ARCHIVES

OF

BIOCHEMISTRY

AND

Subunits

BIOPHYSICS

177,

364-378

(1976)

of Fatty Acid Synthetase

Complexes

Comparative Study of Enzyme Activities and Properties of the Half-Molecular Weight Nonidentical Subunits of Fatty Acid Synthetase Complexes Obtained from Rat, Human, and Chicken Liver and Yeast’-*

AsAF A. QURE~HI,FRANK A. LORNITZO, ROBERT A.JENIK, AND JOHN w. PORTER Lipid

Metabolism

Laboratory, Chemistry,

Veterans University

Administration of Wisconsin, Received

April

Hospital, Madison,

and Department Wisconsin 53706

of Physiological

22, 1976

Rat, human, and chicken liver and yeast fatty acid synthetase complexes were dissociated into half-molecular weight nonidentical subunits of molecular weight 225,000-250,000 under the same conditions as used previously for the pigeon liver fatty acid synthetase complex [Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem. 250, 4520-45291. The separation of the half-molecular weight nonidentical subunits I and II of each fatty acid synthetase was then achieved by affinity chromatography on Sepharose l -aminocaproyl pantetheine. The separations required, as with the pigeon liver fatty acid synthetase, a careful control of temperature, ionic strength, pH, and column flow rate for success, along with the freezing of the enzyme at -20°C prior to the dissociation of the complex and the loading of the subunits onto the column. The separated subunit I (reductase) from each fatty acid synthetase contained P-ketoacyl and crotonyl thioester reductases. Subunit II (transacylasel contained acetyl- and malonyl-coenzyme A: pantetheine transacylases. Each subunit of each complex also contained activities for the partial reactions, P-hydroxyacyl thioester dehydrase (crotonase), and palmitoyl-CoA deacylase. The specific activities of a given partial reaction did not vary in most cases more than twofold from one fatty acid synthetase species to another. The rat and human liver fatty acid synthetases required a much higher ionic strength for stability of their complexes and for the reconstitution of their overall synthetase activity from subunits I and II than did the pigeon liver enzyme. On reconstitution by dialysis in high ionic strength potassium phosphate buffer of subunits I and II of each complex, 6585% of the control fatty acid synthetase activity was recovered. The rat and human liver fatty acid synthetases cross-reacted on immunoprecipitation with antisera. Similarly, chicken and pigeon liver fatty acid synthetases crossreacted with their antisera. There was, however, no cross-reaction between the mammalian and avian liver fatty acid synthetases and the yeast fatty acid synthetase did not cross-react with any of the liver fatty acid synthetase antisera.

We recently reported the two nonidentical

weight subunits of pigeon liver fatty acid synthetase by affinity chromatography on Sepharose l -aminocaproyl pantetheine and the enzymatic activities of each of these subunits (l-3). In the present paper, we report an extension of this study to fatty acid synthetases from rat, human, and chicken liver and yeast. In this study we show that each of the fatty acid synthetases examined dissociates in low ionic

the separation of half-molecular

‘This paper is No. 6 in a series. Paper No. 5 appeared in (1975) Biochem. Biophys. Res. Comnun. 66, 344-351. 2 This investigation was supported in part by a research grant (AM-013831 from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service. 364 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

SUBUNITS

OF

FATTY

ACID

strength Tris-glycine-EDTA buffer to nonidentical half-molecular weight subunits. These subunits were then separated by techniques similar, but with slight modifications, to those reported for the pigeon liver enzyme complex (3) and the subunit location of the partial reactions and the acyl binding sites for each fatty acid synthetase were determined. Of special interest in these studies is the yeast fatty acid synthetase complex which has been reported to exist as a complex of molecular weight 2.3 million. This complex has been dissociated into subunits of molecular weight 200,000 to 250,000 by Sumper et al. (4) by freezing and thawing in the presence of high salt concentrations. Similar results were obtained by Schweizer et al. (5). We have found that the yeast complex can also be dissociated into 250,000 molecular weight subunits under the same mild conditions used to dissociate the pigeon liver enzyme complex (6). The subunits are then separated as indicated above. In addition to the above studies we report in this paper on the properties of the subunits on immunodiffusion, their stability in dithiothreitol and P-mercaptoethanol, and their ability to reassociate to enzymatically active fatty acid synthetases. MATERIALS

AND

METHODS

Fleischmann’s dry bakers’ yeast, rats, pigeons, and chickens were purchased locally. Sepharose Eamino-n-caproic acid was prepared according to the method of Larsson and Mosbach (7) and pantethine, obtained in crystalline form from Sigma Chemical Company, was reduced to pantetheine with sodium amalgam prior to use. The 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride used in the preparation of the affinity column gel was obtained from Sigma and the Aldrich Chemical Company. Protein was determined by light absorption at 280 nm and with Folin’s reagent after precipitation with trichloroacetic acid. Acetyl-CoA,:’ malonyl-CoA, and palmitoyl-CoA were obtained from P-L Biochemicals and [l-‘“Clacetyl-CoA, 12-“Clmalonyl-CoA, and [l-‘lClpalmitoyl-CoA were purchased from New England Nuclear. S-acetoacetyl-N-acetyl cysteamine was obtained from Sigma and m-S-P-hydroxybutyryl-l\r-acetyl cysteamine was prepared according to the method of Kumar et al. (8). An additional chromatographic step on silica gel H thin-layer

fatty

“Abbreviations used: CoA, coenzyme A; acid synthetase; DEAE, diethylaminoethyl.

