Eur. J. Biochem. 98, 165-172 (1979)

Purification and Properties of Long-Chain Acyl-Coenzyme-A Synthetase from Rat Liver Taka0 TANAKA, Kohei HOSAKA, Minoru HOSHIMARU, and Shosaku N U M A Department of Medical Chemistry, Kyoto University Faculty of Medicine (Received February 27, 1979)

Long-chain acyl-coenzyme-A synthetase from the microsomes as well as from the mitochondrial fraction of rat liver has been purified to homogeneity as evidenced by dodecylsulfate/polyacrylamide gel electrophoresis, amino-terminal analysis and the elution profile at the final chromatography step. The purification procedure involves resolution of the cellular particles with Triton X-100 and chromatography on Blue-Sepharose, hydroxyapatite and phosphocellulose. The purified enzymes from both sources have a specific activity of 26-29 units/mg protein at 35 'C, which is more than 100-fold higher than those of long-chain acyl-CoA synthetases of animal and bacterial origin hitherto reported. The purified enzymes exhibit a molecular weight of approximately 76 000 as estimated by dodecylsulfate/polyacrylamide gel electrophoresis and catalyze the activation of saturated fatty acids with 10-18 carbon atoms and unsaturated fatty acids with 16-20 carbon atoms most efficiently. The purified enzyme from the microsomes and that from the mitochondrial fraction, which are obtained by essentially identical procedures, are indistinguishable from each other with respect to all molecular and catalytic properties examined, including molecular weight, amino acid composition, amino-terminal residue, heat stability, specific activity, pH optimum and substrate specificity regarding fatty acid, acyl acceptor and nucleoside 5'-triphosphate.

The importance of long-chain acyl-coenzyme-A derivatives as intermediates and regulators of lipid metabolism has prompted a number of investigators to study long-chain acyl-CoA synthetase (AMPforming), which was first demonstrated by Kornberg and Pricer [ l ] in particulate preparations from guinea pig liver. Subsequent work has extended the study to several different sources [2- 131. It has been reported that this enzyme in rat liver exhibits a bimodal intracellular localization, comparable specific activities being measured in the microsomes and in the mitochondria [14- 161. Recently, acyl-CoA synthetase I from Candida lipolytica, a hydrocarbon-utilizing yeast, has been Abbreviation. Dansyl, 5-dimethylaminonaphthalene-I-sulpho-

purified to homogeneity in this laboratory [17]. However, only limited purification of long-chain acyl-CoA synthetase from other sources has been achieved apparently because of the instability of the enzyme and of the difficulty in resolving the enzyme from particulate preparations [8,12,13]. In the present investigation, long-chain acyl-CoA synthetase has been isolated in homogeneous form from the microsomes as well as from the mitochondrial fraction of rat liver, and the molecular and catalytic properties of the purified enzymes have been studied.

MATERIALS AND METHODS

nyl.

Reagents and Preparations

Enzymes. Acyl-CoA synthetase or acid :CoA ligase (AMPforming) (EC 6.2.1.3); adenylate kinase or ATP: A M P phosphotransferase (EC 2.7.4.3); pyruvate kinase or ATP: pyruvate 2-0phosphotransferase (EC 2.7.1.40); lactate dehydrogenase or L-lactate: NAD' oxidoreductase (EC 1 .I .1.27); catalase or hydrogenperoxide: hydrogen-peroxide oxidoreductase (EC 1.11. I .6); R N A polymerase or nucleosidetriphosphate:RNA nucleotidykransferase (EC 2.7.7.6).

Triton X-100 was a product of Rohm and Haas (Philadelphia, U.S.A.) and had an average molecular weight of 628. Dimethyl sulfoxide was purchased from Wako Pure Chemicals (Kyoto, Japan), and poly(ethyleneglycol) 6000 (average molecular weight, 7800 - 9000) from Nakarai Chemicals (Kyoto, Japan).

