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Comparative studies of the rat and pigeon liver fatty acid synthetases
ARCHIVES
OF
BIOCHEMISTRY
AND
Comparative
Studies Fatty
DAVID Lipid
N. BURTON,
Metabolism Physiological
126,
BIOPHYSICS
Laboratory, Chemistry,
141-154
of the Acid
ARNE Veterans University Received
(1968)
Rat
and
Pigeon
Liver
Synthetases’
G. HAAVIK, Administration of Wisconsin, January
AND
JOHN
Hospital Madison,
W. PORTER
and the Department Wisconsin 63705
of
2, 1968
The fatty acid synthetase of rat liver has been prepared in an essentially homogenous state by a similar, but nonidentical, procedure to that used for the purification of the pigeon liver fatty acid synthetase. Both of these complexes synthesize free palmitic acid from acetyland malonyl-CoA in the presence of NADPH. Enzymic activities for the decarboxylation of malonyl-CoA and the synthesis of HMG-CoA and triacetic acid lactone are also associated with both fatty acid synthetases. The properties of the rat liver fatty acid synthetase have been investigated, and compared to those of the pigeon liver enzyme. Close similarities were observed in molecular weight, thiol dependency, binding stoichiometry for acetyland malonylCoA, 4’-phosphopantetheine content, sulfhydryl and flavin content, inhibition by sulfhydryl binding reagents, pH optimum and amino acid composition. It is suggested, therefore, that the complexes have very similar structures. However, marked differences in the ease of dissociation in phosphate buffers below 0.5 M were observed, together with differences in stability of the enzymes on electrophoresis and DEAEcellulose chromatography. These results indicate that some differences exist in the binding of subunits in the two complexes.
Homogenous enzyme complexes synthesizing long-chain fatty acids have been isolated from yeast (1) and pigeon liver (2). Hsu et al. of this laboratory (2) prepared the soluble fatty acid synthetase of pigeon liver in a homogenous form with high specific act,ivity. This enzyme converts acetylCoA and malonyl-CoA, in the presence of NADPH, to palmitic acid with an overall stoichiometry of 1 acetyl-CoA + 7 malonylCoA + 14 Hf + 14 NADPH + 1 palmitic acid + 7 CoA + 7 CO2 + 6 HzO. The purified enzyme complex also contains activities for malonyl-CoA decarboxylase and the synthesis of P-hydroxy, P-methyl glutaryl CoA and 3,5diketohexanoic acid.
The purified enzyme has a molecular weight of 4.5 X lo5 by sedimentation-equilibrium analysis (3-5) and it contains 64-66 moles of thiol per mole of enzyme (6). Stability of the enzymic activity is dependent on maintenance of thiol reduction. On thiol oxidation enzymic activity is lost and the formation of half molecular weight subunits occurs (4, 5). Recent investigations in this laboratory on the content of the pigeon liver complex in various nutritional states (7) and the effect of experimental diabetes on enzyme activity for the synthesis of fatty acids by t,he rat liver enzyme2 have led to studies on the isolation of the rat liver enzyme complex. This complex has been isolated in an essentially homogenous and highly active state. The results of investigations on the properties of the rat liver enzyme complex
1 This investigation was supported in part by research grants, A-1383 from the National Instit,ute of Arthritis and Metabolic Diseases of the Nat,ional Institutes of Health, United States Public Health Service and No. 66-725 from the American Heart Association.
2 Dahlen, J., Kennan, Arch. Biochem. Biophys. 141
A. H., l&51
and Porter, (1968).
J. W.,
BURTON,
142
HAAVIK,
and a comparison of these properties to those of the pigeon liver enzyme are also reported in this paper. From these data we conclude that there is a striking similarity between the two complexes in overall physical and enzymic properties. There appear, however, to be marked differences in the stability of the complexes on protein
dilution and on decrease of ionic strength. These results suggestdifferences in the forces maintaining the integrity of the two complexes. EXPERIMENTAL
METHODS
Materials. Experimental materials were obtained from the following sources: I-“C-acetic anhydride and 2-l*C-malonic acid from New England Nuclear Corporation; NADPH and ATP from Sigma Chemical Company; S-Palmityl-CoA and CoA from P-L Biochemicals; 2-mercaptoethanol and dithiothreitol from Calbiochem; hydrolyzed starch from the Connaught Laboratories, Toronto; and Sephadex from the Pharmacia Company, Uppsala, Sweden. All reagents used were of analytical grade. Preparation of coenzyme-A esters. Nonradioactive acetyl-CoA and 1-I%-acetyl-CoA were prepared by the method of Simon and Shemin (8) and nonlabeled and 2-14C-malonyl-CoA were synthesized as described by Trams and Brady (9). The CoA esters were purified by the paper chromatographic systems of Brodie and Porter (10). Bu$ers. All buffers contained 1 mM EDTA and either 1 mM dithiothreitol or 1 mM &mercaptoethanol, unless otherwise specified. Assay for protein. Assays for the protein concentration of solutions were carried out by the method of Gornall, Bardawill, and David (11). Assay for fatty acid synthetase activity. The assay system used was that of Hsu, Wasson and Porter (2), as modified by Butterworth et al. (7). Specific activity of the enzyme is expressed as m#moles of lJ”C-acetyl-CcA incorporated into fatty acids per minute per milligram of protein. Determination of radioactivity. All measurements of radioactivity were obtained with a Packard liquid scintillation spectrometer. Preparation of liver supernatant solutions. Rat and pigeon liver supernatant solutions were prepared by the method of Wakil, Porter, and Gibson (12) as modified by Hsu, Wasson, and Porter (2). Male rats weighing about 150 g each were starved 43 hours, then fed 48 hours on a fat-free diet (13) before they were killed by decapitation. Both rat and pigeon liver supernatant solutions were stored at - 15” under nitrogen.
AND PORTER Puri$cation of fatty acid synthetase. Rat liver fatty acid synthetase was prepared by a procedure similar to that used for pigeon liver fatty acid synthetase by Hsu, Wasson, and Porter (2). HOWever, several important modifications were used in the preparation of the rat liver enzyme. First ammonium sulfate fractionation. Frozen rat liver supernatant solution (40 ml) was allowed to thaw at room temperature, and then stirred gently under a stream of nitrogen. Saturated ammonium sulfate solution, pH 7, containing 3 mM EDTA and 1 mM &mercaptoethanol was added to a saturation of 20%. After stirring for 15 minutes, the mixture was centrifuged and the precipitate discarded (RI). The supernatant solution was brought to 33% of saturation in ammonium sulfate. The precipitated protein was collected by centrifugation and retained (Rt) for further fractionation. Calcium phosphate gel adsorption. The RZ protein was dissolved in approximately 25 ml of 5 rnM potassium phosphate buffer, pH 7, and treated with calcium phosphate gel as described by Hsu, Wasson, and Porter (2). The supernatant solution from this treatment was termed ReG. DEAE-Cellulose chromatography. RzG protein was adsorbed onto a column of DEAE-cellulose (10.3 X 3.5 cm) which had previously been washed with 0.05 M potassium phosphate buffer, pH 7. Most of the adsorbed protein was washed off the column with 0.05 M potassium phosphate buffer, pH 7. Washing with thi buffer was continued unt,il the light absorption at 230 mcchad fallen to approximately 0.05 unit. The enzyme was eluted from the column with 0.16 M potassium phosphate buffer, pH 7. Fractions exhibiting absorbancies greater than 0.40 unit were combined and retained. Second ammonium sulfate fractionation. The material eluted from DEAE-cellulose was brought to 33yo of saturation with ammonium sulfate and the precipitated protein was collected by centrifugation (R3). Sephadex gel filtration. RI protein was dissolved in the minimum volume of 0.5 M potassium phosphate buffer, containing 1 mM EDTA and dithiothreitol, and applied to a column of Sephadex G-100 (22 X 1.0 cm) previously equilibrated with the same buffer. The effluent was collected in 0.5ml fractions. The enzyme was usually obtained in approximately 2 ml. Analytical ultracentrifugation and molecular weight determination. Velocity sedimentation experiments were carried out in a Spinco Model E ultracentrifuge. The rotor temperature was 20” and the rotor speed was 59,780 rpm. Sedimentation coefficients were corrected for solvent viscosity and density at 20”.
FATTY
ACID
The diffusion constant of the rat liver fatty acid synthetase was measured with a Spinco Model H instrument at 1”. Photographs of the diffusing protein-buffer boundary were taken at intervals over a 5-day period. The diffusion constant was calculated from measurements of the boundary. Photographs of the boundary were enlarged about ten times for this purpose. The diffusion constant was corrected to 20” and corrections were made for solvent density and viscosity. The molecular weight was calculated from the ratio of sedimentation and diffusion constants. A partial specific volume of 0.744 cm3 g-1 was used for the rat liver fatty acid synthetase. Sucrose density gradient centrifugation. Density gradient centrifugation was carried out by the method of Martin and Ames (14) as described by Yang et al. (4), except that centrifugation was performed in a Spinco SW5OL rotor at 40,000 rpm for 24 hours. Linear 15-35$& sucrose gradients, which contained either 0.5 M or 0.05 M potassium phosphate buffer, pH 7, and 1 mM EDTA and dithiothreitol, were used. Starch gel electrophoresis. Starch gel electrophoresis was performed with a vertical electrophoresis apparatus supplied by Buchler Instruments, Inc., New Jersey. The Tris-borate-EDTA system of Boyer, Fainer, and Naughton was employed (15). Electrophoresis was carried out at 4” and 5 V per vertical cm for 16 hours. Gels were sliced in half and stained with 0.1% Buffalo Black NBR in methanol-acetic acid. Destaining was effected with methanol: acetic acid: water (5:1:5).
