s u e e x t r a c t s has been repor ted (82-85). This nuc leo t idase has, i n f a c t , been u t i l i z e d f o r f o rma t ion o f dephosphocoenzyme A on a p r e p a r a t i v e sca le (84 , 8 6 ) . P r o s t a t i c phosphatase w i l l a l s o remove the 3 ' -phosphate o f coenzyme A ( 8 4 ) . However, our r e s u l t s are not y e t c o n c l u s i v e . Time l i m i t a t i o n s have prevented a more thorough i n v e s t i g a t i o n o f t h i s p o s s i b i l i t y . At any ra te i t is q u i t e apparent t ha t knowledge o f the mechanism o f t h i s enzyme has not a s s i s t e d us in e l u c i d a t i n g the mechanism o f ace toace ta te fo rmat ion as was o r i g i n -a l l y hoped. 0-HYDROXYBUTYRYL DEHYDROGENASE FROM C. KLUYVERI A l l o f our evidence on the mechanism o f ace toace ta te f o r m a t i o n by l i v e r e x t r a c t s was compat ib le w i t h the o p e r a t i o n o f the HMG-CoA pathway. In an at tempt to o b t a i n f u r t h e r conc lus i ve evidence f o r the o b l i g a t o r y fo rmat ion o f HMG-CoA as an i n t e r m e d i a t e , we began to seek some means by which the HMG-CoA pathway could be s p e c i f i c a l l y b locked. One l o g i c a l approach would be to remove HMG-CoA as i t formed by c o n v e r t i n g i t to some i n e r t m e t a b o l i t e . I f ace toace ta te is formed v i a HMG-CoA, the i n t r o d u c t i o n o f such a " s h u n t " i n t o the assay system should prevent ace toace ta te fo rma-t i o n . In the search f o r a means o f shun t ing ou t HMG-CoA, three enzymes were cons idered: Q -methylg lutaconase (58) which ca ta lyzes the i n t e r -74. conversion of HMG-CoA and ^-methylglutaconyl-CoA (reaction 13); the HMG-CoA deacylase of chicken liver described by Dekker et al (80) (reaction 14); and HMG-CoA reductase, described by Rudney and co-workers (87-89) and also by Knappe et a] (90), which catalyzes the conversion of HMG-CoA to mevalonate in the presence of TPNH (reaction 15). The fi r s t of these, HMG-CoA (b -methyl glutaconyl -CoA + H20 (13) HMG-CoA + H20 »- HMG + CoASH (14) HMG-CoA + 2 TPNH >• mevalonate + 2 TPN + CoASH (15) (9-methylglutaconase, was not suitable for our purposes, since the equilib-rium of the hydration-dehydration reaction lies in the direction of forma-tion of HMG-CoA, rather than its removal. The second possibility, the HMG-CoA deacylase, was an attractive possibility, but i t , too, proved to be unsuitable. The deacylase preparations described by Dekker et a_[ (80) contain considerable HMG-CoA cleavage enzyme, and the pH of the catalytic assay system would favor the cleavage reaction rather than deacylation of HMG-CoA. In addition, the deacylase preparations would undoubtedly con-tain "inhibitor enzyme" activity, which would make interpretation of results virtually impossible. The third enzyme mentioned above, HMG-CoA reductase, appeared to be the answer to our problem, since i t has been considerably purified from yeast (89,90) and the equilibrium of the reaction lies far in the direction of mevalonate formation. In view of these considerations, the effect of purified yeast HMG-CoA reductase on acetoacetate formation in the catalytic assay system was determined (Table XXVIl). As the data in the table show, addition of 75. TABLE XXVI1. E f f e c t o f TPNH and HMG-CoA reductase on acetoaceta te fo rma t ion by beef i i v e r enzymes Experiment No. Addi t i o n s Acetoaceta te yumo I es None 1.54 2 TPNH genera t ing system plus HMG-CoA reductase 0.21 3 TPNH genera t ing system w i t h o u t HMG-CoA reductase 0.12 Standard c o n d i t i o n s o f assay system I were employed, w i t h o t h e r a d d i t i o n s as no ted . 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 system used was a beef l i v e r ammonium s u l f a t e 30-60% s a t . f r a c t i o n (3.0 mg p r o t e i n ) . The TPNH genera t ing system cons is ted o f 1 /umole o f TPN, 30/ttmoles glucose 6-phosphate, and 1 u n i t o f glucose 6-phosphate dehydrogenase (Sigma Chemical Co. , Type I I I ) . HMG-CoA reductase was p u r i f i e d f rom baker ' s yeast as descr ibed by Knappe e_t aj_ (90); 15 m i l l i u n i t s were added, where no ted . HMG-CoA reductase and a source o f TPNH to the c a t a l y t i c assay system p r o -duced almost complete i n h i b i t i o n o f ace toace ta te fo rma t ion (Experiment 2), as had been a n t i c i p a t e d . However, a ra the r s u r p r i s i n g obse rva t i on was t h a t a d d i t i o n o f the p u r i f i e d yeast enzyme appeared to be unnecessary (Experiment 3). Presumably e i t h e r the l i v e r enzyme f r a c t i o n used o r the C_. k l u y v e r i e x t r a c t conta ined HMG-CoA reductase. To c o n f i r m t h a t the i n h i b i t i o n o f ace toaceta te syn thes is was due to reduc t ion o f HMG-CoA to mevalonate, an a t tempt was made to i s o l a t e the mevalonate f rom the r e a c t i o n m ix tu re as the hydroxamic a c i d d e r i v a t i v e , as descr ibed by Lynen and Grassl (91). No mevalonate could be d e t e c t e d . Spect rophotometr ic assays <Df the l i v e r and b a c t e r i a l e x t r a c t s f o r HMG-CoA reductase a c t i v i t y were n e g a t i v e . I t t h e r e f o r e became apparent t h a t the i n h i b i t i o n o f ace to -ace ta te f o rma t i