FAS,

SYNTHETASE

COMPLEXES

365

plates with 10% methanol:SO% chloroform as the solvent was used in the purification of the latter compound. S-crotonyl-N-acetyl cysteamine was prepared according to the method of Kumar et al. (81, with an additional purification on a florisil (60-100 mesh) column. Preparation of fatty acid synthetase complexes. Rat liver fatty acid synthetase was prepared as previously described by Burton et al. (9) and chicken liver fatty acid synthetase was prepared according to the method of Yun and Hsu (10). Human liver fatty acid synthetase was prepared by the procedure used routinely for the preparation of pigeon liver enzyme (11). In the final step, however, enzyme in 0.5 M potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, and 10% glycerol was passed through Bio-Gel A-l.5 m. Two separate preparations were made from two entire human livers weighing 3800 and 1450 g, respectively. The 3800-g liver was obtained from a 58-year-old male following death from a heart attack. The specific activity of this preparation was 1132 nmol of NADPH oxidizediminlmg of protein. The 1450-g liver was obtained from a male aged 64 who died of kidney failure. This preparation yielded a specific activity of 480 nmol of NADPH oxidized/min/mg of protein. This enzyme was not used in further studies reported in this paper. The human liver enzyme was stored at -20°C in the presence of 20% glycerol and 10 mM dithiothreitol. Yeast fatty acid synthetase was prepared according to the method of Lynen (121, but with some modifications. Four hundred fifty grams of dried yeast were poured into 675 ml of deionized water. The volume was then brought to 1450 ml and the yeast was allowed to swell for 45 min. Then the volume was brought to 1650 ml with 1 M potassium phosphate buffer, pH 7.0, to bring the ionic concentration to 0.2 M. The cell suspension was kept at O4°C while it was passed through a French press at 2500 psi. All subsequent steps were carried out at O4°C. The cell debris was removed by centrifuging at 10,OOOg for 20 min. The residue was washed in 1 liter of 0.2 M potassium phosphate buffer, pH 7.0, and the wash was combined with the supernatant solution. The combined wash and supernatant solution were brought to 35% saturation with solid ammonium sulfate. After centrifuging at 10,OOOg for 20 min, the supernatant solution was brought to 50% saturation with solid ammonium sulfate. The 35-50% ammonium sulfate residue was dissolved in deionized water to make 163 ml and then dialyzed overnight in 6 liters of 2 mM L-cysteine hydrochloride:2 mM dipotassium phosphate. The dialysate was brought to pH 5.5 and treated with just sufficient calcium phosphate gel (11) to adsorb the enzyme activity. The pH was then adjusted to 6.7 with 0.2 N ammonia and the mixture was stirred for 10 min and centrifuged. The gel was extracted with several 400-ml portions of 66

366

QURESHI

mM potassium phosphate buffer, pH 6.5, and the extracts were combined. The enzyme was concentrated by precipitating between 35 and 50% saturation with solid ammonium sulfate. From this point, the ultracentrifugation procedure of Lynen was followed (12). The final product was stored at -20°C in 10 mM dithiothreito1:O.l M potassium phosphate:1 mM EDTA buffer, pH 7.0. The yeast fatty acid synthetase complex was also prepared, for purposes of comparison, by a method similar to that used in the purification of pigeon liver fatty acid synthetase by Hsu et al. (11). Dry yeast, 24 g, was inoculated into 12 liters of deionized water containing 40 g of Bactopeptone (Difco Laboratories), 12 g of sodium pyrophosphate, 120 g of Bacto yeast extract (Difco Laboratories), and 200 g of dextrose, pH 7.0 (13). The yeast was grown for 12 h at room temperature with aeration of the media. The cells were then harvested in a Sorvall centrifuge at 20,OOOg for 15 min. The cells were resuspended in 0.1 M potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol buffer, pH 7.0, and centrifuged once more. After resuspending in the above buffer, the cells were ruptured by passing through a French press a total of four times at 5000 psi. The temperature of the solution was maintained at 0-4°C during this operation. The cell debris was centrifuged at 20,OOOg for 15 min and then the supernatant solution was centrifuged at 100,OOOg for 1 h. The following purification was performed at room temperature unless indicated otherwise. After centrifugation at lOO,OOOg, a 25-50% ammonium sulfate fraction was prepared from the supernatant solution. The precipitated protein was taken up in 0.1 M potassium phosphate, 1 mM EDTA, 10 mM dithiothreitol, pH 7.0, buffer (120 ml), and frozen at -20°C. After thawing the ionic strength was decreased to 5 mM potassium phosphate, pH 7.0, and 1 mg of calcium phosphate gel (11) was added for every 2 mg of protein determined by the Biuret method. Protein, 1000 mg, was diluted to 50 ml with 5 mM potassium phosphate, pH 7.0. The calcium phosphate gel was added simultaneously with another 200 ml of 5 mM potassium phosphate buffer with stirring. It is crucial that the final protein concentration not exceed 4 mg/ml and that the mixture be centrifuged immediately after the addition of the calcium phosphate gel. Failure in observing these two points resulted in the adsorption of the fatty acid synthetase by the gel. The mixture was centrifuged until a gravitational force of 4000g was attained. The supernatant solution was then poured equally onto two DEAE-cellulose columns, 10 x 3.5 cm, previously washed with 40 mM potassium phosphate, 1 mM EDTA, and 1 mM P-mercaptoethanol, pH 7.0. The columns were then washed with 500 ml of 40 mM potassium phosphate, 1 mM EDTA, 1 mM P-mercaptoethanol, pH 7.0, and then the fatty acid synthetase was eluted with 250 mM potassium phosphate, 1 mM EDTA, 1 mM p-mercaptoethanol, pH

ET

AL.