Analyses
on Fatty Acid Synthetuse
(a) Assay of jlavin content. The flavin content of the purified fatty acid synthetase was assayed with Lactobacillus casei (16). (b) Analysis for sulfhydryl content. The sulfhydryl content of the enzyme complex was determined via titration with 5,5’-dithiobis-(2-nitrobenzoic acid) in the presence of 4 M guanidine hydrochloride (6). (c) Analysis for taurine. Aliquots of rat liver fatty acid synthetase (6.99 mg, 0.4 ml) in 0.01 M Tris-HCI buffer, were subjected to performic acid oxidation by the procedure of Moore (17). After oxidation, the protein was hydrolyzed with 6 N hydrochloric acid under reduced pressure at 110” for 22 hours. Amino acid analysis was carried out on a Beckman Model 120 amino acid analyzer fitted with a high sensitivity cuvette according to the method of Moore and Stein (18). The amount of taurine present is a direct measure of the amount of 4’-phosphopantetheine in the enzyme. (d) Amino acid analysis. An aliquot of rat liver fatty acid synthetase (6.99 mg, 0.4 ml) was ly-
SYNTHETASES
143
ophilized and subjected to hydrolysis with 6 N hydrochloric acid in vacua at 110” for 22 hours. The hydrolysate was analyzed on a Beckman Model 120 amino acid analyzer fitted with a high sensitivity cuvette. Assay for binding of acetate and malonate moieties of I-%7-acetyl-CoA and d-I%?-malonyl-CoA to enzyme. The reaction mixture contained protein, 2-5 mg; potassium phosphate buffer, pH 7, 100 p moles; and I-i4C-acetyl-CoA or 2-14C-malonyl-CoA, in a IO-20-fold molar excess over the amount of enzyme; all in a final volume of 1 ml. The mixture w&s incubated at 0” for various time periods from 30 seconds to 8 minutes. At this temperature the loss of protein-bound radioactivity through deacylation is minimized. The reaction was stopped by the addition of 0.03 ml of 60% perchloric acid, and the precipitated protein was washed free of excess radioactive substrate with 0.2 N acetic acid. The protein residue was dissolved in 1 ml of 1 N sodium hydroxide, and aliquots were taken for protein and radioactivity measurements. Assay for malonyl-CoA decarboxylase activity. Malonyl-CoA decarboxylase activity was meaeured through a determination of the fatty acid synthetase activity when acetyl-CoA was omitted from the incubation mixture. Under these conditions fatty acid synthesis depends on the prior decarboxylation of malonyl-CoA to acetyl-CoA. Assay for the formation of TAL and HMG-CoA. Reaction mixtures contained potassium phosphate buffer, pH 7, 150 rmoles; EDTA, 5 pmoles; 2mercaptoethanol, 5 pmoles; malonyl-CoA, 100 m&moles; l-14C-acetyl-CoA, 100 mfimoles and 150,000 dpm; and fatty acid synthetase 25 pg of protein; in a final volume of 1.5 ml. Incubations were carried out at 38” for 20 minutes. The reaction was terminated by the addition of 1 N potassium hydroxide when assays were made for HMG-CoA. HMG-CoA was hydrolyzed to HMG by this treatment. The mixture was then acidified, lyophilized, and the dry residue was extracted with acetone. When assays were made for TAL, the reaction was stopped with 1 N hydrochloric acid. TAL was then extracted with diethyl ether. The extracted products were methylated with diazomethane in ether. The methyl ester of HMG and methylated TAL were analyzed by gas-liquid chromatography on a 2Oyc butanediol succinate column, using argon as the carrier gas at a flow rate of 100 ml/minute. The gas chromatograph, Barber-Colman Model 10, was operated at a column temperature of 150” for the methyl ester of HMG-CoA and at 200” for the methylated TAL. Each radioactive compound was collected on elution from the column on ether-soaked glass wool
144
BURTON,
HAAVIK,
AND
TABLE PURIFICATION
OF RAT Protein
Fraction
Liver supernatant R2 DEAE eluate Sephadex G-100
Rat 1620 498 23 15
(w)
AND PIGEON
I LIVER
Enzyme activity Pigeon
Rat
1807 607 84 49
9300 5700 1280 1170
Pigeon (miiliunits)
a mpmoles of acetyl-CoA incorporated/min/mg synthetase activity were performed as described
PORTER
10246 9639 5022 3856
FATTY
ACID
Purification
Rat Pigeon (mpmoles/min/mg)
Rat
Pigeon
5.7 11.5 56.2 77.0
2.0 9.9 13.5
2.8 10.6 13.8
of protein. Assays under “Experimental
in a, t’ube cooled in Dry Ice. The collected material was washed into a counting vial with scintillation fluid, and the amount of radioactivity was determined with a liquid-scintillation spectrometer. RESULTS
Pu+jication of the enzyme. The specific activities obtained for the rat liver fatty acid synthetase purified by the procedure described in the “Experimental Methods” section are compared in Table I with those obtained for the pigeon liver fatty acid synthetase. The similarity in values obtained for the two enzyme complexes is evident. To obtain this similarity in activities some modifications in procedure were made in the purification of the rat liver fatty acid synthetase. The main differences between the two procedures of purification were the following: (a) A lower concentration of ammonium sulfate was used to precipitate the rat liver enzyme from the supernatant solution than was required for the pigeon liver enzyme; 20-33 % as compared to 25-40 %. (b) A lower molarity of potassium phosphate buffer was required to elute the rat liver enzyme from DEAE-cellulose than was required for the pigeon liver enzyme; 0.16 M compared to 0.25 M. If 0.25 M buffer was used to elute the rat liver enzyme, other proteins beside the fatty acid synthetase were removed from the column. (c) A buffer concentration of 0.5 M was used for the Sephadex G-100 gel filtration step in the case of the rat liver enzyme, compared to 0.2 N buffer with the pigeon liver system. This concentration of buffer was necessary to preserve the structural integrity of the rat liver multienzyme complex. A further
SYNTHETASES
Specific activitya
5.7 15.9 60.0 78.4 of each fraction methods.”
for
fatty
acid
discussion of this problem will be given later.3 The specific activities of both rat and pigeon liver enzymes (Table I) were almost identical at each step of purification, but the overall yield of enzyme protein was considerably lower in the case of the rat liver system, mainly because of the severe loss of protein during calcium phosphate gel treatment. However, it was found that this step was indispensable to the purification of the enzyme. Stability of the enzyme on storage. The rat liver fatty acid synthetase was found to be stable for at least 2 weeks when stored at 4” as a solution of moderate protein concentration (lo-20 mg/ml) in a mediti consisting of 0.5 M potassium phosphate buffer, pH 6.8, and containing 1 mu EDTA and 1 mu dithiothreitol. However, when stored at a low protein concentration (50 pg/ml) in the same buffer, approximately 50 % of the enzyme activity was lost in 24 hours. ~ltracentrifugat~on of the fatty acid synthetase. The rat liver fatty acid synthetase sediments essentially as a single component in the Spinco Model E analytical ultracentrifuge when dissolved in 0.5 M potassium phosphate buffer (Fig. 1). The ~S’20,~value is 12.3 S. The trace of a slower sedimenting component which is visible very probably represents a dissociation product which is produced in larger amounts in buffers of lower concentration? Sucrose density gradient centrifugation. Figure 2 shows the sucrose density gradient centrifugation of the rat liver fatty acid 3 See section on Purity of the Fatty
“Additional Studies on the Acid Synthetase Complex.”
FATTY
ACID
SYNTHETASES
FIG. 1. Sedimentation pattern of freshly prepared rat were made at 20” in a medium containing 0.5 M potassium 1 mM dithiothreitol. The rotor speed was 59,780 rpm, and graphs were made at 8-minute intervals.
145
liver fatty acid synthetase. Measurements phosphate buffer, pH 6.8, 1 mM EDTA and protein concentration was 6 mg/ml. I’hot,o-
FIG. 2. Sucrose density gradient centrifugation of rat liver fatty acid synthetase. One mg of eiizyme was subjected to a 23.5.hour centrifugation at 40,000 r-pm in a SWBOL rotor in a 5 ml, 15357c sitcrose gradient containing 0.5 1\1 potassium phosphate, pH 6.8, and 1 mM EDTA and dithiothreitol. Fatty acid synthetase activity is plotted here as the number of dpm which the whole of each fraction would incorporate into nalmitic acid from l-I%!-acetvl-CoA under the conditions of assay described in the section on “Experimental Methods.”
synthetase performed in a medium containing 0.5 M buffer. The enzyme sedimented essentially as a single component, and protein and fatty acid synthetase activity were coincident. When density gradient centrifugation was carried out in a medium containing 0.05 M phosphate buffer, two protein peaks mere found, fatty acid synthetase activity being associated wit’h the fastermoving component.” Stawh gel electrophoresis. The behavior of rat liver fatty acid synthetase on starch gel electrophoresis is different from that of the pigeon liver enzyme, which produces a single protein band under the conditions described under “Experimental Methods” (4). The rat liver fatty acid synthetase produces two protein bands under the same conditions. Later in this paper evidence
will be presented that one of these components represents a dissociation product of the intact multienzyme complex. DEAE - Cellulose chromatography. Although the pigeon liver fatty acid synthetase can be eluted from DEAE-cellulose as a single symmetrical band of protein (a), this is not the case with t.he rat liver enzyme. In the latter case, the purified enzyme appears to be irreversibly bound to the DEAE-cellulose, although the partially purified enzyme can be eluted from this adsorbent. This is evident from the fact that DEAE-cellulose chromatography is a step used in the procedure for purification of the enzyme. Additional studies on the purity of the fatty acid synthetase complex. It w-as reported
earlier in this paper that the final step in the purification of the rat liver fatty acid
146
BURTON,
HAAVIK,
AND
PORTER
FIG. 3. Sedimentation patterns obtained with rat liver fatty acid synthetase dialyzed against 0.05 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 5 mM dithiothreitol (upper) and then dialyzed against 0.5 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 5 mM dithiothreitol (lower). The rotor speed was 59,780 rpm, protein concentrations were 6.95 mg/ml (upper) and 7.58 mg/ml (lower), and photographs were made at g-minute int,ervals.
synthetase is Sephadex G-100 gel filtration in a medium containing 0.5 M potassium phosphate buffer. The use of this buffer was necessary to prevent the dissociation of the rat liver fatty acid synthetase. When 0.2 M potassium phosphate buffer was employed in this step (as in the preparation of pigeon liver fa.tty acid synthetase) the rat liver enzyme dissociated into two protein species. In the presence of 0.5 M buffer a single protein species having an A?!&O,~value of 12.3 was obtained (Fig. 1). Figure 3 presents the sedimentation pattern obtained after rat liver fatt.y acid synthetase, prepared in 0.5 M buffer as described in “Experimental Methods” was dialyzed against 0.05 M potassium phosphate buffer. The sedimentation pattern after dialysis of the enzyme back to 0.5 M buffer concentration is also presented. In 0.05 M phosphate buffer two species of S2c,U values, 14.1 and 10.0, were present. It is evident from the data of Fig. 3 that a reversible partial dissociation of the complex into smaller component,s occurs in the presence
of 0.05 M buffer. Concomitant with the appearance of the slower sedimenting component fatty acid synthetase activity also declined. The specific activity of the enzyme as originally prepared was 62 mpmoles/ min/mg; after dialysis to 0.05 M buffer concentration it was 49.8 mpmoles/min/mg; and after dialysis back to 0.5 M buffer concentration it was 66.7 mrmoles/min/mg. The pigeon liver fatty acid synthetase exhibits a single protein species having an S20,m value of 14.5 S in 0.25 M potassium phosphate buffer (19). We have found that the S20,w value of this protein is lowered to 12.2 S at a buffer concentration of 0.5 M. This behavior parallels that of the rat liver enzyme under similar conditions, as described above, except that only one protein species is present throughout in the case of the pigeon liver system. Figure 4 shows the separation obtained between the two components of the partially dissociated rat liver fatty acid synthetase complex on sucrose gradient centrifugation. The “heavy” and “light” components sepa-
FATTY
0
2
4
6
8 FRACTlON
IO
12
14
ACID
16
I8
147
SYNTHETASES
20
NUMBER
FIG. 4. The enzyme was dialyzed against 0.05 M potassium phosphate buffer, pH 6.8, cont,aining 1 mM EDTA and dithiothreitol, and then an aliquot (lOOnl,4.5 mg protein) was applied to a 5 ml, 15-357, sucrose gradient made in the same buffer. The sample was centrifuged for 23.5 hours at 40,000 rpm in the SW5OL rotor. After fractions were collected, the t,ubes indicated were pooled, and used as heavy and light components, after concentration with Carbowax.