on was due to some r e a c t i o n o t h e r than reduc t ion o f HMG-CoA 76. to mevalonate. A sys temat ic i n v e s t i g a t i o n i n t o the basis o f these observa t ions led to the d iscovery t h a t an extremely a c t i v e acetoacety1-CoA reductase o r ^ - h y d r o x y b u t y ry 1 dehydrogenase i n the C_. k l u y v e r i e x t r a c t was respons ib le f o r the observed i n h i b i t i o n o f ace toace ta te f o r m a t i o n . In b r i e f , i n h i b i t i o n o f ace toace ta te f o rma t i on had occur red not because HMG-CoA was being "shunted o u t " to mevalonate, but because acetoacety l -CoA was being reduced to (3 -hydroxybu ty ry l -CoA. A s u r p r i s i n g f i n d i n g was t h a t the enzyme is v i r t u a l l y a b s o l u t e l y s p e c i f i c f o r TPNH (Fi gure 7). This i s . i n c o n t r a s t t o the (3-hydroxy butyry I dehydrogenase f rom l i v e r (92), which is a b s o l u t e l y s p e c i f i c f o r DPNH, and the hear t enzyme (93). which is on ly 1/10 as a c t i v e w i t h TPNH as w i t h DPNH. Spect rophotometr ic examinat ion o f the product o f the r e a c t i o n showed on ly abso rp t i on due to the t h i o e s t e r bond and to the adenine moiety o f the coenzyme A. When the d e p r o t e i n i z e d r e a c t i o n m ix tu re was sub jec ted to paper chromatography, a new t h i o e s t e r spot appeared in the p o s i t i o n repor ted by Stern e t al_ (30) f o r hydroxy butyry 1 -CoA (Table XXVI11). I t is q u i t e p o s s i b l e t h a t t h i s enzyme is i d e n t i c a l w i t h the ^ - h y d r o x y -p rop iony l dehydrogenase descr ibed by Vagelos and Earl (94). These workers repor ted t h a t C. k l u y v e r i e x t r a c t s con ta in an enzyme which ca ta lyzes the r e v e r s i b l e o x i d a t i o n o f ^ -hyd roxyprop iony l -CoA to malonyl semialdehyde-CoA in the presence o f TPN. Fur ther i n v e s t i g a t i o n o f t h i s enzyme w i t h respect to s u b s t r a t e s p e c i f i c i t y might shed f u r t h e r l i g h t on t h i s q u e s t i o n . I t i s e n t i r e l y reasonable t ha t t h i s enzyme may p lay an impor tant r o l e in f a t t y ac id metabol ism in t h i s organism and t h i s problem is c e r t a i n l y wor th f u r t h e r i n v e s t i g a t i o n . U n f o r t u n a t e l y , t ime has marched away. o II UJ o z < CD or o 03 < 0 5 10 TIME (minutes) Figure 7. P y r i d i n e n u c l e o t i d e s p e c i f i c i t y o f the (3 - hydroxy b u t y r y l dehydrogenase o f C_. k l u y v e r i . The e x p e r i -mental c u v e t t e (d = 0.5 cm) conta ined Tr is -HCl b u f f e r , pH 7 .5 , 100/umoles; and DPNH o r TPNH, as no ted , 0 . 3 / i m o l e s . At the f i r s t ar row, 0.02 ml o f C. k l u y v e r i e x t r a c t (0.35 mg p r o t e i n ) was added. At the second ar row, 0.5 /Jmoles o f acetoacety1-CoA was added to i n i t i a t e the r e a c t i o n . To ta l volume, 1.5 ml; temperature, 2 4 ° . The c o n t r o l cuve t te con-ta ined b u f f e r and enzyme. TABLE XXVII I . Chromatography o f products o f the @-hydroxybutyry1 dehydrogenase o f C. k l u y v e r i System Rf values o f t h i o e s t e r s Cont ro l 0 . 5 9 Experimental 0 . 4 5 , 0 . 5 8 , 0 . 6 8 Acetyl-CoA 0 . 4 7 Acetoacety l -CoA 0 . 5 8 The r e a c t i o n m ix tu re conta ined Tr is -HCl b u f f e r , pH 7 . 5 . 3 0 ytimoles; acetoacety l -CoA, 0 .6 /Umoles ; TPNH, 1 . 4 / i m o l e s , and C. k l u y v e r i e x t r a c t , 0 . 0 2 ml ( 0 . 3 5 mg p r o t e i n ) ; t o t a l volume 0 . 3 m l . The c o n t r o l conta ined a l l a d d i t i o n s except enzyme. Incubat ion was f o r 2 0 minutes a t 3 8 ° . The r e a c t i o n was stopped by a c i d i f i c a t i o n to pH 4 and hea t ing in a b o i l i n g water bath f o r 3 m inutes . 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 e n t i r e supernatant s o l u t i o n s were then s t reaked on Whatman 3MM paper, and sub jec ted ascending chromatography in the e t h a n o l / a c e t a t e system ( 9 5 ) f o r 8 hours a t 4 ° . Coenzyme A t h i o e s t e r s were detected by u l t r a v i o l e t abso rp t i on and the delayed n i t r o p r u s s i d e r e a c t i o n ( 9 5 ) . 79. DISCUSSION The experiments repor ted in t h i s t hes i s c l e a r l y i m p l i c a t e HMG-CoA as an in te rmed ia te in ace toaceta te f o r m a t i o n . By the use o f techniques i n -v o l v i n g neu t ra l heat t rea tment , i t is p o s s i b l e to o b t a i n p repara t ions c o n t a i n i n g the HMG-CoA cleavage enzyme which have l o s t over 90% o f t h e i r a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y . This a c t i v i t y can be complete ly res to red by adding back HMG-CoA condensing enzyme. The z i n c - e t h a n o l f r a c t i o n s c l e a r l y c o n t a i n HMG-CoA condensing enzyme. These f r a c t i o n s are unable to syn thes ize acetoaceta te alone and 93.2% o f the o r i g i n a l ace to -a c e t a t e - s y n t h e s i z i n g a c t i v i t y is res to red upon adding back p u r i f i e d HMG-CoA cleavage enzyme or heated e x t r a c t s o f l i v e r . In a d d i t i o n , the experiments i n v o l v i n g ca lc ium phosphate gel column f r a c t i o n a t i o n c l e a r l y i n d i c a t e the separa t ion o f 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 