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Apo- and holofatty acid synthetases from pigeon liver
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Apo- and Holofatty
181,
580-590
(1977)
Acid Synthetases
from Pigeon
Liver’
MANOK KIM, ASAF A. QURESHL2 ROBERT A. JENIK, FRANK A. LORNITZO, AND JOHN W. PORTER Lipid
Metabolism
Laboratory, Veterans Administration Chemistry, University of Wisconsin, Received
September
Hospital, and Department Madison, Wisconsin 53706
of Physiological
10, 1976
Two forms (an apo- and a holoenzyme) of the fatty acid synthetase complex from pigeon liver were separated by affinity chromatography on a Sepharose+aminocaproyl pantetheine column. The difference between these enzymes is the presence or absence of the prosthetic group, 4’-phosphopantetheine. Due to the absence of the prosthetic group, apofatty acid synthetase lacks the overall ability to synthesize fatty acids, and it has no /3-ketoacyl synthetase (condensing enzyme) activity. These two forms of enzyme were shown to be homogeneous and they behaved identically on DEAE-cellulose chromatography, gel filtration, sucrose density gradient centrifugation, disc gel electrophoresis, and immunodiffusion. The isolation and purification of an apoacyl carrier protein are also reported. The apoacyl carrier protein lacks p-alanine and it has no sulfhydryl group, indicating therefore that the acyl carrier protein from apofatty acid synthetase does not have a 4’-phosphopantetheine group. The transfer of 4’-phosphopantetheine from CoA to apofatty acid synthetase was effected by an enzyme system present in the supernatant of pigeon liver homogenate. The resulting product of this reaction was the holofatty acid synthetase. The reverse reaction, the formation of apo- from holofatty acid synthetase, was also demonstrated. The physiological significance of this system is suggested from studies carried out on fasting and refeeding of pigeons. At early times of refeeding (O-4 h) there is a large amount of apoenzyme. The amount of holofatty acid synthetase increases after 4 h of refeeding and the apofatty acid synthetase decreases. When pigeons are refasted, after refeeding for 48 h, the amount of apoenzyme increases and the holoenzyme decreases.
The presence of two enzymes in Escherichia coli which are responsible for the interconversion of holo- and apoacyl carrier protein by the removal or addition of the 4’-phosphopantetheine prosthetic group has been reported (1, 2). Similarly, Tweto et al. (3) have shown that prosthetic group turnover occurs in rat liver fatty acid synthetase, thereby suggesting that the addition and removal of 4’-phosphopantetheine plays a regulatory role in the activity of this enzyme. Recently Yu and
Burton (4, 5) demonstrated indirectly the presence of an apoenzyme in rat liver. They also showed that a crude supernatant liver enzyme system converted some of the inactive apoenzyme to enzyme having fatty acid synthetase activity in the presence of coenzyme A and ATP. This result suggested the existence of an enzyme capable of carrying out the transfer of the 4’-phosphopantetheine group from CoA to apoacyl carrier protein. More recently Roncari (6), using an enzyme preparation from liver of fasted rats, has shown an inactivation of rat and human liver fatty acid synthetases which is accompanied by the release of the 4’-phosphopantetheine prosthetic group. The further study of the enzyme systems responsible for the removal and addition of 4’-phosphopantetheine to the fatty acid
1 This investigation was supported in part by a grant (AM-01383) from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service. 2 Present address: Waisman Center, Neurochemistry Section, 2605 Marsh Lane, Madison, Wis. 53706. 580 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003-9661
APO- AND
HOLOFATTY
synthetase complex requires the development of a method for the separation of apo and holo forms of fatty acid synthetase from each other. Such a method, namely, the separation of apo- and holofatty acid synthetases by affinity chromatography, was recently reported by us (7). This method gives a quantitative separation of the two forms. Hence, it can be used to determine the extent of conversion of one form to another and for the preparation of pure apo- and holoenzyme for use as a substrate. This method also provides a dependable procedure for the complete removal of endogenous fatty acid synthetase from a liver supernatant solution that is to be assayed for the interconversion of apoand holoenzyme species. In the present study we report further on the separation of apo- and holofatty acid synthetases and on the properties of these two forms of enzyme. We also present further evidence for an enzyme system in eukaryotes capable of transferring 4’-phosphopantetheine to apoacyl carrier protein of fatty acid synthetase (4’-phosphopantetheine transferases) and for an enzyme system that removes the prosthetic group from acyl carrier protein (4’-phosphopantetheine transferase) and for an enzyme the results of studies on the effect of nutritional state on the variation in quantity of these two forms of enzyme in liver. These results suggest that the apo to holo interconversion may play a short-term regulatory role in fatty acid synthesis. MATERIALS
AND
METHODS
Acetyl-CoA, malonyl-CoA, and palmitoyl-CoA were obtained from P-L Biochemicals, and [lWlacetyl-CoA, [2-Wlmalonyl-CoA, [Wpantothenic acid, [‘Qsodium bicarbonate, and [l-14ClpalmitoylCoA were purchased from New England Nuclear. S-acetoacetyl-N-acetyl cysteamine, NADPH, and DEAE-cellulose were obtained from Sigma.3 DL-S-/3hydroxybutyryl-N-acetyl cysteamine was prepared as described by Kumar et al. (8) with an additional thin-layer chromatographic purification on silica gel H with a 10% methanol, 90% chloroform solvent system. S-crotonyl-N-acetyl cysteamine was pre3 Abbreviations used: DEAE-, diethylaminoethyl; FAS, fatty acid synthetase; ACP, acyl carrier protein.
ACID
SYNTHETASES
581
pared according to the method of Kumar et al. (81 with an additional purification on a Florisil (60-100 mesh) column (2 x 30 cm) with anhydrous benzene as the eluting agent. Sepharose-e-amino n-caproic acid was prepared according to the method of Larsson and Mosbach (9). Crystalline pantethine, obtained from Sigma, was reduced to pantetheine with sodium amalgam prior to use. The l-ethyl-3-(3dimethyl-aminopropyl) carbodiimide-HCl used in the preparation of the Sepharose-c-aminocaproyl pantetheine column was purchased from Sigma and Aldrich Chemical Co. The pigeons used in this study were fed Purina pigeon grain. Purificatiolt offatty acid synthetase. Pigeon liver fatty acid synthetase was purified according to the method of Hsu et al. (101, except that the final ammonium sulfate fractionation was 25 to 40% instead of 26 to 32%. Assay for partial and overall reactions of fatty acid synthesis. Acetyl-CoA and malonyl-CoA transacylase activities were assayed as previously described (ll), and P-ketoacyl thioester reductase, fl-hydroxybutyryl thioester dehydrase, and crotonyl thioester reductase were assayed as described by Kumar et al. (81, with the modifications described by Lornitzo et al. (12). The condensation reaction (pketoacyl synthetase) and palmitoyl-CoA deacylase were assayed as described previously (8). The assay for overall fatty acid synthetase activity was performed as reported by Kumar et al. (8). Protein estimation. Protein was estimated by the methods of Lowry et al. (13) and Murphy and Kies (14). Protein was also determined by light absorbance at 280 nm. Chromatography on Sepharose-E-aminocaproyl pantetheine. DEAE-cellulose-purified fatty acid synthetase was subjected to chromatography on Sepharose-e-aminocaproyl pantetheine as reported previously (7). The apo and holo fractions separated by this technique were then subjected to the analyses reported in the following sections. DEAE-cellulose chromatography. DEAE-cellulose anion exchanger (0.93 mequiv/g, coarse mesh) was prepared for use by soaking overnight in 1 M potassium phosphate, pH 7.0, and then washing thoroughly with deionized water. The anion exchanger was finally resuspended in 40 mxu potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Two milligrams (1 ml) of either holo- or apofatty acid synthetase was diluted to 20 ml with deionized water to bring the ionic strength to 20 mM potassium phosphate, and the enzyme was adsorbed on a DEAE-cellulose column (1 x 10 cm) at room temperature. The column was then washed with 30 ml of 40 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. The apo- or holofatty acid synthetase was eluted from the column with a linear gradient, 40 ml each of 0.05 to 0.3 M potassium phosphate, pH 7.0, contain-
582
KIM
ET AL.