acid synthetase on starch gel electrophoresis, performed as described under “Experimental Methods.” It is evident that the mobilities of the “light” component and the faster moving band from the untreated fatty acid synthetase are the same, indicating that the presence of two protein species in the untreated enzyme is a reflection of dissociation due to the low ionic strength of the buffer used in the starch gel electrophoresis, rather than to the presence of an impurity. In confirmation of this conclusion, it was found that the “light” component could be converted to the “heavy” component by incubation at 38” in 0.5 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and dithiothreitol. After such an incubation the “light” component sedimented in the same position as the “heavy” component on sucrose density gradient centrifugation. Parallel studies of enzyme activity showed that the “light” component TABLE
II
EFFECTS OF DITHIODIGLYCOLLIC ACID AND POTASSIUM MALEATE ON THE ACTIVITY OF THE RAT LIVER Enzyme
FATTY
ACID
preparation
Control Dithiodiglycollic acid treated” Dithiodiglycollic acid treated and reconstituteda Control Maleate treatedb
FIG. 5. Starch gel electrophoresis of rat liver fatty acid synthetase. (a) “Light” component from sucrose density gradient separation (Fig. 4) ; (b) untreated rat liver fatty acid synthetase. Approximately 100 pg of protein was applied to each slot of the starch gel block, and electrophoresis was carried out as described in “Experimental Methods.”
rated by this procedure were reserved for further study. Figure 5 shows the behavior of the “light” component and the untreated rat liver fatty
SYNTHETASE
Specific activity (mfimoles/min/mg) 81 1.9 46 90 1.55
a Rat liver fatty acid synthetase was prepared as described in “Methods,” and dialyzed against 0.5 M potassium phosphate buffer, pH 8, containing 1 rnM EDTA and 2 mu dithiothreitol. An equal volume of 0.2 M dithiodiglycollic acid, pH 8, was added, and the mixture was incubated at 4” for 18 hours. After analytical ultracentrifugation the protein solution was dialyzed against 0.5 M potassium phosphate buffer, pH 7, containing 1 mM EDTA and dithiothreitol. Then it was incubated with 0.01 M dithiothreitol in the same buffer at 38” for 2.5 hours. b Rat liver fatty acid synthetase prepared as described in “Methods” was dialyzed against 0.1 M maleic acid and 0.1 M potassium chloride for 42 hours at 4”.
148
BURTON,
HAAVIK,
possessed only a fraction of the activity exhibited by the original preparation. However, incubation of the “light” component at 38” in 0.5 M potassium phosphate buffer, pH 6.8, containing 1 rnM EDTA and dithiothreitol restored more than 50 % of the original enzyme activity. Quite possibly, the small activity shown by the “light” component. is due to some reassociation to the intact complex during the period of assay, which is performed at 38”. Dissociation of fatty acid synthetase with maleate, cLithiocLigLycollic acid, or palmitylCoA. Butterworth et al. (.5) have demonstrated that the pigeon liver fatty acid synthetase can be dissociated into half molecular weight subunits by disulfide exchange or by reaction of sulfhydryl groups with potassium maleate. These treatments resulted in loss of overall enzyme activity. Table II shows the effect of treatment of the rat liver fatty acid synthetase with dithiodiglycollic acid and potassium maleate, as described in the legend to the table. Both treatments inhibited enzyme activity almost completely, but parallel studies using analytical ultracentrifugation showed that the
AND
PORTER
enzyme was only partially dissociated to a slower moving component (30-40 %), in contrast to the behavior of the pigeon liver enzyme. Enzyme activity was restored by reduction of the disulfide bond between dithiodiglycollic acid and the enzyme with dithiothreitol. The inactivation by maleat,e is irreversible. l?igure 6 and Table III show the effect of micellar concentrations of palmityl-CoA on the electrophoretic mobility and enzyme activity of the rat liver fatty acid synthetase. Clearly the structural integrity of the complex is disrupted and enzymic activity is lost in this process. Molecular weight cletemination,. The molecular weight of the rat liver fatty acid synthetase in 0.5 RI potassium phosphate buffer is calculated from sediment’ation-diff usion analyses to be 5.4 X lo5 g/mole. These data demonstrate the close similarity between the rat and pigeon liver fatty acid synthetases. The latter has an S~O,~,of 14.5 (19) and a molecular weight of 4.5 X lo5 as measured by the technique of Yphantis (3, 4). A previous determination of the molecular weight of the pigeon liver fatty
FIG. 6. Effect of palmityl-CoA on rat liver fatty acid svnthetase. Rat liver fatty acid synthetase was mixed with palmityl-CoA, as indicated, in 0.2 M potassium phosphate buffer, pH 6.8. 100 pg of each protein mixture was applied t,o each slot and electrophoresis was performed as described in “Experimental Methods.” Control-slots 1 and 7; slots 2-6 (left to right), molar excess of palmityl-CoA, 300, 250, 200, 150, and 100, respectively.
FATTY TABLE EFFECT
RAT
LIVER
FATTY
Molar except of palmityl-CoA OYer enzyme
ACID
TABLE
ON
THE
ACTIVITY
and fatty acid synthetase as described in “Methods.”
Aspartic Threonine Serine
LIVER
acid
Proline Glutamic
Glycine
incorporated Palmityl-CoA activity
AMINO
Amino acid
98.5 64.9 39.0 24.5 8.6 8.4
and enzyme were mixed in the proportions
PARTIAL PIGEON
Specific activity” (m~moles/min/rrg)
0 mpmoles of l-IT-acetyl-CoA fatty acids/min/mg of protein.
cated, assayed
OF
SYNTHETASE
0 100 150 200 250 300
into
149
SYNTHETASES
III
CoA
OF PALMITYL
ACID
indiwas
acid synthetase by Ehrenberg’s method (20) and from separately determined sedimentation and diffusion coefficients, gave values of 5.67 and 5.33 X 105, respectively (19).
Alanine Valine Tsoleucine Leucine Tyrosine Phenylalanine
acid
ACID
IV
COMPOSITION
FATTY
ACID
OF
R.VJTAND
SYNTHETASFW
Rat (moles/mole of enzyme)
Pigeon (moles/mole of enzyme)
280 189 248 194 355 274 306 256 141 456 76 118
254 137 193 137 344 240 250 240 165 340 76 99
a Values for the pigeon liver fatty acid synthetase were taken from Hsu et al. (2) and corrected to a molecular weight of 450,000 g/mole.
tal Methods.“) Under these conditions taurine would arise from the terminal pComponents of Rat and Pigeon Liver Fatty mercaptoethylamine residue of 4’-phosphoAcicl Synthetases pantetheine. (a) F&win. content. Like the pigeon liver Duplicate assays of the rat liver fatty fatby acid synthetase, but unlike the yeast acid synthetase yielded a value of 1.05 moles enzyme (1, al), the rat liver enzyme con- 4’-phosphopantetheine per mole of enzyme tains negligible amounts of flavin, when (540,000 g). This compares very closely with determined by microbiological assay (16). the value of 1.12 moles per mole of enzyme The values obtained ranged from 0.006 to (450,000 g) obtained for the pigeon liver 0.024 moles of flavin per mole of enzyme. complex by Jacob, Bubterworth and Porter.4 (b) Amino acids. Table IV present’s a Characteristics of fatty acid synthetase comparison of the conbents of neutral and reaction. The dependence of fatty acid acidic amino acids of the rat and pigeon synthesis by the rat liver enzyme system liver fatty acid synthetases. It may be seen on pH is demonstrated in Fig. 7. The pH t’hat the amino acid contents of these com- optimum was found to be 6.S which complexes are very similar. pares closely to the value of 6.7 reported (c) Sulfhydryl content. The sulfhydryl con- for the pigeon liver fatty acid synthetase b! tent,s of the rat and pigeon liver enzyme Hsu, Wasson, and Porter (2). systems are quite high. The rat liver enzyme Figure 8 shows the dependence of fatty was found t’o contain 9&92 such groups acid synthetase activity on time and protein per mole (based on a molecular weight of concentration. The reaction is linear up to 540,000) in three separate assays. The 6 minutes and up to 10 pg of protein. The pigeon liver fatty acid synthetase has 64-66 K,,, for acetyl-CoA is 4.4 PM and the prod(based upon a molecular weight of 450,000) uct of the reaction has been identified as (6). free palmitic acid by radioactively moni(cl) 4’-Phosphopantetheinecontent. The 4’tored gas liquid chromatography (Fig. 9). phosphopantetheine content of the fatty Binding of acetate and maZon.atemoieties of acid synthetase was obtained by assaying the performic acid oxidized and acid hydro4 Jacob, E. J., Butterworth, P. H. W., and Porter, J. W. Arch. Biochem. Riophys. 124,392 (1968). lyzed enzyme for taurine. (See “Experimen-
150
BURTON,
HAAVIK,
1 -14C-ucetyl-CoA tend 2J4C malony WoA to emyme. The binding of the acetyl and malonyl groups of acetyl- and malonyl-CoA to the rat liver fatty acid synthetase is illustrated in Fig. 10. Binding was performed at O”, since loss of bound substrate by deacylation was minimal at this temperature. The amount of substrate bound varied with the molar excess of substrate over enzyme. Fig.
PH
7. Dependence of fatty acid synthetase activity on pH. Assay conditions were as described in “Experimental Methods” except that the pH was varied as shown. 4.03 pg of enzyme protein was used in each assay. FIG.
AND
PORTER
10 shows binding data obtained using molar excesses between lo- and 20-fold. Under these conditions the enzyme bound close to 2 moles of substrate per mole. Assay for malonyl-CoA decarboxylase activity. Purified rat liver fatty acid synthetase was assayed for malonyl-CoA decarboxylase activity as described in “Experimental Methods.” The specific activity of the enzyme under these conditions was 4.7, compared to a value of 77, when both acetyland malonyl-CoA were present as substrates. Formation of /3-hydroxy-P-methyl glutarylCoA and TAL by the fatty acid synthetase. Gas-liquid chromatographic assay for the formation of TAL by rat and pigeon liver fatty acid synthetases provided proof that both enzyme complexes synthesized small amounts of this compound, from acetyland malonyl-CoA in the absence of NADPH. The rat liver enzyme incorporated 11.52 mpmoles lJ4C-acetyl-CoA/min/mg into TAL, and the corresponding value for the pigeon liver fatty acid synthetase was 7.05 mpmoles/min/mg, under the same conditions. The two fatty acid synthetases were also shown to produce small amounts of HMGCoA. The latter compound was converted to the methyl ester and assayed by gasliquid chromatography.
