' “ 'T""‘_‘f"'.'n STUDIES ON THE ISOLATION AND ‘ ‘ Summon 0F AcmL 00A SYNTHETASEiFROM i ______ f MITOCHONDRIA OF momma ' ' ‘ ‘ BOVINE MAMMARY GLAND Thesis for the Degree of Ph. D. MiCHIGAN STATE UNIVERSITY SHAHiDA-QURESHI' 1971 ' LIBRA R 1/ Michigan State University This is to certify that the thesis entitled STUDIES ON THE ISOLATION AND PURIFICATION OF ACETYL COA SYNTHETASE FROM MITOCHONDRIA OF LACTATING BOVINE MAMMARY GLAND presented by Shahida Qureshi has been accepted towards fulfillment of the requirements for _Eh...D..__degree in Mary and Dairy Science fl/W17‘77 623615: ‘ 4/ Date November: 1971 fl¢€¢(cceq//{ 1‘ 0-7639 l Purifyi mammary methods adsorpt ce11u101 CarbOXyn P‘lOO a; gradient gel, and Th than 90 l folioWed f0llOWed finally c potasSiur ABSTRACT STUDIES ON THE ISOLATION AND PURIFICATION OF ACETYL COA SYNTHETASE FROM MITOCHONDRIA OF LACTATING BOVINE MAMMARY GLAND BY Shahida Qureshi This work was undertaken to develop a method for purifying acetyl-CoA synthetase from lactating bovine mammary gland mitochondria. The different purification methods studied were ammonium sulphate fractionation, adsorption on alumina CY gel, chromatography on TEAE cellulose using KHCO3 or Tris-HCl buffers, DEAE cellulose, carboxymethyl cellulose, Sephadex G-100 and G-200, Bio-gel P-lOO and P-ZOO, ultracentrifugation in sucrose density gradient, adsorption chromatography on calcium phosphate gel, and polyacrylamide gel electrophoresis. The method that yielded an enzyme preparation more than 90 per cent pure was ammonium sulphate fractionation followed by chromatography on DEAE cellulose (DE-23), followed by chromatography on DEAE cellulose (DE-52), and finally chromatography on calcium phosphate gel using potassium phosphate buffers. The enzyme was concentrated, transf Subst: most a and ma cofact 2.24 x acetat is 63, Acetyl Chondr "as sh and ge; in the prepare of this Shahida Qureshi transferred to Tris-HCl buffer, and stored at -60° C. Substrate specificity studies showed the enzyme to be most active on acrylate followed by acetate, propionate, and maleate. Michaelis-Menten constants for the various 4 4 cofactors and substrate were 6.51 x 10- M, 2.92 x 10- M, 4 M, and 6.1 x 10'4 M for Mg, COA, ATP, and 2.24 x 10’ acetate, respectively. The molecular weight of the enzyme is 63,000. AMP at high levels inhibited enzyme activity. Acetyl-CoA synthetase, from bovine mammary gland mito- chondria, exhibits a strong tendency to aggregate. This was shown by gel filtration, sedimentation equilibrium, and gel electrophoresis studies. The enzyme is not active in the aggregated form. The presence of salt in the enzyme preparation tends to prevent aggregation. The exact nature of this phenomenon is not understood clearly at present. STUDIES ON THE ISOLATION AND PURIFICATION OF ACETYL COA SYNTHETASE FROM MITOCHONDRIA OF LACTATING BOVINE MAMMARY GLAND BY Shahida Qureshi A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Departments of Biochemistry and Dairy Science 1971 To my parents, Zakia Khatoon and Abdul Shakoor Quraishi, whose many sacrifices have so meaningfully enriched my life, and my husband, Wahid Hosain, whose patience, understanding and encouragement made this work possible. ii __I — heart advise Patien Sible, he Sho‘ enCOun for gr‘ the De Hafs' E on my c GQnts E AC KNOWLEDGMENTS I would like to take this Opportunity to extend my heart felt gratitude to Dr. Robert M. Cook, my academic advisor, without whose encouragement, guidance, and patient understanding this work would not have been pos- sible. I also appreciate the kindness and understanding he showed for the difficulties a foreign student may encounter. Thanks are due also to Dr. William W. Wells for graciously consenting to be my academic advisor in the Department of Biochemistry. I thank Dr. Loren Bieber, Dr. Steven Aust, Dr. Harold Hafs, and Dr. Edward Convey for kindly consenting to serve on my committee. I am also grateful to the graduate stu- dents and staff in the departments of Biochemistry and Dairy Science for helpful discussions and assistance with biochemical techniques--Dr. D. I. Marinez, Dr. Ronald Slabaugh, Dr. James Johnson, Dr. Gary Gerard, Dr. Richard Walters, and Dr. George Stancel, just to name a few. The assistance of the Dairy Science Department in the form of a research assistantship in the years 1966- 1967 is gratefully acknowledged. Also, the assistantship iii ship Raec some husbe SO pa of th- provided by NIH grant AM12277 is kindly acknowledged. I am indebted to the Institute of International Education for providing a travel grant through a Fulbright Scholar- ship. Technical assistance provided by Rosemary Parker, Raechel Smith, and David Zulke is appreciated. I am indebted to my brother, Aslam, for drawing some of the figures included in this thesis. Last, but not least, many thanks are due to my husband, Wahid, and my brother, Asad Quraishi, for being so patient, understanding, and helpful during the course of this study. iv VITAE The author, Shahida Qureshi, was born in Alwar, India on March 16, 1940. In 1947 her family migrated to Karachi, Pakistan. She graduated from Government Girls High School in 1954. She spent two years in Central Government College for Women and then transferred to D. J. Government Science College where she graduated in 1958, with Zoology and Chemistry as majors. She obtained an M.Sc. in Chemistry (organic) from the University of Karachi in 1960. She then joined the Pakistan Council of Scientific and Industrial Research as a research assistant. She came to the United States of America on August 10, 1965, and joined the Agricultural Biochemistry and Soils Department of the University of Idaho as a graduate student. In 1966 she transferred to Michigan State University and joined the Biochemistry and Dairy Science Departments to work for her doctorate. On December 20, 1969, she married Wahid Hosain Qureshi. LI LI. LIE Ch}. III. TABLE OF CONTENTS LIST OF TABLES O O O O O I O O O O 0 LIST OF FIGURES . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . CHAPTER I. INTRODUCTION . . . . . . . . . II. LITERATURE REVIEW . . . . . . . Acetyl-CoA Synthetase . . . . . General. . . . . . . . . Mechanism . . . . .' . . . Molecular Weight. . . . . . Multiple Forms . . . . . . Intracellular Localization . . Intramitochondrial Localization. Medium Chain Fatty Acid Acyl-CoA Synthetase . . . . Long Chain Fatty Acid Acyl-CoA Synthetase . . GTP— —dependent Fatty Acid Synthetase Methods Used to Purify Acyl -CoA Synthetases . . . . . . . . III. EXPERIMENTAL PROCEDURE . . . . . . Materials and Methods . . . . . Reagents . . . . . . . . Spectrophotometery . . . . . Enzyme Assay . . . . . . . Protein Determination . . . Sucrose Density Centrifugation ElectrOphoresis . . . . . Ultracentrifugation. . . . Iso-Electric Point Determination vi Page viii ix xii DJ OOKOCDQWW Hra 12 l3 l4 l7 l7 l7 l8 l8 19 22 22 24 24 Chapter Page Fractionation of Bovine Mammary Gland . . 26 Isolation of Mitochondria . . . . . 26 Ammonium Sulphate Fractionation . . . 27 Triethyl-Aminoethy1-Ce11ulose Chromatography . . . . . . . . 28 Diethylaminoethyl-Cellulose Chromatography . . . . . . . . 31 Carboxymethyl-Cellulose Chromatography . . . . . . . . 32 IV. RESULTS . . . . . . . . . . . . . 33 v. DISCUSSION 0 O I O I O O 0 O O O O 110 LITERATURE CITED 0 O O O O O O I O O O O 119 vii LI ST OF TABLES Table Page 1. PrOperties of acetyl~CoA synthetase . . . . 6 2. Purification of acetyl-CoA synthetase (COW 330) I O O C I O O I O O O O 34 3. Purification of acetyl-CoA synthetase (COW 329) O O O O O O O O O O O O 42 4. Purification of acetyl-CoA synthetase (COW 444) o o o o o o o o o o o o 51 5. Purification of acetyl-CoA synthetase (Cow 445) . . . . . . . . . . . . 74 6. Purification of acetyl-CoA synthetase (COW 1063) o o o o o o o o o o o 79 7. Substrate specificity of acetyl-CoA synthetase O O O O O O O O O O O 90 viii LIST OF FIGURES Glutathione standard curve . . . . . . . Fractionation of bovine mammary gland tissue . acetyl-CoA synthetase on using KHCO3 buffers and a Rechromatography of acetyl-CoA synthetase on using KHCO3 buffers and a acetyl-CoA synthetase on using KHCO3 buffers without acetyl-CoA synthetase on acetyl-CoA synthetase on acetyl-CoA synthetase on acetyl-CoA synthetase on carboxymethyl cellulose . . . . . . . Effect of Tris-HCl concentration on acetyl- activity . . . . . . . Effect of KCl concentration on acetyl-CoA synthetase activity . . . . . . . u Effect of RF on acetyl-CoA synthetase Figure 1. 2. 3. Chromatography of TEAE cellulose KCl gradient. 4. TEAE cellulose KCl gradient. 5. Chromatography of TEAE cellulose a KCl gradient 6. Chromatography of Bio-Gel P-lOO 7. Chromatography of Bio-Gel P-200 8. Chromatography of Sephadex G-100 9. Chromatography of 10. CoA synthetase ll. 12. activity . . 13. Chromatography of acetyl-CoA synthetase on TEAE cellulose using Tris buffers and a KCl gradient. ix Page 21 30 37 39 41 44 46 48 50 53 55 58 60 | '| I [I -l l Illa-ll." III-10].! I III. .lllll Ill] 1.