ing 1 rn~ EDTA and 1 mM dithiothreitol. Eluate fractions of 1 ml were collected. Bio-Gel filtration of apo- and holofatty acid synthetases. A Bio-Gel A-1.5m column (1.2 x 26 cm) was equilibrated with 0.2 M potassium phosphate, pH 7.0,1 mM EDTA, and 2 mM 2-mercaptoethanol. Four milligrams of either apo- or holofatty acid synthetase was loaded onto the column and eluted with the above buffer. One-milliliter fractions were collected at a flow rate of 1 ml/5 min. Sucrose density gradient centrifugation. Sucrose density gradient centrifugation of apo- and holofatty acid synthetases was performed with 5 linear gradient of 5 to 20% sucrose (19 ml of each solution) in 0.2 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. Centrifugation was carried out at 58,000g for 24 h at 20°C. Onemilliliter fractions were collected for each fatty acid synthetase. Preparation of antiserum and Ouchterlony double diffusion analysis. Rabbit antiserum to DEAE-cellulose-purified pigeon liver fatty acid synthetase was prepared according to the method of Collins et al. (15). Ouchterlony micro-double diffusion analysis with rabbit antiserum and preparations of pigeon liver apo- and holofatty acid synthetases was then carried out using 0.5% agarose gel. High voltage electrophoresis of [Wlacetyl-labeled apo- and holofatty acid synthetuse peptic peptides. One milligram of apo- or holofatty acid synthetase was incubated with [I-Wacetyl-CoA and then treated with pepsin as described previously (12). The pepsin-digested protein was subjected to high voltage paper electrophoresis on Whatman 3 MM chromatography paper at pH 3.7 for 90 min at 2500 V. One-centimeter strips were cut from the paper, placed in vials containing 15 ml of dioxane scintillation solution, and assayed for radioactivity in a Packard Tri-Carb liquid scintillation spectrometer. Isolation of apo- and holoacyl carrier protein. The apo- and holofatty acid synthetase fractions were separately combined after affinity chromatography and then dialyzed in Tris-glycine (5~35 mM) containing 5 mM 2-mercaptoethanol at 0°C and pH 8.3 for 4 h with two changes of buffer. The dialyzed enzyme was then subjected to ultrafiltration using a Diaflo PM-10 membrane. The filtrates contained approximately 80% of the acyl carrier protein, apoacyl carrier protein when apofatty acid synthetase was used, and holoacyl carrier protein when holofatty acid synthetase was used. The filtrates were lyophilized and the residues were dissolved in water andpurified by passing through Sephadex G-10, G-25, and G-50 columns (1.1 x 25 cm). The acyl carrier proteins were eluted with 10 mM potassium phosphate, pH 7.0, containing 5 mM 2-mercaptoethanol. All steps involving acyl carrier protein were carried out with quartz-distilled water.
Transfer of the [‘YJlacetyl group from acetyl-CoA to holo- and apoacyl carrier protein. Apo- and holoacyl carrier proteins (0.5 mg each) obtained from the Sephadex G-50 column were incubated with [l14C]acetyl-CoA and pigeon liver fatty acid synthetase (10 pg). The product of this reaction was purified as described previously (16). Amino acid analyses of the acyl carrier proteins. Each acyl carrier protein was hydrolyzed with constant-boiling hydrochloric acid under reduced pressure at 110°C for 22 h. Amino acid analysis was then carried out on a Beckman Model 120 amino acid analyzer equipped with a high-sensitivity cuvette and a recorder scale expansion according to the method of Spackman et al. (17) as modified by Gerritaen et al. (18). Titration of sulfhydryl groups. Each acyl carrier protein (4-5 nmol) was incubated 30 min at 30°C in 20 mM 2-mercaptoethanol to reduce disulfide bonds (19) and then dialyzed for 4 h at 0°C in deionized water with three changes. The determination of sulfhydryl groups was carried out by the spectrophotometric method of Ellman (20). Polyacrylamide disc gel electrophoresis. Five percent polyacrylamide gels, prepared by the method of Hedrick and Smith (21), were used for disc gel electrophoresis. Preparation of an enzyme fraction containing 4’phosphopantetheine transfemse activity. This enzyme system was prepared free of fatty acid synthetase as described previously (7). An alternative method of removing fatty acid synthetase involved the heating of 1 ml of the 100,oOOg supernatant of pigeon liver (S,) for 2.5 min at 56°C. The denatured protein was removed by centrifugation and the supernatant solution was assayed for fatty acid synthetase activity. This heated supernatant fraction was devoid of fatty acid synthetase activity. A ‘2140% ammonium sulfate-precipitated fraction of the heated supernatant solution was prepared and then treated in the same way as previously reported (7). In both cases the enzyme fraction was prepared from the liver of a pigeon refed for 12 h following a 48-h fast. Preparation of an enzyme fraction containing 4’phosphopantetheine hydrolase activity. A 55-75% ammonium sulfate-precipitated fraction was prepared from the 100,OOOg supernatant solution of liver homogenate from a pigeon that had been refed for 48 h and then refasted for 12 h (22). Incorporation of [%lpantothenic acid into pigeon liver fatty acid synthetase on refeeding fasted birds. [‘ClPantetheine-labeled pigeon liver fatty acid synthetase was obtained from birds fasted 48 h and then refed a fat-free diet (short-grain rice) for 0, 2, 4, 12, 24, or 48 h. The pigeons were injected intravenously with [‘Y!]pantothenic acid (0.025 mCi/pigeon) 4 h prior t.0 sacrifice.
APO- AND
HOLOFATTY
RESULTS
Separation of ApoSynthetases
and Holofatty
Acid
DEAE-cellulose-purified [14Clpantetheine-labeled fatty acid synthetase from 48-h refed pigeons was separated into apo- and holofatty acid synthetases by chromatography on Sepharose-•-aminocaproyl pantetheine as reported previously (7). In the present study 20 mg of fatty acid synthetase protein (13, 500 dpm) were chromatographed on 4 g of gel in an 8.5-mm x lo-cm column. The apofatty acid synthetase (2.7 mg of protein) was eluted in 15 ml of 0.06 M potassium phosphate buffer, pH 7.0, containing 2 mM dithiothreitol, and the holofatty acid synthetase (16.6 mg of protein) was eluted in 40 ml of 100 mM Tris-100 mM phosphate buffer, pH 7.0, containing 2 mM dithiothreitol. Assays were then carried out for fatty acid synthetase and palmitoyl-CoA deacylase activities for both forms of fatty acid synthetase, and a 200~1 aliquot of each fraction was assayed for radioactivity. Radioactive pantetheine (13,000 dpm) and overall fatty acid synthetase activity were found only in association with the active (holo) fatty acid synthetase. The palmitoyl-CoA deacylase activity was associated with both active (holo) and inactive (apo) fatty acid synthetases. Table I shows the results obtained in assays of apo- and holofatty acid synthetases for all of the partial reactions of fatty acid synthesis and for the overall reaction. It can be seen that apofatty acid synthetase lacks only the overall fatty acid synthetase and /3-ketoacyl synthetase (condensing enzyme) activities. Physical Properties of APO- and Holofatty Acid Syn thetases DEAE-cellulose column chromatography. The apo- and holofatty acid synthe-
tases were adsorbed separately on a DEAE-cellulose column and eluted as described in the methods section. In each separation, the protein was eluted as a single sharp band (Figs. 1A and B), thus indicating the homogeneity of the apo- and holofatty acid synthetases. Again, the apofatty acid synthetase lacks both overall
ACID
583
SYNTHETASES
fatty acid synthetase and /3-ketoacyl synthetase activities. Bio-Gel filtration and sucrose density gradient centrifugation. The size and homogeneity of the apo- and holofatty acid synthetases were determined by filtration over a Bio-Gel A-1.5m column (Figs. 2A and B). The proteins were eluted in the same fractions, indicating that they ,have similar molecular weights. The size of each complex was verified further by sucrose density gradient centrifugation as reported in the methods section. Again, both apo- and holofatty acid synthetases migrated similarly. Immunochemical
studies
Apo- and holofatty acid synthetases cross-react with antiserum made to DEAE-cellulose-purified fatty acid syntbetase. In con&nation of previous results (7) it was shown that apo-, holo- and BioGel-purified fatty acid synthetases gave reactions of complete identity, thus illustrating that they are immunologically identical. High
Electrophoresis of Voltage [“+Z’]Acetyl-Labeled APO- and Holofatty Acid Synthetase Peptic Peptides
The high voltage electrophoresis at pH 3.7 of [Wlacetyl-labeled peptic peptides of holo- and apofatty acid synthetases is shown in Figs. 3A and B. The holofatty acid synthetase peptides gave peaks corresponding to the AZ, B1, and B, components (4’-phosphopantetheine and hydroxyl and cysteine binding sites, respectively) reported by Kumar et al. (8). The [l14Clacetyl-labeled peptic peptides of apofatty acid synthetase showed a radioactive peak for only the B, (hydroxyl) component. The lack of the cysteine site in the apofatty acid synthetase confirms earlier studies that the prosthetic group, 4’-phosphopantetheine, is necessary for the transfer of acetyl groups to the cysteine site and for pketoacyl synthetase activity. The Isolation of Apo- and Holoacyl rier Proteins
Car-
The lack of the 4’-phosphopantetheine site in the peptic peptides of apofatty acid
584
KIM TABLE
ET AL.