166

Blue-Sepharose (CL-6B) and Sephadex G-10 were obtained from Pharmacia (Uppsala, Sweden), hydroxyapatite (Hypatite C) from Clarkson Chemical Co. (Williamsport, U.S.A.), and phosphocellulose (P-11) from Whatman (Kent, England). Pig-muscle adenylate kinase, rabbit-muscle pyruvate kinase, rabbit-muscle lactate dehydrogenase and bovine-liver catalase were products of Boehringer (Mannheim, F.R.G.). Escherichia coli RNA polymerase was a generous gift from Professor A. Ishihama. [U-'4C]Palmitic acid was purchased from the Radiochemical Centre (Amersham, England), and crystalline bovine serum albumin, ITP and arachidonic acid from Sigma (St Louis, U.S.A.). Dodecanedioic acid and hexadecanedioic acid were kindly provided by Okamura Oil Mill Ltd. (Osaka, Japan). 16-Hydroxypalmitic acid and nonadecanoic acid were obtained from Aldrich (Milwaukee, U.S.A.), and other fatty acids from Nakarai Chemicals (Kyoto, Japan). Palmitoyl-CoA was prepared by the procedure of Young and Lynen [18], and palmitoyl hydroxamic acid by the method of Dittmer and Wells [19]. All other reagents, including CoA, nucleoside 5'-triphosphates and their analogues, were obtained as described previously [17].

Preparation of Particulate Fractions Male Wistar-strain rats weighing 180 - 200 g, which were maintained on a balanced diet (Clea, Tokyo, Japan), were used. After being deprived of food for 12 h, rats were sacrificed by decapitation followed by exsanguination. The livers were quickly removed and homogenized in 3 vol. of 0.25 M sucrose by three down-and-up strokes of the Teflon pestle in the glass tube. The homogenate was fractionated essentially according to the procedure of De Duve et al. [20] as follows. The whole homogenate was centrifuged at 600 x g for I0 min, and the pellet was washed twice by rehomogenization in 3 vol. of 0.25 M sucrose and recentrifugation as before. The supernatant and washings were combined and subjected to successive centrifugation at 13000 x g for 20 min and at 230000 x g for 60 min to yield the mitochondria1 fraction (which includes peroxisomes and lysosomes) and microsomes, respectively ; each fraction was washed once with a small volume of 0.25 M sucrose, and the washing was combined with the supernatant at each stage. Assay of Acyl-CoA Synthetase Activity The activity of acyl-CoA synthetase was assayed at 35 "C either by the isotopic method or by the spectrophotometric method essentially according to the procedure described previously [171; the spectrophotometric assay was used only for the purified

Long-Chain Acyl-Coenzyme-A Synthetase from Rat Liver

enzyme. The standard reaction mixture for the isotopic assay (total volume, 0.2 ml) contained 0.1 M Tris-HC1 buffer pH 8.0, 1.6 mM Triton X-100, 5 mM dithiothreitol, 0.15 M KCl, 15 mM MgC12, 10 mM ATP, 1 mM potassium [U-'4C]palmitate (0.2 Ciimol), 1 mM CoA and enzyme; the time of reaction was 2 min. The standard reaction mixture for the spectrophotometric assay (total volume, 1 ml) was composed of the same ingredients at the same concentrations as that for the isotopic assay, except that ['4C]palmitate was replaced by 0.1 mM nonlabelled potassium palmitate and the concentration of CoA was reduced to 0.6 mM, and that 0.2 mM potassium phosphoenolpyruvate, 0.15 mM NADH, 45 pg adenylate kinase/ml, 30 pg pyruvate kinaselm1 and 30 pg lactate dehydrogenasei ml were added to measure the formation of AMP; the oxidation of NADH at 334 nm was followed with a recording spectrophotometer (Eppendorf model 1101M, Hamburg, F.R.G.). Under the standard conditions used, both assays gave essentially identical activities. All assays were carried out within the range where the reaction proceeded linearly with time and the initial rate of reaction was proportional to the amount of enzyme added. One unit (U) of acyl-CoA synthetase activity is defined as that amount which catalyzes the formation of 1 pmol palmitoyl-CoA or AMP per min under the standard assay conditions. Electrophoretic Analysis Electrophoresis on discontinuous sodium dodecylsulfate/polyacrylamide slab gel containing 8 '%, acrylamide was performed as described by King and Laemmli [21]. After electrophoresis at room temperature, gels were stained for protein with 0.1 % Coomassie brilliant blue dissolved in isopropyl alcohol/ acetic acidiwater (25/10/65, by vol.) and were then destained with methanol/acetic acidiwater (1 5/10/15, by vol.). Analysis of Amino Acid Composition and Amino Terminus Amino acid compositions were determined by the method of Spackman et al. [22] with an amino acid analyzer (ATTO model MLC-703, Tokyo, Japan) ; samples (100- 150 pg) were hydrolyzed with 6 M HC1 under vacuum at 110°C for 20 h. Amino-terminal analysis by dansylation was performed according to the procedure of Gray [23] with the use of the solvent systems described by Hartley [24]. Determinations Protein was determined by the method of Lowry et al. [25] with bovine serum albumin as the standard