‘ ‘rll Ii Figure 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. Rechromatography of acetyl-CoA synthetase on TEAE cellulose using Tris buffers and a KCl gradient . . . . . . . . . . . Sedimentation equilibrium study of acetyl—CoA synthetase . . . . . . . . . . . Schematic presentation of polyacrylamide gel electrophoresis of acetyl-CoA synthetase. . Sucrose density gradient centrifugation of acetyl-CoA synthetase using t-RNA as a reference . . . . . . . . . . . . Chromatography of acetyl-CoA synthetase on Sephadex 6-200 0 o o o o o I o o o Iso-electric focusing of acetyl-CoA synthetase. Chromatography of acetyl-CoA synthetase on TEAE cellulose using Tris buffers and a KCl gradient . . . . . . . . . . . Chromatography of acetyl-CoA synthetase on calcium phosphate gel. (Enzyme prepared from mitochondria of Cow 445) . . . . . Chromatography of acety1-CoA synthetase on [DE-’23 cellulose o o o o o o o o o o Rechromatography of acetyl-CoA synthetase on DE-SZ CGIIUIOSQ o o o o o o o o o o Chromatography of acetyl-CoA synthetase on calcium phosphate gel. (Enzyme prepared from mitochondria of Cow 1063) . . . . . Sucrose density gradient centrifugation of acetyl-CoA synthetase (using ovalbumin as a reference) . . . . . . . . . . . Effect of time on the linearity of the acetyl- CoA synthetase reaction . . . . . . . Effect of protein concentration on the linearity of the acetyl-CoA synthetase reaction . . . . . . . . . . . . Page 62 64 66 68 71 73 76 78 82 84 86 89 92 94 Figure Page 28. Effect of pH on acetyl-CoA synthetase aCtiVitY O I O O O O O C O C O O 96 29. Effect of Mg concentration on acetyl~CoA synthetase activity . . . . . . . . 98 30. Effect of CoA concentration on acetyl-CoA synthetase activity . . . . . . . . 100 31. Effect of ATP concentration on acetyl-CoA synthetase activity . . . . . . . . 102 32. Effect of acetate concentration on acetyl- CoA synthetase activity . . . . . . . 104 33. Effect of AMP concentration on acetyl—CoA synthetase activity . . . . . . . . 106 34. Schematic presentation of polyacrylamide gel electrophoresis of purified acetyl- CoA synthetase . . . . . . . . . . 108 xi ATP GTP CTP ITP UTP CoA GSH 2-ME DEAE TEAE BSA NADH EDTA Tris P-P LIST OF ABBREVIATIONS adenosine triphosphate guanosine triphosphate cytidine triphosphate inosine triphosphate uridine triphosphate adenosine monOphOSphate Coenzyme A Michaelis-Menten constant glutathione 2-mercaptoethanol diethylaminoethyl triethylaminoethyl bovine serum albumin trichloroacetic acid nicotinamide adenine dinucleotide, reduced form ethylene diamine tetraacetic acid tris (hydroxymethyl) amino methane pyrophosphate xii CHAPTER I INTRODUCTION In ruminant animals the microbial population in the rument ferments carbohydrates to acetate, propionate, butyrate, carbon dioxide, and methane. Acetate is pro- duced in the largest amount and is the principal source of energy in the ruminant. In fed ruminants the concen- tration of acetate in whole blood is usually 1 to 2 mM. Acetate not only is a major source of energy in ruminants, but it plays a major role in lipogenesis, comparable to the one played by glucose in nonruminants. The activity of ATP-citrate lyase is negligible in ruminants. There- fore, glucose can only supply limited amounts of acetyl—CoA for fatty acid synthesis. Before being utilized by the cell acetate must be activated (covalently linked to the thiol group of coenzyme A). This reaction is catalyzed by the thioki- nase, acetyl-CoA synthetase (acetate: CoA ligase (AMP) (6.2.1.1)l. Mg++ O = H CH -COOH + ATP + COA-SH ‘—— CH -C-SCOA + AMP + P-Pi 3 3 Acetyl CoA synthetase is widely distributed in plants, animals, and microorganisms. Kinases such as thioki- nases are generally considered to be nonequilibrium enzymes and, therefore, they can function in metabolic control processes. It is proposed that the thiokinase, acetyl-CoA synthetase, is an important rate limiting step in acetate metabolism in ruminants. In order to fully elucidate the role acetyl-CoA synthetase plays in con- trol, the enzyme needs to be purified so that its physical and catalytic properties can be determined. The purpose of this work is to study methods for purifying acetyl-CoA synthetase from lactating bovine mammary gland mitochondria. The lactating mammary gland was chosen as a source of the enzyme because of the high rate of acetate utili- zation for both oxidative and synthetic reactions and because milk synthesis is of major economic importance. CHAPTER II LITERATURE REVIEW Acetyl-CoA Synthetase General The enzyme acetyl-CoA synthetase belongs to the general class of enzymes [acid: CoA ligase (AMP) (6.2.1)] which catalyze the activation of a free fatty acid to its corresponding thioester according to the following general reaction: ++ Mg , ___3 Fatty ac1d + ATP + CoA-SHier——-Fatty acyl-COA + AMP + P-Pi Acetyl-CoA synthetase is widely distributed in nature. It has been partially purified from many sources like yeast, bacteria, higher plants, mammalian organs, and pigeon liver. The activation of acetate was reported for the first time by Nachmansohn and Machado (1943). They prepared the activating system from rat brain homogenate and used it for acetylating choline. Lipmann and Tuttle (1945) showed that when ATP and acetate were incubated with fresh pigeon liver extracts and hydroxylamine added in low concentrations, an appreciable amount of acethydroxamate accumulated in the reaction mixture. Using the same enzyme source Stern and Ochoa (1950) observed citrate synthesis from acetate, ATP, and oxalo- acetate. The reaction required Mg++ and CoA and was thought to proceed through two steps: (1) ATP + acetate >"active acetate" (2) "active acetate j>citrate + oxaloacetate Lynen et al. (1951) identified "active acetate" as acetyl-CoA, thus elucidating the function of CoA as an acyl carrier._ The enzyme was partially purified, for the first time from beef heart and pig heart mitochondria (Hele, 1954; Beinert gE_§l,, 1953). About the same time, partial purification of acetyl-CoA synthetase was also achieved from spinach leaf mitochondria (Millerd gt_31., 1954) and later on from yeast (Berg, 1956). It was finally crystallized from beef heart mitochondria with 9% recovery and 64 fold purification (Webster, 1965). The enzyme was also studied in rabbit heart myocardium (Severin, 1967), rat liver (Aas, 1968), potato tuber (Huang et al., 1970), and yeast (DeVincenzi, 1970). The purified enzyme showed instability after dilution or dialysis and lost activity if kept at 4°C. (Hele, 1954; Webster, 1963). It was also reported to be heat labile (Huang gg_al., 1970). Crude extracts of spinach leaf enzyme required GSH but the purified enzyme did not (Millerd §£;§1,, 1954). The beef heart enzyme was found to be more stable in the presence of 2—mercapto— ethanol (Webster, 1965). The rabbit heart enzyme was reported to have 5 sulfhydryl groups which were not required for enzyme activity (Severin, 1967). The formation of acetyl-CoA from acetate, ATP, and CoA was reversible; if P-Pi was added acetyl—CoA dis- appeared (Hele, 1954; Beinert gt_al., 1953). The enzyme activated acetate, prOpionate, and acrylate (Hele, 1954; Webster, 1963; Aas, 1968). Other acids like butyrate, fluoroacetate, succinate, acetoacetate, formate, malonate, glycine, cyanoacetate, oxaloacetate, and glycollate were not activated (Webster, 1963; Hele, 1954; Huang gt_al., 1970). The spinach enzyme did not activate propionate, but butyrate, succinate, valerate, and caproate were activated at slow rates. The Michaelis—Menten constants, for different sub- strates, for the enzyme from various sources are given in Table 1. The absolute requirements of Mg++ was established by Berg (1956). In addition to that a mnoaxoo.v mnoaxom.m mnoaxnm.a mnoaxo~.~ mud vloaxmv.m muoaxmm.m mnoaxnm.a mloaxoo.m etc vnoaxmv.m muoaxmm.m muoaxno.a mnoaxoo.~ mud vuoaxom.m maoaxmm.m ouoaxom.~ mnoaxoo.~ mn< vuoaxmv.m mnoaxnm.m mnoaxoo.m mnoaxnm.a Hn< 03H Ifllm 98% 338 Nuoaxoo.a muoaxoo.m mExncm venouomco maoaxoo.a enoaxoo.m oemucm canouom onma .HuchCH>mo ammo» Ade< ozv enoaxoa.n vnoaxoa.m fl.uo?om.m onmfl .umuumm .oufiE anew: ummm muoaxov.v moma ..MMIMM mm< um>wa umm mnoaxov.a enoaxoo.m vnoaxoo.v mnoaxa.a vnoaxoo.m mmma .mmma .umumomz .Ouwe uunmn moom mnoaxm.m muoax~¢.H vmma .mam: .Oqu uummc umom «Hum: mad <00 onwazuod mumcoHooum mumumod wocmummwm mousow :3 Ex ommuozuchm «oouaaumod no mofiuuoooum second divalent cation and a monovalent cation were also . + . . . + needed. Tris, NH: and K were stimulating while Na and Li+ were inhibitory (Webster, 1965). Mechanism The mechanism of acetyl-CoA formation from acetate, ATP, and CoA, in the presence of Mg++ was studied by Berg (1956), using the yeast enzyme. The reaction was diphasic; the initial step was the formation of acetyl-AMP, which then reacted with CoA to form acetyl—CoA. (1) acetate + ATP + CoA-SH :FZifacetyl-AMP + P-Pi (2) acetyl-AMP + CoA F==3'acety1-C0A + AMP Mg++ was required for the first partial reaction. Acetyl- AMP was chemically synthesized which, upon incubation with the enzyme, P-Pi and Mg++, gave rise to ATP and acetate. AMP and acetyl-CoA were formed when acetyl-AMP was incu- bated with CoA. The attempt to isolate acetyl-AMP from the reaction mixture was not successful and, therefore, the intermediate was considered to be tightly bound to the enzyme. Webster (1962) did, however, isolate acetyl-14C- AMP from the reaction mixture containing 14C-labelled acetate, ATP, MgClz, and an excess of acetyl-CoA syn- thetase. . Berg (1956) showed that the yeast enzyme followed a Bi Uni Uni Bi Ping Pong mechanism. One of the five dif- ferent forms of the potato enzyme was reported to follow an 150 Bi Uni Uni Bi Ping Pong mechanism and at least two catalyzed the enzymatic activation of acetate by ordered Ter Ter mechanism (Huang e£_31., 1970). To quote the authors, "A single reaction is catalyzed by an enzyme which exists in different forms and each form of the enzyme catalyzes