I
ENZYME ACTIVITIEEI OF Am- AND HOLOFATTY ACID SYNTHETASEB Reaction Enzyme source DEAE" HoloFAS ApcFAS (nmol of product formedlminlmg of protein) Fatty acid synthesis Acetyl-CoA transacylase Malonyl-CoA transacylase P-Ketoacyl synthetase fl-Ketoacyl reductase P-Hydroxyacyl dehydrase Enoyl reductase Palmitoyl-CoA deacylase -.
85-100
122-130
-
350
402
370
100
135
130
4.3
4.7
-
142
214
187
270
316
292
240 43
277 57
259 61
’ The DEAE-cellulose-purified fatty acid synthetase was obtained from 48-h refed pigeons.
--
synthetase was confirmed by the isolation and purification of apoacyl carrier protein (Fig. 4B). The elution profiles, on Sephadex G-50, of apo- and holoacyl carrier proteins (Figs. 4A and B) indicate similar molecular weights. The inability of apoacyl carrier protein to accept the [14C]acetyl group in the presence of pigeon liver fatty acid synthetase further indicates that apoacyl carrier protein lacks 4’-phosphopantetheine (Figs. 4C and D). This conclusion is supported by the finding that apoacyl carrier protein lacks both P-alanine and the sulfhydryl group associated with holoacyl carrier protein (Table II). Otherwise the amino acid compositions of the two acyl carrier proteins are very similar. Disc Gel Electrophoresis of APO-, Holo-, and DEAE-Cellulose-Purified Fatty Acid Synthetases
The migrations of the apo-, holo-, and DEAE-cellulose-purified fatty acid synthe-
FRACTK)N NUMBERS
FIG. 1. Comparison of the behavior of (A) holofatty acid synthetase and (B) apofatty acid synthetase on DEAE-cellulose chromatography. A DEAE-cellulose column (1 x 10 cm) was equilibrated with 0.04 M potassium phosphate, pH 7.0, and then 2 mg of enzyme protein in 0.02 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 1 mre dithiothreitol, was loaded onto the column. The enzyme was eluted with a linear gradient, 0.05 to 0.3 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol. Light absorbance at 280 nm (O-O); fatty acid synthetase activity in nanomoles of NADPH oxidized per minute per milliliter (0-O); P-ketoacyl thioester reductase activity in nanomoles of NADPH oxidized per minute per milliliter (0 - -0); acetyl-CoA transacylase activity in counts per minute of [Wlacetyl groups transferred to pantetheine per minute per milliliter (A-. -A); and pketoacyl synthetase activity in counts per minute of 14Cof potassium bicarbonate incorporated into malonyl-CoA per minute per milliliter (A-A).
APO- AND
HOLOFATTY
ACID
585
SYNTHETASES
A H
HOLO-
FAS
6.0 9
il x -E -4.0
”
-2.0
T 0 x
-g
z \
I E B - 3.0 u z Q 2 -1.5 E4
f 3
?+ N ” 58
0.6-
0.4-
h c!a 0.3-
0.2-
0
IO FRACTION
20 NUMBERS
FIG. 2. Comparison of (A) holofatty acid synthetase and (B) apofatty acid synthetase on BioGel A-1.5m gel filtration. Protein, 4 mg, was loaded onto a Bio-Gel column (1.2 x 26 cm) previously equilibrated with 0.2 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. Elution was effected with the same buffer and l-ml fractions were collected at room temperature. Light absorbance at 280 mn (O-O); fatty acid synthetase activity in nanomoles of NADPH oxidized per minute per milliliter (0-O); p-ketoacyl thioester reductase activity in nanomoles of NADPH oxidized per minute per milliliter 0 - -0); palmitoyl-CoA deacylase activity, counts per minute of [14C1palmitate formed per minute per milliliter (O- - -0); /3-ketoacyl synthetase activity, counts per minute of [WI of potassium bicarbonate incorporated into malonyl-CoA per minute per milliliter (A-A).
tases were nearly identical on disc gel electrophoresis (Fig. 5), thus indicating a close similarity between these proteins. Similarly, the apo- and holoacyl carrier proteins migrated nearly the same distance on disc gel electrophoresis. The above results suggest that the only difference between the apo- and holofatty acid synthetases is the presence or absence of the 4’phosphopantetheine prosthetic group on the acyl carrier protein. 3a
20
20 IO ORIGIN IO DISTANCE FROM THE ORIGIN IN cm
30
FIG. 3. Analysis of [lJ4C]acetyl-bound peptic peptides from (A) holofatty acid synthetase and (B) apofatty acid synthetase by high voltage paper electrophoresis at pH 3.7. See the experimental section for details.
Enzymatic Interconversion of ApoHolofatty Acid Synthetases
and
Conversion of apo- to holofatty acid synthetase. The apofatty acid synthetase is
converted to holofatty acid synthetase by incubation with CoA, ATP, and a 4’-phos-
586
KIM
ET AL.
0 FRACTCN
NUMBER
10 FRAcnm
20 MJ.mER
FIG. 4. Gel filtration of (A) holo- and (B) apoacyl carrier protein on a Sephadex G-50 column. The filtrate from ultrafiltration on a PM-10 membrane of dissociated holo- or apofatty acid synthetase was lyophilized and the residue was dissolved in 1 ml of 10 mM potassium phosphate buffer, pH 7.0, containing 20 mM 2-mercaptoethanol and incubated at 30°C for 20 min. This solution was passed through a Sephadex G-50 column (1.1 x 25 cm). Elution was carried out with 10 mM potassium phosphate buffer, pH 7.0, and 5 mM 2-mercaptoethanol, and l-ml fractions were collected at a rate of 1 ml/3 min. Fractions 18-24 were pooled and lyophilized, and the residue was dissolved in 1 ml of 10 mM potassium phosphate buffer. Sephadex G-50 filtration of [‘Qacetyl-labeled acyl carrier protein (Cl and (D). [WAcetyl groups were transferred to acyl carrier protein as reported in the methods section. Aliquots of 1.2 ml were collected. Light absorbance at 280 nm (O---O); radioactivity in counts per minute per milliliter (O- - - -0):
phopantetheine transferase enzyme frac- Nutritional Studies, tion prepared as described in the methods section. The maximum activity for the The amount of DEAE-cellulose-purified transferase was found in the 21-40% am- fatty acid synthetase, the specific activity monium sulfate precipitate of the 100,OOOg for fatty acid synthesis, and the amount of supernatant of liver homogenate of 12-h [14Clpantothenic acid incorporated into enrefed pigeons. The fatty acid synthetase of zyme are plotted as a function of time of this fraction was removed prior to incuba- refeeding in Fig. 6. It is evident that tion by affinity chromatography as de- [14Clpantothenic acid is not incorporated scribed previously (7). The amount of con- into fatty acid synthetase during the early version of apo- to holofatty acid synthetase hours of refeeding. It is also evident that after incubation with the transferase frac- the increase in specific activity for the tion is reported in Table III. The separa- fatty acid synthetase parallels the intion of the two forms was achieved by af- creased amount of [14C]pantothenic acid finity chromatography, and then the incorporated into enzyme. The amounts of amount of apoenzyme was determined fol- apo- and holofatty acid synthetases preslowing DEAE-cellulose chromatography. ent at the various times of refeeding are Conversion of holo- to apofatty acid synplotted in Fig. 7. The data clearly indicate the&se. The conversion of holo-.to apofatty an increased amount of apofatty acid synacid synthetase in the presence of an am- thetase during the early hours of refeedmonium sulfate-precipitated fraction of pi- ing. However, as the time of refeeding progeon liver supernatant solution was also gresses, there is a decrease in apofatty acid demonstrated (Table IV). However, the synthetase and an increase in the amount extent of conversion of holo- to apo- was of holoenzyme. In a related study, the efless than the conversion of apo- to holoen- fects of both refeeding and refasting on the zyme . amounts of apo- and holofatty acid synthe-
APO- AND TABLE
HOLOFATTY
II
AMINO ACID COMPOSITIONS OF Hou)CARRIER PROTEINS Amino acid residue
HoloACP”
p-Alanine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.80 7.9 3.6 13.7 11.5 5.7 13.6 5.5 3.8 1.1 1.9 3.7 1.8 1.9 3.8 1.3 1.4
Sulfhydryl groups/ mole of prot,ein
AND APOACYL ApoACP”
1 8 4 14 12 6 14 6 4 1 2 4 2 2 4 1 1
0
0
7.6 3.7 13.4 12.4 6.6 14.2 5.7 3.8 0.9 1.6 3.6 1.8 1.7 3.8 1.2 1.3
8 4 13 12 7 14 6 4 1 2 4 2 2 4 1 1
1
0
a Values are residues per mole of protein.