70-
b ; I
50-
0
: g 407 z z5
0
30-
% zo2
100 80 60
0
40 0
0
IOm
60- :,i... 0
20 2
4
6
6
TIME IN MINUTES
IO
12
2
6 IO 14 ,q OF PROTEIN
FIG. 8. Dependence of fatty acid synthetase activity on time and protein concentration. The standard assay system was used, except that time and protein concentration were varied as shown. For the time study 3.42 pg of enzyme protein was used; a IS-minute assay period was employed for the protein dependence study.
FATTY
0
5
ACID
151
SYNTHETASES
IO 15 TIME IN MINUTES
20
25
Fro. 9. Identification of product of reaction catalyzed by rat liver fatty acid synthetase. The product of the reaction, carried out by the normal assay method, was extracted from the reaction mixture with petroleum ether. The solvent was removed under Ns and a solution of diazomethane in diethyl ether was added. After reaction excess ether and diazomethane were removed under Nz, and ,the methyl esters were mixed with a standard mixture of nonradioactive fatty acid methyl esters. This mixture was subjected to radioactively monitored gas-liquid chromatography on a 6’-butanediol succinate column supported on chromosorb W. The operating temperature was 198” and the rate of flow of the carrier gas (helium) was 95 ml/minute. The instrument used was a Barber-Colman Selecta-System Series 5090. DISCUSSION
FIG. 10. Binding of I-14C-acetyl-Cob and 2J4Cmalonyl-CoA to rat liver fatty acid synthetase at 0”. Fatty acid synthetase was incubated at 0” with l-I%-acetyl or 2-14C-malonyl-CoA for the time periods indicated. The protein was precipitated with 60% perchloric acid, and excess substrate was removed by repeated washing with 0.2 N acetic acid. Residual protein was dissolved in I N NaOH and aliquots were taken for radioactivity and protein determinations.
It is now well established that all the enzymic components required for the biosynthesis of long-chain fatty acids from acetyland malonyl-CoA by yeast (1, 21) and pigeon liver (2) reside in single stable protein complexes. In the present paper it has been demonstrated that the rat liver fatty acid synthetase is also a multienzyme complex. This enzyme system was isolated in 0.5 M potassium phosphate buffer as an essentially homogenous protein with respect to molecular weight. The enzyme sedimented as a single protein species on analytical ultracentrifugation and sucrose density gradient centrifugation. Protein and enzyme activity peaks were also coincident in the latter system. The rat liver fatty acid synthetase yielded
152
BURTON,
HAAVIK,
two bands on st,arch gel electrophoresis. However, it was proved that one of these bands was a dissociation product of the complex. Hence it is concluded that the rat liver fatty acid synthetase was also obtained as an electrophoretically homogenous species. The procedure used to purify the rat liver fatty acid synthetase is very similar to the procedure used to purify the analogous enzyme system from pigeon liver. Only minor variations exist between the two procedures. However, these variations are vital for the isolation of a homogenous preparation. The properties of the pigeon and rat liver fatty acid synthetases are very similar in many respects. Thus the two enzymes had almost the same specific activities at each step in the purification procedure. They also have approximately the same molecular weight. The rat liver fatty acid synthetase exhibits a molecular weight of 5.4 X lo5 g/mole (in 0.5 M buffer) as calculated from separately determined sedimentation and diffusion constants. This compares quite closely to the best estimate of the molecular weight of the pigeon liver enzyme, which is 4.5 X lo5 g/mole (4). Another indication of the similarity of the two enzymes is found in the fact that the S20,W of both complexes is altered on changing the concentration of buffer in which the protein is dissolved. The pigeon liver fatty acid synthetase exhibits an SzO,W value of 14.5 in 0.25 M potassium phosphate buffer (19)) but in 0.5 M buffer the A!&,~ is 12.2. Similarly, the rat liver enzyme shows a change in &I,~ from 14.1 S to 12.3 S when the buffer concentration is changed from 0.05 M t0
0.5
M.
The rat liver fatty acid synthetase exhibits essentially the same specific enzyme activity when assayed in 0.5 M potassium phosphate as it does when assayed in 0.1-0.2 M buffer. This result may be interpreted as meaning that both 14.1 and 12.3 S species possess the same enzymic activity, and that the change in SZoZwvalue probably reflects a conformational rather tha,n a molecular weight change. Further support for this hypothesis is found in the fact that the pigeon liver enzyme exists as a single protein
AND
PORTER
species under both sets of conditions. If a molecular weight change were involved in the change in &Y,,~ values, it would be expected that two protein species would be present when the complex had the lower S20,W value. Thus in terms of sedimentation behavior and molecular weight, the pigeon and rat liver fatty acid synthetases appear to be very similar. The similarity between the two fatty acid synthetases is found in other properties of these complexes. Both fatty acid synthetases contain negligible quantities of flavin, in contrast to the yeast system and the flavin-dependent acyl-CoA dehydrogenase of the fatty acid oxidation cycle. The sulfhydryl contents measured in the presence of the dissociating agent guanidine hydrochloride were found to be 90-92 and 64-66 groups per mole for the rat and pigeon liver systems, respectively (based on a molecular weight of 540,000 for the rat and 450,000 for the pigeon liver fatty acid synthetases). Again in contrast to the yeast system, which produces stearyl- and palmityl-CoA both rat and pigeon liver (22, 23) fatty acid synthetases produce free palmitic acid. The rat liver enzyme complex binds the acetate and malonate moieties of acetyland malonyl-CoA to approximately the same extent as the pigeon liver system and both complexes appear to contain only 1 mole of 4’-phosphopantetheine per mole of enzyme. (The latter finding must be considered when a mechanism of action for this enzyme is postulated.) Both enzymes possess some malonyl-CoA decarboxylase activity and each enzyme synthesizes HMG-CoA and triacetic acid lactone from acetyl- and malonyl-CoA in the absence of KADPH. The synthesizing activity for the formation of TAL purifies with the pigeon liver fatty acid synthetase.5 Similar analyses have not been made for the rat liver enzyme system. The pH optima and the overall amino acid composition of the two enzymes are also very similar. The most important difference between the two enzyme systems concerns their dissociation. The pigeon liver fatty acid 5 Nixon, observations.
J. E.,
and Porter,
J. W.,
Unpublished
FATTY
ACID
SYNTHETASES
153
synthetase has been shown to be stable for sulfhydryl groups (5). Similar treatment, of at least 5 days when stored in a medium the rat liver fatty acid synthetase with containing 0.2 M potassium phosphate dithiodiglycollic acid or potassium maleate buffer, 1 mM EDTA and 1 mu dithiothreitol resulted in the complete loss of enzymic (4). However, if P-mercaptoethanol is used activity. However, in each case dissociation in place of dithiothreitol, the complex disso- into smaller subunits proceeded only to the ciates into approximately half molecular extent of 3&40 %, and in addition, it is likely weight subunits over a period of several that a proportion of this dissociation was days. An equilibrium of about equal propor- due to the use of buffer concentrations betions of original complex and half molecular low 0.5 M. It appears probable from this weight units is finally reached (5). This result that sulfhydryl groups are either less processis referred to as ‘
154
BURTON,
HAAVIK,
AND
ACKNOWLEDGMENTS The attthors wish to express t,heir thanks to Professor R. M. Bock for useful discussions and for obtaining some of the analytical ultracentrifugation data, and to Professor H. F. Detttsch for the determination of the diffusion constant of the rat liver fatty acid synthetase. We are also grateful to Professor Theo Gerritsen and Mr. J. Campbell for taurine and amino acid analyses, and to Mr. G. Velasquez for excellent technical assistance in the performance of ultracentrifugation analyses. We wish to thank Dr. Maria Burger of the Wisconsin Alumni Research Foundation for performing assays for riboflavin content of the rat liver fatty acid synthetase. REFERENCES 1. LYNEN, F., HOPPER, I., LORCH, E., KIRSCHNER, K., HAGEN, A., AND SCHWEITZER, E., Proc. 6th International Congress Biochem., 1964, New York, VII, S-2. 2. Hsu, R. Y ., WASSON, G., AND PORTER, J. W., J. Biol. Chem. MO, 3736 (1965). 3. YPHANTIS, D. A., Biochemistry 3, 297 (1965). 4. YANG, P. C., BUTTERWORTH, P. H. W., BOCK, R. M., BND PORTER, J. W., J. Biol. Chem. 242,350l (1967). 5. BUTTER~ORTH, P. H. W., YANG, P. C., BOCK, R. M., :IND PORTICR, J. W., J. Biol. Chem. 242, 3508 (1967). 6. BUTTERWORTH, P. H. W., BAUM, H., AND PORTER, J. W., drch. Biochem. Biophys. 118, 716 (1967). 7. BUTTICR~ORTH, P. H. W., GUCHHAIT, R. B., BAUM, H., OLSON, E. B., MARGOLIS, S. A.,
8. 9. 10. 11. 12.
13. 14. 15. 16.
17. 18. 19.
20. 21. 22. 23.
PORTER
AND PORTER, J. W., Arch. Biochem. Biophys. 116, 453 (1966). SIMON, E. J., .IND SHEMIN, D. J., J. Amer. Chem. Sot. 76, 2.520 (1953). TR.IMS, E. G., .\ND BRADY, R. O., J. Amer. Chem. Sot. 82, 2972 (1960). BRODIF:, J. D., .IND PORTER, J. W., Biochem. Biophys. Res. Commun. 3, 173 (1960). GORNALL, A. G., B.~RD.IWILL, C. J., AND DAVID, ibf. >I., J. Biol. Chem. 177, 751 (1949). W.IKIL, S. J., PORTER, J. W., .IND GIBSON, Il. M., Biochim. Biophys. Acta 24, 453 (1957). KORNACKE:R, iU. S., AND LO~ENSTEIN, J. M., Biochem. J. 94,209 (1965). ?rlSRTIN, R. G., .IND Awns, B. N., J. Biol. Chem. 236, 1372 (1961). BOYIZR, S. H., F.IINER, D. C., AND N;BUGHTON, XI. A., Science 140, 1228 (1963). “Official Methods of Analysis of the Association of Official Agricultural Chemists,” p. 773,10th Ed. 1965. W. Horwitz, Ed. Assoc. Official Agric. Chem., Washington, D. C. &lOORI?, S., J. Biol. Chem. 238, 235 (1963). MOORF:, S., .&ND STEIN, W. H., Anal Chem.. 30, 1190 (1958). YANG, P. C., BOCK, R. M., Hsu, R. Y., AND PORTEZR, J. W., Riochem. Biophys. Acta 110, 608 (1965). EHRICNRERG, A., dcta Chem. &and. 11, 1257 (19,57). LYNEN, F., Fed. Proc. 20,941 (1961). PORTER, J. W., .IND TI~TZ, A., Biochim. Biophys. Acta 26, 41 (1957). BRISSSLER, R., .IND W.IKIL, S. J., J. Biol. Chem. 237, 1441 (1962).