the reaction by a different mechanism." Farrar (1970) carried out kinetic studies on acetyl—CoA synthetase isolated and partially purified from beef heart mitochondria. The data were consistent with a Bi Uni Uni Bi Ping Pong mechanism as proposed earlier by Berg (1956). According to this mechanism acetate and ATP were added to the enzyme first. Pyro— phosphate was released. Next CoA was added, and finally AMP and acetyl-CoA were released. Molecular Weight The molecular weight first reported for the par- tially purified acetyl-CoA synthetase from beef heart mitochondria was between 40,000 and 80,000 (Hele, 1954). Webster (1963) reported a sedimentation constant of 4.458 which corresponded to a molecular weight of 70,000. However, the sedimentation constant reported for the freshly purified crystalline enzyme was 3.55 which agreed well with the value, 3.868, for the rabbit heart myocar— dium enzyme (Severin, 1967). The molecular weight calcu— lated from the sedimentation equilibrium experiment was 30,570 at the meniscus and 55,740 at the bottom of the cell. The weight average molecular weight was calcu— 1ated to be 35,790 while the z—average molecular weight was 71,000. The molecular weight calculated by the bind- ing studies of acetyl—AMP to the enzyme was between the range of 31,000—34,000, assuming a 1:1 stoichiometry be- tween enzyme and acetyl—AMP (Webster, 1962). The other molecular weight values reported for acetyl-CoA synthetase were 59,500 for the potato enzyme (Huang gt_al., 1970) and 130,000 for the yeast enzyme (DeVincenzi, 1970). Multiple Forms Multiple forms of acetyl—CoA synthetase were re— ported in yeast (DeVincenzi, 1970) and potato tuber (Huang, 1970). The enzyme activity was reported to be associated with mitochondria both in yeast and potato tuber as well as with yeast microsomes. Three different forms of acetyl-CoA synthetase were reported in yeast. Two forms were associated with the microsomes of the standing and 24-hour-old aerobic culture while the third was localized in the mitochondria of 48—hour-old culture. The three forms had the same molecular weight, electro- phoretic mobility, pH Optimum and were inhibited by Na+ to the same extent. They differed, however, in their catalytic properties, namely, Km values, for acetate and ATP, and catalytic behavior toward propionate. 10 The potato enzyme existed in five different forms. They had identical molecular weight, pH Optimum, and were specific for acetate. They had similar Km values for ATP and Mg++ but different values for acetate and COA. Intracellular Localization Acetyl-CoA synthetase activity was found in the cytosol Of adipose and mammary gland tissue Of ruminants (Hanson gt_31., 1967). The enzyme was localized predomi— nantly in the mitochondria in lung and liver, while in kidney it was equally divided between mitochondria and cytosol. In heart and mammary gland two-thirds Of the enzyme activity was in the cytosol and one-third in the mitochondria (Quraishi gt_gl., 1971). Aas and Bremer (1968) have reported the distribution Of acetyl-COA synthetase in rat liver. They found that 51% Of the total activity was associated with the mito- chondria and about 22% was found in the particle free supernatent. Intramitochondrial Localization TO investigate the submitochondrial localization Of these enzymes, the mitochondria were disrupted and the enzyme activity determined in relation tO marker enzymes (Aas and Bremer, 1968). L-glutamate dehydrogenase is for the matrix, D-B-hydroxybutyrate dehydrogenase and ll carnitine-palmitoyl transferase for the inner membrane, and long-chain acyl-CoA synthetase for the outer mem- brane. Acetyl-CoA synthetase activity was localized in the matrix Of the mitochondria. Medium-Chain Fatty Acid acyl-COA Synthetase Acyl-CoA synthetase (ATP dependent) (EC.6.2.1.2) catalyzes the activation of medium-chain (C4-C12) fatty acids and does not activate acetate. It was isolated and partially purified for the first time from beef liver mitochondria (Mahler e£_31,, 1953). The enzyme was also isolated from hog liver (Jenck et_213, 1957), beef heart mitochondria (Webster, 1965), ox liver particles (Bar-tana gE_§1., 1968), and from beef liver (Graham gt_§1., 1969). The enzyme required a divalent metal ion for maximal activity but no monovalent cation was required (Webster, 1965). AMP and ADP inhibited the reaction. The enzyme was heat unstable and was sensitive tO extremes Of pH (Mahler gt_§l., 1953). The enzyme prepared from ox liver acetone powder separated intO two enzymatically active pro- tein peaks upon chromatography on Bio-Gel and DEAE-Sephadex columns (Bar-tana gt_§l., 1968). One Of the enzymes catalyzed the reaction by "Bi Uni Uni Bi Ping Pong" mechanism, as suggested by Berg (1956) for acetyl—CoA synthetase, while the other followed an ordered Ter Ter mechanism. The octanoyl-CoA synthetase from beef liver 12 also followed a Bi Uni Uni Bi Ping Pong mechanism (Graham g£_al., 1969). The medium-chain fatty acyl-COA synthetase is localized in the matrix Of the mitochondria (Aas gt_31., 1968). Long-Chain Fatty Acid acyl—COA Synthetase Kornberg and Pricer (1953) were first to demonstrate the long-chain fatty acyl-COA synthetase (acid: CoA ligase (AMP), EC 6.2.1.3; trivial name: Palmitoyl-COA synthetase) in guinea-pig and rat liver microsomes as well as the cytosol. The preparation was active on fatty acids ranging from C to C22. Acetate, propionate, and 5 butyrate were not activated. Maximum enzyme activity was Observed using C12. The enzymatic activation followed the identical over-all equation as for acetate or medium- chain fatty acid activation. Farstad e£_§13 (1966) presented evidence for the bimodal distribution Of palmitoyl-CoA synthetase, i.e., both in mitochondria and microsomes Of rat liver. Approx- imately 70% Of the total palmitoyl-CoA synthetase activity is found in microsomes and approximately 30% in the mito- chondria. The enzyme is firmly bound tO membranes both in microsomes and in mitochondria. Allman et a1. (1966) have shown that the enzyme is located in the outer membrane Of beef liver mitochondria. l3 Lippel and Beattie (1970) also investigated the sub- mitochondrial localization Of palmitoyl-CoA synthetase. The activity was found mostly to reside in the outer mitochondrial membrane and some in the inner membrane, the ratio between the two being (O.M./I.M.) 26. Skrede and Bremer (1970) confirmed the above findings of Lippel and Beattie (1970). GTP-Dependent Fatty Acid Synthetase Rossi and Gibson (1964) were first to demonstrate the GTP-specific thiokinase from beef liver mitochondria. The enzyme catalyzes the activation Of fatty acids accord- ing to the following reaction: GTP + R.COOH + COA-SH : R-CO-S-COA + GDP + Pi 4 to C12 were active sub- strates for the GTP-kinase system. CTP, UTP, and ATP Fatty acids of chain length C were inactive, but ITP replaced GTP. The same enzyme was reported by Galzigna g£_31. (1966) from rat liver mitochondria. This enzyme cat- alyzed the activation of both short- and long-chain fatty acids. They found that the GTP—dependent acti- vation was sensitive to P1 and F‘. The difference between the substrate specificity Of the beef liver enzyme and that of rat liver was shown to be due to the use of organic solvent in isolating the beef liver enzyme. It 14 was shown by Sartorelli (1967) that lecithin was bound to the GTP—specific enzyme and its removal with organic solvents affected the enzyme activity as well as its substrate specificity. The enzyme was further purified by Galzigna gt_gl. (1967). They reported one band on polyacrylamide gel electrOphroesis. The molecular weight was estimated to be around 20,000 and the sedi- mentation coefficient was 1.08. Rossi gt_al. (1970) identified 4'-phospho— pantetheine as the cofactor which was required for enzyme activity. The cofactor was bound tO the apo- enzyme in a molecular ratio Of 1:1 by weak secondary bonds. The GTP-dependent fatty acid synthetase is located in the outer membrane Of beef liver mitochondria (Allman gt_al., 1966). Methods Used to Purify_acyl—COA Synthetases Several different methods have been used tO partially purify acyl-CoA synthetases. Fractional pre- cipitation by protamine was used for the yeast enzyme (Berg, 1956), ammonium sulphate precipitation in the case Of heart and liver enzymes (Mahler gt_al., 1953; Hele, 1954) and various column chromatography techniques (Webster, 1963 and 1965; Severin gt_§l., 1967; Huang g£_31., 1970; Farrar, 1970). The acid pH step, adsorption by calcium phosphate gel, and hydroxylapatite have also 15 been used tO purify both ATP-dependent and GTP-dependent acyl-COA synthetases (Galzigna g£_313, 1967; Huang gt_gl., 1970). The most extensively purified enzyme among the acyl-COA synthetases is acetyl-COA synthetase, which was isolated and purified from bovine heart mitochondria (Webster, 1965). The various steps used tO purify this enzyme were: ammonium sulphate precipitation, adsorption by alumina Cy-gel, chromatography using Sephadex and TEAE-cellulose columns, and finally crystallization. That the enzyme protein was not homogeneous was evident by the type Of data Obtained by the sedimentation equile ibrium experiment. The enzyme showed four protein bands upon gel electrophoresis (personal communication). Farrar (1970) has purified acetyl-CoA synthetase from beef heart mitochondria using a different procedure. After the initial ammonium sulphate precipitation steps and the enzyme was taken