tase were determined. The amount of apofatty acid synthetase increased as the pigeons were refasted, and the amount of holofatty acid synthetase and its activity decreased. DISCUSSION
In the course of studies in this laboratory on the effect of insulin on the synthesis of fatty acid synthetase it was demonstrated that immunoprecipitable protein was synthesized almost immediately on refeeding fasted rats a fat-free diet (23,24). However, enzyme activity for fatty acid synthesis did not increase until approximately 4 h after refeeding was started. This suggested that a nonenzymatically active protein was synthesized before the enzymatically active protein was formed. Similar conclusions were reached by Tweto and Larrabee (25) and by Yu and Burton (4, 5) from related studies. The data of Yu and Burton (4) on the appearance of immunoprecipitable protein and enzyme activity in fasted-refed animals showed that the incorporation of [14Clpantetheine closely agreed with the appearance of enzyme activity for fatty acid synthesis. The incorporation of [14Clpante-
ACID
SYNTHETASES
587
theine into the fatty acid synthetase does not occur until approximately 4 h after the start of refeeding, whereas the incorporation of [3H]amino acids into immunoprecipitable protein commences with the time of refeeding. These studies suggested that the difference between the inactive and active enzymes might be a 4’-phosphopantetheine group. Such a difference had been suggested from previous experiments by Tweto and Larrabee (25) which showed that the turnover of the 4’-phosphopantetheine group is much faster than the turnover of the fatty acid synthetase complex. It was also shown in previous studies by Elovson and Vagelos (1) with E. coli that apo- and holoacyl carrier proteins exist in this organism. It is obvious that a method of separation of apo- and holofatty acid synthetases would be desirable so that direct proof could be provided that an apoenzyme is formed prior to the formation of holo- or active fatty acid synthetase. Such a method for the separation of apo- and holofatty acid synthetases has recently been reported by us (7). In the present paper we report further on the separation of the apoand holoenzymes and on the properties of each. In the affinity chromatographic method DEAE-cellulose-purified pigeon liver fatty acid synthetase is subjected to chromatography on Sepharose+aminocaproyl pantetheine. The apofatty acid synthetase is separated from the holoenzyme by eluting with 60 mM potassium phosphate, pH 7.0. When assays were carried out on the apoenzyme, it was found that it had no activity for the synthesis of fatty acids. It was also lacking in P-ketoacyl synthetase activity and it contained no radioactivity from the [14Clpantothenic acid injected into the pigeons. All of the other partial reactions carried out by the fatty acid synthetase complex were present in this protein (Table I). It has been found that the apoenzyme and the holofatty acid synthetase complex behave in the same way on DEAB-cellulose chromatography, on gel filtration, on sucrose density gradient centrifugation, and on disc gel electrophoresis. It is evident, therefore, that the apoenzyme has approximately the same charge and molec-
588
KIM
FIG. 5. Polyacrylamide synthetase (1); holofatty protein (4); and apoacyl (5:35:1 mM), pH 8.3, at 3 acetic acid and destained TABLE
(5%) disc gel electrophoresis of DEAE-cellulose-purified fatty acid acid synthetase (2); apofatty acid synthetase (3); holoacyl carrier carrier protein (5). The gels were run in Tris-glycine-EDTA buffer mA/gel for 5 h. The gels were stained with 0.5% amido schwarz in 7% in 7% acetic acid. TABLE
III
CONVERSION OF APO- TO HOLOFATTY ACID SYNTHETASE IN THE PRESENCE OF TRANSFERASE~ Complete sysFatty Complete sysacid syntern minus CoA tern thetase and ATP Protein (mid ApoFAS HoloFAS
2.3 0.153
ET AL.
Total units 0 20
Protein
(rng) 0.664 1.677
Total units 0 415
a The incubation was carried out as reported previously (7). APO- and holofatty acid synthetases were then separated by affinity chromatography on Sepharose-e-aminocaproyl pantetheine (0.6 g; 5 mm x 5 cm). The conditions for adsorbing and eluting the proteins were the same as reported previously (7). The amount of apofatty acid synthetase was determined after DEAE-cellulose chromatography of the apo fraction obtained from the affinity column.
ular weight as the enzymatically active enzyme complex. It has also been shown that this protein behaves the same way on immunodiffusion as enzymatically active fatty acid synthetase, thus showing that apofatty acid synthetase has the same an-
IV
CONVERSION OF HOLO- TO APOFAWY ACID SYNTHETASE IN THE PRESENCE OF 4’PHOSPHOPANTETHEINE HYDROLASE” Fatty acid synthetase
ApoFAS HoloFAS
Complete SYStern drnnps; hy-
Complete tern
sys-
Protein (mn)
Units of activity
PI-Otein (mg)
Units of activity
0 2.0
0 2272
0.28 1.7
0 485
a The incubation system consisted of holofatty acid synthetase, 2 mg of protein; 1 rnM MgCl,; 5 rnM ATP, a 55-75% (NH&SO, precipitate of liver supernatant protein (hydrolase fraction) obtained from 48-h refed, 12-h fasted pigeons, 4 mg of protein; and 0.2 M potassium phosphate buffer, pH 7.0. Samples were incubated at 32°C for 20 min and frozen overnight, and then the proteins were separated by affinity chromatography as described in the footnote to Table III.
tigenicity. Analysis of acyl carrier protein from the nonenzymatically active protein for /3-alanine showed the absence of this component, suggesting that the apofatty acid synthetase lacks the 4’-phosphopan-
APO- AND
HOLOFATTY
16
FIG. 6. The dependence (2) specific activity of fatty milligram of protein (0-O); tase on the time of refeeding DEAE-cellulose step of the
24
ACID
SYNTHETASES
589
52 40 I
of (1) total fatty acid synthetase from two pigeon livers (O-O); acid synthetase in nanomoles of palm&ate formed per minute per and (3) incorporation of [Wlpantetheine into fatty acid synthebirds after a 48-h fast (A- - -A). Enzyme was purified through the standard procedure (10
FIG. 7. The dependence of the quantities of apoand holoenzymes on the time of refeeding. Apofatty acid synthetase protein in milligrams (+O); holofatty acid synthetase protein in milligrams (O---O); fatty acid synthetase activity in nanomoles of palmitate formed per minute per milligram of holofatty acid synthetase (A-A); fatty acid synthetase activity in nanomoles of palmitate formed per minute per milligram of apofatty acid synthetase (A--A).
tetheine prosthetic group. This conclusion was supported by the isolation of apoacyl carrier protein. Tables III and IV present data showing that a system exists in the supernatant solution of pigeon liver homogenate which is capable of effecting the interconversion of apo- and holofatty acid synthetases. The apo- to holoenzyme conversion occurs in the presence of CoA and ATP. In this reaction, a 4’-phosphopantetheine group is
transferred from CoA to the apofatty acid synthetase with the formation of the enzymatically active fatty acid synthetase complex. Presumably, this is the mechanism by which enzymatically inactive apoenzyme is converted to holoenzyme during the synthesis of the enzymatically active fatty acid synthetase complex in the liver cell. The conversion of holo- to apoenzyme was effected in the presence of Mg2+ and ATP with a pigeon liver supernatant fraction containing 4’-phosphopantetheine hydrolase activity. As can be seen from Table IV the amount of holo- converted to apofatty acid synthetase is small, but the activity of the remaining holoenzyme is decreased fivefold from the starting materia14 It is of interest to note that the activities of the 4’-phosphopantetheine transferase and hydrolase vary with the nutritional state of the pigeon. For 4’-phosphopantetheine transferase activity, the birds were refed for 12 h after a 48-h fast; no activity was detected in fasted pigeons. The 4’-phosphopantetheine hydrolase ac4 This decrease in activity has been found to be due to a kinase present in the enzyme fraction containing the hydrolase. This result has suggested to us the possibility that phosphorylation-dephosphorylation may also play a role in regulating the activity of the fatty acid synthetase complex (22).
KIM
ET AL.
tivity was detected in refasting pigeons. Roncari (6) has obtained similar results with the hydrolase from rat liver where hydrolase activity is high in fasted rats but not detectable in rats fed ad Zibitum. The separation and identification of apoand holofatty acid synthetases has suggested the possibility that these forms may also be involved in the short-term regulation of fatty acid synthetase activity in liver. Hence, assays were carried out to determine the quantities of apo- and holoenzyme in pigeon liver during a period of fasting and refeeding. The results (Fig. 7) clearly show that there is a change in the quantity of apo- and holoenzyme during refeeding. The quantity of apoenzyme first increases and then declines on refeeding fasted birds, and then it increases when the birds are subjected to a period of fasting. This change in quantities of apo- and holoenzyme reinforces the suggestion that the interconversion of these forms may represent a physiologically significant short-term mechanism of control of fatty acid synthetase activity. The separation of apofatty acid synthetase from rat liver fatty acid synthetase has also been accomplished by using the same procedure as reported above for the pigeon liver enzyme. However, the separation of apoenzyme from rat liver fatty acid synthetase has been complicated by the presence of a 7s component in association with the DEAE-cellulose-purified enzyme (15). ACKNOWLEDGMENTS We wish to thank Professor Harold Deutsch of the Department of Physiological Chemistry, University of Wisconsin, for the amino acid analyses. REFERENCES 1. EL~VSON, J., AND VAGEL~~, P. R. (1968)J. Biol. Chem. 243, 3603-3611. 2. VAGELOS, P. R., AND LARRABEE, A. R. (1967) J. Biol. Chem. 242, 1776-1781. 3. TWETO, J., LIBERATI, M., AND LARRABEE, A. R. (1971) J. Biol. Chem. 246, 2468-2471.