7.0. The pooled fractions were concentrated by precipitation at 60% saturation with ammonium sulfate. The precipitate was dissolved in 0.2 M potassium phosphate, 1 mM EDTA, 10 mM dithiothreitol, pH 7.0, and dialyzed overnight with the same buffer at 4°C. Yeast fatty acid synthetase was further purified by Bio-Gel A-l.5 m filtration. Elution of the enzyme was effected with 0.2 M potassium phosphate, 1 mM EDTA, 2 mM dithiothreitol, or p-mercaptoethanol buffer, pH 7.0. The specific activity of the fatty acid synthetase was 290 nmol of NADPH oxidizedlminimg of protein. Identical results were obtained in subsequent studies on the separation of subunits with yeast fatty acid synthetase obtained by both methods of purification. Dissociation of fatty acid synthetase complexes. Dissociation of each of the fatty acid synthetase complexes was carried out by dialysis in 5 mM Tris: 35 mM glycine: 1 rnM EDTA: 2 mM P-mercaptoethano1 at WC, pH 8.3, for 4 to 5 h with three changes of buffer. Affinity chromatography. The preparation of the affinity column gel was carried out as previously reported (l-3). +Aminocaproic acid was bound to Sepharose via the c-amino group and pantetheine was then esterified through the thio group to the caproic acid. The subunits of the dissociated complex were then separated by affinity chromatography as reported previously for the pigeon liver enzyme (3) but with modifications reported in the Results section. Assays for overall and partial reactions of fatty acid synthesis. Fatty acid synthetase activity was assayed spectrophotometrically as described by Kumar et al. (6, 8) except that assays on the dissociated fatty acid synthetase complexes were carried out at 5-10°C. Acetyl-CoA:pantetheine transacylase, malonyl-Cokpantetheine transacylase, and palmitoylCoA deacylase activities were assayed as previously described (l-3). Acetyland malonyl-CoA were chromatographed on paper by the isobutyric acid-sodium isobutyrate, pH 4.3, procedure (P-L system No. IV-A) to remove CoA prior to use. P-Ketoacyl thioester reductase was also assayed as described previously (81, except that the level of thioester substrate was 2 mM rather than 10 mM. P-Hydroxybutyryl thioester dehydrase was assayed according to the spectrophotometric method of Kumar et al. (8) at a substrate concentration of 270 mM and a level of protein of approximately 15-30 pg/ml. Crotonyl thioester reductase was assayed spectrophotometrically according to the method of Kumar et al. (8) at a substrate level of 4 mM and a concentration of protein between 15 and 20 pg/ml. Preparation of antisera. Rabbit antisera to DEAE-cellulose-purified pigeon, chicken, human, and rat liver fatty acid synthetases and pigeon liver subunit I were prepared according to the method of Collins et al. (14). Ouchterlony microdouble diffusion with rabbit antiserum and preparations of rat,

SUBUNITS

OF

FATTY

ACID

human, chicken, yeast, and pigeon liver fatty acid synthetases and their corresponding subunits I and II were carried out in 0.5% agarose (14). Binding of acetyl groups. The binding of acetyl groups to protein was carried out on each fatty acid synthetase complex before and after dissociation according to the method of Kumar et al. (6) except that acetyl binding to the dissociated complexes was effected in the dissociation buffer (5 mM Tris:35 mM glycine:l mM EDTA:2 mM p-mercaptoethanol). RESULTS

Dissociation of Rat, Human, and Chicken Liver and Yeast Fatty Acid Synthetases High-voltage electrophoresis patterns of the [l-14Clacetyl-labeled peptides resulting

M!

FIG. 1. Covalent

DISTANCE

SYNTHETASE

COMPLEXES

367

from pepsin digestion of labeled fatty acid synthetase complexes (rat, human, and chicken liver and yeast enzymes) before (A, B, C, D) and after dissociation (A’, B’, C’, D’) are shown in Fig. 1. The complexes were labeled with [l-‘4Clacetyl groups as reported in the Methods section prior to pepsin digestion. [l-14ClAcetyl groups were bound to the three sites, AZ, B,, and BZ, corresponding to 4’-phosphopantetheine, serine, and cysteine, respectively, in the intact complex, whereas the nonidentical half-molecular weight subunits (A’, B’, C’, and D’ of Fig. 1) bound acetyl groups at the A, and B, sites but not at the B, (cysteine) site. Previously, Kumar et al.

FROM

ORIGIN

IN Cm

binding of the acetyl group from [l-Wlacetyl-CoA to undissociated (A) rat, (B) human, (C) chicken, and (D) yeast fatty acid synthetases and Tris-glycine-EDTA dissociated (A’) rat, (B’) human, (C’) chicken, and CD’) yeast fatty acid synthetases at 0°C and pH 8.3. These figures show the electrophoretic patterns of the radioactive peptides obtained from the peptic digests of acid-precipitated proteins containing covalently bound [‘Clacetyl groups. A,, B,, and B, represent the peptides obtained from the regions around the three binding sites of fatty acid synthetase: A,, 4’-phosphopantetheine; B, , hydroxyl; and B, , cysteine. Other details are described in (6, 8).

FIG. 2. Sucrose density gradient centrifugation of each of the fatty acid synthetase complexes, (A) rat, (B) human, (C) chicken, and (D) yeast, at 20°C in a Spinco Model L3-50 ultracentrifuge SW27 rotor at 58,000g. The sucrose gradient was 5 to 20% (w/v) containing (A) 0.5 M potassium phosphate, 1 mM EDTA, 2 mM fi-mercaptoethanol, pH 7.0, and it was centrifuged for 40 h; (B) 0.5 M potassium phosphate, 1 mM EDTA, 2 mM p-mercaptoethanol, 5% glycerol, pH 7.0, and centrifuged for 40 h; (C) 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 2 mM P-mercaptoethanol, 5% glycerol, centrifuged for 30 h; and (D) 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM P-mercaptoethanol, and centrifuged for 32 h. The dissociated fatty acid synthetase complexes, (A’) rat, (B’) human, (C!‘) chicken, and CD’) yeast, were subjected to a 5 to 20% (w/v) sucrose gradient at 4°C and 58,OOOg. The gradients contained (A’! 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 1 rnM P-mercaptoethanol; (B’) 0.2 M potassium phosphate, 1 mM EDTA, 1 mM P-mercaptoethanol, pH 7.0, 10% glycerol; CC’) 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 2 mM @mercaptoethanol, 5% glycerol; (D’) 0.1 M potassium phosphate, pH 7.0, 1 mM EDTA, 5 mM P-mercaptoethanol. (A’) was centrifuged for 40 h, (B’) for 40 h, (C’) for 30 h, and (D’) for 32 h. One and one-half to two milligrams of protein were used in each of the gradients. Fraction 0 is the bottom and fraction 40 is the top of the gradient. Light absorption at 280 nm (C-O); fatty acid synthetase activity in units/ml x lo-* (0-O); oketoacyl thioester reductase activity in units/ml x 10m2 (A--A). A unit is defined as 1 nmol of NADPH oxidized per minute. 368