system i n t o two f r a c t i o n s , each o f which has l i t t l e o r no a b i l i t y to form ace toace ta te by i t s e l f . One f r a c t i o n when supplemented w i t h HMG-CoA condensing enzyme gave an almost q u a n t i t a t i v e recovery o f a c e t o a c e t a t e - s y n t h e s i z i n g a c t i v i t y . The second f r a c t i o n when supplemented w i t h HMG-CoA cleavage enzyme l i k e w i s e gave an almost complete recovery o f a c t i v i t y . Combining the two f r a c t i o n s a lso res to red the a b i l i t y to form ace toace ta te . The ques t ion now is whether the HMG-CoA pathway is the on ly mechanism f u n c t i o n i n g in the c a t a l y t i c assay system. The q u a n t i t a t i v e nature o f the recovery achieved on adding condensing enzyme to heated e x t r a c t s , and upon adding cleavage enzyme to the z i n c - e t h a n o l f r a c t i o n s s t r o n g l y i nd i ca tes t h a t most, i f not a l l , o f the ace toaceta te formed by l i v e r e x t r a c t s in the c a t a l y t i c assay system proceeds through t h i s mechanism. This mat te r is discussed f u r t h e r below. The HMG-CoA pathway as a mechanism f o r ace toace ta te f o rma t i on is a 80. very a t t r a c t i v e one, s ince i t p rov ides an exp lana t i on f o r many o f the known p r o p e r t i e s o f 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 system. The t h i o l and d i v a l e n t c a t i o n requirements f o r ace toace ta te fo rma t ion from ace ty l phosphate and coenzyme A in the c a t a l y t i c assay system (35,47) are r e a d i l y exp la ined by the requirement o f the HMG-CoA cleavage enzyme f o r these f a c t o r s (37). The r e l a t i v e e f f i c a c y o f var ious t h i o l s in a c t i v a t i n g the a c e t o a c e t a t e - a c t i v a t -ing system o f beef l i v e r e x t r a c t s c l o s e l y p a r a l l e l s t h e i r e f f i c a c y in a c t i v a t i n g the HMG-CoA cleavage enzyme (37). Bachhawat e t aj_ (37) found M g + + to be the most e f f e c t i v e o f the d i v a l e n t ca t ions in a c t i v a t i n g the cleavage enzyme, w i t h M n + + p a r t i a l l y e f f e c t i v e and C o + + i n e f f e c t i v e . This is i n c o n t r a s t w i t h our r e s u l t s w i t h 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 system ( c f . F igure 1). However, these workers were us ing very h igh concen t ra t i ons o f metal i o n . We have found t h a t h igh concen t ra t ions o f the ca t i ons are i n h i b i t o r y ( c f . F igure 1), and a lso t h a t i n o r d e r to produce maximal assay c o n d i t i o n s the metal ion c o n c e n t r a t i o n i n the di r e c t HMG-CoA cleavage enzyme assay must be reduced by o n e - h a l f o r more f rom tha t repor ted by Bachhawat e t aj_ (37). This may e x p l a i n the d isc repancy . The HMG-CoA pathway is a l so c o n s i s t e n t w i t h the heat l a b i l i t y o f 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 system, s ince the HMG-CoA condensing enzyme i s very s e n s i t i v e to even b r i e f h e a t i n g . This has been discussed in d e t a i l e a r l i e r . The HMG-CoA pathway a lso prov ides an e x p l a n a t i o n f o r the long-known "asymmetr ic l a b e l l i n g " o f m e t a b o l i c a l l y - f o r m e d ace toace ta te . When, f o r example, o c t a n o a t e - l - C ^ is incubated w i t h a l i v e r enzyme p r e p a r a t i o n ( e . g . s l i c e s , homogenates, m i t o c h o n d r i a , e t c . ) , the ace toaceta te formed conta ins more isotope in the carboxyl group than in the carbonyl group. The e a r l i e s t exp lanat ions o f t h i s phenomenon invoked the ex is tence o f two 81. d i f f e r e n t forms o f " a c t i v e a c e t a t e " . Lynen ( c f . re ference (32)) and Be ine r t and Stans ly (96) extended t h i s theory by p o s t u l a t i n g t h a t an S-acyl ^ - k e t o -t h i o l a s e func t i oned as an a l t e r n a t e form o f " a c t i v e a c e t a t e " . However, a s imp ler and more l o g i c a l exp lana t i on f o r the asymmetric l a b e l l i n g is a v a i l a b l e i f the HMG-CoA pathway f u n c t i o n s as the mechanism o f l i v e r k e t o -genes is . When octanoate-l-c'** is incubated w i t h l i v e r p r e p a r a t i o n s , one " t u r n " o f the f a t t y ac id |3-oxidation " c y c l e " ( o r , more p r o p e r l y , ^ - o x i d a t i o n " s p i r a l " ) w i l l re lease a c e t y l - l - C l i f - C o A , thus l a b e l l i n g the " a c e t y l p o o l " . Ox ida t ion o f the r e s u l t i n g hexanoate would f i n a l l y y i e l d unlabel led ace to -acety l -CoA. Condensation o f t h i s un label led acetoacety1-CoA w i t h l a b e l l e d acetyl -CoA from the " a c e t y l p o o l " ( r e a c t i o n 11) would form HMG-S-c'^-CoA, which wou ld , v i a the HMG-CoA cleavage enzyme r e a c t i o n ( r e a c t i o n 12), g ive r i s e to ace toace ta te label led e x c l u s i v e l y i n the carboxyl group. However, some isotope would be incorpora ted i n t o acetoacety1-CoA through e q u i l i b r a -t i o n w i t h the l a b e l l e d " a c e t y l p o o l " v i a ^ - k e t o t h i o l a s e ; the a c e t o a c e t y l -CoA thus formed would ca r ry iso tope in both the carboxyl