4. Yu, H. L., AND BURTON, D. N. (1974) Arch. Biochem. Biophys. 161, 297-305. 5. Yu, H. L., AND BURTON, D. N. (1974) Biochem. Biophys. Res. Commun. 61, 483-488. 6. &NCARI, D. A. K. (1975)Canad. J. Biochem. 53, 135-142. 7. QURESHI, A. A., KIM, M., LORNITZO, F. A., JENIK, R. A., AND PORTER, J. W. (1975) Biothem. Biophys. Res. Commun. 64, 836-844. 8. KUMAR, S., DORSEY, J. A., MUESING, R. A., AND PORTER, J. W. (1970) J. Biol. Chem. 245,47324744. 9. LARSSON, P., AND MOSBACH, H. (1971) Biotechnol. Bioeng. 13, 393-398. 10. Hsu, R. Y., WASSON, G., AND PORTER, J. W. (1965) J. Biol. Chem. 240, 3736-3746. 11. LORNITZO, F. A., QURESHI, A. A., AND PORTER, J. W. (1974) J. Biol. Chem. 249, 1654-1656. 12. LORNITZO, F. A., QURESHI, A. A., AND PORTER, J. W. (1975) J. Biol. Chem. 250, 4520-4529. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 14. MURPHY, J. G., AND KIES, M. W. (1960)Biochim. Biophys. Actu 45, 382-384. 15. COLLINS, J. M., CRAIG, M. C., NEPOKROEFF, C. M., KENNAN, A. L., AND PORTER, J. W. (1971) Arch. Biochem. Biophys. 143, 343-353. 16. QURESHI, A. A., LORNITZO, F. A., Hsu, R. Y., AND PORTER, J. W. (1976)Arch. B&hem. Biophys. 177, 379-393. 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30, 1190-1206. 18. GERRITSEN, I., REHBERG, M. L., AND WAISMAN, H. A. (1965) Anal. Biochem. 11, 460-466. 19. MAJERUS, P. W., ALBERTS, A. W., AND VAGELOS, P. R. (1964) Proc. Nat. Acad. Sci. USA 51, 1231-1238. 20. ELLMAN, G. L. (1970) Arch. Biochem. Biophys. 82, 70-77. 21. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 22. QURESHI, A. A., JENIK, R. A., KIM, M., LORNITZO, F. A., AND PORTER, J. W. (1975) Biothem. Biophys. Res. Commun. 66, 344-351. 23. NEPOKROEFF, C. M., LAKSHMANAN, M. R., NESS, G. C., MUESING, R. A., KLEINSEK, D. A., and PORTER, J. W. (1974)Arch. B&hem. Biophys. 162, 340-344. 24. LAKSHMANAN, M. R., NEPOKROEFF, C. M., KIM, M., AND PORTER, J. W. (1975)Arch. B&hem. Biophys. 169, 737-745. 25. TWETO, J., AND LARRABEE, M. (1972) J. Biol. Chem. 247, 4990-4904.
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Apo- and Holofatty
181,
580-590
(1977)
Acid Synthetases
from Pigeon
Liver’
MANOK KIM, ASAF A. QURESHL2 ROBERT A. JENIK, FRANK A. LORNITZO, AND JOHN W. PORTER Lipid
Metabolism
Laboratory, Veterans Administration Chemistry, University of Wisconsin, Received
September
Hospital, and Department Madison, Wisconsin 53706
of Physiological
10, 1976
Two forms (an apo- and a holoenzyme) of the fatty acid synthetase complex from pigeon liver were separated by affinity chromatography on a Sepharose+aminocaproyl pantetheine column. The difference between these enzymes is the presence or absence of the prosthetic group, 4’-phosphopantetheine. Due to the absence of the prosthetic group, apofatty acid synthetase lacks the overall ability to synthesize fatty acids, and it has no /3-ketoacyl synthetase (condensing enzyme) activity. These two forms of enzyme were shown to be homogeneous and they behaved identically on DEAE-cellulose chromatography, gel filtration, sucrose density gradient centrifugation, disc gel electrophoresis, and immunodiffusion. The isolation and purification of an apoacyl carrier protein are also reported. The apoacyl carrier protein lacks p-alanine and it has no sulfhydryl group, indicating therefore that the acyl carrier protein from apofatty acid synthetase does not have a 4’-phosphopantetheine group. The transfer of 4’-phosphopantetheine from CoA to apofatty acid synthetase was effected by an enzyme system present in the supernatant of pigeon liver homogenate. The resulting product of this reaction was the holofatty acid synthetase. The reverse reaction, the formation of apo- from holofatty acid synthetase, was also demonstrated. The physiological significance of this system is suggested from studies carried out on fasting and refeeding of pigeons. At early times of refeeding (O-4 h) there is a large amount of apoenzyme. The amount of holofatty acid synthetase increases after 4 h of refeeding and the apofatty acid synthetase decreases. When pigeons are refasted, after refeeding for 48 h, the amount of apoenzyme increases and the holoenzyme decreases.
The presence of two enzymes in Escherichia coli which are responsible for the interconversion of holo- and apoacyl carrier protein by the removal or addition of the 4’-phosphopantetheine prosthetic group has been reported (1, 2). Similarly, Tweto et al. (3) have shown that prosthetic group turnover occurs in rat liver fatty acid synthetase, thereby suggesting that the addition and removal of 4’-phosphopantetheine plays a regulatory role in the activity of this enzyme. Recently Yu and
Burton (4, 5) demonstrated indirectly the presence of an apoenzyme in rat liver. They also showed that a crude supernatant liver enzyme system converted some of the inactive apoenzyme to enzyme having fatty acid synthetase activity in the presence of coenzyme A and ATP. This result suggested the existence of an enzyme capable of carrying out the transfer of the 4’-phosphopantetheine group from CoA to apoacyl carrier protein. More recently Roncari (6), using an enzyme preparation from liver of fasted rats, has shown an inactivation of rat and human liver fatty acid synthetases which is accompanied by the release of the 4’-phosphopantetheine prosthetic group. The further study of the enzyme systems responsible for the removal and addition of 4’-phosphopantetheine to the fatty acid
1 This investigation was supported in part by a grant (AM-01383) from the National Institute of Arthritis and Metabolic Diseases of the National Institutes of Health, United States Public Health Service. 2 Present address: Waisman Center, Neurochemistry Section, 2605 Marsh Lane, Madison, Wis. 53706. 580 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0003-9661
APO- AND
HOLOFATTY
synthetase complex requires the development of a method for the separation of apo and holo forms of fatty acid synthetase from each other. Such a method, namely, the separation of apo- and holofatty acid synthetases by affinity chromatography, was recently reported by us (7). This method gives a quantitative separation of the two forms. Hence, it can be used to determine the extent of conversion of one form to another and for the preparation of pure apo- and holoenzyme for use as a substrate. This method also provides a dependable procedure for the complete removal of endogenous fatty acid synthetase from a liver supernatant solution that is to be assayed for the interconversion of apoand holoenzyme species. In the present study we report further on the separation of apo- and holofatty acid synthetases and on the properties of these two forms of enzyme. We also present further evidence for an enzyme system in eukaryotes capable of transferring 4’-phosphopantetheine to apoacyl carrier protein of fatty acid synthetase (4’-phosphopantetheine transferases) and for an enzyme system that removes the prosthetic group from acyl carrier protein (4’-phosphopantetheine transferase) and for an enzyme the results of studies on the effect of nutritional state on the variation in quantity of these two forms of enzyme in liver. These results suggest that the apo to holo interconversion may play a short-term regulatory role in fatty acid synthesis. MATERIALS
AND
METHODS
Acetyl-CoA, malonyl-CoA, and palmitoyl-CoA were obtained from P-L Biochemicals, and [lWlacetyl-CoA, [2-Wlmalonyl-CoA, [Wpantothenic acid, [‘Qsodium bicarbonate, and [l-14ClpalmitoylCoA were purchased from New England Nuclear. S-acetoacetyl-N-acetyl cysteamine, NADPH, and DEAE-cellulose were obtained from Sigma.3 DL-S-/3hydroxybutyryl-N-acetyl cysteamine was prepared as described by Kumar et al. (8) with an additional thin-layer chromatographic purification on silica gel H with a 10% methanol, 90% chloroform solvent system. S-crotonyl-N-acetyl cysteamine was pre3 Abbreviations used: DEAE-, diethylaminoethyl; FAS, fatty acid synthetase; ACP, acyl carrier protein.