T. Tanaka, K. Hosaka, M. Hoshimaru, and S. Numa

161

as described previously [26]. CoA, 4'-phosphopantetheine and pantetheine were determined as reported previously [17].

RESULTS Purification oj' Acyl-CoA Synthetase f r o m Rat Liver Microsomes The procedure of a typical purification of acylCoA synthetase from rat liver microsomes is described below. All operations were carried out at 0-4°C. Resolution of Microsomes. Microsomes derived from 30 livers were suspended in 840 ml of a mixture containing Triton X-100, potassium phosphate buffer pH 7.4, 2-mercaptoethanol and EDTA, the final concentrations of which were 5 mM, 50 mM, 5 mM and 1 mM, respectively, to give a protein concentration of 4.5 mg/ml. The mixture was allowed to stand for 1 h and was then centrifuged at 230000xg for 1 h. The resulting supernatant was collected (775 ml). Blue-Sepharose Chromatography. The 230000 x g supernatant was diluted with 5 volumes of a solution containing 2 mM Triton X-100 and 5 mM 2-mercaptoethanol. The diluted solution was applied to a Blue-Sepharose column (2.6 x 18 cm) equilibrated with 20 mM potassium phosphate buffer pH 7.4 containing 2 mM Triton X-100 and 5 mM 2-mercaptoethanol (buffer A). The column was washed with five column volumes of buffer A and then with five column volumes of buffer A containing 10 mM ATP. Elution was carried out with buffer A containing 10 mM ATP and 0.8 M NaCl at a flow rate of approximately 15 ml/h; 15-ml fractions were collected. The enzyme emerged in a single peak as shown in Fig. 1. The fractions exhibiting high enzyme activities (fractions 54- 67) were combined (210 ml). Hydroxyapatite Chromatography. To concentrate the pooled enzyme solution from the Blue-Sepharose chromatography step, 0.7 volume of 50 (w/v) poly(ethyleneglycol) 6000 was added. After gentle stirring for 30 min, the resulting precipitate was collected by centrifugation at 230000 x g for 30 min and then loosely homogenized in 50 ml of 20 mM potassium phosphate buffer pH 7.4 containing 5 mM Triton X-100 and 5 m M 2-mercaptoethanol. The mixture was gently stirred for 30 min and then freed from insoluble material by centrifugation at 230 000 x g for 30min. This was passed through a Sephadex G-10 column ( 5 x 25 cm) equilibrated with buffer A. All protein-containing fractions were combined (165 ml) and applied to a hydroxyapatite column (2 x 16 cm) equilibrated with buffer A. The column was washed with two column volumes of buffer A and then eluted with a linear concentration gradient established between six column volumes of buffer A and the same volume of 0.35 M potassium phosphate buffer pH 7.4

0

40

20

60

80

Fraction number

m

Fig. 1. B/ite-St~p/iu,.rJ.,i, i~hroniutogruphq.qf acyl-C'(iA . s ~ ~ n t / ~ c ,fi.oni tu.~c the microsomes. For experimental details, see the text. The diluted 230000 x g supernatant was applied, and the column was washed with buffer A ; all the enzyme activity applied was adsorbed to the column, while 90 'i: of the protein applied was found in the effluent. Buffer A containing 10 m M ATP was applied at arrow a. and buffer A containing 10 mM ATP and 0.8 M NaCl at arrow b. The volume of each fraction was 15 ml. ( 0 ) Enzyme activity per ml effluent (assayed by the isotopic method with the standard reaction protein mixture); (0)