SUBUNITS

OF FATTY

ACID

(6, 8) showed that acetyl groups are bound to the B, (cysteine) site of pigeon liver fatty acid synthetase only when the subunits are associated as a complex. Therefore, the above results indicate that the complexes were completely or nearly completely dissociated by 4-h dialysis in Trisglycine-EDTA-&mercaptoethanol at 0°C. Further evidence of the dissociation of the fatty acid synthetase complexes in the presence of Tris-glycine-EDTA buffer was obtained when each was subjected to sucrose density gradient centrifugation (Fig. 2). Only 14s peaks of protein were present when freshly prepared (A) rat, (B) human, and (C) chicken liver fatty acid synthetases were centrifuged. In the case of (D), the yeast fatty acid synthetase, two species active for fatty acid synthesis were found. One form was the 2.3 x lo6 molecular weight complex (activity not shown in Fig. 2D) and the second was a 14s species. Only t’he 14s species was used for further studies. Centrifugation of the Tris-glycineEDTA-treated complexes of (A’) rat, (B’) human, (C’) chicken, and (D’) yeast fatty acid synthetases showed the presence of mainly a 9s peak, thereby providing additional evidence that each complex is almost completely dissociated into the two subunits under the conditions used. Separation of Subunits Fatty Acid Synthetase

I and II of Each

Glass-jacketed columns of 21 cm x 8.5 mm were packed with 4-6 g of Sepharose Eaminocaproyl pantetheine. Dissociated fatty acid synthetase, 4 mg of protein per each gram of Sepharose l -aminocaproyl pantetheine, was added to the column and the binding of the subunits was carried out as previously reported (l-3). Each enzyme was stored at -20°C for 2-3 weeks prior to dissociation in Tris-glycine-EDTA buffer. The conditions for the separation of the subunits were modified somewhat from that of subunits of pigeon liver fatty acid synthetase (l-3). In all cases, subunit I (reductase) was eluted at 0°C and subunit II (transacylase) was eluted at 25°C. For rat (Fig. 3A), subunit I was eluted with 0.2 M Tris-0.2 M potassium phosphate, 2 mM P-mercaptoethanol, pH 8.4; subunit II

SYNTHETASE

COMPLEXES

369

(transacylase) was eluted when the above buffer was raised to pH 10 by the addition of ammonia. The human fatty acid synthetase subunits (Fig. 3B) were eluted from the column under essentially the same conditions as used for the rat enzyme except that the elution buffer contained 5% glycerol. Subunit I of chicken liver fatty acid synthetase was eluted with 0.1 M Tris-0.1 M potassium phosphate, 2 mM pmercaptoethanol, 5% glycerol, pH 8.4, while subunit II was eluted with 0.1 M Tris-0.1 M potassium phosphate, 2 mM pmercaptoethanol, 5% glycerol, pH 10.0 (Fig. 30. Yeast subunit I (Fig. 3D) was eluted with 0.1 M Tris-0.1 M potassium phosphate, 2 mM ,&mercaptoethanol, pH 8.4, and subunit II was eluted with the same buffer with the pH adjusted to 10 by the addition of ammonia. When subunit II was eluted from the column, 100 ~1 of 2 M potassium monophosphate was added to each collection tube containing rat or human transacylase subunits and 100 ~1 of 1 M potassium monophosphate was added to each collection tube containing subunit II from chicken or yeast enzyme. The addition of potassium monophosphate served to lower the pH from 10.0 to 7.5 when l-ml fractions were collected. It is evident (Fig. 3) that subunits I and II of each dissociated fatty acid synthetase were separated on affinity chromatography. There was a small amount of contamination of each subunit by the other after carrying out the affinity separation, as had been observed in previous studies with pigeon liver fatty acid synthetase subunits.4 Therefore, the purification of each 4 The present and previous studies (1, 3) clearly demonstrate the separation of two nonidentical halfmolecular weight subunits from fatty acid synthetase complexes of mammalian and avian liver and yeast on affinity gel Sepharose c-aminocaproyl pantetheine. A further study of this procedure is presently being carried out with the aim of eliminating the l&20% contamination of each subunit by the other. If we are successful, the need for the sucrose density gradient centrifugation step will be eliminated. Also, some modifications of the method are being tested in an effort to increase the yield of the transacylase (subunit II). Finally, modifications are also being tested which may improve the reproducibility of the subunit separation and minimize the

370

QURESHI

ET

AL.