up in 0.02 M KHCO NiCl 3' 2' acetyl-adenylate added to it before the RNA-pH precipi- tation step. Yeast RNA was added tO the enzyme and the pH Of the solution lowered to 4.7, and kept there for 3 minutes. The cloudy solution was then centrifuged and the precipitate taken up in KHCO and the pH raised 3 to 6.2. Insoluble material was removed by centrifu— gation. After two more steps Of ammonium sulphate pre- cipitation the protein was chromatographed on TEAE- collulose column using a linear KCl gradient l6 (0 to 0.25 M KCl) in KHCO3. The eluted enzyme was pre- cipitated by ammonium sulphate. The purification pro- cedure resulted in 41.6 fold purification Of the enzyme with 2.3% yield. The specific activity Of the purified enzyme was, however, less than one-third Of the value reported by Webster (1965) for both the crystalline enzyme and that from the Sephadex column. Gel elec- trophoresis Of the purified enzyme revealed several protein bands (personal communication). The potato enzyme (Huang e£_§l., 1970) has been partially purified by ammonium sulphate precipitation, DEAE-cellulose, and hydroxylapatite column chromatog- raphy. However, the enzyme was far from being a homo— geneous protein; tO quote the authors, "a homogeneous potato acetyl-CoA synthetase is at present unavailable." Also, the attempts to purify the long-chain fatty acyl-COA synthetase have not been successful (Farstad, 1968). It is evident that none Of the enzyme preparations Of acyl-CoA synthetase, from various sources, have been purified to homogeneity. Therefore, the present knowledge about their physical properties is somewhat speculative, and, hence, further studies on purification are needed. CHAPTER III EXPERIMENTAL PROCEDURE Materials and Methods Reagents All chemicals were purchased from commercial sources. Acetyl-COA, COA, ATP, Tris (Trizma base), and other nucleotides were purchased from Sigma Chemical Company, St. Louis, Missouri. TEAE-cellulose was pur- chased from Brown Company, Berlin, New Hampshire. DEAE- celluloses (DE-23 and DE-52) were purchased from Whatmann, W and R Balston Ltd, England. Ovalbumin, Sephadex gels, and Sephadex columns were purchased from Pharmacia Fine Chemical Inc., Piscataway, New Jersey. Bio-gels and cellex-CM were Obtained from Bio-Rad Laboratories, Richmond, California. Calcium phosphate gel was pre- pared according tO the method Of Miller gt_§1. (1965). Chemicals for gel electrophoresis were purchased from Canal Industrial Corporation, Rockville, Maryland. 2-Mercaptoethanol was from Eastman Organic Chemicals, Rochester, New York. Ammonium sulphate used throughout the experiment was a special enzyme research grade from 17 18 General Biochemicals, Chagrin Falls, Ohio. Ampholine, Carrier Ampholytes was from LKB-Produkter AB, Bromma, Sweden. The dialysis tubing was purchased from Union Carbide Corporation, New York, New York. Spectrophotometery The Coleman Junior Spectrophotometer, the Beckman DB-G Spectrophotometer, and the Gilford Model 2400 Spec— trophotometer equipped with Gilford automatic sample changer were used for spectrophotometric measurements. Enzyme Assay Acetyl-coenzyme A synthetase activity was measured by the acetate-dependent disappearance Of the free sulfhydryl group Of coenzyme A as described by Mahler, Wakil, and Bock (1953). In a total volume Of 0.20 ml, the complete reaction mixture contained 5 umoles Of K-acetate, 1.1 umOles Of ATP, 1.5 umoles Of MgClZ, 0.17 umoles Of CoA-SH, l6 umoles Of Tris (hydroxy- methyl) aminO methane hydrochloride buffer. From 25 to 4 ug Of enzyme protein were used. Blank tubes did not contain acetate. All tubes were preincubated for one minute at 37°. After the enzyme addition the incubation period was three minutes at 37°. In some assays the incubation period was for 10 minutes. The reaction was terminated by the addition Of 2.8 m1 Of the nitroprusside color reagent 19 prepared by the method Of Grunert and Phillips (1951). The Optical density was read after 30 seconds at 520 mu. The difference in Optical density between the blank and the complete reaction mixture is the measure Of enzyme activity. Enzyme concentration is adjusted to give a difference in Optical density between 0.075 and 0.250. Within this range A00 is proportional to 520 enzyme concentration. Under the assay conditions a difference Of 0.185 in optical density corresponds tO the disappearance Of 0.10 pm Of COA. Glutathione was used as the reference standard (Figure 1). One unit Of enzyme activity is defined as the amount which catalyzes the disappearance Of 1 umole Of coenzyme-A per hour under standard assay conditions. The AO.D. was con- verted to units by multiplying by a factor Of 10.81 for a 3-minute assay, and by 3.243 for a lO-minute assay. Specific activity is expressed in units Of enzyme activity per mg Of protein. Protein Determination Protein was measured by the Lowry (1951) method using bovine serum albumin (BSA) as a reference standard. The purified enzyme protein was measured by the absorbance at 280 and 260 um according tO the method Of Warburg and Christian (1941). 20 FIGURE l.--G1utathione standard curve. 21 O. D. 520"“) Pmoles of GSH/ lube Yfilere at 21 ffact assay: 22 Sucrose Density Centrifugation The molecular weight Of acetyl-COA synthetase was determined by the method Of Martin and Ames (1961) using a Beckman Model L2 65B preparative ultracentrifuge. Linear sucrose gradient Of from 5 to 20 per cent sucrose containing 0.05 M Tris-HCl (pH 7.5) in a volume Of 4.4 ml was prepared in cellulose nitrate tubes. 43.8 ug of enzyme protein in 0.1 ml of the same buffer were applied tO the tOp Of the gradient and centrifuged at 50,000 rpm (246,000 xg) for 10 hours at 4 C? in a Beckman SW-56 rotor. Ovalbumin was used as a marker. At the end Of the run fractions were collected by punctur- ing the bottom Of the tube. Eight drops per fraction were collected. Ovalbumin was measured by the absorption at 210 mu using BSA as the standard (Tombs, 1959). The fractions from the tube containing the enzyme were assayed for activity. The above technique was also employed to determine the sedimentation coefficient Of acetyl-COA synthetase using E. COli t-RNA as a marker. This centrifugation was carried out in a Beckman SW-39 rotor at 35,000 rpm for 28 hours. t-RNA was determined by measuring the absorption at 260 mu. Electrophoresis Polyacrylamide Disc gel (5.5% acrylamide) electro- phoresis was performed by the method Of Davis (1964). The fro the par Tri. diar par1 metl four 4 mg was were meri; 23 The acrylamide and bisacrylamide were recrystallized from chloroform and acetone respectively, according tO the method Of Loening (1957). The 5.5% gel was pre- pared as follows: one part solution A [IN HCL, 48 ml; 1 l Tris, 36.3 g; Temed (N, N, N , N - tetra methyl ethylene diamine), 0.23 ml; water tO 100 ml] was mixed with two parts Of solution C [Acrylamide, 22.2 g, BIS (N, N1 - methylenebisacrylamide), 0.30 9; H20 to make 100 ml], four parts H20 and one part Of solution B [Riboflavin, 4 mg/100 ml]. One end Of each tube (5 mm ID x 75 mm) was sealed using parafilm. 1.6 m1 Of the above solution were transferred to each tube. The gel solution poly- merized within 25-40 minutes under fluorescent light. The protein sample was made denser by adding a few crystals Of sucrose. In addition, 5 ul Of 0.05% bromo- phenol blue dye were also added to the sample. The buffer used was 0.025 M Tris-HCL - 0.20 M Glycine, pH 8.3. All gel tubes were pre-electrOphoresed for 15 minutes before the sample addition. The electrOphoresis was carried out with a current Of 6 ma/tube for 30' at 4° C. After electrophoresis the gel columns were stained for proteins either with 0.5% Amido-black in 7% acetic acid for 1 hour and then destained electrOphoretically, or with coomassie blue according to the method Of Chrambach gt_gl. (1967). The gels were kept in 10% TCA for 15 minutes and then put in the staining solution [0.4 g Of coc fox and TCA WEI men Tri; enz; Ultz form equi Inte. rotoi Celli 24 coomassie blue dye in 100 m1 of 20% methanol and 10% TCA] for 12 hours. The gels were then rinsed with 33% methanol and 10% TCA for 6 hours and finally transferred to 10% TCA for 10-12 hours. TO locate the enzyme activity the gel columns that were not stained for protein were sliced into 2 mm seg- ments and each segment was placed in 0.1 m1 Of 0.2 M Tris-HCl buffer, pH 8.6. The extract was assayed for enzyme activity the next day. Ultracentrifugation The sedimentation equilibrium experiment was per- formed using a Spinco Model E analytical ultracentrifuge equipped with mechanical speed control and Rayleigh Interference Optics. Double sector cells with an An-D rotor were used. The enzyme protein from a TEAE- cellulose column was diluted to 0.5 mg/ml with 0.2 M Tris-HCl buffer, pH 8.6. After dialysis against 100 volumes Of the same buffer for 5 hours, 0.11 ml (50 pg protein) Of the sample were transferred to the cell. The centrifugation was carried out at 20,410 rpm at 4° C. for 18 hours, according to thantis (1964). Protein was determined by 280/260 ratio. Iso-Electric Point Determination The isoelectric focusing experiment was carried out to achieve further purification Of acetyl-CoA syn 25 synthetase isolated from the TEAE cellulose column, and to determine the Isoelectric point of the enzyme. For this purpose 110 ml column was used at a temperature Of 4° C. The experiment was carried out according tO the procedure outlined in the LKB brochure.1 The anode solution