0

10

20 Fraction number

30

40

Fig. 2. Hydro,~yupulitrchromutogruphy of ucyl-CoA synthetuse fioni the microsomes. For experimental details, see the text. The concentrated and gel-filtered enzyme solution was applied, and the column was washed with buffer A ; all the enzyme activity applied was adsorbed to the column, while 9.8 mg protein was found in the effluent. The gradient elution was initiated at the arrow. The volume of each fraction was 15 ml. ( 0 ) Enzyme activity per ml effluent (assayed by the isotopic method with the standard reaction protein mixture) ; (0)

containing 2 mM Triton X-100 and 5 mM 2-mercaptoethanol; the flow rate was approximately 20 ml/h, and 15-ml fractions were collected. The enzyme appeared in a single peak, which coincided with the main protein peak (Fig. 2). The fractions having specific activities higher than 8 U/mg were combined (105 ml). Phosphocellulose Chromatography. The pooled enzyme solution from the hydroxyapatite chromatography step was concentrated to 50 ml by ultrafiltration with a Diaflo membrane filter PM-30 (Amicon Far

Long-Chain Acyl-Coenzyme-A Synthetase from Rat Liver

168

Table 1. The overall purification was 84-fold with a yield of 5 % . The purified enzyme could be stored at -70°C for at least four months without loss of activity.

Purification of Acyl-CoA Synthetase from the Mitochondria1 Fraction qf Rat Liver

0

30

10 20 Fraction number

Fig . 3. Pk ospliocellulose chroma tography o j acyl- CoA .synthe taw ,from the microsomes. For experimental details, see the text. The concentrated and gel-filtered enzyme solution was applied, and the column was washed with buffer A ; all the enzyme activity applied was adsorbed to the column, while 3.7 mg protein was found in the effluent. The gradient elution was started at the arrow. The volume of each fraction was 10 ml. ( 0 ) Enzyme activity per ml effluent (assayed by the isotopic method with the standard reaction mixture); (0) protein; (A)specific activity

Table 1. Purification of acyl-CoA synthetase ,from rat liver microsomes 30 livers were used, Enzyme activity was determined by the isotopic method with the standard reaction mixture Fraction

Microsomes 230000 x g supernatant Blue-Sepharose H ydroxyapatite Phosphocellulose

Protein

Total activity

Specific activity

Yield

mg

U

U/mg

"/,

3780 2460 56.2 15.6 2.2

1290 1230 349 162 63.1

0.34 0.50 6.21 10.4 28.7

100 95 21 13 5

East, Tokyo, Japan). The concentrated enzyme solution was passed through a Sephadex G-10 column (5 x 25 cm) equilibrated with buffer A. The proteincontaining fractions combined (165 ml) were applied to a phosphocellulose column (2 x 10 cm) equilibrated with buffer A. The column was washed with two column volumes of buffer A and then eluted with a linear concentration gradient made between ten column volumes of buffer A and the same volume of 0.35 M potassium phosphate buffer pH 7.4 containing 2 mM Triton X-100 and 5 mM 2-mercaptoethanol at a flow rate of approximately 10 ml/h; 30-ml fractions were collected. The enzyme emerged in a single peak, which coincided with the nearly single protein peak (Fig. 3). The fractions exhibiting maximal and essentially constant specific activities (27 - 30 Ujmg) were combined (50 ml). The results of the purification of acyl-CoA synthetase from the microsomes are summarized in