and carbonyl carbons. Condensation o f t h i s s y m m e t r i c a l l y - l a b e l l e d acetoacety1-CoA w i t h l a b e l l e d acetyl -CoA f rom the " a c e t y l p o o l " , f o l l owed by cleavage o f the HMG-CoA thus formed, would g i ve r i s e to ace toace ta te l a b e l l e d in both the carboxyl and carbonyl carbons. The net r e s u l t would be the fo rma t ion o f ace toaceta te l a b e l l e d in both p o s i t i o n s , but w i t h the m a j o r i t y o f the iso tope i n the carboxyl group, i . e . , asymmetr ica l ly l a b e l l e d . The longer the f a t t y ac id cha in being o x i d i z e d , the g rea te r the complement o f symmet r i ca l l y l a b e l l e d acetoacetyI-CoA would be. The end r e s u l t would be a r a t i o o f label in the carboxyl and carbonyl groups o f ace toace ta te approaching u n i t y . The o v e r a l l p i c t u r e is summarized in F igure 8. In a d d i t i o n to the above p o i n t s , the MMG-CoA pathway can a lso be OH 0 CH -C-CH2-C-SCoA • * CH,-COOH [HMG-CoA cleavage enzyme 0 0 CH.-C-CH--C00H + CH,-*C-SCoA 3 2 * J Figure 8. The HMG-CoA mechanism as an explanation for the asymmetric isotope labelling of enzymatically-formed aceto-acetate. For details, see text. 83. used to e x p l a i n the lack o f accumulat ion o f ace toaceta te du r i ng o x i d a t i o n o f f a t t y ac ids by e x t r a h e p a t i c t i s s u e s . L i v e r , the on ly one o f the t i ssues tes ted which forms apprec iab le amounts o f ace toace ta te , is a l so the on ly mammalian t i s s u e which conta ins r e a d i l y de tec tab le l eve l s o f the HMG-CoA condensing enzyme. The f a c t t h a t e x t r a h e p a t i c t i ssues lack t h i s enzyme may w e l l be the reason f o r the i n a b i l i t y o f e x t r a h e p a t i c t i ssues to cause acetoaceta te accumula t ion , ra the r than the f a c t t h a t these t i ssues can c a t a l y z e the f u r t h e r o x i d a t i o n o f ace toace ta te , which has been genera l l y considered as the reason. The s i m i l a r i t i e s between the p r o p e r t i e s o f the a c e t o a c e t a t e - s y n t h e s i z -ing system and those o f the HMG-CoA condensing and cleavage enzymes p rov ide a s t rong i n d i c a t i o n t h a t they may be i d e n t i c a l . The evidence presented i n t h i s thes is s t r o n g l y i nd i ca tes t ha t t h i s is so. In a recent paper, H i rd and Symons (49) have presented evidence which s t r o n g l y i nd i ca tes t h a t ace toaceta te fo rma t ion f rom f a t t y ac ids by i n t a c t c e l l p repara t ions f rom sheep omasum and rumen e p i t h e l i u m proceeds l a r g e l y , i f not e n t i r e l y , v i a HMG-CoA. When butyrate-l-c'** was incubated w i t h t h e i r enzyme p r e p a r a t i o n s , 75% o f the c'^ i ncorpora ted i n t o the ace toace ta te which accumulated in the medium was located in the carboxyl group. They a lso incubated t h e i r enzyme p repara t ions w i t h unlabel led b u t y r a t e in the presence o f a c e t a t e - 1 -c'^ o r a source o f a c e t a t e - l - C ^ ( 1 a c t a t e - 2 - C ^ \ o c t a n o a t e - l - C 1 * * ) . When the accumulated acetoaceta te was analyzed f o r iso tope d i s t r i b u t i o n , between 75 and 80% o f the r a d i o a c t i v i t y was found in the carboxyl group. In the f i r s t - m e n t i o n e d set o f exper iments , u t i l i z i n g b u t y r a t e - l - C ^ \ the asymmetric label Hmg can be exp la ined through a p a r t i a l e q u i l i b r a t i o n o f isotope v i a the t h i o l a s e r e a c t i o n , as shown by F igure 9, w i t h o u t invok ing the HMG-CoA pathway. Thus, the a s y m m e t r i c a l l y - l a b e l l e d ace toace ta te could Sk. •COOH CH,-CH 9 -CH 0 - i 3 2 2 * A , j p - o x i d a t i o n CoASH .9 2 CH,-C-SCoA 3 * P «9 CH -C-CH.-c'-SCoA 3 2«« fl-ketothiolase — ff-ketothiolase{ * To 0 -CH,-C-CH,-C-SCoA + CoASH CH,-C-CH0-C-SCoA I i I I *9 ^ CH^C-CH2-C00H F igure 9 . Asymmetric l a b e l l i n g o f ace toaceta te formed enzymati ca l ly f rom buty r a t e - l - C ^ . For e x p l a n a t i o n , see t e x t . have been formed by a d i r e c t d e a c y l a t i o n o f acetoacety 1-CoA. The r e s u l t s o f the second set o f exper iments , u t i l i z i n g unlabel led b u t y r a t e and a c e t a t e - 1 - C a s s u b s t r a t e s , however, v i r t u a l l y exclude the d i r e c t d e a c y l a t i o n as a major pathway f o r ace toaceta te fo rma t ion in these t i s s u e p r e p a r a t i o n s . I f the ace toaceta te is formed by d i r e c t d e a c y l a t i o n o f acetoacety l -CoA, the on ly way in which the a c e t a t e - l - c ' ^ cou ld have been incorpora ted i n t o the acetoaceta te would be v i a the f3 -ke to th io lase r e a c t i o n , as shown in F igure 10, and t h i s could g ive r i s e on ly to symmetr ic-a l l y - l a b e l l e d ace toace ta te . I f , on the o t h e r hand, a c e t a t e - l - c ' ^ cou ld be incorpora ted both v i a the ^ - k e t o t h i o l a s e e q u i l i b r i u m and v i a the HMG-CoA pathway, the s i t u a t i o n would be e s s e n t i a l l y t h a t represented in F i gu re 8, 85. CH--COOH. 3 * CH3-CH2-CH2-COOH CH.