was prepared by diluting 0.1 ml Of H2804 tO 10 ml with water. The cathode solution was 0.4 ml Of ethylenediamine and 12 g Of sucrose dissolved in 14 m1 Of water. The anode was located at the top and the cathode at the bottom. Twenty-four tubes were prepared by mixing different amounts of the dense and less dense solution. Each tube contained 4.6 ml. The dense solution was prepared by dissolving 1.9 m1 Of 40% ampholyte, pH 3 to 10, and 28 g Of sucrose in 42 m1 of water. The less dense solution was prepared by diluting 0.6 m1 Of the ampholyte to 60 m1 Of water. Two mg Of enzyme protein from the TEAE-cellulose fraction were added to tube #11. The sucrose density column was then prepared by carefully layering these tubes. The experiment was carried out for 42 hours using a potential Of 300 volts. At the end Of the experiment 2 m1 fractions were collected. Aliquots Of the fractions were analyzed for acetyl-COA synthetase activity. Also, the pH of every fifth fraction was determined. In one experiment the ampholytes were lLKB Instruments Inc., 12221 Parklawn Drive, Rock- ville, Maryland 20852. nus the 10M oxi and 444 fina fed , grair 4° C. dmmon (1965i ISola 109 a 26 removed, from each fraction by dialysis, and protein was determined by 280/260 ratio. Fractionation of Bovine Mammary Gland The mammary gland tissue used in these studies was obtained from five different lactating Holstein cows. The cows were fed normal rations and were slaughtered at peak lactation. Acetyl-COA synthetase is known to have highest activity at peak lactation. The activity diminishes considerably when the cows dry up. The mammary gland tissue from cows 329 and 330 was used in the early studies. These cows were fed a high grain and low roughage ration. Cow 330 was also fed magnesium oxide. The second enzyme preparation was from cow 444 and cow 445. Cow 445 was fed hay and concentrates while 444 was fed only concentrates. The tissue used in the final studies was Obtained from cow 1063. This cow was fed a normal ration of corn silage, alfalfa hay, and grain. All Of the fractionation steps were conducted at 4° C. The enzyme isolation procedure used through #5 ammonium sulphate fraction was that described by Webster (1965). Isolation of Mitochondria Mammary gland tissue was immediately chilled in ice after slaughter. After removal Of fat and connective ti: gr< hOI wi‘ sec MS} The la) was in bri fro: an a inta acti equi; Show was 4 when denCe Ammoh 27 tissue, the tissue was cut into thin long strips and ground in a meat grinder. One kg of the tissue was then homogenized in 2 liters of 0.13 M KCl, adjusted to pH 8 with KOH, using a one-gallon waring blender at high speed for 20 seconds and then at low speed for 20 seconds. The homogenate was then centrifuged in an MSE six-liter centrifuge at 1200 xg for 15 minutes. The 1200 xg supernatant was filtered through several layers of cheese cloth and centrifuged in a Sorvall RC-ZB at 20,200 xg for 25 minutes. The top fluffy layer was discarded and the mitochondrial pellet was taken up in 0.13 M KCl [1 gm (wet weight) per 7 m1 Of KCl] and briefly homogenized. The resulting suspension was frozen in plastic bottles at -20° C. Before freezing an aliquot of the 20,200 g pellet was characterized for intactness, Observed by NADH uptake, phosphorylating activity and purity (KCN sensitive) using a polarograph equipped with a clark oxygen electrode. The preparations showed good phosphorylating activity and oxygen uptake was inhibited by KCN. There was slight electron transport when NADH was the substrate. This data is taken as evi- dence that 20,200 xg pellet is respiring mitochondria. Ammonium Sulfate Fractionation The mitochondria were thawed rapidly by swirling in a water bath at 40° C. After each thaw the pH was adjusted to 8 with l N NH4OH. This process was repeated 28 three times. After the last thaw the mitochondrial sus— pension was centrifuged at 20,200 xg for 45 minutes. The supernatant was adjusted to pH 8 and to 0.1 M in 2-mercaptoethanol. TO the fraction thus obtained, the mitochondrial extract, 21 gms Of ammonium sulphate per 100 ml were added slowly and with stirring. The solution was adjusted tO pH 8 with IN NH4OH, stirred for one hour and then centrifuged at 20,200 xg for 10 minutes. An additional 23.5 gms Of ammonium sulphate were added to each 100 m1 Of the supernatant. The precipitate Obtained was recentrifuged to remove excess ammonium sulphate, and was then taken up in 0.02 M KHCO and stored at -60° C. 3 (Figure 2). TEAE-Cellulose Chromatography TEAE-cellulose was washed according to Whatman's procedure1 and then equilibrated in 0.05 M Tris-HCl buffer, pH 7.8. In some cases 0.005 M Tris-HCl buffer was used. (Also in the initial studies 0.02 M KHCO3 buffer was used.) The column dimensions were 1.7 cm x 42 cm. The column was washed overnight with the buffer at a flow rate of 30 ml/hr. About 200 mg Of ammonium sulphate precipitate were dialyzed against 0.05 M Tris-HCl buffer, pH 7.8. The lAdvanced Ion-exchange Celluloses Laboratory Manual (H. Reeve AngeIand CO. Ltd., I4 New Bridge, London, England.) 29 (CF\ f\U use has «Hum HE N\Wmmmwu m 0 MN Emu/N Cum UTNHCUDOEOC mm}. QSWWHQ .OSmmHu cacao MHMEEME mafi>on mo COHuMGOfipOMHmII.N mmeHm c a 3 it m. COOOEOC NC HUN 2 MH.O m0 Emu HO \mlfl 95.2%: 30 .dsm 9mm and + s mmsmwuucmu as ooa\eomlemzv m o.ma sea 9mm .osm mum + s dmum eommxvmzv weaves .msm 9mm hum s + ommDMHuucou sommlemzv m m.mv emcee 9mm .dsm mum s + > mmum Hem: O moomm as a: nexus .dsm 9mm m-m + + use OH . m x oo~.o~ as ooa\¢OmmA¢mzv m mm 664 9mm + .dsm sum use OH . m x oo~.o~ as ooa\eom~1smzv m m.~m sea 8mm .uxm onaz mum A.pxm chase .dsm .7 CHE mv .msm . m x oo~.om um nonmauusmo moEHu m 3mcu one museum HUM 2 ma.o ca ocmmmsm Rouse. 9mm + 8mm CHE mm I m x oom.o~ .msm nae OH I m x coma "ommswfluucoo mos mumsmmoeoc was new idea He «\msmmau m H. Hos s mH.o cw pmnwsmmoeon mm3 manna» panda mumEEmz in CO ‘59 ra' Wa: tej Was lin 31 protein sample was added to the column and washed with the starting buffer until the optical density Of the effluent buffer read below 0.1 at 280 mu. The protein was then eluted from the column using a linear KCl gradient Of 0.0 to 0.6 M in 0.05 M Tris-HCl buffer, in a total volume Of 600 ml. The enzyme eluted at a KCl concentration Of 0.26 M. Fractions having specific activity Of about 80 or above were combined and concen- trated using the Dia-flo cell. The concentrated protein was stored at -60° C. DEAE-Cellulose Chromatography The DEAE—cellulose was washed2 as described in the Whatmann brochure and suspended as a thick slurry in 0.005 M Tris-HCl buffer, 3 mM in 2—ME, pH 7.5. The column dimensions were 1.7 cm x 42 cm. The column was equilibrated overnight with the same buffer, at a flow rate Of 15-20 ml/hr. The ammonium sulphate precipitate was dialyzed for 30 minutes, diluted to 10 mg Of pro- tein/ml, and added to the column. The column was then washed with 140 m1 Of buffer and then eluted using a linear KCl gradient. Six m1 fractions Of eluate were Ibid. 32 collected. The tubes with the highest enzyme activity were pooled and concentrated in a Dia-flow cell. The concentrated enzyme protein was stored at —60° C. Carboxymethyl-Cellulose Chromatography Carboxymethyl-cellulose (Cellex-CM) was prepared by washing first with 0.5 M NaOH - 0.5 M NaCl then with 1 N HCl and finally with water until free Of acid. The cellulose was then suspended in 0.01 M potassium phos- phate buffer, pH 7.5, which was used throughout the experiment. The column dimensions were 0.9 x 26 cm. The column was equilibrated overnight with the buffer at a flow rate Of 6 ml/hr. About 50 mg of ammonium sulphate precipitate were dialyzed against the buffer. This was diluted to give 5 mg/ml of enzyme protein and then added to the column. The column was eluted with 0.01 M potassium buffer, pH 7.5. Three ml fractions Of the eluate were collected. CHAPTER IV RESULTS Mammary gland tissue from five lactating holstein cows were used for these studies. When experiments on purification had exhausted the supply Of mitochondria, another cow was selected for slaughter and a fresh supply Of mitochondria was prepared. In the initial studies attempts were made to purify acetyl-CoA synthetase accord- ing to the procedure described by Webster (1965) for purification Of acetyl-CoA synthetase from bovine heart mitochondria. The procedure involved after the ammonium sulphate fractionation steps final purification by column chromatography using TEAE cellulose and KHCO buffers. 3 This procedure did not give extensive purification using mitochondria prepared from Cow 330 (Table 2). Column chromatography using TEAE cellulose gave less than a two-fold increase in specific activity over the #9 ammonium sulphate precipitate (Table 2). The #9 precipitate was only slightly more active than the #5 precipitate. The enzyme eluted in two peaks when 33 34 mm mmm.mm m.hN vov.H vm Aammv m* as www.mv m.ma omm.