OF
BIOCHEMISTRY
AND
Comparative
Studies Fatty
DAVID Lipid
N. BURTON,
Metabolism Physiological
126,
BIOPHYSICS
Laboratory, Chemistry,
141-154
of the Acid
ARNE Veterans University Received
(1968)
Rat
and
Pigeon
Liver
Synthetases’
G. HAAVIK, Administration of Wisconsin, January
AND
JOHN
Hospital Madison,
W. PORTER
and the Department Wisconsin 63705
of
2, 1968
The fatty acid synthetase of rat liver has been prepared in an essentially homogenous state by a similar, but nonidentical, procedure to that used for the purification of the pigeon liver fatty acid synthetase. Both of these complexes synthesize free palmitic acid from acetyland malonyl-CoA in the presence of NADPH. Enzymic activities for the decarboxylation of malonyl-CoA and the synthesis of HMG-CoA and triacetic acid lactone are also associated with both fatty acid synthetases. The properties of the rat liver fatty acid synthetase have been investigated, and compared to those of the pigeon liver enzyme. Close similarities were observed in molecular weight, thiol dependency, binding stoichiometry for acetyland malonylCoA, 4’-phosphopantetheine content, sulfhydryl and flavin content, inhibition by sulfhydryl binding reagents, pH optimum and amino acid composition. It is suggested, therefore, that the complexes have very similar structures. However, marked differences in the ease of dissociation in phosphate buffers below 0.5 M were observed, together with differences in stability of the enzymes on electrophoresis and DEAEcellulose chromatography. These results indicate that some differences exist in the binding of subunits in the two complexes.
Homogenous enzyme complexes synthesizing long-chain fatty acids have been isolated from yeast (1) and pigeon liver (2). Hsu et al. of this laboratory (2) prepared the soluble fatty acid synthetase of pigeon liver in a homogenous form with high specific act,ivity. This enzyme converts acetylCoA and malonyl-CoA, in the presence of NADPH, to palmitic acid with an overall stoichiometry of 1 acetyl-CoA + 7 malonylCoA + 14 Hf + 14 NADPH + 1 palmitic acid + 7 CoA + 7 CO2 + 6 HzO. The purified enzyme complex also contains activities for malonyl-CoA decarboxylase and the synthesis of P-hydroxy, P-methyl glutaryl CoA and 3,5diketohexanoic acid.
The purified enzyme has a molecular weight of 4.5 X lo5 by sedimentation-equilibrium analysis (3-5) and it contains 64-66 moles of thiol per mole of enzyme (6). Stability of the enzymic activity is dependent on maintenance of thiol reduction. On thiol oxidation enzymic activity is lost and the formation of half molecular weight subunits occurs (4, 5). Recent investigations in this laboratory on the content of the pigeon liver complex in various nutritional states (7) and the effect of experimental diabetes on enzyme activity for the synthesis of fatty acids by t,he rat liver enzyme2 have led to studies on the isolation of the rat liver enzyme complex. This complex has been isolated in an essentially homogenous and highly active state. The results of investigations on the properties of the rat liver enzyme complex
1 This investigation was supported in part by research grants, A-1383 from the National Instit,ute of Arthritis and Metabolic Diseases of the Nat,ional Institutes of Health, United States Public Health Service and No. 66-725 from the American Heart Association.
2 Dahlen, J., Kennan, Arch. Biochem. Biophys. 141
A. H., l&51
and Porter, (1968).
J. W.,
BURTON,
142
HAAVIK,
and a comparison of these properties to those of the pigeon liver enzyme are also reported in this paper. From these data we conclude that there is a striking similarity between the two complexes in overall physical and enzymic properties. There appear, however, to be marked differences in the stability of the complexes on protein
dilution and on decrease of ionic strength. These results suggestdifferences in the forces maintaining the integrity of the two complexes. EXPERIMENTAL
METHODS
Materials. Experimental materials were obtained from the following sources: I-“C-acetic anhydride and 2-l*C-malonic acid from New England Nuclear Corporation; NADPH and ATP from Sigma Chemical Company; S-Palmityl-CoA and CoA from P-L Biochemicals; 2-mercaptoethanol and dithiothreitol from Calbiochem; hydrolyzed starch from the Connaught Laboratories, Toronto; and Sephadex from the Pharmacia Company, Uppsala, Sweden. All reagents used were of analytical grade. Preparation of coenzyme-A esters. Nonradioactive acetyl-CoA and 1-I%-acetyl-CoA were prepared by the method of Simon and Shemin (8) and nonlabeled and 2-14C-malonyl-CoA were synthesized as described by Trams and Brady (9). The CoA esters were purified by the paper chromatographic systems of Brodie and Porter (10). Bu$ers. All buffers contained 1 mM EDTA and either 1 mM dithiothreitol or 1 mM &mercaptoethanol, unless otherwise specified. Assay for protein. Assays for the protein concentration of solutions were carried out by the method of Gornall, Bardawill, and David (11). Assay for fatty acid synthetase activity. The assay system used was that of Hsu, Wasson and Porter (2), as modified by Butterworth et al. (7). Specific activity of the enzyme is expressed as m#moles of lJ”C-acetyl-CcA incorporated into fatty acids per minute per milligram of protein. Determination of radioactivity. All measurements of radioactivity were obtained with a Packard liquid scintillation spectrometer. Preparation of liver supernatant solutions. Rat and pigeon liver supernatant solutions were prepared by the method of Wakil, Porter, and Gibson (12) as modified by Hsu, Wasson, and Porter (2). Male rats weighing about 150 g each were starved 43 hours, then fed 48 hours on a fat-free diet (13) before they were killed by decapitation. Both rat and pigeon liver supernatant solutions were stored at - 15” under nitrogen.
AND PORTER Puri$cation of fatty acid synthetase. Rat liver fatty acid synthetase was prepared by a procedure similar to that used for pigeon liver fatty acid synthetase by Hsu, Wasson, and Porter (2). HOWever, several important modifications were used in the preparation of the rat liver enzyme. First ammonium sulfate fractionation. Frozen rat liver supernatant solution (40 ml) was allowed to thaw at room temperature, and then stirred gently under a stream of nitrogen. Saturated ammonium sulfate solution, pH 7, containing 3 mM EDTA and 1 mM &mercaptoethanol was added to a saturation of 20%. After stirring for 15 minutes, the mixture was centrifuged and the precipitate discarded (RI). The supernatant solution was brought to 33% of saturation in ammonium sulfate. The precipitated protein was collected by centrifugation and retained (Rt) for further fractionation. Calcium phosphate gel adsorption. The RZ protein was dissolved in approximately 25 ml of 5 rnM potassium phosphate buffer, pH 7, and treated with calcium phosphate gel as described by Hsu, Wasson, and Porter (2). The supernatant solution from this treatment was termed ReG. DEAE-Cellulose chromatography. RzG protein was adsorbed onto a column of DEAE-cellulose (10.3 X 3.5 cm) which had previously been washed with 0.05 M potassium phosphate buffer, pH 7. Most of the adsorbed protein was washed off the column with 0.05 M potassium phosphate buffer, pH 7. Washing with thi buffer was continued unt,il the light absorption at 230 mcchad fallen to approximately 0.05 unit. The enzyme was eluted from the column with 0.16 M potassium phosphate buffer, pH 7. Fractions exhibiting absorbancies greater than 0.40 unit were combined and retained. Second ammonium sulfate fractionation. The material eluted from DEAE-cellulose was brought to 33yo of saturation with ammonium sulfate and the precipitated protein was collected by centrifugation (R3). Sephadex gel filtration. RI protein was dissolved in the minimum volume of 0.5 M potassium phosphate buffer, containing 1 mM EDTA and dithiothreitol, and applied to a column of Sephadex G-100 (22 X 1.0 cm) previously equilibrated with the same buffer. The effluent was collected in 0.5ml fractions. The enzyme was usually obtained in approximately 2 ml. Analytical ultracentrifugation and molecular weight determination. Velocity sedimentation experiments were carried out in a Spinco Model E ultracentrifuge. The rotor temperature was 20” and the rotor speed was 59,780 rpm. Sedimentation coefficients were corrected for solvent viscosity and density at 20”.
FATTY
ACID
The diffusion constant of the rat liver fatty acid synthetase was measured with a Spinco Model H instrument at 1”. Photographs of the diffusing protein-buffer boundary were taken at intervals over a 5-day period. The diffusion constant was calculated from measurements of the boundary. Photographs of the boundary were enlarged about ten times for this purpose. The diffusion constant was corrected to 20” and corrections were made for solvent density and viscosity. The molecular weight was calculated from the ratio of sedimentation and diffusion constants. A partial specific volume of 0.744 cm3 g-1 was used for the rat liver fatty acid synthetase. Sucrose density gradient centrifugation. Density gradient centrifugation was carried out by the method of Martin and Ames (14) as described by Yang et al. (4), except that centrifugation was performed in a Spinco SW5OL rotor at 40,000 rpm for 24 hours. Linear 15-35$& sucrose gradients, which contained either 0.5 M or 0.05 M potassium phosphate buffer, pH 7, and 1 mM EDTA and dithiothreitol, were used. Starch gel electrophoresis. Starch gel electrophoresis was performed with a vertical electrophoresis apparatus supplied by Buchler Instruments, Inc., New Jersey. The Tris-borate-EDTA system of Boyer, Fainer, and Naughton was employed (15). Electrophoresis was carried out at 4” and 5 V per vertical cm for 16 hours. Gels were sliced in half and stained with 0.1% Buffalo Black NBR in methanol-acetic acid. Destaining was effected with methanol: acetic acid: water (5:1:5).