ACID
SYNTHETASES
581
pared according to the method of Kumar et al. (81 with an additional purification on a Florisil (60-100 mesh) column (2 x 30 cm) with anhydrous benzene as the eluting agent. Sepharose-e-amino n-caproic acid was prepared according to the method of Larsson and Mosbach (9). Crystalline pantethine, obtained from Sigma, was reduced to pantetheine with sodium amalgam prior to use. The l-ethyl-3-(3dimethyl-aminopropyl) carbodiimide-HCl used in the preparation of the Sepharose-c-aminocaproyl pantetheine column was purchased from Sigma and Aldrich Chemical Co. The pigeons used in this study were fed Purina pigeon grain. Purificatiolt offatty acid synthetase. Pigeon liver fatty acid synthetase was purified according to the method of Hsu et al. (101, except that the final ammonium sulfate fractionation was 25 to 40% instead of 26 to 32%. Assay for partial and overall reactions of fatty acid synthesis. Acetyl-CoA and malonyl-CoA transacylase activities were assayed as previously described (ll), and P-ketoacyl thioester reductase, fl-hydroxybutyryl thioester dehydrase, and crotonyl thioester reductase were assayed as described by Kumar et al. (81, with the modifications described by Lornitzo et al. (12). The condensation reaction (pketoacyl synthetase) and palmitoyl-CoA deacylase were assayed as described previously (8). The assay for overall fatty acid synthetase activity was performed as reported by Kumar et al. (8). Protein estimation. Protein was estimated by the methods of Lowry et al. (13) and Murphy and Kies (14). Protein was also determined by light absorbance at 280 nm. Chromatography on Sepharose-E-aminocaproyl pantetheine. DEAE-cellulose-purified fatty acid synthetase was subjected to chromatography on Sepharose-e-aminocaproyl pantetheine as reported previously (7). The apo and holo fractions separated by this technique were then subjected to the analyses reported in the following sections. DEAE-cellulose chromatography. DEAE-cellulose anion exchanger (0.93 mequiv/g, coarse mesh) was prepared for use by soaking overnight in 1 M potassium phosphate, pH 7.0, and then washing thoroughly with deionized water. The anion exchanger was finally resuspended in 40 mxu potassium phosphate buffer, pH 7.0, containing 1 mM EDTA. Two milligrams (1 ml) of either holo- or apofatty acid synthetase was diluted to 20 ml with deionized water to bring the ionic strength to 20 mM potassium phosphate, and the enzyme was adsorbed on a DEAE-cellulose column (1 x 10 cm) at room temperature. The column was then washed with 30 ml of 40 mM potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. The apo- or holofatty acid synthetase was eluted from the column with a linear gradient, 40 ml each of 0.05 to 0.3 M potassium phosphate, pH 7.0, contain-
582
KIM
ET AL.
ing 1 rn~ EDTA and 1 mM dithiothreitol. Eluate fractions of 1 ml were collected. Bio-Gel filtration of apo- and holofatty acid synthetases. A Bio-Gel A-1.5m column (1.2 x 26 cm) was equilibrated with 0.2 M potassium phosphate, pH 7.0,1 mM EDTA, and 2 mM 2-mercaptoethanol. Four milligrams of either apo- or holofatty acid synthetase was loaded onto the column and eluted with the above buffer. One-milliliter fractions were collected at a flow rate of 1 ml/5 min. Sucrose density gradient centrifugation. Sucrose density gradient centrifugation of apo- and holofatty acid synthetases was performed with 5 linear gradient of 5 to 20% sucrose (19 ml of each solution) in 0.2 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. Centrifugation was carried out at 58,000g for 24 h at 20°C. Onemilliliter fractions were collected for each fatty acid synthetase. Preparation of antiserum and Ouchterlony double diffusion analysis. Rabbit antiserum to DEAE-cellulose-purified pigeon liver fatty acid synthetase was prepared according to the method of Collins et al. (15). Ouchterlony micro-double diffusion analysis with rabbit antiserum and preparations of pigeon liver apo- and holofatty acid synthetases was then carried out using 0.5% agarose gel. High voltage electrophoresis of [Wlacetyl-labeled apo- and holofatty acid synthetuse peptic peptides. One milligram of apo- or holofatty acid synthetase was incubated with [I-Wacetyl-CoA and then treated with pepsin as described previously (12). The pepsin-digested protein was subjected to high voltage paper electrophoresis on Whatman 3 MM chromatography paper at pH 3.7 for 90 min at 2500 V. One-centimeter strips were cut from the paper, placed in vials containing 15 ml of dioxane scintillation solution, and assayed for radioactivity in a Packard Tri-Carb liquid scintillation spectrometer. Isolation of apo- and holoacyl carrier protein. The apo- and holofatty acid synthetase fractions were separately combined after affinity chromatography and then dialyzed in Tris-glycine (5~35 mM) containing 5 mM 2-mercaptoethanol at 0°C and pH 8.3 for 4 h with two changes of buffer. The dialyzed enzyme was then subjected to ultrafiltration using a Diaflo PM-10 membrane. The filtrates contained approximately 80% of the acyl carrier protein, apoacyl carrier protein when apofatty acid synthetase was used, and holoacyl carrier protein when holofatty acid synthetase was used. The filtrates were lyophilized and the residues were dissolved in water andpurified by passing through Sephadex G-10, G-25, and G-50 columns (1.1 x 25 cm). The acyl carrier proteins were eluted with 10 mM potassium phosphate, pH 7.0, containing 5 mM 2-mercaptoethanol. All steps involving acyl carrier protein were carried out with quartz-distilled water.
Transfer of the [‘YJlacetyl group from acetyl-CoA to holo- and apoacyl carrier protein. Apo- and holoacyl carrier proteins (0.5 mg each) obtained from the Sephadex G-50 column were incubated with [l14C]acetyl-CoA and pigeon liver fatty acid synthetase (10 pg). The product of this reaction was purified as described previously (16). Amino acid analyses of the acyl carrier proteins. Each acyl carrier protein was hydrolyzed with constant-boiling hydrochloric acid under reduced pressure at 110°C for 22 h. Amino acid analysis was then carried out on a Beckman Model 120 amino acid analyzer equipped with a high-sensitivity cuvette and a recorder scale expansion according to the method of Spackman et al. (17) as modified by Gerritaen et al. (18). Titration of sulfhydryl groups. Each acyl carrier protein (4-5 nmol) was incubated 30 min at 30°C in 20 mM 2-mercaptoethanol to reduce disulfide bonds (19) and then dialyzed for 4 h at 0°C in deionized water with three changes. The determination of sulfhydryl groups was carried out by the spectrophotometric method of Ellman (20). Polyacrylamide disc gel electrophoresis. Five percent polyacrylamide gels, prepared by the method of Hedrick and Smith (21), were used for disc gel electrophoresis. Preparation of an enzyme fraction containing 4’phosphopantetheine transfemse activity. This enzyme system was prepared free of fatty acid synthetase as described previously (7). An alternative method of removing fatty acid synthetase involved the heating of 1 ml of the 100,oOOg supernatant of pigeon liver (S,) for 2.5 min at 56°C. The denatured protein was removed by centrifugation and the supernatant solution was assayed for fatty acid synthetase activity. This heated supernatant fraction was devoid of fatty acid synthetase activity. A ‘2140% ammonium sulfate-precipitated fraction of the heated supernatant solution was prepared and then treated in the same way as previously reported (7). In both cases the enzyme fraction was prepared from the liver of a pigeon refed for 12 h following a 48-h fast. Preparation of an enzyme fraction containing 4’phosphopantetheine hydrolase activity. A 55-75% ammonium sulfate-precipitated fraction was prepared from the 100,OOOg supernatant solution of liver homogenate from a pigeon that had been refed for 48 h and then refasted for 12 h (22). Incorporation of [%lpantothenic acid into pigeon liver fatty acid synthetase on refeeding fasted birds. [‘ClPantetheine-labeled pigeon liver fatty acid synthetase was obtained from birds fasted 48 h and then refed a fat-free diet (short-grain rice) for 0, 2, 4, 12, 24, or 48 h. The pigeons were injected intravenously with [‘Y!]pantothenic acid (0.025 mCi/pigeon) 4 h prior t.0 sacrifice.