FIG. 3. Separation of the nonidentical half-molecular weight subunits of (A) rat, (B) human, (Cl chicken, and (D) yeast fatty acid synthetases. The details of loading and elution are given in the Results section. Light absorption at 280 nm (O-01; the actual amount of pketoacyl thioester reductase (A-A) or acetyl-CoA:pantetheine transacylase (0-O) present, in milligrams of enzyme protein in each fraction, based on enzymatic activity versus the activity found in dissociated fatty acid synthetase. One milligram of P-ketoacyl thioester reductase activity represents 140 nmol of NADPH oxidized/min/ml, and 1 mg of acetylCoA:transacylase activity represents 340 nmol of product formedlminiml.

subunit was effected by the methods of reassociation of unlike subunits and subsequent sucrose density gradient centrifugation (l-3). In this purification step, p-mercaptoethanol was added to subunit I and dithiothreitol was added to subunit II as reported previously for the pigeon liver fatty acid synthetase subunits (3). The profiles of the separation of each subunit from small amounts of contaminating subunit on sucrose density gradients are shown in Fig. 4. It should be noted (see enzyme acneed for the stringent controls on flow rate, temperature, preparation of gel, and prior handling of enzyme preparation that are now required.

tivities) that in each centrifugation the major subunit occurs in excess of the minor subunit in the 14s peak. In the centrifugation of subunit I in each case there was an excess of this subunit (P-ketoacyl thioester reductase activity) over subunit II, and in the centrifugation of subunit II there was an excess of this protein (acetyland malonyl-CoA transacylase activities) over subunit I in the 14s peak, even though the amount of I and II should be equal in the 14s complex. This indicates that the 9s subunits are capable, to some extent, of forming 14s complexes with themselves under these conditions. As previously noted with pigeon liver fatty acid

SUBUNITS

OF

FATTY

ACID

SYNTHETASE

COMPLEXES

FIG. 4. Purification of subunits I and II by sucrose density gradient centrifugation after separation by chromatography on Sepharose-•-aminocaproyl pantetheine. The conditions for the reassociation with the contaminating subunit were as follows: (A) rat subunit I, 0.5 M potassium phosphate, pH 7.0, 1 mM EDTA, and 10 mM dithiothreitol; (B) human subunit I, 0.5 M potassium phosphate, pH 7.0, 1 mM EDTA, 10 mM dithiothreitol, and 5% glycerol; (C) chicken subunit I, 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 10 mM dithiothreitol, and 5% glycerol; (D) yeast subunit I, 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, and 10 mM dithiothreitol. (A’) rat subunit II, (B’) human subunit II, CC!‘) chicken subunit II, and CD’) yeast subunit II were reassociated as described above for their respective subunits I, except that pmercaptoethanol was used instead of dithiothreitol. Reassociation was carried out by dialyzing at room temperature for 2 h in the respective buffer. Two buffer changes were made before subjecting the reassociated enzyme complexes to sucrose density gradient centrifugation. Sucrose density gradient centrifugation was the same as that reported for the fatty acid synthetase complexes in Fig. 2 except that the temperature of the run was 10°C instead of 20°C.

372

QURESHI

synthetase subunits (31, this phenomenon was not observed at low protein concentrations. Reassociation of the Subunits after Separation by Affinity Chromatography

ET AL.

thiothreitol, pH 7.0, 75% of the activity of the control was recovered. In the case of human liver fatty acid synthetase subunits, a recovery of 65% of the activity of the control was achieved when reassociation was carried out with 0.5 M potassium phosphate, 1 mM EDTA, 10 mM dithiothreitol, pH 7.0, and 16-20% glycerol.

Table I shows the recovery of activity for each fatty acid synthetase after reassociation of the separated subunits. Rat liver subunits I and II were reassociated in 0.5 Disc Gel Electrophoresis of Fatty Acid Synthetase Complexes and Their SubM potassium phosphate, 1 mM EDTA, 10 units mM dithiothreitol, pH 7.0, at room temperDisc gel electrophoresis was carried out ature, and chicken liver subunits I and II were reassociated in 0.2 M potassium phos- on the native fatty acid synthetase comphate, 1 mM EDTA, 10 mM dithiothreitol, plexes, subunits I and II, and reconstituted pH 7.0, containing 10% glycerol. A 75 to fatty acid synthetase complexes according to the method of Hedrick and Smith (15). A 85% recovery of overall fatty acid synthesingle major band was obtained in each tase activity was achieved on recombination of rat and chicken liver subunits into case for the separated subunits I and II their respective complexes. When the (Fig. 5). However, the variations in ionic yeast fatty acid synthetase subunits I and strength and glycerol in the samples reII were dialyzed together in 0.2 M potas- sulted in gross differences in migration sium phosphate, 1 mM EDTA, 10 mM di- distances of the bromphenol blue marker

a

bc

d

e

f

g

h

FIG. 5. Disc gel electrophoresis of subunits I (reductase) and II (transacylase) of different fatty acid synthetases. (a) Pigeon liver, 100 Kg of subunit I, (bl100 pg of subunit II; Cc) rat liver, 80 wg of subunit I, (d) 80 pg of subunit II; (e) human liver, 50 pg of subunit I, (D 50 pg of subunit II; (g) yeast, 50 Kg of subunit I; and (h) 50 Kg of subunit II. Samples in (a) and (b) were loaded onto the gel in 0.2 M potassium phosphate, 1 mM EDTA, pH 7.0; in (c) and (d) in 0.5 M potassium phosphate, 1 mM EDTA, pH 7.0; in (e) and (0 in the same buffer as in Cc) and Cd) plus 20% glycerol; and in (g) and (h) in the same buffer as (a) and (b). Electrophoresis was carried out for 4-5 h at a current of 3mA/gel. Bromphenol blue was used as a marker.

SUBUNITS

OF

FATTY

ACID

and each of the proteins. The patterns obtained were identical to those reported for pigeon liver fatty acid synthetase subunits in a previous paper (3). Immunodiffusion of the Fatty thetases and Their Subunits

Acid

Syn-

Subunits I and II of each fatty acid synthetase cross-reacted with the antiserum to the respective complex, indicating that there are antigenic sites on each half of the complex. Each of the five fatty acid synthetases and their subunits was also tested for cross-reaction with each of the rabbit antisera prepared against pigeon, chicken,

SYNTHETASE

rat, and human liver fatty acid synthetases and pigeon liver subunit I (Table II). Only pigeon and chicken liver fatty acid synthetases and subunits and their antisera and rat and human liver fatty acid synthetases and subunits and their antisera cross-reacted (Fig. 6). Yeast fatty acid synthetase did not cross-react with any of the antisera tested. Antisera prepared to pigeon liver subunit I cross-reacted with fatty acid synthetase complexes from pigeon and chicken. Pigeon liver subunit I antisera also cross-reacted with subunit II from pigeon and chicken (Fig. 6B). All reactions gave sharp single bands.