-C-SCoA 3 * p 1 - k e t o t h i o l a s e CH3-C-CH2-C-SCoA T. +9 -° CH--C-CH--C-SCoA (+ CoASH) J * z « P ' ,9 CH,-C-CH9-C-SCoA CoASH CH,-C-CHo-C00H F igure 10. I n c o r p o r a t i o n o f a c e t a t e - l - C i n t o ace to -ace ta te enzymat i ca l l y formed v i a d i r e c t d e a c y l a t i o n o f a c e t o a c e t y l -CoA. For e x p l a n a t i o n , see t e x t . 1 and the acetoaceta te would be l a b e l l e d predominant ly i n the carboxy l group. Since the ace toace ta te formed in t h i s se t o f experiments conta ined three to f o u r t imes as much c'^ i n the carboxyl group as in the carbonyl group, i t would appear t h a t the ace toaceta te was indeed formed v i a HMG-CoA. On the basis o f t h e i r d a t a , H i rd and Symons were ab le to c a l c u l a t e t ha t 75 to 80% o f the ace toace ta te could o n l y have been formed v i a the HMG-CoA pathway. The o t h e r 20 to 25% o f the ace toace ta te could have been formed v i a a d i r e c t d e a c y l a t i o n o f acetoacety l -CoA, o r by the HMG-CoA pathway a f t e r e q u i l i b r a t i o n o f a c e t y l - l - C ^ -CoA and acetoacety 1-CoA v i a the 3 - k e t o t h i o l a s e r e a c t i o n , o r by a combinat ion o f bo th . 86. In contrast to the evidence described above, Stern and co-workers have presented evidence for the presence of a specific acetoacety!-CoA deacylase in liver extracts, using partially purified beef liver enzymes (47) and sonic extracts of rat liver mitochondria (45). After treatment with iodoacetamide to inactivate the HMG-CoA condensing and cleavage enzymes, these extracts were able to synthesize acetoacetate from substrate concentrations of acetoacety1-CoA. Unlike the acetoacetate-synthesizing system of beef liver extracts described in this thesis, the deacylase system described by the Cleveland group requires neither divalent cation nor thiol. Stern apparently holds the opinion that although both the HMG-CoA condensing and cleavage enzymes are present in liver extracts, and their levels of activity are sufficiently high to. account for all of the acetoacetate formed, these enzymes may not be involved in acetoacetate formation. The evidence presented in this thesis, and the evidence of other workers which has also been described here, does not definitely ex-clude the possibility of the existence of a specific acetoacety1-CoA deacylase. However, there is l i t t l e evidence that such a deacylase plays a major role in acetoacetate formation. It is quite possible that this deacylase does exist, and may account for a small part of the acetoacetate formed by liver extracts. For example, prolonged treatment of the aceto-acetate-synthesizing system with iodoacetamide or the addition of high concentrations of EDTA to the catalytic assay system do not completely block acetoacetate formation. The existence of a specific acetoacety1-CoA deacylase might account for the small amount of residual activity (less than 10%) under these conditions. However, the presence both of iodoacetamide and of substrate concentrations of acetoacetyI-CoA, as in the stoichiometric assay used by Stern (45,47), could hardly be considered as physiological, 87. and i t would be rather risky to base conclusions as to the physiological mechanism of acetoacetate formation on results obtained under such condi-tions. In addition, the specific deacylase does not explain the very definite divalent cation and thiol requirements for acetoacetate synthesis in the catalytic assay system. Another point is that there is no adequate explanation as to why the HMG-CoA condensing and cleavage enzymes would not couple to provide a pathway for acetoacetate formation, when i t has now been established that both enzymes are very definitely present. Segal and Menon (48) have also presented evidence which they claim indicates the formation of acetoacetate via direct deacylation of aceto-acetyl-CoA. They incubated rat liver mitochondrial preparations with 14 unlabel led acetoacety1-CoA and acetyI-2-C -CoA for short periods, and analyzed the acetoacetate which formed for isotope content; they found that very l i t t l e radioactivity had been incorporated. Analysis of residual acetoacetyl-CoA showed that i t , too, contained very l i t t l e isotope. They also reported that their mitochondrial preparations contained virtually no -p-ketothiolase activity. The validity of their results can be criticized on several points. Starting with 1 /umole of acetoacety1-CoA, at pH 7.9, 0.41 ^imole of acetoacetate formed chemically, in the absence of enzyme, and 0.52/Umoles in the presence of enzyme (100 ^g. of mitochondrial protein) after ten minutes incubation. This extremely high rate of chemical deacyla-tion does not correspond with the known stability characteristics of acetoacetyI-CoA (97). They reported that they used the Walker method (69) for determination of acetoacetate, and make no mention of any modification of the method. We have found that the original procedure, as published by Walker, is excellent when pure solutions of acetoacetate are being assayed, but is very unreliable when trichloroacetic acid supernatant 88. s o l u t i o n s from t i s s u e p repara t ions are being assayed. Under these cond i -t i o n s , a 20% v a r i a t i o n in the assay o f two i d e n t i c a l samples was q u i t e common. Another weak p o i n t i n t h e i r