~ oma “some he mm mvm.am m.mH HGH.m was lemme ms om «Hm.em m.m oeo.m oom.m A.dsmv as ooa ~H~.mm m.e omm.HH oofl.m A.uxm obese ma m8 0 w muses \us\maosa s as >um>oomm Hmuoe mufl>wu04 cwwuonm OEDHO> cowuomum oamaommm Hence Em mum u mwuocosoouflfi mo_»:mw03 #03 ox m.m n panama ocmam mHmEEmz nomm sous mmmumnuasm «oonasumom no coaumoamausmun.m mamas 35 chromatographed on TEAE cellulose (Figure 3). When the tubes with peak enzyme activity were combined and rechromatographed, the enzyme eluted in three separate peaks (Figure 4). In an attempt tO separate the enzyme protein from the non-enzyme protein a column was run in which a KCl gradient was not used. The enzyme protein eluted with the major protein peak (Figure 5). These studies exhausted the supply Of the #9 precipitate prepared from Cow 330 (Table 2). Mitochondria were prepared from Cow 329 and the enzyme was purified by ammonium sulphate fractionation through the #7 precipitate (Table 3). The #9 precipitate was not prepared because previous data showed that a major increase in specific activity was not Obtained. Further purification Of the #7 precipitate was attempted using Bio-gel P-lOO (Figure 6) or Bio-gel P-200 (Figure 7), or Sephadex G-100 (Figure 8) or Carboxymethyl cellulose (Figure 9). None Of these column chromatography techniques using various buffers gave a significant purification. Another preparation Of mitochondria was Obtained from Cow 444 (Table 4). The effect of Tris-HCl concen- tration on enzyme activity is shown in Figure 10. Optimum enzyme activity was Obtained over a Tris-HCl concentration range of 0.04 M to 0.2 M. Potassium chloride did not increase the activity Of the enzyme (Figure 11). 36 FIGURE 3.--Chromatography Of acetyl-CoA synthetase on TEAE cellulose using KHCO3 buffers and a KCl gradient. The buffer used throughout this procedure was 0.02 M KHCO - 0.5 mM EDTA - 3 mM 2-ME, pH 8. 500 mg Of protein 313,500 units Of enzyme) were diluted, dialyzed, and added to the column. The protein was eluted by washing the column with a linear KCl gradient Of 0.05 M to 1.4 M in 440 ml Of the buffer. The two peaks Of enzyme activity were combined and chromato- graphed (Figure 4). (————), protein mg/ml; (-.-.-), specific activity. 37 LKSEQ‘ bxkxbflmii. w w m IO .. I00 pilr p.L/. .. u ’1 p m 33‘25 15.3% II FRACT ION NUMBER 38 FIGURE 4.--Rechromatography Of acetyl-CoA synthetase on TEAE—cellulose using KHCO3 buffers and a KCl gradient. The combined enzyme fractions (Figure 3, 120 mg protein) were rechromatographed. A linear KCl gradient of 0.05 M to 0.5 M was used in a total volume of 420 ml. (-—-—4, protein mg/ml; (-.-.-), specific activity. 39 \stkb‘ bxkxb ii. a m a m 3.0 l.0 )- prhppvh uh J4 e. M. b. m $.35 eEStI 30405060 FRACTION NUMBER 20 40 FIGURE 5.--Chromatography Of acetyl—CoA synthetase on TEAE cellulose using KHCO3 buffers without a KCl gradient. About 500 mg Of enzyme protein were added to the column. Elution was carried out using 0.02 M KHCO - 0.5 mM EDTA - 3 mM 2-ME buffer, pH 8. A KCl gradient was not used. (——-—0, protein mg/ml; (-.-.-), specific activity. xkxéku‘ uxkxug Ii. 41 0 O 0 w m m m m a 2 .. q u 1 1 q u q u r w .. m L .. m 1 i\\\‘ liH..\l..l.l.l.l.!I.l J m I.I.IHH..H.H§I IIIHH iiliflfliii. .2! I. !L.......... 1 n b I b-Wp-L-bm-Wrflh‘wPLL’Tab O 6 a. 3 l SEES SE II FRACTION NUMBER 42 m.mm mmm.m H.5H m.hmv m.~a Aemmv h* m.nm Nam.ma H.ma cum OOH Aammv ma «.mm mmm.m m.m Hos.a cam A.dsmv as OOH mm~.ea m smm.~ mms x.uxm obese ms OE OE w muses \un\maosn as muo>ooom Hence wua>wuo¢ Camacho oEdHO> sowuomum . Hmuoe camaoomm Em Ava u masosonoouwe mo ucmfloz um3 ox v u unmwm3 cacao unseen: Amwm 300v mmmumnuchm coolamumom mo cowum0flmaH5m|1.m flames 43 FIGURE 6.--Chromatography Of acetyl—CoA synthetase on Bio-Gel P-100. The buffer used was 0.05 M KHCO3 - 0.005 M Tris-HCl, pH 7.6. The column dimensions were 2.5 x 34 cm. (--——0, protein mg/ml; (----), units/ml. 44 E. \225 ....... s m n..... m 1... 40 '5'0 40 CE\OEV 5305 ll Fracrion Number ‘33 _ 45 FIGURE 7.—-Chromatography Of acetyl—COA synthetase Bio—Gel P-200. The column dimensions were 1.8 x 70 cm. The buffer is described before (Figure 6). (————), protein mg/ml; (---—), units/m1. on 46 _E\wu_C—d ....... 2 4 Mo w 3 n. w — W — — _ 1.5 .— - s s 3 2 4-5 - A_E\OEV £22.. I Fraction Number 47 41585:: . Til; “dime cflmuonm . Alli .m mm .moomm z mo.o mm3 pom: Hommsn one .80 em x m.~ mnm3 mcoflmcoEwo sEsHoo one .ooalw xeomcmmm so mwmumnuc>m moulamumom mo mammnmoumfioucuul.m mmDOHm 48 to s 30 AA 1 Iw/suNn ------- I!) N g :9 2 "I T I I I I 4.5 _ 3'5 " l ‘0 ‘0' no- N I'- ' (IN/5w )Nlaioad — FRACTION NUMBER 49 FIGURE 9.--Chromatography Of acetyl-CoA synthetase on carboxymethyl cellulose. The column dimensions were 0.9 x 26 cm. 0.01 M potassium phosphate buffer, pH 7.5 was used tO eluate the column. b—————), protein mg/ml; ( ----- ), units/ml. 50 - 32 1.30 ‘r -20 _s\m:z: ....... 6 2 cl 1 _ _ 16" I-2- 8 3522.90 I FRACTION NUMBER 51 em oam.a o.mma em «.ma A> assHoOV mmOHdHHOOIm¢MB om ome.e m.ms am am A>HIH massaoov mmoHsHHmonmame mm aam.m m.m ems «a nanny me u u . mam.a osm.a A.dsmc as ooH mem.ma H.v mme.m omH.H 1.uxm obese me me OE w when: \un\maosn as >Hm>ooom Hmuoa mua>auo¢ cflmuoum oEsHo> cowuomum . . Hence oemwomdm 0 end I mfluocO£OOpHE mo ucmwo3 903 ax m.H u unmflms cacao unseen: Avvv 300V mmmuonucmw douramumom mo GOABMOHmausmll.v mnmfie 52 .mpw>fluom mmmuwnucmm ¢Oolamumom so coaumuucmocoo Homlmflue mo uommmmll.oa mmDon 53 (dwozs) 'a'ov — (dwozs) '0 'o -------- O In 0 s c? S? l l r V N \O p, co .«6 O". 1 1 l l r O o a on v ‘2' '? '7' ° C.’ o [ms-Hellmm)xlo' 54 FIGURE 11.-—Effect Of KCl concentration on acetyl-COA synthetase activity. 55 s h N N I O 6 c e a e D \Q s s O a. I O s 4 s a s s s s e s a s s s ss 0 s s5 I. m s \I s s e e P, "” . _ mow? _ F _ I s III—I — — 9 J . l otoocoam Aieommv .00 < [Kcl] (m M) x IO 56 Studies using KF as an inhibitor Of ATPase indicated that this enzyme was not interfering with the enzyme assay (Figure 12). A major purification using TEAE cellulose was achieved when Tris-HCl buffer was used instead Of KHCO3 buffer. In contrast with the results in Figure 5, the enzyme protein separated from the major protein peak when a Tris-HCl buffer was used in the absence of a KCl gradient (Figure 13). The enzyme isolated from four such columns was combined, concentrated, and rechromatographed on TEAE cellulose (Figure 14). Since a major purification of the enzyme was achieved (Figure 14, Table 4), further studies on the purity Of the preparation were carried out. One protein band was Observed upon polyacrylamide gel electrophoresis using 10 ug Of protein and amido black dye. However, sedimentation equilibrium studies indicated the presence Of at least two molecular species, one with a molecular weight Of 49,000 and one with a molecular weight Of 84,500 (Figure 15). Polyacrylamide gel electrophoresis using 100 ug Of protein showed the presence of four protein bands (Figure 16). Enzyme activity was found in the third protein band (Figure 16). Using sucrose density gradient centrifugation, the sedimentation coefficient was estimated to be 4.5 S and the molecular weight was estimated to be 62,000 (Figure 17). This did not correspond to any Of the values Obtained from the sedimentation equilibrium study. 57 FIGURE 12.——Effect Of KF on acetyl-COA synthetase activity. 58 60 80 40 [K FJIm M) 2:23.9oq .14 .. .12 - -I0 59 0:8 .HE ooo mo HUM Hmmcfla s anew: OMOHSHHOO mmme .ESE 5-1 :5... £38. .TL .05 onsu nouns pounmum mma ucowpmnm oEsHo> annoy M GA poms was 2 m.o on 2 Ho.o mo ucmflomnm .o.s mm .Homumawe z moo.o mes Hannah mcfiusam one .ucwflomum HUM m was mummmsn MHHB co mmmumsuc>m «Donawumom mo mammnmoumEOH£U|l.mH mmoon 60 IW/Sl/Nfl --- L9 O N mwmiaz 29.541“. On. ON. O: OO. Om 00 05 OO Om O? On ON 0. O CO on O? On ON O. o "darling/q 1 q M ii a d a d q q q d J ¢ ‘3 ’I I 7 N. '0. was: no — V, 61 FIGURE 14.——Rechromatography Of acetyl-CoA synthetase on TEAE cellulose using Tris buffers and a KCl gradient. The column dimensions were 0.9 x 20 cm. The linear KCl gradient used was 0.1 M to 0.6 M in a total volume Of 100 ml. 3 ml fractions Of the eluate were collected. The gradient was started at tube 1. (-—-—9, protein mg/ml; (----), units/m1. 62 \E\ MKSS III 50 1 300 .. 250 .I 200 5 I 00 _ p n n h 2.6 _ 2.4 I. 2.2 . 20_ . 8. 6. A 2 O. 3 ashes 336% II 40 20 25 30 35 FRACTION NUMBER IS 63 .mnsomoonm Hopcmaflummxm may CH OOQHHOMOU mum maflmwoo mce .UOESMMM mm3 mEsHO> OHMHommm Hafiunmm How mn.o mo 05Hc> a .O ow um Emu oav.vm um .mnson ma CH oocommu mos ESHHQHHHSOM .mmmumcecmm oflo> one .oonooaaoo oHoB oposao onp mo mnoanoonm HE H .CEDHOO onu on emcee mums afimuond no as m .o.s mm .HO& 2 H.o . Homumeue z moo.o mm3 pom: Hommsn one .80 OH x N.H ouo3 mcowmnofiwo GEOHOO one .oomlw xooonmom no omouonun>m dOUIHMBooo mo mnmoumouoEOHnUIl.mH mmeHm 71 [s i f. IW/Sllflfl ---- Ma) 91: '0'0 N_. 6.. Ho: 33.". I'm) 092 '0'0 — 72 .mm .AOIOIOV “HE\MUHGO .AIIIIV “HE\mE snououm .Alllllv .ousooooum Hmucofifinomxm on» CH oonwuomoo out mawouoo one .omouonucem noulaeuooo mo meansOOm OHHuooHoIOmHll.mH mmDon 73 3 our (nu/ow; III/310w NO 0 0? 23322299 15 mwmzaz 29.55.“. mm o. N. v. @. "val/sum 8 N. n. 74 s~.H smm mam om.o om Hum ouonmmonm Edwoamu OOH osm.~ own ma v.m .ememnnsd. mmoHsHHmoumame mm oom.m mma no em mmoasaamonmame mm oom.~a am «no on .emm. ms am mma.