Analyses
on Fatty Acid Synthetuse
(a) Assay of jlavin content. The flavin content of the purified fatty acid synthetase was assayed with Lactobacillus casei (16). (b) Analysis for sulfhydryl content. The sulfhydryl content of the enzyme complex was determined via titration with 5,5’-dithiobis-(2-nitrobenzoic acid) in the presence of 4 M guanidine hydrochloride (6). (c) Analysis for taurine. Aliquots of rat liver fatty acid synthetase (6.99 mg, 0.4 ml) in 0.01 M Tris-HCI buffer, were subjected to performic acid oxidation by the procedure of Moore (17). After oxidation, the protein was hydrolyzed with 6 N hydrochloric acid under reduced pressure at 110” for 22 hours. Amino acid analysis was carried out on a Beckman Model 120 amino acid analyzer fitted with a high sensitivity cuvette according to the method of Moore and Stein (18). The amount of taurine present is a direct measure of the amount of 4’-phosphopantetheine in the enzyme. (d) Amino acid analysis. An aliquot of rat liver fatty acid synthetase (6.99 mg, 0.4 ml) was ly-
SYNTHETASES
143
ophilized and subjected to hydrolysis with 6 N hydrochloric acid in vacua at 110” for 22 hours. The hydrolysate was analyzed on a Beckman Model 120 amino acid analyzer fitted with a high sensitivity cuvette. Assay for binding of acetate and malonate moieties of I-%7-acetyl-CoA and d-I%?-malonyl-CoA to enzyme. The reaction mixture contained protein, 2-5 mg; potassium phosphate buffer, pH 7, 100 p moles; and I-i4C-acetyl-CoA or 2-14C-malonyl-CoA, in a IO-20-fold molar excess over the amount of enzyme; all in a final volume of 1 ml. The mixture w&s incubated at 0” for various time periods from 30 seconds to 8 minutes. At this temperature the loss of protein-bound radioactivity through deacylation is minimized. The reaction was stopped by the addition of 0.03 ml of 60% perchloric acid, and the precipitated protein was washed free of excess radioactive substrate with 0.2 N acetic acid. The protein residue was dissolved in 1 ml of 1 N sodium hydroxide, and aliquots were taken for protein and radioactivity measurements. Assay for malonyl-CoA decarboxylase activity. Malonyl-CoA decarboxylase activity was meaeured through a determination of the fatty acid synthetase activity when acetyl-CoA was omitted from the incubation mixture. Under these conditions fatty acid synthesis depends on the prior decarboxylation of malonyl-CoA to acetyl-CoA. Assay for the formation of TAL and HMG-CoA. Reaction mixtures contained potassium phosphate buffer, pH 7, 150 rmoles; EDTA, 5 pmoles; 2mercaptoethanol, 5 pmoles; malonyl-CoA, 100 m&moles; l-14C-acetyl-CoA, 100 mfimoles and 150,000 dpm; and fatty acid synthetase 25 pg of protein; in a final volume of 1.5 ml. Incubations were carried out at 38” for 20 minutes. The reaction was terminated by the addition of 1 N potassium hydroxide when assays were made for HMG-CoA. HMG-CoA was hydrolyzed to HMG by this treatment. The mixture was then acidified, lyophilized, and the dry residue was extracted with acetone. When assays were made for TAL, the reaction was stopped with 1 N hydrochloric acid. TAL was then extracted with diethyl ether. The extracted products were methylated with diazomethane in ether. The methyl ester of HMG and methylated TAL were analyzed by gas-liquid chromatography on a 2Oyc butanediol succinate column, using argon as the carrier gas at a flow rate of 100 ml/minute. The gas chromatograph, Barber-Colman Model 10, was operated at a column temperature of 150” for the methyl ester of HMG-CoA and at 200” for the methylated TAL. Each radioactive compound was collected on elution from the column on ether-soaked glass wool
144
BURTON,
HAAVIK,
AND
TABLE PURIFICATION
OF RAT Protein
Fraction
Liver supernatant R2 DEAE eluate Sephadex G-100
Rat 1620 498 23 15
(w)
AND PIGEON
I LIVER
Enzyme activity Pigeon
Rat
1807 607 84 49
9300 5700 1280 1170
Pigeon (miiliunits)
a mpmoles of acetyl-CoA incorporated/min/mg synthetase activity were performed as described
PORTER
10246 9639 5022 3856
FATTY
ACID
Purification
Rat Pigeon (mpmoles/min/mg)
Rat
Pigeon
5.7 11.5 56.2 77.0
2.0 9.9 13.5
2.8 10.6 13.8
of protein. Assays under “Experimental
in a, t’ube cooled in Dry Ice. The collected material was washed into a counting vial with scintillation fluid, and the amount of radioactivity was determined with a liquid-scintillation spectrometer. RESULTS
Pu+jication of the enzyme. The specific activities obtained for the rat liver fatty acid synthetase purified by the procedure described in the “Experimental Methods” section are compared in Table I with those obtained for the pigeon liver fatty acid synthetase. The similarity in values obtained for the two enzyme complexes is evident. To obtain this similarity in activities some modifications in procedure were made in the purification of the rat liver fatty acid synthetase. The main differences between the two procedures of purification were the following: (a) A lower concentration of ammonium sulfate was used to precipitate the rat liver enzyme from the supernatant solution than was required for the pigeon liver enzyme; 20-33 % as compared to 25-40 %. (b) A lower molarity of potassium phosphate buffer was required to elute the rat liver enzyme from DEAE-cellulose than was required for the pigeon liver enzyme; 0.16 M compared to 0.25 M. If 0.25 M buffer was used to elute the rat liver enzyme, other proteins beside the fatty acid synthetase were removed from the column. (c) A buffer concentration of 0.5 M was used for the Sephadex G-100 gel filtration step in the case of the rat liver enzyme, compared to 0.2 N buffer with the pigeon liver system. This concentration of buffer was necessary to preserve the structural integrity of the rat liver multienzyme complex. A further
SYNTHETASES
Specific activitya
5.7 15.9 60.0 78.4 of each fraction methods.”
for
fatty
acid
discussion of this problem will be given later.3 The specific activities of both rat and pigeon liver enzymes (Table I) were almost identical at each step of purification, but the overall yield of enzyme protein was considerably lower in the case of the rat liver system, mainly because of the severe loss of protein during calcium phosphate gel treatment. However, it was found that this step was indispensable to the purification of the enzyme. Stability of the enzyme on storage. The rat liver fatty acid synthetase was found to be stable for at least 2 weeks when stored at 4” as a solution of moderate protein concentration (lo-20 mg/ml) in a mediti consisting of 0.5 M potassium phosphate buffer, pH 6.8, and containing 1 mu EDTA and 1 mu dithiothreitol. However, when stored at a low protein concentration (50 pg/ml) in the same buffer, approximately 50 % of the enzyme activity was lost in 24 hours. ~ltracentrifugat~on of the fatty acid synthetase. The rat liver fatty acid synthetase sediments essentially as a single component in the Spinco Model E analytical ultracentrifuge when dissolved in 0.5 M potassium phosphate buffer (Fig. 1). The ~S’20,~value is 12.3 S. The trace of a slower sedimenting component which is visible very probably represents a dissociation product which is produced in larger amounts in buffers of lower concentration? Sucrose density gradient centrifugation. Figure 2 shows the sucrose density gradient centrifugation of the rat liver fatty acid 3 See section on Purity of the Fatty
“Additional Studies on the Acid Synthetase Complex.”
FATTY
ACID
SYNTHETASES
FIG. 1. Sedimentation pattern of freshly prepared rat were made at 20” in a medium containing 0.5 M potassium 1 mM dithiothreitol. The rotor speed was 59,780 rpm, and graphs were made at 8-minute intervals.
145
liver fatty acid synthetase. Measurements phosphate buffer, pH 6.8, 1 mM EDTA and protein concentration was 6 mg/ml. I’hot,o-
FIG. 2. Sucrose density gradient centrifugation of rat liver fatty acid synthetase. One mg of eiizyme was subjected to a 23.5.hour centrifugation at 40,000 r-pm in a SWBOL rotor in a 5 ml, 15357c sitcrose gradient containing 0.5 1\1 potassium phosphate, pH 6.8, and 1 mM EDTA and dithiothreitol. Fatty acid synthetase activity is plotted here as the number of dpm which the whole of each fraction would incorporate into nalmitic acid from l-I%!-acetvl-CoA under the conditions of assay described in the section on “Experimental Methods.”
synthetase performed in a medium containing 0.5 M buffer. The enzyme sedimented essentially as a single component, and protein and fatty acid synthetase activity were coincident. When density gradient centrifugation was carried out in a medium containing 0.05 M phosphate buffer, two protein peaks mere found, fatty acid synthetase activity being associated wit’h the fastermoving component.” Stawh gel electrophoresis. The behavior of rat liver fatty acid synthetase on starch gel electrophoresis is different from that of the pigeon liver enzyme, which produces a single protein band under the conditions described under “Experimental Methods” (4). The rat liver fatty acid synthetase produces two protein bands under the same conditions. Later in this paper evidence
will be presented that one of these components represents a dissociation product of the intact multienzyme complex. DEAE - Cellulose chromatography. Although the pigeon liver fatty acid synthetase can be eluted from DEAE-cellulose as a single symmetrical band of protein (a), this is not the case with t.he rat liver enzyme. In the latter case, the purified enzyme appears to be irreversibly bound to the DEAE-cellulose, although the partially purified enzyme can be eluted from this adsorbent. This is evident from the fact that DEAE-cellulose chromatography is a step used in the procedure for purification of the enzyme. Additional studies on the purity of the fatty acid synthetase complex. It w-as reported
earlier in this paper that the final step in the purification of the rat liver fatty acid
146
BURTON,
HAAVIK,
AND
PORTER
FIG. 3. Sedimentation patterns obtained with rat liver fatty acid synthetase dialyzed against 0.05 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 5 mM dithiothreitol (upper) and then dialyzed against 0.5 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and 5 mM dithiothreitol (lower). The rotor speed was 59,780 rpm, protein concentrations were 6.95 mg/ml (upper) and 7.58 mg/ml (lower), and photographs were made at g-minute int,ervals.
synthetase is Sephadex G-100 gel filtration in a medium containing 0.5 M potassium phosphate buffer. The use of this buffer was necessary to prevent the dissociation of the rat liver fatty acid synthetase. When 0.2 M potassium phosphate buffer was employed in this step (as in the preparation of pigeon liver fa.tty acid synthetase) the rat liver enzyme dissociated into two protein species. In the presence of 0.5 M buffer a single protein species having an A?!&O,~value of 12.3 was obtained (Fig. 1). Figure 3 presents the sedimentation pattern obtained after rat liver fatt.y acid synthetase, prepared in 0.5 M buffer as described in “Experimental Methods” was dialyzed against 0.05 M potassium phosphate buffer. The sedimentation pattern after dialysis of the enzyme back to 0.5 M buffer concentration is also presented. In 0.05 M phosphate buffer two species of S2c,U values, 14.1 and 10.0, were present. It is evident from the data of Fig. 3 that a reversible partial dissociation of the complex into smaller component,s occurs in the presence
of 0.05 M buffer. Concomitant with the appearance of the slower sedimenting component fatty acid synthetase activity also declined. The specific activity of the enzyme as originally prepared was 62 mpmoles/ min/mg; after dialysis to 0.05 M buffer concentration it was 49.8 mpmoles/min/mg; and after dialysis back to 0.5 M buffer concentration it was 66.7 mrmoles/min/mg. The pigeon liver fatty acid synthetase exhibits a single protein species having an S20,m value of 14.5 S in 0.25 M potassium phosphate buffer (19). We have found that the S20,w value of this protein is lowered to 12.2 S at a buffer concentration of 0.5 M. This behavior parallels that of the rat liver enzyme under similar conditions, as described above, except that only one protein species is present throughout in the case of the pigeon liver system. Figure 4 shows the separation obtained between the two components of the partially dissociated rat liver fatty acid synthetase complex on sucrose gradient centrifugation. The “heavy” and “light” components sepa-
FATTY
0
2
4
6
8 FRACTlON
IO
12
14
ACID
16
I8
147
SYNTHETASES
20
NUMBER
FIG. 4. The enzyme was dialyzed against 0.05 M potassium phosphate buffer, pH 6.8, cont,aining 1 mM EDTA and dithiothreitol, and then an aliquot (lOOnl,4.5 mg protein) was applied to a 5 ml, 15-357, sucrose gradient made in the same buffer. The sample was centrifuged for 23.5 hours at 40,000 rpm in the SW5OL rotor. After fractions were collected, the t,ubes indicated were pooled, and used as heavy and light components, after concentration with Carbowax.