evidence is t h a t a cons iderab le 'amount o f the acetoacety1-CoA (0.4 / imole) i n i t i a l l y present is not accounted f o r , e i t h e r per se o r as ace toace ta te . In a d d i t i o n , t h e i r s tatement t h a t the m i tochondr ia l p repara t ions used i n t h e i r experiments conta ined l i t t l e o r no p - k e t o t h i o l a s e is d i f f i c u l t to unders tand. I f i t had been p o s s i b l e to o b t a i n an a c e t o a c e t a t e - s y n t h e s i z i n g l i v e r f r a c t i o n complete ly o r l a r g e l y f r e e o f t h i o l a s e , the problem o f the enzymatic mechanism o f ace toace ta te syn thes is would undoubtedly now be on ly o f h i s t o r i c a l i n t e r e s t . The main s tumbl ing b lock in the study o f the mechanism o f ketogenesis i n l i v e r e x t r a c t s has always been t h a t i n a l l the l i v e r f r a c t i o n s o b t a i n e d , both crude and p u r i f i e d , the disappearance o f acetoacety1-CoA v i a the ^ - k e t o t h i o l a s e r e a c t i o n proceeded a t a ra te many times g r e a t e r than the r a t e a t which acetoacety1-CoA was hydro lyzed to ace toace ta te . From t h e i r d a t a , i t can be c a l c u l a t e d t h a t ace toaceta te was formed " e n z y m a t i c a l l y " a t a r a t e o f 6.6 /limoles per mg p r o t e i n per hour. This would make t h e i r m i tochondr ia l p r e p a r a t i o n the most a c t i v 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 f r a c t i o n ever prepared - i t would be approx imate ly 16 t imes as a c t i v e as an i d e n t i c a l l i v e r p r e p a r a t i o n repor ted by Stern and M i l l e r (45). On the basis o f these c o n s i d e r a t i o n s , i t would appear t ha t a l o g i c a l e x p l a n a t i o n for the lack o f 14 i n c o r p o r a t i o n o f iso tope f rom acetyl-2-C -CoA i n t o ace toaceta te in t h e i r experiments would be t h a t there was l i t t l e o r not ace toace ta te formed e n z y m a t i c a l l y . I t is the o p i n i o n o f t h i s author t h a t the repo r t o f Segal and Menon (48) must be viewed ra the r s k e p t i c a l l y , a t l e a s t u n t i l f u r t h e r experiments are c a r r i e d out under very c a r e f u l l y c o n t r o l l e d c o n d i t i o n s . From the r e s u l t s repor ted here , t h i s l abo ra to ry has been led to the 89. conc lus ion t h a t c e r t a i n l y most, i f not a l l , o f the ace toace ta te formed by l i v e r p repara t ions proceeds through HMG-CoA. The r o l e o f a s p e c i f i c acetoacety l -CoA deacylase must be r e l a t i v e l y minor , i f i t is invo lved a t a l l . I t s p rec i se and q u a n t i t a t i v e r o l e , i f any, must awa i t f u t u r e s t u d i e s . Our conclus ions are thus very s i m i l a r to those o f H i rd and Symons (49) who used sheep omasum and rumen e p i t h e l i u m f o r t h e i r s t u d i e s . We are thus a lso in agreement w i t h Lynen e t al_ (35) • even though we have been unable to reproduce t h e i r r e s u l t s . F i n a l l y , i t can be sa id t ha t the HMG-CoA pathway is p a r t i c u l a r l y a t t r a c t i v e f rom a p h y s i o l o g i c a l p o i n t o f v iew, s ince i t prov ides a biochemical e x p l a n a t i o n f o r the long-known p h y s i o l o g i c a l r e l a -t i onsh ips between the metabol ism o f ace toace ta te , c h o l e s t e r o l , f a t t y ac ids and branched-chain f a t t y a c i d s . 90. BIBLIOGRAPHY 1. P e t t e r s , W., Prager 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 i s c h e Hei lkunde; 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 Pub l i shers I n c . , New York, 1957. 2. Gerhardt , C , Wien. med. Presse, 6_, No. 28, 673 (1865); 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. 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Chem. jM3_, 291 (1958). 92. Waki1, S. J . , D. E. Green, S. Mi i and H. R. Mahler, J . B i o l . Chem. 207, 631 (1954). 93. S t e r n , J . R., B iochim. Biophys. Acta 26, 448 (1957). 94. Vagelos, P. R., and J . M. E a r l , J . B i o l . Chem. 2J4, 2272 (1959). 95. Stadtman, E. R., J . B i o l . Chem. \£6, 535 (1952). 96. B e i n e r t , H., and P. G. S t a n s l y , J . B i o l . Chem. 204, 67 (1953). 97. S t e r n , J . R., J . B i o l . Chem. 221. 33 (1956). Reprinted from Vol. 4, No. 2,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Copyright © by Academic Prefs Inc. Printed in U.S.A. ENZYMES OF ACETOACETATE FORMATION Ian C. Caldwell and George I. Drummond Department of Pharmacology, School of Medicine, University of British Columbia, Vancouver, Canada. Received January 20, 196I Two mechanisms have been proposed for the formation of acetoacetate. Using extracts prepared from acetone powders of beef liver Lynen and coworkers (1958) obtained evidence that acetoacetate formation occurred via HMG-CoA as intermediate and involved the HMG-CoA cleavage and HMG-CoA condensing enzymes as shown in reactions (l) and (2). (1) Ac-CoA + AcAc-CoA + Ry) > HMG-CoA + CoASH (2) HMG-CoA > Acetoacetate + Ac-CoA In the assay system used, acetyl-CoA was generated from catalytic amounts of CoA in the presence of excess acetyl phosphate and bacterial phosphotransacetylase. Acetoacetyl-CoA is formed by thiolase present in liver fractions and