APO- AND
HOLOFATTY
RESULTS
Separation of ApoSynthetases
and Holofatty
Acid
DEAE-cellulose-purified [14Clpantetheine-labeled fatty acid synthetase from 48-h refed pigeons was separated into apo- and holofatty acid synthetases by chromatography on Sepharose-•-aminocaproyl pantetheine as reported previously (7). In the present study 20 mg of fatty acid synthetase protein (13, 500 dpm) were chromatographed on 4 g of gel in an 8.5-mm x lo-cm column. The apofatty acid synthetase (2.7 mg of protein) was eluted in 15 ml of 0.06 M potassium phosphate buffer, pH 7.0, containing 2 mM dithiothreitol, and the holofatty acid synthetase (16.6 mg of protein) was eluted in 40 ml of 100 mM Tris-100 mM phosphate buffer, pH 7.0, containing 2 mM dithiothreitol. Assays were then carried out for fatty acid synthetase and palmitoyl-CoA deacylase activities for both forms of fatty acid synthetase, and a 200~1 aliquot of each fraction was assayed for radioactivity. Radioactive pantetheine (13,000 dpm) and overall fatty acid synthetase activity were found only in association with the active (holo) fatty acid synthetase. The palmitoyl-CoA deacylase activity was associated with both active (holo) and inactive (apo) fatty acid synthetases. Table I shows the results obtained in assays of apo- and holofatty acid synthetases for all of the partial reactions of fatty acid synthesis and for the overall reaction. It can be seen that apofatty acid synthetase lacks only the overall fatty acid synthetase and /3-ketoacyl synthetase (condensing enzyme) activities. Physical Properties of APO- and Holofatty Acid Syn thetases DEAE-cellulose column chromatography. The apo- and holofatty acid synthe-
tases were adsorbed separately on a DEAE-cellulose column and eluted as described in the methods section. In each separation, the protein was eluted as a single sharp band (Figs. 1A and B), thus indicating the homogeneity of the apo- and holofatty acid synthetases. Again, the apofatty acid synthetase lacks both overall
ACID
583
SYNTHETASES
fatty acid synthetase and /3-ketoacyl synthetase activities. Bio-Gel filtration and sucrose density gradient centrifugation. The size and homogeneity of the apo- and holofatty acid synthetases were determined by filtration over a Bio-Gel A-1.5m column (Figs. 2A and B). The proteins were eluted in the same fractions, indicating that they ,have similar molecular weights. The size of each complex was verified further by sucrose density gradient centrifugation as reported in the methods section. Again, both apo- and holofatty acid synthetases migrated similarly. Immunochemical
studies
Apo- and holofatty acid synthetases cross-react with antiserum made to DEAE-cellulose-purified fatty acid syntbetase. In con&nation of previous results (7) it was shown that apo-, holo- and BioGel-purified fatty acid synthetases gave reactions of complete identity, thus illustrating that they are immunologically identical. High
Electrophoresis of Voltage [“+Z’]Acetyl-Labeled APO- and Holofatty Acid Synthetase Peptic Peptides
The high voltage electrophoresis at pH 3.7 of [Wlacetyl-labeled peptic peptides of holo- and apofatty acid synthetases is shown in Figs. 3A and B. The holofatty acid synthetase peptides gave peaks corresponding to the AZ, B1, and B, components (4’-phosphopantetheine and hydroxyl and cysteine binding sites, respectively) reported by Kumar et al. (8). The [l14Clacetyl-labeled peptic peptides of apofatty acid synthetase showed a radioactive peak for only the B, (hydroxyl) component. The lack of the cysteine site in the apofatty acid synthetase confirms earlier studies that the prosthetic group, 4’-phosphopantetheine, is necessary for the transfer of acetyl groups to the cysteine site and for pketoacyl synthetase activity. The Isolation of Apo- and Holoacyl rier Proteins
Car-
The lack of the 4’-phosphopantetheine site in the peptic peptides of apofatty acid
584
KIM TABLE
ET AL.
I
ENZYME ACTIVITIEEI OF Am- AND HOLOFATTY ACID SYNTHETASEB Reaction Enzyme source DEAE" HoloFAS ApcFAS (nmol of product formedlminlmg of protein) Fatty acid synthesis Acetyl-CoA transacylase Malonyl-CoA transacylase P-Ketoacyl synthetase fl-Ketoacyl reductase P-Hydroxyacyl dehydrase Enoyl reductase Palmitoyl-CoA deacylase -.
85-100
122-130
-
350
402
370
100
135
130
4.3
4.7
-
142
214
187
270
316
292
240 43
277 57
259 61
’ The DEAE-cellulose-purified fatty acid synthetase was obtained from 48-h refed pigeons.
--
synthetase was confirmed by the isolation and purification of apoacyl carrier protein (Fig. 4B). The elution profiles, on Sephadex G-50, of apo- and holoacyl carrier proteins (Figs. 4A and B) indicate similar molecular weights. The inability of apoacyl carrier protein to accept the [14C]acetyl group in the presence of pigeon liver fatty acid synthetase further indicates that apoacyl carrier protein lacks 4’-phosphopantetheine (Figs. 4C and D). This conclusion is supported by the finding that apoacyl carrier protein lacks both P-alanine and the sulfhydryl group associated with holoacyl carrier protein (Table II). Otherwise the amino acid compositions of the two acyl carrier proteins are very similar. Disc Gel Electrophoresis of APO-, Holo-, and DEAE-Cellulose-Purified Fatty Acid Synthetases
The migrations of the apo-, holo-, and DEAE-cellulose-purified fatty acid synthe-
FRACTK)N NUMBERS
FIG. 1. Comparison of the behavior of (A) holofatty acid synthetase and (B) apofatty acid synthetase on DEAE-cellulose chromatography. A DEAE-cellulose column (1 x 10 cm) was equilibrated with 0.04 M potassium phosphate, pH 7.0, and then 2 mg of enzyme protein in 0.02 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 1 mre dithiothreitol, was loaded onto the column. The enzyme was eluted with a linear gradient, 0.05 to 0.3 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 1 mM dithiothreitol. Light absorbance at 280 nm (O-O); fatty acid synthetase activity in nanomoles of NADPH oxidized per minute per milliliter (0-O); P-ketoacyl thioester reductase activity in nanomoles of NADPH oxidized per minute per milliliter (0 - -0); acetyl-CoA transacylase activity in counts per minute of [Wlacetyl groups transferred to pantetheine per minute per milliliter (A-. -A); and pketoacyl synthetase activity in counts per minute of 14Cof potassium bicarbonate incorporated into malonyl-CoA per minute per milliliter (A-A).
APO- AND
HOLOFATTY
ACID
585
SYNTHETASES
A H
HOLO-
FAS
6.0 9
il x -E -4.0
”
-2.0
T 0 x
-g
z \
I E B - 3.0 u z Q 2 -1.5 E4
f 3
?+ N ” 58
0.6-
0.4-
h c!a 0.3-
0.2-
0
IO FRACTION
20 NUMBERS
FIG. 2. Comparison of (A) holofatty acid synthetase and (B) apofatty acid synthetase on BioGel A-1.5m gel filtration. Protein, 4 mg, was loaded onto a Bio-Gel column (1.2 x 26 cm) previously equilibrated with 0.2 M potassium phosphate, pH 7.0, containing 1 mM EDTA and 2 mM 2-mercaptoethanol. Elution was effected with the same buffer and l-ml fractions were collected at room temperature. Light absorbance at 280 mn (O-O); fatty acid synthetase activity in nanomoles of NADPH oxidized per minute per milliliter (0-O); p-ketoacyl thioester reductase activity in nanomoles of NADPH oxidized per minute per milliliter 0 - -0); palmitoyl-CoA deacylase activity, counts per minute of [14C1palmitate formed per minute per milliliter (O- - -0); /3-ketoacyl synthetase activity, counts per minute of [WI of potassium bicarbonate incorporated into malonyl-CoA per minute per milliliter (A-A).
tases were nearly identical on disc gel electrophoresis (Fig. 5), thus indicating a close similarity between these proteins. Similarly, the apo- and holoacyl carrier proteins migrated nearly the same distance on disc gel electrophoresis. The above results suggest that the only difference between the apo- and holofatty acid synthetases is the presence or absence of the 4’phosphopantetheine prosthetic group on the acyl carrier protein. 3a
20
20 IO ORIGIN IO DISTANCE FROM THE ORIGIN IN cm
30
FIG. 3. Analysis of [lJ4C]acetyl-bound peptic peptides from (A) holofatty acid synthetase and (B) apofatty acid synthetase by high voltage paper electrophoresis at pH 3.7. See the experimental section for details.
Enzymatic Interconversion of ApoHolofatty Acid Synthetases
and
Conversion of apo- to holofatty acid synthetase. The apofatty acid synthetase is
converted to holofatty acid synthetase by incubation with CoA, ATP, and a 4’-phos-
586
KIM
ET AL.