Crude acyl-CoA synthetase in the mitochondrial fraction of rat liver, in contrast to the enzyme in the microsomes, was very unstable. At 0 "C, approximately 90% of the enzyme activity in the mitochondrial fraction, which was suspended in 50 mM potassium phosphate buffer pH 7.4 containing 2 mM dithiothreitol and 1 mM EDTA, was lost in 8 h, while essentially no loss of the activity in the microsomes occurred for at least 24 h. The addition of dimethyl sulfoxide at a final concentration of 20-35% (v/v) protected the enzyme in the mitochondrial fraction from inactivation; at O T , 85-95% of the activity was preserved after 8 h. Therefore, all solutions used for the purification of acyl-CoA synthetase from the mitochondrial fraction contained 25 (v/v) dimethyl sulfoxide. Prior to the purification, the mitochondrial fraction was treated with dimethyl sulfoxide; 840 ml of a suspension of the particles (protein concentration, 8.2 mgiml), which were derived from 50 livers, was allowed to stand for 1 h in the presence of 25 % (v/v) dimethyl sulfoxide, 50 mM potassium phosphate buffer pH 7.4, 5 mM 2-mercaptoethanol and 1 mM EDTA, and was then centrifuged at 230000 x g for 1 h. The particles precipitated were subjected to Triton X-100 treatment and high-speed centrifugation followed by chromatography on Blue-Sepharose, hydroxyapatite and phosphocellulose in essentially the same manner as described for the purification of the enzyme from the microsomes; although the starting material contained a larger amount of protein than that used for the purification of the microsomal enzyme, the volume of the enzyme preparation at each step, as well as the sizes of the chromatography columns, was essentially unaltered. The results of a typical purification of acyl-CoA synthetase from the mitochondrial fraction are summarized in Table 2. The overall purification was 125-fold with a yield of 6 %. The purified enzyme could be stored at - 70 "C for at least four months without loss of activity. Purity and Molecular Weight The purified acyl-CoA synthetase preparation from the microsomes as well as from the mitochondrial fraction was essentiaily homogeneous as evidenced by dodecylsulfate/polyacrylamide slab gel electrophoresis (Fig. 4). Comparison of the electrophoretic

T. Tanaka, K. Hosaka, M . Hoshimaru, and S. Numa

169

Table 2. Purifi'cation qf'acyl-CoA synthetase from the mitochondrial ,fraction qf rat liver 50 livers were used. Enzyme activity was determined by the isotopic method with the standard reaction mixture Fraction

Protein

Total activity

Specific activity

Yield

Table 3. Amino acid compositions of acyl-CoA synthetases ,froni tltc microsomes andjrom the mitochondria1 fiaction Duplicate analyses were made as described in Materials and Methods, and the mean values are given. No corrections have been made for losses during hydrolysis or for incomplete hydrolysis. Half-cystine and tryptophan were not determined Amino acid

Amino acid in acyl-CoA synthetase from ~~~~~

Mitochondria1 fraction 230000 X:I supernatant Blue-Sepharoae Hydroxyapatite Phosphocellulose

6890 4030 528 86 33

1450 1050 528 157 86 5

021 0 26 100 183 26 2

100 12 36 11 6

microsomes

mitochondrial fraction

mo1/100 inol ~~

Origin

4

165000 -155000 --95m

-68m

-57200

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

9.3 5.3 6.9 13.5 4.0 9.8 7.3 6.1 2.2 5.6 9.4 3.2 4.5 1.8 6.5 4.6

~

9.4 5.5 6.0 13.0 4.6 9.4 7.4 6.9 2.3 5.9 10.2 2.0 4.8 1.5 6.3 4.5

-39000 Amino Acid Composition and Amino Terminus Dye

A

B

C

Fig. 4. Dodec~l.su~ute/pol~ucrylamide gel electrophoresis of acylC'oA .syntlietases from the microsomes and ,from the mitochondria1 ,fraction. For experimental details, see Materials and Methods. The amount of the purified enzymes used was as follows: (A) 4 pg enzyme from the microsomes, (B) 4 p g enzyme from the mitochondria] fraction, (C) 2 pg enzyme from the microsomes plus 2 pg enzyme from the mitochondrial fraction. The marker polypeptides employed were subunits of E. coli RNA polymerase ( M , 165000, 155000, 95000 and 39000), bovine serum albumin (hl,68000) and bovine liver catalase ( M , 57200). Bromophenol blue served as the tracking dye

mobility of the enzymes, which were treated with dodecylsulfate and 2-mercaptoethanol, with those of markers showed that the molecular weight of both enzymes was approximately 76 000. Further evidence for the homogeneity of the two enzyme preparations was provided by the demonstration of a single aminoterminal residue (see below) as well as by the fact that the major enzyme-containing fractions from the final phosphocellulose chromatography step exhibited essentially constant specific activities (see Fig. 3).

Table 3 compares the amino acid compositions of the purified acyl-CoA synthetases from the microsomes and from the mitochondrial fraction, revealing a close resemblance. Amino-terminal analysis by dansylation showed that aspartic acid (or asparagine) occupied the amino-terminal position of both enzymes; the dansyl-aspartic acid derivative was detected with essentially no accompanying traces of other amino acid derivatives.