in the bacterial extract. Using a similar assay system, and also substrate amounts of acetoacetyl-CoA, Drummond and Stern (i960) suggested that acetoacetate formation in extracts prepared from fresh beef liver could take place by a direct deacylation of aceto-acetyl-CoA (reaction 3). (3) AcAc-CoA + H20 > Acetoacetate + CoASH Very recently, Segal and Menon (i960) have reported that acetoacetate synthesis in mitochondrial rat liver preparations occurs exclusively by this latter mechanism. We now wish to report that acetoacetate formation in extracts of fresh beef liver requires two enzymes each of which have been obtained relatively free of the other. The data strongly suggest that the HMG-CoA pathway accounts for all the acetoacetate formed in these liver extracts as measured in the catalytic assay system. When a number of beef liver fractions were subjected to heating at 50°, aceto-127 Vol.4, No. 2,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS acetate synthesis was largely destroyed. Activity vas completely restored by addition of purified yeast HMG-CoA condensing enzyme (Table I). Lynen and co-workers (1958) concluded from similar observations that loss in acetoacetate synthesis was due to heat lability of the condensing enzyme. These fractions all contain HMG-CoA cleavage enzyme as determined by direct assay and this enzyme is stable to heat in all fractions except the 20-35$ ethanol (the most purified aceto-acetate synthesizing preparation of Stern et al (i960)). In accord with this, addition of yeast condensing enzyme gave only very small recovery of activity in this heated preparation (Table I). With the exception of the 20-35/4 ethanol, acetoacetate synthesis in most of the unheated fractions is increased by addition of condensing enzyme, presumably because this enzyme is limiting. Vieland and co-workers (i960) have made similar observations in crude rat liver extracts. The results indicate that loss of acetoacetate forming activity by heat may indeed be due to destruction of the condensing enzyme, and that in the 20-35$ ethanol fraction the cleavage enzyme is also largely removed by heat. In an effort to obtain further information regarding the mechanism involved, our most purified preparation (20-35$ ethanol) was subjected to several fraction-ation procedures. By precipitation with zinc ion, followed by increasing concen-trations of ethanol, several fractions were obtained in which acetoacetate synthesizing activity was virtually absent (Table II). The fractions obtained by ethanol precipitation showed no increase in activity when assayed with excess yeast condensing enzyme. Three of the fractions, however, showed a very great increase in activity when assayed in the presence of excess cleavage enzyme (Table II, column 5). This strongly indicated that these fractions contained the condensing enzyme largely free of cleavage enzyme. The latter enzyme was shown by separate specific assay to be present only in very small amounts in two of the fractions (Table II, column 2). The cleavage enzyme is known to be precipitated by zinc (see footnote Table II). Furthermore acetoacetate synthesizing activity in the zinc-ethanol fractions was now restored by adding a heat-treated preparation (Table II, last column). 128 Vol. 4, No. 2,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table I Effect of yeast HMG-CoA condensing enzyme on acetoacetate synthesis in heated beef liver fractions (1) (2) (3) (4) (5) Fraction Protein HMG-CoA cleavage activity Aceto-acetate synthesis (4) + yeast condensing enzyme Recovery mg/ml units/ml units/ml units/ml per cent Crude KHCO3 extract Heated KHCO3 extract 62 31.6 - 8.3 0.76 13.7 14.7 107 30-60$ (NH4>2 S04 Heated 30-60$ (NH4) 2 S04 74.0 46.2 61.3 57.0 43.6 2.8 35.2 20-35$ ethanol Heated 20-35$ ethanol 44 18.2 64.0 4.34 25.4 1.4 24.9 4.2 16.6 Extract acetone pwdr. beef liver Heated acetone pwdr. extract 30.2 20.2 36.2 35.4 14.3 1.22 22.0 22.0 100 Extract of acetone pwdr. pigeon liver Heated acetone pwdr. pigeon liver 31.4 24.0 - 48.7 5.75 100.0 96.1 96.1 The crude bicarbonate extract, the 30-60$ saturated (NH4>2 SO4, and the 20-35$ ethanol fractions were prepared by the method of Stern et al (i960) and represent three of the steps in the purification of the acetoacetate synthesizing system. The acetone powder extracts were prepared by the method of Lynen and coworkers (1958). Heat treatment was carried out by adjusting the fractions (2 ml) to pH 7.5 and stirring in a bath at 50° for 8 minutes, followed by cooling and centrifugation. Protein was determined by the method of Warburg and Christian (1941). Acetoacetate synthesis was assayed by the method of Stern et al (i960) except that all components were reduced by one-fourth and acetoacetate was determined by the method of Walker (1954). Specific activity is defined as units per mg protein. least HMG-CoA condensing enzyme was purified by the method of Ferguson and Rudney (1959). The ammonium sulfate precipitate obtained after protamine treatment was exhaustively dialyzed against .02 M Tris, pH 7.0. An amount was added which catalyzed the formation of at least 4 jimoles of acetoacetate in the presence of excess cleavage enzyme. HMG-CoA was prepared by the procedure of Hilz et al (1958). HMG-CoA cleavage enzyme was