acid synthetase on starch gel electrophoresis, performed as described under “Experimental Methods.” It is evident that the mobilities of the “light” component and the faster moving band from the untreated fatty acid synthetase are the same, indicating that the presence of two protein species in the untreated enzyme is a reflection of dissociation due to the low ionic strength of the buffer used in the starch gel electrophoresis, rather than to the presence of an impurity. In confirmation of this conclusion, it was found that the “light” component could be converted to the “heavy” component by incubation at 38” in 0.5 M potassium phosphate buffer, pH 6.8, containing 1 mM EDTA and dithiothreitol. After such an incubation the “light” component sedimented in the same position as the “heavy” component on sucrose density gradient centrifugation. Parallel studies of enzyme activity showed that the “light” component TABLE
II
EFFECTS OF DITHIODIGLYCOLLIC ACID AND POTASSIUM MALEATE ON THE ACTIVITY OF THE RAT LIVER Enzyme
FATTY
ACID
preparation
Control Dithiodiglycollic acid treated” Dithiodiglycollic acid treated and reconstituteda Control Maleate treatedb
FIG. 5. Starch gel electrophoresis of rat liver fatty acid synthetase. (a) “Light” component from sucrose density gradient separation (Fig. 4) ; (b) untreated rat liver fatty acid synthetase. Approximately 100 pg of protein was applied to each slot of the starch gel block, and electrophoresis was carried out as described in “Experimental Methods.”
rated by this procedure were reserved for further study. Figure 5 shows the behavior of the “light” component and the untreated rat liver fatty
SYNTHETASE
Specific activity (mfimoles/min/mg) 81 1.9 46 90 1.55
a Rat liver fatty acid synthetase was prepared as described in “Methods,” and dialyzed against 0.5 M potassium phosphate buffer, pH 8, containing 1 rnM EDTA and 2 mu dithiothreitol. An equal volume of 0.2 M dithiodiglycollic acid, pH 8, was added, and the mixture was incubated at 4” for 18 hours. After analytical ultracentrifugation the protein solution was dialyzed against 0.5 M potassium phosphate buffer, pH 7, containing 1 mM EDTA and dithiothreitol. Then it was incubated with 0.01 M dithiothreitol in the same buffer at 38” for 2.5 hours. b Rat liver fatty acid synthetase prepared as described in “Methods” was dialyzed against 0.1 M maleic acid and 0.1 M potassium chloride for 42 hours at 4”.
148
BURTON,
HAAVIK,
possessed only a fraction of the activity exhibited by the original preparation. However, incubation of the “light” component at 38” in 0.5 M potassium phosphate buffer, pH 6.8, containing 1 rnM EDTA and dithiothreitol restored more than 50 % of the original enzyme activity. Quite possibly, the small activity shown by the “light” component. is due to some reassociation to the intact complex during the period of assay, which is performed at 38”. Dissociation of fatty acid synthetase with maleate, cLithiocLigLycollic acid, or palmitylCoA. Butterworth et al. (.5) have demonstrated that the pigeon liver fatty acid synthetase can be dissociated into half molecular weight subunits by disulfide exchange or by reaction of sulfhydryl groups with potassium maleate. These treatments resulted in loss of overall enzyme activity. Table II shows the effect of treatment of the rat liver fatty acid synthetase with dithiodiglycollic acid and potassium maleate, as described in the legend to the table. Both treatments inhibited enzyme activity almost completely, but parallel studies using analytical ultracentrifugation showed that the
AND
PORTER
enzyme was only partially dissociated to a slower moving component (30-40 %), in contrast to the behavior of the pigeon liver enzyme. Enzyme activity was restored by reduction of the disulfide bond between dithiodiglycollic acid and the enzyme with dithiothreitol. The inactivation by maleat,e is irreversible. l?igure 6 and Table III show the effect of micellar concentrations of palmityl-CoA on the electrophoretic mobility and enzyme activity of the rat liver fatty acid synthetase. Clearly the structural integrity of the complex is disrupted and enzymic activity is lost in this process. Molecular weight cletemination,. The molecular weight of the rat liver fatty acid synthetase in 0.5 RI potassium phosphate buffer is calculated from sediment’ation-diff usion analyses to be 5.4 X lo5 g/mole. These data demonstrate the close similarity between the rat and pigeon liver fatty acid synthetases. The latter has an S~O,~,of 14.5 (19) and a molecular weight of 4.5 X lo5 as measured by the technique of Yphantis (3, 4). A previous determination of the molecular weight of the pigeon liver fatty
FIG. 6. Effect of palmityl-CoA on rat liver fatty acid svnthetase. Rat liver fatty acid synthetase was mixed with palmityl-CoA, as indicated, in 0.2 M potassium phosphate buffer, pH 6.8. 100 pg of each protein mixture was applied t,o each slot and electrophoresis was performed as described in “Experimental Methods.” Control-slots 1 and 7; slots 2-6 (left to right), molar excess of palmityl-CoA, 300, 250, 200, 150, and 100, respectively.
FATTY TABLE EFFECT
RAT
LIVER
FATTY
Molar except of palmityl-CoA OYer enzyme
ACID
TABLE
ON
THE
ACTIVITY
and fatty acid synthetase as described in “Methods.”
Aspartic Threonine Serine
LIVER
acid
Proline Glutamic
Glycine
incorporated Palmityl-CoA activity
AMINO
Amino acid
98.5 64.9 39.0 24.5 8.6 8.4
and enzyme were mixed in the proportions
PARTIAL PIGEON
Specific activity” (m~moles/min/rrg)
0 mpmoles of l-IT-acetyl-CoA fatty acids/min/mg of protein.
cated, assayed
OF
SYNTHETASE
0 100 150 200 250 300
into
149
SYNTHETASES
III
CoA
OF PALMITYL
ACID
indiwas
acid synthetase by Ehrenberg’s method (20) and from separately determined sedimentation and diffusion coefficients, gave values of 5.67 and 5.33 X 105, respectively (19).
Alanine Valine Tsoleucine Leucine Tyrosine Phenylalanine
acid
ACID
IV
COMPOSITION
FATTY
ACID
OF
R.VJTAND
SYNTHETASFW
Rat (moles/mole of enzyme)
Pigeon (moles/mole of enzyme)
280 189 248 194 355 274 306 256 141 456 76 118
254 137 193 137 344 240 250 240 165 340 76 99
a Values for the pigeon liver fatty acid synthetase were taken from Hsu et al. (2) and corrected to a molecular weight of 450,000 g/mole.
tal Methods.“) Under these conditions taurine would arise from the terminal pComponents of Rat and Pigeon Liver Fatty mercaptoethylamine residue of 4’-phosphoAcicl Synthetases pantetheine. (a) F&win. content. Like the pigeon liver Duplicate assays of the rat liver fatty fatby acid synthetase, but unlike the yeast acid synthetase yielded a value of 1.05 moles enzyme (1, al), the rat liver enzyme con- 4’-phosphopantetheine per mole of enzyme tains negligible amounts of flavin, when (540,000 g). This compares very closely with determined by microbiological assay (16). the value of 1.12 moles per mole of enzyme The values obtained ranged from 0.006 to (450,000 g) obtained for the pigeon liver 0.024 moles of flavin per mole of enzyme. complex by Jacob, Bubterworth and Porter.4 (b) Amino acids. Table IV present’s a Characteristics of fatty acid synthetase comparison of the conbents of neutral and reaction. The dependence of fatty acid acidic amino acids of the rat and pigeon synthesis by the rat liver enzyme system liver fatty acid synthetases. It may be seen on pH is demonstrated in Fig. 7. The pH t’hat the amino acid contents of these com- optimum was found to be 6.S which complexes are very similar. pares closely to the value of 6.7 reported (c) Sulfhydryl content. The sulfhydryl con- for the pigeon liver fatty acid synthetase b! tent,s of the rat and pigeon liver enzyme Hsu, Wasson, and Porter (2). systems are quite high. The rat liver enzyme Figure 8 shows the dependence of fatty was found t’o contain 9&92 such groups acid synthetase activity on time and protein per mole (based on a molecular weight of concentration. The reaction is linear up to 540,000) in three separate assays. The 6 minutes and up to 10 pg of protein. The pigeon liver fatty acid synthetase has 64-66 K,,, for acetyl-CoA is 4.4 PM and the prod(based upon a molecular weight of 450,000) uct of the reaction has been identified as (6). free palmitic acid by radioactively moni(cl) 4’-Phosphopantetheinecontent. The 4’tored gas liquid chromatography (Fig. 9). phosphopantetheine content of the fatty Binding of acetate and maZon.atemoieties of acid synthetase was obtained by assaying the performic acid oxidized and acid hydro4 Jacob, E. J., Butterworth, P. H. W., and Porter, J. W. Arch. Biochem. Riophys. 124,392 (1968). lyzed enzyme for taurine. (See “Experimen-
150
BURTON,
HAAVIK,
1 -14C-ucetyl-CoA tend 2J4C malony WoA to emyme. The binding of the acetyl and malonyl groups of acetyl- and malonyl-CoA to the rat liver fatty acid synthetase is illustrated in Fig. 10. Binding was performed at O”, since loss of bound substrate by deacylation was minimal at this temperature. The amount of substrate bound varied with the molar excess of substrate over enzyme. Fig.
PH
7. Dependence of fatty acid synthetase activity on pH. Assay conditions were as described in “Experimental Methods” except that the pH was varied as shown. 4.03 pg of enzyme protein was used in each assay. FIG.
AND
PORTER
10 shows binding data obtained using molar excesses between lo- and 20-fold. Under these conditions the enzyme bound close to 2 moles of substrate per mole. Assay for malonyl-CoA decarboxylase activity. Purified rat liver fatty acid synthetase was assayed for malonyl-CoA decarboxylase activity as described in “Experimental Methods.” The specific activity of the enzyme under these conditions was 4.7, compared to a value of 77, when both acetyland malonyl-CoA were present as substrates. Formation of /3-hydroxy-P-methyl glutarylCoA and TAL by the fatty acid synthetase. Gas-liquid chromatographic assay for the formation of TAL by rat and pigeon liver fatty acid synthetases provided proof that both enzyme complexes synthesized small amounts of this compound, from acetyland malonyl-CoA in the absence of NADPH. The rat liver enzyme incorporated 11.52 mpmoles lJ4C-acetyl-CoA/min/mg into TAL, and the corresponding value for the pigeon liver fatty acid synthetase was 7.05 mpmoles/min/mg, under the same conditions. The two fatty acid synthetases were also shown to produce small amounts of HMGCoA. The latter compound was converted to the methyl ester and assayed by gasliquid chromatography.