TABLE FATTY

ACID

SYNTHETASE SUBUNITS

Protein

ACTIVITIES IN THE I AND II OF ENZYME

I

UNDISSOCIATED COMPLEX AND OF AVIAN AND MAMMALIAN

sample

Freshly Subunit Subunit

prepared I II

fatty

Control Mixture

fatty acid synthetase of subunits I and II

NADPH

acid synthetase

373

COMPLEXES

IN SEPARATED AND LIVER AND YEAST”

oxidation

RECOMBINED

(nmol/min/mg

Pigeon”

Chicken

Rat

Human

Yeast

1220 -

568 -

1029 -

1132 -

818 -

980 780

372 278

607 518

582 444

362 232

(’ Subunits I and II were combined in the range of 0.4 to 0.5 mg of protein of each subunit and then dialyzed at room temperature for 3-5 h, with a change of buffer after every hour, in 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 10 mM dithiothreitol; 0.2 M potassium phosphate pH 7.0, 1 mM EDTA, 10 mM dithiothreitol, 10% glycerol; 0.5 M potassium phosphate, pH 7.0,1 mM EDTA, 10 mM dithiothreitol; 0.5 M potassium phosphate, pH 7.0, 1 mM EDTA, 10 mM dithiothreitol, 20% glycerol; and 0.2 M potassium phosphate, pH 7.0, 1 mM EDTA, 20 mM dithiothreitol, respectively, for pigeon, chicken, rat, and human liver and yeast enzyme. b These values are from a previous paper (3).

TABLE OUCHTERLONY

Fatty

acid

DOUBLE-DIFFUSION

synthetase

Antisera ____-

Pigeon Chicken Rat Human Yeast Pigeon subunit I OR II Chicken subunit I or II Rat subunit I or II Human subunit I or II Yeast subunit I or II ” + indicates

II

ANALYSIS OF RAT, HUMAN, CHICKEN, FATTY ACID SYNTHETASES

cross-reaction

Pigeon _____+ ‘J + 0 0 0 + + 0 0 0 and 0 indicates

Chicken + + ‘0 0 0 + + 0 0 0 the absence

to fatty

acid

AND

PIGEON

LIVER

AND

YEAST

synthetases

Rat

Human

0 0 + + 0 0 0 + + 0

0 0 + + 0 0 0 + + 0

of cross-reaction.

Pigeon

subunit + + 0 0 0 + + 0 0 0

I

374

QURESHI

ET AL.

3-

-2

3-

3-

-2

3I I

I FIG. 6. Immunodiffusion of pigeon and human liver fatty acid synthetases and subunits I and II. The center wells contained (A) antisera to DEAE-cellulose-purified pigeon liver fatty acid synthetase and (B) antisera to pigeon liver fatty acid synthetase subunit I. Wells marked 1, 2, and 3 contained pigeon liver fatty acid synthetase prepared by Bio-Gel filtration and pigeon liver fatty acid synthetase subunits I and II, respectively. The center wells in (C) and (D) contained antisera to DEAE-cellulose-purified human liver fatty acid synthetase. Wells marked 1, 2, and 3 in (C) contained Bio-Gel-purified human liver fatty acid synthetase and human liver fatty acid synthetase subunits I and II, respectively. In (D), wells marked 1 and 3 contained human and pigeon liver fatty acid synthetases purified by Bio-Gel filtration, and well 2 contained DEAE-cellulose-purified rat liver fatty acid synthetase.

Partial Reactions of Separated Subunits The specific activities of the partial reactions obtained with purified subunits I and II and the dissociated and undissociated fatty acid synthetase complexes are reported in Table III. p-Ketoacyl thioester

reductase and crotonyl thioester reductase activities were found only in subunit I, while acetyl-CoA:pantetheine and malonyl-CoA:pantetheine transacylase activities were found only in subunit II, as found previously with the pigeon liver fatty acid synthetase. The other partial reactions, p-

SUBUNITS

OF

FATTY

ACID

SYNTHETASE

TABLE PARTIAL

Enzyme source

Fatty acid synthetase

AcetylCoA transacylase

REACTIONS

III

OF FATTY

MalonylCoA transacylase

ACID

P-Ketoacyl thioester synthetase

(nmol of product Undissociated FAS Pigeon liver Chicken liver Rat liver Human liver Yeast Dissociated FAS Pigeon liver Chicken liver Rat liver Human liver Yeast Subunit I Pigeon liver Chicken liver Rat liver Human liver Yeast Subunit II Pigeon liver Chicken liver Rat liver Human liver Yeast

375

COMPLEXES

SYNTHETASE”

P-Ketoacyl thioester reductase

P-Hydroxyacyl thioester dehydrase

formedlminimg

of protein)

Enoylacyl thioester reductase

PalmitoylCoA deacylase

1220 568 1029 1132 818

370 228 590 246 196

201 176 222 192 132

4.7 1.7 3.9 1.2 0.4

130 118 248 108 84

466 263 576 202 327

120 168 237 124 148

46 32 39 36 44

-0 -

170 160 297 136 83

100 99 109 87 56

-

70 57 157 52 27

265 104 311 175 279

120 88 167 100 110

18 12 18 21 19

-

-

-

-

156 103 311 99 51

270 178 282 158 234

246 159 370 214 196

16 13 17 23 23

-

350 204 577 262 147

210 146 215 129 92

-

-

273 162 260 164 241

-

16 12 19 22 20

0 The conditions of assay for each partial reaction are reported h Fatty acid synthetase assay was carried out at 5-10°C using and 100 pM NADPH; lo-15 pg of protein was used per assay.