assayed by the system of Bachhawat et al (1955) except that all components were reduced by one—half and the Walker method used for acetoacetate determination. The per cent recovery is calculated on the basis of acetoacetate formed in the original unheated extracts in the presence of added condensing enzyme, because this enzyme is limiting. The total recovery of units was 93.2$ of the original 20-35$ ethanol when these fractions were supplemented with a heat treated acetone powder extract. Clearly, recovery of the condensing enzyme was virtually quantitative. Identical results 129 Table IX Effect of HMG-CoA condensing and cleavage enzymes on acetoacetate formation in zinc-ethanol fraotions of beef liver (1) Fraction (2) Total protein (3) HMG-CoA cleavage activity (4) Acetoacetate synthesis total activity (5) (4) + condensing enzyme (6) (4) + cleavage enzyme (7) (4) -1- heated beef acetone pwdr. extract mg units units units units units 20-35$ ethanol 440 640 253 249 455 455 Extract of in"1"1" ppt. 53.3 - 13.0 12.5 - 39.0 0-6.2$ EtQH 44.5 - 14.2 8.7 94.7 92.5 6.2-11.8$ EtOH 60.0 18.2 8.0 10.6 147.0 147.0 11.8-16.6$ EtOH 81.0 20.0 20.3 15.2 133.0 133.0 16.6-30$ EtOH 21.5 - 1.7 1.2 - 10.7 Total recovery $ 60.0 - 12.5 10.5 - 93.2 20-35$ ethanol of beef liver fraction (44 mg/ml) (10 ml) in .02 MKPO4 pH 7.5 was diluted with 10 ml water and, while stirring in ice, 2 ml M K succinate pH 6.0 was added, followed by 8 ml of 0.1 M zinc acetate. The heavy precipitate was removed by centrifugation. The supernatant was fractionated with ethanol between the limits indicated in the table. The 0-6.2$ fraction was obtained at 0°, the 6.2-11.8 and the 11.8-16.6$ fractions at -5° C and the remaining fraction at -15°. All precipitates were taken up in 0.02 M Tris pH 7.5 containing 0.1$ glutathione. Only a small amount of the zinc precipitate dissolved. It was recentrifuged and the precipitate discarded. All fractions were dialyzed overnight against 6 ^  of 0.02 M phosphate pH 7.5 contair' g 1 mM EDTA and 1 mM cysteine, and for a further 5 hours against 4 1_ of the same buffer without EDTA. Acetoacetate synthesis was measured as described in Table I. HMG-CoA cleavage enzyme was purified from liver by the method of Lynen and coworkers (1958) and sufficient enzyme was added to catalyze the formation of at least 2 umoles of acetoacetate per hour in the presence of excess yeast condensing enzyme. The heated beef liver acetone powder extract was that shown in Table I. Recoveries are calculated on the basis of the original 20-35$ ethanol assayed with added cleavage enzyme, since, in this fraction this enzyme is limiting. * Lynen and coworkers (1958) found that the cleavage enzyme was solubilized from a zinc precipitate only by extraction with high phosphate concentration. In a more recent experiment we have confirmed that this enzyme can indeed be recovered from the zinc precipitate. Vol. 4, No. 2,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS were obtained when the heated 30-6OJ& ammonium sulfate fraction was used. The heated 20-35# ethanol preparation, however, did not restore activity obviously because the cleavage enzyme had been largely destroyed. Addition of purified cleavage enzyme to the original 20-35$ ethanol increases acetoacetate synthesis, indicating that i n this fraction, cleavage enzyme i s limiting (see Table II). The condensing enzyme i n these fractions can also be demonstrated fay the optical test system described by Ferguson and Rudney (1959). The enzyme i s quite stable even after repeated freezing and thawing. The condensing enzyme from yeast, i n contrast to the findings of the above authors, i s also stable. This may be due to exhaustive dialysis i n dilute buffer using deionized glass d i s t i l l e d water. It thus seems likely that acetoacetate synthesis i n these liver- fractions occurs by the combined action of HMJ-CoA condensing and cleavage enzymes. Bach enzyme has been obtained largely free of the other. The quantitative nature of the recovery on combination of the two enzymatic entities indicates that this system accounts for most, i f not a l l , of the acetoacetate formed i n these extracts as measured In the catalytic assay system. This work was supported by the National Research Council of Canada. Vo are Indebted to Canada Backers, Ltd., Vancouver for generous supplies of fresh beef liver. REFERENCES Bachhawat, B., Robinson, V.G., and Coon, 11.J., J . Biol. Chem., 216. 727 (1955) Drummond, G.I., and Stern, J.R., J. Biol. Chem., 235. 318 (i960) Ferguson, J.J. Jr., and Rudney, H., J. Biol. Chem., 234. 1072 (1959) Hilz, H., Khappe, J., Ringleman, E., and Lynen, P., Biochem. Z., 329. 476 (1958) Lynen, F.t Henning, U., Bublitz, C , Sorbo, G., and Kroplin-Bueff, L., Biochem. Z., 330, 269 (1958) Segal, H.L., and lie non, G.K.K., Biochem. and Biophys. Research Communications, 3, 406 (1960) Stern, J.R., Drummond, G.I., Coon, M.J., and del Campillo, A., J. Biol. Chem. 235. 313 (1960) 131 Vol.4, No. 2,1961 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Walker, P.G., Biochem. J., 58, 699 (1954) Warburg, 0., and Christian, W., Biochem. Z., 310. 384 (1941-42) Vieland, 0., Loffler, G., Weiss, L., and Neufeldt, I., Biochem. Z ., 333. 10 (i960) 132 

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