~n an was owe ..msmv as con oma.ma m.m mms.H 0mm ..uxm ones. me me me w when: \un\maosn as muo>ooom Hmuoe mua>auo¢ caououm oEsHo> coauooum . . Hmuoe oeufioomm m m.em u canononoouafi mo unmwos uoz on ¢H.H u unmwoz cacao unseen: .mvv 300. omouonucem «OOIHmuoom mo sowumowmwusmll.m mqmmm.m omo.v A.uxm ouwzv m* me 05 w mafia: \Hn\waosn HE hnm>oomm Hmuoe mufl>fiuo¢ aflwumum mEsHo> nowuomum camaommm a a B m nmv u mfiuwcozooufla mo panama umz mx ma.m u unuflmz vcmam mHmEEmz Ammoa 300v mmmumnucmm «ooIflmumom mo cofluMOflmwnsmII.m mam¢a 80 purification of the enzyme from Cow 445. Purification of the #5 precipitate could not be achieved with new lots of TEAE cellulose from other sources. Approximately 2 g of the #5 precipitate (Table 6) were consumed in this attempt. However, a preparation of DEAE cellulose (DE-23) was found to give good resolution of the enzyme (Figure 22). The enzyme from the DE-23 column was rechromato- graphed on DE-52 column (Figure 23). DE-52 is a micro- granular material that Huang and Stumpf (1970) found would give good resolution of acetyl-CoA synthetase isozymes from potato. Further resolution was not achieved. However, when the enzyme from the DE-52 column was diluted with 0.001 M potassium phosphate buffer and chromatographed on calcium phosphate gel, extensive purification was achieved (Figure 24). The enzyme eluted in two peaks. The fractions containing enzyme activity from the first peak were combined and rechromatographed on calcium phosphate gel to determine whether or not the enzyme would eluate at the same place. The protein did appear at the same place. However, the enzyme activity was lost. Acetyl-CoA synthetase activity in the second peak (Figure 24) appeared to fall on the protein curve. The Specific activity varied from 200 to 424, but most of the tubes had a specific activity of 325 to 400. The 81 FIGURE 22.—~Chromatography of acetyl—CoA synthetase on De-23 cellulose. The column was washed first with 140 ml of 3 mM Z—ME - 0.005 M Tris-HCl buffer, pH 7.5; then with 160 ml of 3 mM 2-ME - 0.01 M Tris—HCl buffer, pH 7.5. The activity was eluted with 600 ml of a linear KCl gradient of 0 to 0.6 M in 3 mM Z-ME - 0.01 M Tris-HCl buffer, pH 7.5. (-—-—)I protein mg/ml; (----), units/ml. 82 _E\m._._z: IIIII 3.0 2 8 4 O M 2 8 4 O 7 6 6 6 5 4 4 4 - 1 A 1 I- d - I d d d 4 q d d 1 .- u fi"".ll|'.l'---II A“ b r — k P — b - - b — _ 6. A 2 O. 8 6 A 2 O 9 8 7 2 2 2 Z I I I I I 2.8 ' :59... 25.85. I20 I30 I40 IIO I00 30 40 50 60 70 80 90 I0 20 0 FRACTION NUMBER 83 FIGURE 23.v-Rechromatography of acetyl-CoA synthetase on DE-52 cellulose. The column was washed first with 75 ml of 0.005 M Tris-HCl — 3 mM 2—ME buffer, pH 7.5. The activity was eluted with 400 ml of a linear KCl gradient of 0.15 M to 0.4 M in, 3 mM 2-ME - 0.01 M Tris-HCl buffer, pH 7.5. 0-———), protein mg/ml; (----), units/ml. 84 Jlao -I I70 -I60 d I50 - I40 -I I30 _E\m._..za IIIII O 0 4 "I20 -I|0 O 0 9 8 T J 170 <60 ‘50 fi40 ‘30 -20 mo P LJSL — — 2.8 b 2.6- 2.4 I- 2.2 _ 2.0 '- I.8'- . 6. A 4.2.0.33 Eton: z.m...omn_ I20 I00 IIO 80 90 I0 20 30 40 50 60 70 O FRACTION NUMBER 85 .HE\muHGS .AIIIIV «HE\OE cflmuonm .Allllo .5 mm .ummwon mpmnmmonm Enammmuom mo coaumnu Icmocoo mcfimmmuocfi mo ucmflpmum mmflBprm suwz pmnmms mm3 cEdHoo one .Ammoa 300 m0 mHHpconoouHE Eonm Umummmnm mEMNcmv me mumsmmonm ESHOHMO co mwmnmcuamm «COlamumom mo mammumoumfiousoll.vm mmeHm 86 Iw/SlINn «$0232 20:041.. 12?. T 1N. |'_'I2_.I+I2 ono.l+|2no.|+|20¢0.ll+l2no.|+ll 2.0. + I .OO.|III._ 03 con OmN OQN CAN OON OnN OcN OnN ONN O.N OON Om. 00. Os. 0%. On. 0?. On. ON. O: OO. om On On Ow On O? On ON 0. O _ l I. 1 _ _ _ _ _ _ _ _ _ _ 4 d v r _ 1N0. . oI “ in . ~_I _ loo. . . — . w_l . _ loo. . . ON... . lO.. . . VNI o _ l~_. . . . ONT " .— 10.. . _ ~nr w “ Ice . _ mnr . . in: . . . . oc I .. . 10m. _. I a. INN. . . ‘ (I‘ll/5W) NIBLOUd 87 specific activity when plotted gave almost a straight line. These data indicated that the enzyme protein was pure. The enzyme from this second peak was concentrated, divided into small aliquots, and frozen at —60° C. The molecular weight of the enzyme was determined to be 63,000 using sucrose density gradient centrifugation (Martin and Ames, 1961) (Figure 25). This value agreed with the one obtained for the enzyme from Cow 444 (Figure 17). Substrate specificity studies showed that the enzyme was most active with acrylate followed by acetate, propionate and maleate (Table 7). The enzyme was not active with C4 to C8 straight chain fatty acids. Enzyme activity was linear with time and protein concentration (Figures 26 and 27). The effect of pH on enzyme activity is shown in Figure 28. Michaelis-Menten constants for Mg, CoA, ATP, and acetate are presented in Figures 29, 30, 31, and 32, respectively. These values agreed with those reported by others (Table 1). In con— trast with the heart and potato enzymes (Farrar, 1970; Huang gt_§l., 1970) relatively high levels of AMP were required for enzyme inhibition (Figure 33). Of major significance were the characterization studies using polyacrylamide gel electrophoresis. When stained with coomassie blue dye, there were seven protein bands (Figure 34). It appeared that more than 90% of the protein was in the fourth band. This fourth protein band, 5,. I- 88 FIGURE 25.--Sucrose density gradient centrifugation of acetyl-CoA synthetase (using ovalbumin as a reference). The centrifugation was carried out at 50,000 rpm for 10 hours at 4° C. using a 5-20% sucrose gradient. (Details of the experiments are given in the Experimental Procedure.) 120a .o.o IIIII 4. 2 0. 8 "26 ~24 -2.2 ~20 a. 6. _ . 89 . q _ _ _ J 1 Llllll 20 20— I8- 6 IO" 8 p 4 2 .E\ w...z: I4 I2 I0 I6 FRACTION NUMBER 24 22 26 90 TABLE 7.--Substrate specificity of acetyl-CoA synthetase Substrates Tested Relative Activity Acetate 100 Propionate 65 Butyrate 0 Valerate 0 Hexanoate 0 Heptanoate 0 Octanoate 0 Acrylate 151 Maleate 25 Crotonate 0 91 FIGURE 26.—~Effect of time on the linearity of the acetyl-CoA synthetase reaction. A 0.0. (520 IIIII) .20 +- .04 - 92 .24 I' 5 I N I o 1 1 L O I 2 3 TIME (min) p. 93 FIGURE 27.—-Effect of protein concentration on the linearity of the acetyl—CoA synthetase reaction. A 0.0. (520 mp) .28 .24 94 l l 2 4 PROTEIN CONC. l 6 (mg/I'I'IIIXIO2 I I I 95 Hmmmsn mcflomau DIDI D Momma. mumammozm Edflmmmuom Huom mwmuwnucmm «ooIkuwom no mm 00 powwmmII.m~ mmoon 96 I I I N (D '3' "' O O 0 (Wow) '00 v 97 .mump mEMm mgp mo uon xudmlum>mmzwcwq map ma ummcfl one .mufl>fluom mmMOmnucmm doolamumom co coflumnacmocoo m2 m0 uomMMMII.mN mmson 98 Ts. To. x a} w n c n — — q q a 2.70. x 38 u ex ambwoan 9 -l). 0 V N 0. o. 0. (”wozg) '00 v 00 0. 99 .mump mEMm map mo uoam xusmlum>mm3mcflq may ma ummcfi woe .muw>fiuom mmMpmnpcMm doolampmow co GOHuMHucmocoo «00 mo pomMMMII.om mmeHm 100 :25. T48. N 0.. mac 24-0. x NTN "2.x 1 NO. ¢ 0. 10 o (”w 029) '0 '0 V 1 (p Q lOl .mump mEMm msp mo uon xusmlnm>mm3®cflq map ma pmmcfl one .>0H>Huom mmmpmnpcmm floolamuwom so coflpmuucwocoo m94 mo vomMMMII.Hm mmeHm 102 Es. Tim ON Ts. 170. x E} _ _ . q 4 _ 5:10. .. ¢~.~ u 20. N O 3, IO 0. (”w 029) '00 v 00 o. 103 FIGURE 32.-—Effect of acetate concentration on acetyl—CoA synthetase activity. The inset is the Lineweaver-Burk plot of the same data. i0 0 I A 0. 0. (520mp.) .08 .06 .04 .02 104 Km: 6.l x I0'4M m T J l l 5 I o I 2 3 4 5 I/[s] II IO‘3 M" 3 I I3 I 5 0 J I I I0 20 30 Acetate (mM) 40 105 .muH>Huom mmmumsucmm doolamumom so coaumnucmocoo mzd mo pommwmII.mm mmDUHm 106 .25. 7:3; 0. _ O. ON Om o¢ on GO ON 00 Om vOO. 10V ONINIVWBH % 60V 107 .Mpfl>fluoo oEmNco mBOSm m How poo mpoon :Houonm m30£m ¢ How .U 04 yo Hom\oE o mcflms .om How 050 pofinuoo mos can one .Hom on“ mo mou so pouomoa oHoB aflououm Mo on om .poms mo3 .moma .mH>oQ. Bowman Homwsn m.m mm o .omouozpcmm ¢OUIH>uooo UoHMHHDQ mo memouonmouuooao Hom opHanwuoomaom mo coeuoucomoum oaumaonomII.wm mmeHm < II Iflll HIE 109 along with two other bands, contained acetyvaoA synthetase activity. The enzyme activity from the first peak of the calcium phosphate gel column (Figure 24) was lost during rechromatography. Consequently, the nature of the pro- teins that were present in this enzyme peak is not known. CHAPTER V DISCUSSION The data indicate that acetyl-CoA synthetase exists in multiple molecular fonms. The exact nature of these forms is not understood at present. Since it is now known that many enzymes do exist in more than one molecular form, it is not surprising to find multiple forms of acetyl-CoA synthetase in bovine mammary gland. If a single enzyme occurs in different forms in a single organism and each form catalyses the same reaction, then isozymes of the enzyme exist. The term isozyme, then, can be defined as those enzymes that exist in more than one structural form in the same species. This term was coined for the first time by Markert and Mdller (1959) to designate the multiple molecular forms of lactic dehydrogenase and other enzymes found within a single organism. Isozymes are of different kinds, homopolymeric, heteropolymeric, conformational, hybrid, conjugated, and others. On the other hand certain enzyme proteins occur 110 111 in different aggregate forms of a monomer. In such a case either the aggregated or the monomer form could be enzymat- ically active. It is not entirely clear whether the multiple molecular forms of acetyl-CoA synthetase are aggregates of a monomeric unit or whether isozymes exist. Acetyl-CoA synthetase activity did not separate from other proteins but emerged within the void volume when chromatographed on Sephadex G-100 column (Figure 8). The enzyme behaved similarly when Bio-gel P-lOO (Figure 6) and Bio-gel P-200 (Figure 7) columns were used. These results suggest aggregation of the enzyme under the experimental con- ditions. Sedimentation equilibrium studies of the enzyme purified from a TEAE Cellulose column (Figure 13, Figure 14) can be interpreted in three different ways (Figure 15). First that the enzyme may exist in a monomer-dimer equilibrium. The lower molecular weight value of 49,000 obtained for acetyl—CoA synthetase could represent the monomer form of the enzyme while the higher value of 85,000 could represent the dimer form or else some average value of the two species present. However, the molecular weight value of the enzyme from sucrose density gradient centrifugation is estimated to be 62,000 (Figure 17). The minimum molecular weight reported for acetyl-CoA synthetase .. '7“?! 