70-
b ; I
50-
0
: g 407 z z5
0
30-
% zo2
100 80 60
0
40 0
0
IOm
60- :,i... 0
20 2
4
6
6
TIME IN MINUTES
IO
12
2
6 IO 14 ,q OF PROTEIN
FIG. 8. Dependence of fatty acid synthetase activity on time and protein concentration. The standard assay system was used, except that time and protein concentration were varied as shown. For the time study 3.42 pg of enzyme protein was used; a IS-minute assay period was employed for the protein dependence study.
FATTY
0
5
ACID
151
SYNTHETASES
IO 15 TIME IN MINUTES
20
25
Fro. 9. Identification of product of reaction catalyzed by rat liver fatty acid synthetase. The product of the reaction, carried out by the normal assay method, was extracted from the reaction mixture with petroleum ether. The solvent was removed under Ns and a solution of diazomethane in diethyl ether was added. After reaction excess ether and diazomethane were removed under Nz, and ,the methyl esters were mixed with a standard mixture of nonradioactive fatty acid methyl esters. This mixture was subjected to radioactively monitored gas-liquid chromatography on a 6’-butanediol succinate column supported on chromosorb W. The operating temperature was 198” and the rate of flow of the carrier gas (helium) was 95 ml/minute. The instrument used was a Barber-Colman Selecta-System Series 5090. DISCUSSION
FIG. 10. Binding of I-14C-acetyl-Cob and 2J4Cmalonyl-CoA to rat liver fatty acid synthetase at 0”. Fatty acid synthetase was incubated at 0” with l-I%-acetyl or 2-14C-malonyl-CoA for the time periods indicated. The protein was precipitated with 60% perchloric acid, and excess substrate was removed by repeated washing with 0.2 N acetic acid. Residual protein was dissolved in I N NaOH and aliquots were taken for radioactivity and protein determinations.
It is now well established that all the enzymic components required for the biosynthesis of long-chain fatty acids from acetyland malonyl-CoA by yeast (1, 21) and pigeon liver (2) reside in single stable protein complexes. In the present paper it has been demonstrated that the rat liver fatty acid synthetase is also a multienzyme complex. This enzyme system was isolated in 0.5 M potassium phosphate buffer as an essentially homogenous protein with respect to molecular weight. The enzyme sedimented as a single protein species on analytical ultracentrifugation and sucrose density gradient centrifugation. Protein and enzyme activity peaks were also coincident in the latter system. The rat liver fatty acid synthetase yielded
152
BURTON,
HAAVIK,
two bands on st,arch gel electrophoresis. However, it was proved that one of these bands was a dissociation product of the complex. Hence it is concluded that the rat liver fatty acid synthetase was also obtained as an electrophoretically homogenous species. The procedure used to purify the rat liver fatty acid synthetase is very similar to the procedure used to purify the analogous enzyme system from pigeon liver. Only minor variations exist between the two procedures. However, these variations are vital for the isolation of a homogenous preparation. The properties of the pigeon and rat liver fatty acid synthetases are very similar in many respects. Thus the two enzymes had almost the same specific activities at each step in the purification procedure. They also have approximately the same molecular weight. The rat liver fatty acid synthetase exhibits a molecular weight of 5.4 X lo5 g/mole (in 0.5 M buffer) as calculated from separately determined sedimentation and diffusion constants. This compares quite closely to the best estimate of the molecular weight of the pigeon liver enzyme, which is 4.5 X lo5 g/mole (4). Another indication of the similarity of the two enzymes is found in the fact that the S20,W of both complexes is altered on changing the concentration of buffer in which the protein is dissolved. The pigeon liver fatty acid synthetase exhibits an SzO,W value of 14.5 in 0.25 M potassium phosphate buffer (19)) but in 0.5 M buffer the A!&,~ is 12.2. Similarly, the rat liver enzyme shows a change in &I,~ from 14.1 S to 12.3 S when the buffer concentration is changed from 0.05 M t0
0.5
M.
The rat liver fatty acid synthetase exhibits essentially the same specific enzyme activity when assayed in 0.5 M potassium phosphate as it does when assayed in 0.1-0.2 M buffer. This result may be interpreted as meaning that both 14.1 and 12.3 S species possess the same enzymic activity, and that the change in SZoZwvalue probably reflects a conformational rather tha,n a molecular weight change. Further support for this hypothesis is found in the fact that the pigeon liver enzyme exists as a single protein
AND
PORTER
species under both sets of conditions. If a molecular weight change were involved in the change in &Y,,~ values, it would be expected that two protein species would be present when the complex had the lower S20,W value. Thus in terms of sedimentation behavior and molecular weight, the pigeon and rat liver fatty acid synthetases appear to be very similar. The similarity between the two fatty acid synthetases is found in other properties of these complexes. Both fatty acid synthetases contain negligible quantities of flavin, in contrast to the yeast system and the flavin-dependent acyl-CoA dehydrogenase of the fatty acid oxidation cycle. The sulfhydryl contents measured in the presence of the dissociating agent guanidine hydrochloride were found to be 90-92 and 64-66 groups per mole for the rat and pigeon liver systems, respectively (based on a molecular weight of 540,000 for the rat and 450,000 for the pigeon liver fatty acid synthetases). Again in contrast to the yeast system, which produces stearyl- and palmityl-CoA both rat and pigeon liver (22, 23) fatty acid synthetases produce free palmitic acid. The rat liver enzyme complex binds the acetate and malonate moieties of acetyland malonyl-CoA to approximately the same extent as the pigeon liver system and both complexes appear to contain only 1 mole of 4’-phosphopantetheine per mole of enzyme. (The latter finding must be considered when a mechanism of action for this enzyme is postulated.) Both enzymes possess some malonyl-CoA decarboxylase activity and each enzyme synthesizes HMG-CoA and triacetic acid lactone from acetyl- and malonyl-CoA in the absence of KADPH. The synthesizing activity for the formation of TAL purifies with the pigeon liver fatty acid synthetase.5 Similar analyses have not been made for the rat liver enzyme system. The pH optima and the overall amino acid composition of the two enzymes are also very similar. The most important difference between the two enzyme systems concerns their dissociation. The pigeon liver fatty acid 5 Nixon, observations.
J. E.,
and Porter,
J. W.,
Unpublished
FATTY
ACID
SYNTHETASES
153
synthetase has been shown to be stable for sulfhydryl groups (5). Similar treatment, of at least 5 days when stored in a medium the rat liver fatty acid synthetase with containing 0.2 M potassium phosphate dithiodiglycollic acid or potassium maleate buffer, 1 mM EDTA and 1 mu dithiothreitol resulted in the complete loss of enzymic (4). However, if P-mercaptoethanol is used activity. However, in each case dissociation in place of dithiothreitol, the complex disso- into smaller subunits proceeded only to the ciates into approximately half molecular extent of 3&40 %, and in addition, it is likely weight subunits over a period of several that a proportion of this dissociation was days. An equilibrium of about equal propor- due to the use of buffer concentrations betions of original complex and half molecular low 0.5 M. It appears probable from this weight units is finally reached (5). This result that sulfhydryl groups are either less processis referred to as ‘
154
BURTON,
HAAVIK,
AND
ACKNOWLEDGMENTS The attthors wish to express t,heir thanks to Professor R. M. Bock for useful discussions and for obtaining some of the analytical ultracentrifugation data, and to Professor H. F. Detttsch for the determination of the diffusion constant of the rat liver fatty acid synthetase. We are also grateful to Professor Theo Gerritsen and Mr. J. Campbell for taurine and amino acid analyses, and to Mr. G. Velasquez for excellent technical assistance in the performance of ultracentrifugation analyses. We wish to thank Dr. Maria Burger of the Wisconsin Alumni Research Foundation for performing assays for riboflavin content of the rat liver fatty acid synthetase. REFERENCES 1. LYNEN, F., HOPPER, I., LORCH, E., KIRSCHNER, K., HAGEN, A., AND SCHWEITZER, E., Proc. 6th International Congress Biochem., 1964, New York, VII, S-2. 2. Hsu, R. Y ., WASSON, G., AND PORTER, J. W., J. Biol. Chem. MO, 3736 (1965). 3. YPHANTIS, D. A., Biochemistry 3, 297 (1965). 4. YANG, P. C., BUTTERWORTH, P. H. W., BOCK, R. M., BND PORTER, J. W., J. Biol. Chem. 242,350l (1967). 5. BUTTER~ORTH, P. H. W., YANG, P. C., BOCK, R. M., :IND PORTICR, J. W., J. Biol. Chem. 242, 3508 (1967). 6. BUTTERWORTH, P. H. W., BAUM, H., AND PORTER, J. W., drch. Biochem. Biophys. 118, 716 (1967). 7. BUTTICR~ORTH, P. H. W., GUCHHAIT, R. B., BAUM, H., OLSON, E. B., MARGOLIS, S. A.,
8. 9. 10. 11. 12.
13. 14. 15. 16.
17. 18. 19.
20. 21. 22. 23.
PORTER
AND PORTER, J. W., Arch. Biochem. Biophys. 116, 453 (1966). SIMON, E. J., .IND SHEMIN, D. J., J. Amer. Chem. Sot. 76, 2.520 (1953). TR.IMS, E. G., .\ND BRADY, R. O., J. Amer. Chem. Sot. 82, 2972 (1960). BRODIF:, J. D., .IND PORTER, J. W., Biochem. Biophys. Res. Commun. 3, 173 (1960). GORNALL, A. G., B.~RD.IWILL, C. J., AND DAVID, ibf. >I., J. Biol. Chem. 177, 751 (1949). W.IKIL, S. J., PORTER, J. W., .IND GIBSON, Il. M., Biochim. Biophys. Acta 24, 453 (1957). KORNACKE:R, iU. S., AND LO~ENSTEIN, J. M., Biochem. J. 94,209 (1965). ?rlSRTIN, R. G., .IND Awns, B. N., J. Biol. Chem. 236, 1372 (1961). BOYIZR, S. H., F.IINER, D. C., AND N;BUGHTON, XI. A., Science 140, 1228 (1963). “Official Methods of Analysis of the Association of Official Agricultural Chemists,” p. 773,10th Ed. 1965. W. Horwitz, Ed. Assoc. Official Agric. Chem., Washington, D. C. &lOORI?, S., J. Biol. Chem. 238, 235 (1963). MOORF:, S., .&ND STEIN, W. H., Anal Chem.. 30, 1190 (1958). YANG, P. C., BOCK, R. M., Hsu, R. Y., AND PORTEZR, J. W., Riochem. Biophys. Acta 110, 608 (1965). EHRICNRERG, A., dcta Chem. &and. 11, 1257 (19,57). LYNEN, F., Fed. Proc. 20,941 (1961). PORTER, J. W., .IND TI~TZ, A., Biochim. Biophys. Acta 26, 41 (1957). BRISSSLER, R., .IND W.IKIL, S. J., J. Biol. Chem. 237, 1441 (1962).