Stability When purified, acyl-CoA synthetase from the mitochondrial fraction, like the enzyme from the microsomes, was rather stable even in the absence of dimethyl sulfoxide ; both purified enzymes retained their full activity after dialysis at 0 "C for 24 h against 50 mM potassium phosphate buffer pH 7.4 containing 2 mM Triton X-100, 2 mM dithiothreitol and 1 mM EDTA. When the two dialyzed enzymes were incubated at 35"C, no difference was noted in their stability; SO-87%, 63-67% and 41-42% of the initial activity were preserved after 2.5 min, 5 min and 15 min, respectively.

170

Long-Chain Acyl-Coenzyme-A Synthetase from Rat Liver

0

0.1

0.2

[Fatty acid) (mM)

0

ai

0.2

[Fatty acid] (rnM)

Fig. 5. Sirhstrmte spec;ficity ~ / u c J ~ - C synthetaseJrorn OA the rnicrosomes with respect tofutry acid. Enzyme activity was assayed by the spectrophotometric method with 0.58 pg of the purified enzyme and the standard reaction mixture, except that various fatty acids and their analogues were used at the indicated concentrations. (A) Saturated fatty acids; (0) hexanoic acid, (a)octanoic acid, ( 0 )decanoic acid, (0) undecanoic acid. (a)lauric acid, (m) tridecanoic acid, (A) myristic acid, (A) pentadecanoic acid, (A)palmitic acid. (v)heptadecanoic acid, (V)stearic acid. (V)nonadecanoic acid, ( x ) ardchidic acid, (+) docosdnoic acid. (B) Unsaturated fatty aclds, hydroxyfatty acid and dicarbouylic acids; (0) palmitoleic acid, ( 0 )oleic acid, (0)linoleic acid, (H) linolenic acid, (A) arachidonic acid, (A) 16-hydroxypalmitic acid, (v)dodecanedioic acid, (V)hexadecanedioic acid

Cofuctor Requirement, Reaction Products and Stoichiometry The purified acyl-CoA synthetases from the microsomes and from the mitochondrial fraction absolutely required ATP, Mg2+, fatty acid and CoA for their activity. The reaction product formed by the purified enzymes from ['4C]palmitate was identified as palmitoyl-CoA by chromatographic analysis of the thioester as well as of the hydroxamic acid derived from it according to the procedure described previously [27]. When adenylate kinase was omitted from the reaction mixture for the spectrophotometric assay, no oxidation of NADH occurred, indicating that AMP was a reaction product. Furthermore, a stoichiometric relationship was observed between the amounts of palmitic acid or CoA added and that of AMP formed as measured spectrophotometrically. These results confirm that both acyl-CoA synthetases catalyze the stoichiometric conversion of ATP, fatty acid and CoA to AMP, PPi and acyl-CoA. Substrate Specificity and Kinetic Properties Studies were made on the substrate specificity and kinetic properties of the purified acyl-CoA synthetases from the microsomes and from the mitochondrial

fractjon. As shown in Fig.5 and in Fig.6, the two en@'me&exhibited essentially identical substrate specificities %th respect to fatty acid, utilizing saturated fatty acids with 10- 18 carbon atoms and unsaturated fatty acids with 16-20 carbon atoms most efficiently. Saturated fatty acids containing more than 20 or less than 8 carbon atoms (including n-butyric acid and acetic acid) as well as hexadecanedioic acid and dodecanedioic acid were essentially ineffective. The substrate specificities of the two acyl-CoA synthetases with respect to acyl acceptor were likewise indistinguishable; the spectrophotometric assay with the standard reaction mixture was used, except that this substrate was varied. CoA was utilized most efficiently. The V values (percentage of that for CoA) and the apparent K, values for CoA and its effective analogues, as estimated by Lineweaver-Burk plots [28], were as follows: CoA, loo%, 0.026-0.028 mM; dephospho-CoA, 45 - 46 %, 0.23 - 0.29 mM ; 4'-phosphopantetheine, 25 - 26 %, 0.41 - 0.43 mM ; pantetheine, 26 - 27 %, 1.1 - 1.2 mM. N-Acetylcysteamine, L-cysteine, GSH and dithiothreitol at concentrations up to 2 mM were not utilized. The substrate specificities of the two acyl-CoA synthetases with respect to nucleoside 5'-triphosphate were also essentially identical ; the isotopic assay with the standard reaction mixture was used, except