hydroxybutyryl thioester dehydrase and palmitoyl-CoA deacylase, were found in both subunits. The highest specific activities for crotonyl thioester reductase and for the dehydrase are obtained only with freshly prepared nn-S-P-hydroxybutyrylN-acetyl cysteamine and with S-crotonylN-acetyl cysteamine purified by thin-layer chromatography before use (3). The specific activity obtained for P-ketoacyl thioester reductase was similar to that previously reported for the pigeon liver enzyme. The specific activities for p-ketoacyl and crotonyl thioester reductases, the transacylases, and palmitoyl-CoA deacylase in the separated subunits were those expected from the specific activities found for the dissociated fatty acid synthetase. Thus, P-ketoacyl thioester reductase and

in the Methods 33 PM acetyl-CoA,

section. 100 PM malonyl-CoA,

crotonyl thioester reductase and transacylase specific activities in the individual subunits were twice the specific activities found in the equimolar mixture of the dissociated complex. Palmitoyl-CoA deacylase and P-hydroxybutyryl thioester dehydrase activities were the same in subunits I and II and in the complexes. DISCUSSION

It is of interest to note that the procedures for preparing the various fatty acid synthetase complexes do not vary much from one another except for the yeast enzyme, which is precipitated with ammonium sulfate in the absence of EDTA at pH 5.5 (12). However, the yeast fatty acid synthetase can also be prepared by the method used for pigeon liver fatty acid

316

QURESHI

synthetase (11). The specific activities of yeast fatty acid synthetase were found to vary from 2115 down to 240 nmol of NADPH oxidized/min/mg of protein for overall fatty acid synthesis. Such a variation in specific activity for the yeast enzyme was also found by Lynen (12). The absence of binding of acetyl groups to the cysteine site in each Tris-glycineEDTA treated fatty acid synthetase complex (Fig. 1) is evidence for the dissociation of each enzyme complex into subunits I and II. This is true because Kumar et al. (6,8) showed previously that acetyl groups are bound to the cysteine site only when the subunits are associated in the fatty acid synthetase complex. In addition, we showed in a previous paper (3) that the cysteine site is on subunit II, while the 4’phosphopantetheine site, from which the acetyl group is transferred to the cysteine site, is on subunit I. This was shown through incubation of subunit II with [l14C]acetyl-labeled acyl carrier protein (3). Further proof of dissociation of the fatty acid synthetases was obtained through sucrose density gradient centrifugation of the Tris-glycine-EDTA-treated enzyme complexes. The conditions under which the fatty acid synthetases are completely dissociated into half-molecular weight subunits Thus, dissociation is are identical. achieved in Tris:glycine:EDTA:p-mercaptoethanol or dithiothreitol (5:35:1:2 mM) at 0°C (ice bath) for 4 h. This finding is important because the fatty acid synthetase must be quantitatively dissociated before affinity column separation of the subunits can be achieved. However, if dialysis is carried out for a long period of time [36 h at 0°C (ice bath)], the subunits cannot be eluted from the column. It has also been found that freezing at -20°C for several days or more prior to dissociation is necessary for each of the fatty acid synthetases, if separation by affinity chromatography is to be successful. This suggests that all of the fatty acid synthetases have similar conformational requirements for binding to the affinity column. Hence the conditions for loading the column (0°C and a flow rate of 4 ml/h) were the same as reported for the pigeon liver enzyme.

ET AL.

The buffer concentrations used to elute subunits on afinity chromatography and for sucrose density gradient centrifugation for the different fatty acid synthetases are related to the ionic strength requirements for stability of these complexes. These are much higher for the rat and human liver fatty acid synthetases, 0.5 M, than they are for the pigeon and chicken liver enzymes, 0.2 M, potassium phosphate. These ionic strengths apply to both the complex formed from the half-molecular weight subunits and to the enzyme activities in the subunits. In addition, stability of the human liver complex and its subunits requires 10% glycerol (16). In the absence of glycerol, the human liver fatty acid synthetase is inactivated in 2 h. However, all of the enzymes are similar in their requirement for a high salt concentration and a pH of 7.0 for stability and for reassociation and reconstitution of activity. The requirement of glycerol for stability of the human and chicken liver fatty acid synthetases may be related to its effect on the reassociation of pigeon liver fatty acid synthetase subunits at 0°C in 0.1 M KC1 (17). The fatty acid synthetases are identical with respect to the location of partial enzyme activities in subunits I and II. The location of these enzyme activities is reported in the schematic diagram (Fig. 7). It should be noted that the condensation reaction only occurs when both subunits are associated as a complex or when acyl carrier protein is associated with subunit II. The largest deviation occurring in the specific activity of a partial reaction of different complexes was generally not more than a factor of 2, and in mos,t instances the difference was much less. The results of the double-diffusion immunoprecipitation experiments suggest common amino acid sequences or similar native three-dimensional structures for the two mammalian and for the two avian enzymes. The yeast enzyme, on the other hand, reacts with none of the liver enzyme antibodies, even though its general architecture and behavior appear more similar to the avian and mammalian complexes than expected. The cross-reaction between antiserum to pigeon liver subunit I and pigeon and chicken liver subunit II indi-

SUBUNITS

OF

FATTY

ACID

(8,) SlTE

SITE

SITE

FIG. 7. Schematic diagram of the partial enzyme activities found in each subunit of pigeon, rat, human, and chicken liver and yeast fatty acid synthetases.