112 from bovine heart mitochondria is 31,000 to 34,000 (Webster, 1965). If it is assumed that the enzymes from both tissues have similar molecular weights then the mammary gland enzyme could possibly have been aggre- gated to a dimer form under the conditions of the experi- ment to give a value of 62,000. The second possibility suggested by the sedimen- tation equilibrium studies of the mammary gland enzyme is that a high molecular weight protein may be present hw in the enzyme preparation. To test this possibility the enzyme was chromatographed on Sephadex G-200 to separate a high molecular weight impurity from acetyl-CoA synthe- tase (Figure 18). However, the enzyme lost two-thirds of the activity. An inactive protein peak eluted in the void volume and the enzyme right after the void volume. This can be explained either on the basis that Sephadex denatures the enzyme as has been reported for many enzymes or else that the first inactive protein peak that emerged from the column represents an aggregate form of the enzyme which is not active. The concentration of KCl in the buffer was 0.1 M and perhaps not enough to break up the aggregation. The fractions of the eluate from the Sephadex G-200 column were combined but original enzyme activity was not restored. Non-specific aggregation of acetyl-CoA synthetase is another possible interpretation of the sedimentation 113 equilibrium studies. There is not enough evidence, at present, to determine the exact nature of aggregation exhibited by acetyl-CoA synthetase. Disc gel electrophoresis studies of the enzyme purified from Cow 444 mammary gland tissue also supported the hypothesis that the enzyme acetyl-CoA synthetase exists in multiple molecular forms. Staining of the gel with amido black dye showed four protein bands (Figure 16). The enzyme activity was found associated with the third band, next to the fastest moving band. The two slower moving protein bands which represented about 70% of the total protein applied had no enzyme activity. These two protein bands were also not well separated indicating some kind of equilibrium between the proteins. These protein bands possibly could repre- sent aggregate forms of the enzyme which are not active. The hypothesis that acetyl-CoA synthetase has a strong tendency to aggregate, the state in which it is not active, is further supported by the fact that the enzyme is very stable in the presence of ammonium sul- phate and can be stored frozen for a long period of time without losing activity. The presence of the salt is known to prevent aggregation. This might explain why the enzyme remains stable longer in the presence of salt. Also, the activity of the enzyme is found to increase 114 when assayed in a more dilute solution, a condition where the proteins are known to exist in a non-aggregate form. When more enzyme was isolated and purified from the mammary gland tissue of cows 445 and 1063, the purifi- cation was carried a step beyond chromatography on TEAE cellulose. Adsorption chromatography using calcium phos- 1 phate gel gave more than a three-fold purification I (Table 6). Acetyl-CoA synthetase activity which emerged as a single peak from the DE-52 cellulose column . (Figure 23) separated into several protein peaks emerging at different ionic strengths of phosphate buffer. The enzyme activity was found to be associated with two protein peaks (Figure 24). The proteins, com- prising the other inactive peaks, should have very similar overall charges since they emerged as a single peak from the DE-52 cellulose column. It is not known whether they represent impure foreign proteins or inactive forms of acetyl-CoA synthetase. Aggregation of the active enzyme to form an inactive aggregate is possible. This kind of aggregate, however, would also be expected to separate upon centrifugation in a sucrose density gradient. The two sucrose density gradient centrifugation experiments carried out using the enzyme from a TEAE cellulose column (Figure 17) and from a calcium phosphate gel column (second enzyme activity peak, Figure 25) showed only —‘—‘- I” " Li." 115 one activity peak. It can be argued, however, that the conditions were not right for the different forms of the enzyme to separate. Although much of the data suggest that acetyl—CoA synthetase aggregates to yield multiple molecular forms, it is possible that isozymes exist. The enzyme purified using calcium phosphate gel could be separated into seven protein bands using gel electrophoresis. Enzyme activity was found to be associated with three of these protein bands (Figure 34). This data suggest the existence of acetyl-CoA synthetase as isozymes. The earlier report that acetyl-CoA synthetase exists as isozymes in potato tuber strengthens this interpretation (Huang and Stumpf, 1970). Five isozymes of acetyl-CoA synthetase were reported which were separated by initial chromatography on DEAE cellulose and then further purified by adsorption chromatography on hydroxylapatite. Each isozyme catalyzed the activation of acetate and exhibited similar kinetic properties. Unpublished studies in this laboratory indicate that acetyl-CoA synthetase in bovine fetal tissues preferentially activates propionate rather than acetate, a situation in contrast to the adult enzyme. This observation can also be explained on the basis of isozymes. That is, isozymes exist in fetal tissues that preferentially activate propionate, whereas an isozyme in adult tissue predominate that preferentially activates acetate . 116 The nature of the multiple molecular forms of lactic dehydrogenase, glutamate dehydrogenase, adenosine deami- nase, carbonic anhydrase, and acetyl-CoA carboxylase has been studied extensively. Of these enzymes the nature of the multiple forms of acetyl-CoA synthetase most closely resembles that of glutamate dehydrogenase and acetyl-CoA carboxylase. The most extensively studied example of an isozyme is that of lactic dehydrogenase from mammalian tissues. This enzyme exists as a tetramer and is made up of two different types of subunits, M and H kind, which are produced by two different genes. The different combination of these two subunits results in the production of five isozymes. A single subunit is not found to be enzymatically active (Markert, 1968). A single polypeptide chain may also polymerize to yield a series of isozymes of different polymer size, like glutamate dehydrogenase (Bitensky et al., 1965). Polymer : monomer x : monomer y Monomer x is the form of glutamate dehydrogenase which catalyzes the reaction. At high protein concentration the monomer aggregates to form the higher molecular weight polymer while certain small allosteric modifiers influence the formation of y monomer causing the dis- aggregation of the polymeric form of the enzyme. 117 A single polypeptide chain can also fold in dif- ferent conformations giving rise to isozymes called "con- formors," like adenosine deaminase (Murphy gt_21,, 1969) and carbonic anhydrase (Edsall, 1968). These conformers have the same catalytic properties but can be separated by electrophoresis or ion-exchange chromatography. Acetyl-CoA carboxylase provides an example of the phenomenon where the same enzyme can exist in different forms of aggregation (Lane, 1969). Acetyl-CoA carbo- xylase exists as an unbranched, filamentous structure with a sedimentation coefficient of 50-68 S and molecular weight of several million. Lane had carried out extensive studies with avian liver and bovine adipose tissue acetyl-CoA carboxylases. Both exhibit aggregation phe- nomenon. The polymeric form of each carboxylase can be dissociated into protomers of the same molecular weight 410,000 and sedimentation coefficient, 13 to 14 S. Each protomer, in its turn, is composed of four non-identical subunits each of about 100,000 molecular weight. The catalytic properties of acetyl-CoA carboxylase are intimately associated with its state of aggregation. The enzyme is active in the aggregated, polymeric form while the dissociated subunits have no activity. The rat liver acetyl-CoA carboxylase exhibits similar phe- nomenon (Numa et al., 1966). Tricarboxylic acids like 118 citrate and isocitrate activate acetyl-CoA carboxylase by shifting the equilibrium toward the formation of the polymeric form. Acetyl-CoA synthetase from the mitochondria of lactating mammary gland may exhibit an aggregation phe- nomenon similar to that observed for acetyl-CoA carbo- xylase, except that as the extent of aggregation increases enzyme activity decreases. In this sense, acetyl-CoA synthetase is more like glutamate dehydrogenase where the polymeric form is not active but the monomer form is active. Similarly there is a possibility that some small molecules might act as an allosteric modifier of acetyl-CoA synthetase and play an important role in main- taining the prOper equilibrium between the different molecular forms of the enzyme.. In summary, acetyl-CoA synthetase prepared as described herein represents the most highly purified form of the enzyme ever obtained from any mammalian tissue. The enzyme exists in multiple molecular forms. This work provides a sound basis for continued study of the various properties of the enzyme and the role it 3 plays in control of cell metabolism. 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