T. Tanaka, K. Hosaka, M. Hoshimaru, and S. Numa I

171 1

I

B

'f 0

0.1 [Fatty acid] (mM)

0.2

0

0.1

0.2

[Fatty acid] (mM)

Fig. 6 . Suhsrrute .sprc,ific.ityo/'ucyl-CoA .synthetase fkom the mitochondriu1,fruclion n,itl7 re.vprct 10 Jutty mid. For the conditions of enzymc assay as well as for symbols in A and B, see Fig. 5. 0.51 hg of the purified enzymc was used for each assay

that this substrate was varied. Among the different compounds tested, only ATP and dATP were effective. The V value for dATP was 27 - 30 of that for ATP, and the apparent K,,, values for ATP and dATP were 2.3-2.4 mM and 12.5- 13.3 mM, respectively. GTP, UTP, CTP, ITP, dTTP, adenylyl (P,y-methy1ene)diphosphonate and adenylyl imidodiphosphate at concentrations up to 10 mM were ineffective. Both acyl-CoA synthetases exhibited a broad pH optimum ranging from 7.4 to 9.1 ; the isotopic assay with the standard reaction mixture was used, except that the pH was varied with Tris-HC1 buffers (pH 7.48.7), potassium phosphate buffers (pH 5.8 - 7.7) and glycine-NaOH buffers (pH 8.0 - 9.6). DISCUSSION In the present investigation, long-chain acyl-CoA synthetase has been purified to homogeneity both from the microsomes and from the mitochondrial fraction of rat liver. The purified enzymes exhibit a specific activity of 26-29 U/mg at 3.5 "C, which is more than 100-fold higher than those of long-chain acyl-CoA synthetases from animal and bacterial sources hitherto described [8,12,13] and is comparable to that of the pure enzyme from C. lipolytica [17]. Bar-Tana and his associates [13,29,30] have reported that their preparation of long-chain acylCoA synthetase obtained from freeze-dried rat liver microsomes has a specific activity of 0.25 U/mg at

37°C and a molecular weight of 27000 under denaturing conditions. Since this enzyme preparation exhibits a much smaller molecular weight and a much lower specific activity than our purified enzyme preparations do, it might represent either a degradation product of the enzyme or a protein structurally unrelated to our preparations. The purified acyl-CoA synthetase from the microsomes and that from the mitochondrial fraction, which were obtained by essentially identical procedures, were indistinguishable from each other with respect to all molecular and catalytic properties thus far examined. The two enzymes exhibited the same molecular weight, closely resembling amino acid compositions and an identical amino-terminal residue. They showed essentially identical heat stabilities, specific activities, pH optima and substrate specificities regarding fatty acid, acyl acceptor and nucleoside 5 '-triphosphate. Recent work from this laboratory has demonstrated the presence of two distinct long-chain acylCoA synthetases in C. lipolytica and has elucidated their physiological functions [17,27,31,32]. Acyl-CoA synthetase I is responsible for the production of acylCoA to be utilized for the synthesis of cellular lipids, while acyl-CoA synthetase I1 provides acyl-CoA that is exclusively degraded via P-oxidation. As described above, available evidence supports the view that the acyl-CoA synthetase purified from the microsomes and that purified from the mitochondrial fraction are

112

T. Tanaka, K. Hosaka, M. Hoshimaru, and S. Numa: Long-Chain Acyl-Coenzyme-A Synthetase from Rat Liver

identical enzymes. Thus, it is possible that an additional long-chain acyl-CoA synthetase yet unknown is present in animal tissues as well. This investigation was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, the Mitsubishi Foundation, the Foundation for the Promotion of Research on Medicinal Resources and the Japanese Foundation of Metabolism and Diseases.

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T. Tanaka, K. Hosaka, M. Hoshimaru, and S. Numa, Department of Medical Chemistry, Kyoto University Faculty of Medicine, Yoshida, Sakyo-ku, Kyoto-shi, Kyoto-fu, Japan 606