cates that both subunits comprising the fatty acid synthetase complex have some antigenic determinants in common. This is not surprising, for both subunits possess palmitoyl-CoA deacylase and p-hydroxybutyryl thioester dehydrase activities. The genetic studies on yeast fatty acid synthetase carried out by Schweizer and co-workers (5, M-20) suggest that two complex unlinked gene loci code for the polypeptides comprising the fatty acid synthetase multienzyme complex. Schweizer and co-workers favor the hypothesis that the fatty acid synthetase complex is composed of only two multifunctional polypeptide chains coded for by the two gene loci (21, 22). The two reductase activities have been located on separate gene loci, enoyl reductase is on fas 1 and /3-ketoacyl reductase is on fas 2 (5, 18). However, the data presented in this paper show that both of the reductase activities are on subunit I. This finding is not in agreement with Schweizer’s hvnothesis. -died --I which would re-

SYNTHETASE

377

COMPLEXES

quire the two reductase activities to be on different subunits. Therefore, in order to have both reductases on the same subunit and assuming that two multifunctional polypeptide chains are translated from fatty acid synthetase mRNA, these polypeptide chains would have to be posttranslationally cleaved to give individual component enzymes which then associate to form the fatty acid synthetase complex. Possibly, the two multifunctional polypeptides remain in their uncleaved form until they find each other and associate to form some specific intermediate structure. The polypeptide chains would then be in the correct conformation for specific proteolytic cleavage into individual enzyme components. These enzyme components would then assemble into the subunits making up the fatty acid synthetase complex. Similar mechanisms have been proposed by Hershko and Fry (23) for structural proteins. A further indication of homology in the assembly of the fatty acid synthetase complexes is indicated in the reported isolation of acyl carrier protein from subunit I of both pigeon liver (24) and rat liver (25) fatty acid synthetases. Since these reports, we have also isolated acyl carrier protein from subunit I of human and chicken liver and yeast fatty acid synthetases (26). This work is reported in the accompanying paper. The separation of the subunits by the afinity chromatography step should permit the ultimate separation of the individual proteins in subunits I and II that are responsible for catalyzing the different reactions of fatty acid synthesis. ACKNOWLEDGMENTS We wish to thank Gino Saccone for their of the yeast enzyme.

Drs. Bernard Maudinas and assistance in the preparation

REFERENCES 1. LORNITZO,

F. A.,

QURESHI,

A.

A.,

AND

PORTER,

J. W. (19741 J. Biol. Chem. 249, 1654-1656. 2. LORNITZO, F. A., QURESHI, A. A., AND PORTER,

J. W. (1974)

Fed.

Proc.

33, 1506. Abstract.

3. LORNITZO, F. A., QURESHI, A. A., AND PORTER, J. W. (1975) J. Biol. Chem. 250, 4520-4529. 4. SUMPER, M., RIEPERTINGER, C., AND LYNEN: F.

(1969) 5. SCHWEIZER,

FEBS

Lett. 5, 45-49. E., KNEIP, B., CASTORPH,

H.,

AND

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6. 7. 8.

9.

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13. 14.

15. 16. 17.

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HOLZNER, U. (1973) Eur. J. Biochem. 39, 353362. KUMAR, S., MUESING, R. A., AND PORTER, J. W. (1972) J. Biol. Chem. 247, 4749-4752. LARSSON, P., AND MOSBACH, H. (1971) Biotech. Bioeng. 13, 393-398. KUMAR, S., DORSEY, J. A., MUESING, R. A., AND PORTER, J. W. (1970) J. Biol. Chem. 245,47324744. BURTON, D. N., HAAVIK, A. G., AND PORTER, J. W. (1968) Arch. Biochem. Biophys. 126, 141154. YUN, S., AND Hsu, R. Y. (1972) J. Biol. Chem. 247, 2689-2698. Hsu, R. Y., WASSON, G., AND PORTER, J. W. (1965) J. Biol. Chem. 240, 3736-3746. LYNEN, F. (1969) in Methods in Enzymology (Lowenstein, J. M., ed.), Vol. 14, pp. 17-33, Academic Press, New York. QURESHI, A. A., BEYTIA, E., AND PORTER, J. W. (1973) J. Biol. Chem. 248, 1848-1855. COLLINS, J. M., CRAIG, M. C., NEPOKROEFF, C. M., KENNAN, A. L., AND PORTER, J. W. (1971) Arch. Biochem. Biophys. 143, 343-353. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. RONCARI, D. A. K. (1974)Canad. J. Biochem. 52, 221-240. MUESING, R. A., LORINTZO, F. A., KUMAR, S.,

ET

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25.

26.

AL. AND PORTER, J. W. (1975) J. Biol. Chem. 250, 1814-1823. K~~HN, L., CASTORPH, H., AND SCHWEIZER, E. (1972) EUF. J. Biochem. 24, 492-497. TAURO, P., HOLZNER, U., CASTORPH, H., HILL, F., AND SCHWEIZER, E. (1974) Molec. Gen. Genet. 129, 131-148. SCHWEIZER, E., KUHN, L., AND CASTORPH, H. (1971) Hoppe-Seyler’s Z. Physiol. Chem. 352, 377-384. SCHWEIZER, E. (1973) Angew. Chem. Internat. Edit. 12, 341-342. SCHWEIZER, E., HOLZNER, U., MEYER, K. H., TAURO, P., AND SCHWEIZER, M. (1974) in Comparative Biochemistry and Physiology of Transport (Bolis, L., Bloch, K., Luria, S. E., and Lynen, F., eds.), pp. 219-244, North-Holland, Amsterdam. HERSHKO, A., AND FRY, M. (1975) Ann. Rev. Biochem. 44, 775-797. QURESHI, A. A., LORNITZO, F. A., AND PORTER, J. W., 168th Ann. Meet., Amer. Chem. Sot., Div. Biol. Chem., 1974, No. 82. QURESHI, A. A., LORNITZO, F. A., AND PORTER, J. W. (1974)Biochem. Biophys. Res. Commun. 60: 158-165. QURESHI, A. A., LORNITZO, F. A., Hsu, R. Y., AND PORTER, J. W. (1976) Arch. Biochem. Biophys. 177, 379-393.