0 FRACTCN
NUMBER
10 FRAcnm
20 MJ.mER
FIG. 4. Gel filtration of (A) holo- and (B) apoacyl carrier protein on a Sephadex G-50 column. The filtrate from ultrafiltration on a PM-10 membrane of dissociated holo- or apofatty acid synthetase was lyophilized and the residue was dissolved in 1 ml of 10 mM potassium phosphate buffer, pH 7.0, containing 20 mM 2-mercaptoethanol and incubated at 30°C for 20 min. This solution was passed through a Sephadex G-50 column (1.1 x 25 cm). Elution was carried out with 10 mM potassium phosphate buffer, pH 7.0, and 5 mM 2-mercaptoethanol, and l-ml fractions were collected at a rate of 1 ml/3 min. Fractions 18-24 were pooled and lyophilized, and the residue was dissolved in 1 ml of 10 mM potassium phosphate buffer. Sephadex G-50 filtration of [‘Qacetyl-labeled acyl carrier protein (Cl and (D). [WAcetyl groups were transferred to acyl carrier protein as reported in the methods section. Aliquots of 1.2 ml were collected. Light absorbance at 280 nm (O---O); radioactivity in counts per minute per milliliter (O- - - -0):
phopantetheine transferase enzyme frac- Nutritional Studies, tion prepared as described in the methods section. The maximum activity for the The amount of DEAE-cellulose-purified transferase was found in the 21-40% am- fatty acid synthetase, the specific activity monium sulfate precipitate of the 100,OOOg for fatty acid synthesis, and the amount of supernatant of liver homogenate of 12-h [14Clpantothenic acid incorporated into enrefed pigeons. The fatty acid synthetase of zyme are plotted as a function of time of this fraction was removed prior to incuba- refeeding in Fig. 6. It is evident that tion by affinity chromatography as de- [14Clpantothenic acid is not incorporated scribed previously (7). The amount of con- into fatty acid synthetase during the early version of apo- to holofatty acid synthetase hours of refeeding. It is also evident that after incubation with the transferase frac- the increase in specific activity for the tion is reported in Table III. The separa- fatty acid synthetase parallels the intion of the two forms was achieved by af- creased amount of [14C]pantothenic acid finity chromatography, and then the incorporated into enzyme. The amounts of amount of apoenzyme was determined fol- apo- and holofatty acid synthetases preslowing DEAE-cellulose chromatography. ent at the various times of refeeding are Conversion of holo- to apofatty acid synplotted in Fig. 7. The data clearly indicate the&se. The conversion of holo-.to apofatty an increased amount of apofatty acid synacid synthetase in the presence of an am- thetase during the early hours of refeedmonium sulfate-precipitated fraction of pi- ing. However, as the time of refeeding progeon liver supernatant solution was also gresses, there is a decrease in apofatty acid demonstrated (Table IV). However, the synthetase and an increase in the amount extent of conversion of holo- to apo- was of holoenzyme. In a related study, the efless than the conversion of apo- to holoen- fects of both refeeding and refasting on the zyme . amounts of apo- and holofatty acid synthe-
APO- AND TABLE
HOLOFATTY
II
AMINO ACID COMPOSITIONS OF Hou)CARRIER PROTEINS Amino acid residue
HoloACP”
p-Alanine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine
0.80 7.9 3.6 13.7 11.5 5.7 13.6 5.5 3.8 1.1 1.9 3.7 1.8 1.9 3.8 1.3 1.4
Sulfhydryl groups/ mole of prot,ein
AND APOACYL ApoACP”
1 8 4 14 12 6 14 6 4 1 2 4 2 2 4 1 1
0
0
7.6 3.7 13.4 12.4 6.6 14.2 5.7 3.8 0.9 1.6 3.6 1.8 1.7 3.8 1.2 1.3
8 4 13 12 7 14 6 4 1 2 4 2 2 4 1 1
1
0
a Values are residues per mole of protein.
tase were determined. The amount of apofatty acid synthetase increased as the pigeons were refasted, and the amount of holofatty acid synthetase and its activity decreased. DISCUSSION
In the course of studies in this laboratory on the effect of insulin on the synthesis of fatty acid synthetase it was demonstrated that immunoprecipitable protein was synthesized almost immediately on refeeding fasted rats a fat-free diet (23,24). However, enzyme activity for fatty acid synthesis did not increase until approximately 4 h after refeeding was started. This suggested that a nonenzymatically active protein was synthesized before the enzymatically active protein was formed. Similar conclusions were reached by Tweto and Larrabee (25) and by Yu and Burton (4, 5) from related studies. The data of Yu and Burton (4) on the appearance of immunoprecipitable protein and enzyme activity in fasted-refed animals showed that the incorporation of [14Clpantetheine closely agreed with the appearance of enzyme activity for fatty acid synthesis. The incorporation of [14Clpante-
ACID
SYNTHETASES
587
theine into the fatty acid synthetase does not occur until approximately 4 h after the start of refeeding, whereas the incorporation of [3H]amino acids into immunoprecipitable protein commences with the time of refeeding. These studies suggested that the difference between the inactive and active enzymes might be a 4’-phosphopantetheine group. Such a difference had been suggested from previous experiments by Tweto and Larrabee (25) which showed that the turnover of the 4’-phosphopantetheine group is much faster than the turnover of the fatty acid synthetase complex. It was also shown in previous studies by Elovson and Vagelos (1) with E. coli that apo- and holoacyl carrier proteins exist in this organism. It is obvious that a method of separation of apo- and holofatty acid synthetases would be desirable so that direct proof could be provided that an apoenzyme is formed prior to the formation of holo- or active fatty acid synthetase. Such a method for the separation of apo- and holofatty acid synthetases has recently been reported by us (7). In the present paper we report further on the separation of the apoand holoenzymes and on the properties of each. In the affinity chromatographic method DEAE-cellulose-purified pigeon liver fatty acid synthetase is subjected to chromatography on Sepharose+aminocaproyl pantetheine. The apofatty acid synthetase is separated from the holoenzyme by eluting with 60 mM potassium phosphate, pH 7.0. When assays were carried out on the apoenzyme, it was found that it had no activity for the synthesis of fatty acids. It was also lacking in P-ketoacyl synthetase activity and it contained no radioactivity from the [14Clpantothenic acid injected into the pigeons. All of the other partial reactions carried out by the fatty acid synthetase complex were present in this protein (Table I). It has been found that the apoenzyme and the holofatty acid synthetase complex behave in the same way on DEAB-cellulose chromatography, on gel filtration, on sucrose density gradient centrifugation, and on disc gel electrophoresis. It is evident, therefore, that the apoenzyme has approximately the same charge and molec-
588
KIM
FIG. 5. Polyacrylamide synthetase (1); holofatty protein (4); and apoacyl (5:35:1 mM), pH 8.3, at 3 acetic acid and destained TABLE
(5%) disc gel electrophoresis of DEAE-cellulose-purified fatty acid acid synthetase (2); apofatty acid synthetase (3); holoacyl carrier carrier protein (5). The gels were run in Tris-glycine-EDTA buffer mA/gel for 5 h. The gels were stained with 0.5% amido schwarz in 7% in 7% acetic acid. TABLE
III
CONVERSION OF APO- TO HOLOFATTY ACID SYNTHETASE IN THE PRESENCE OF TRANSFERASE~ Complete sysFatty Complete sysacid syntern minus CoA tern thetase and ATP Protein (mid ApoFAS HoloFAS
2.3 0.153
ET AL.
Total units 0 20
Protein
(rng) 0.664 1.677
Total units 0 415
a The incubation was carried out as reported previously (7). APO- and holofatty acid synthetases were then separated by affinity chromatography on Sepharose-e-aminocaproyl pantetheine (0.6 g; 5 mm x 5 cm). The conditions for adsorbing and eluting the proteins were the same as reported previously (7). The amount of apofatty acid synthetase was determined after DEAE-cellulose chromatography of the apo fraction obtained from the affinity column.
ular weight as the enzymatically active enzyme complex. It has also been shown that this protein behaves the same way on immunodiffusion as enzymatically active fatty acid synthetase, thus showing that apofatty acid synthetase has the same an-
IV
CONVERSION OF HOLO- TO APOFAWY ACID SYNTHETASE IN THE PRESENCE OF 4’PHOSPHOPANTETHEINE HYDROLASE” Fatty acid synthetase
ApoFAS HoloFAS
Complete SYStern drnnps; hy-
Complete tern
sys-
Protein (mn)
Units of activity
PI-Otein (mg)
Units of activity
0 2.0
0 2272
0.28 1.7
0 485
a The incubation system consisted of holofatty acid synthetase, 2 mg of protein; 1 rnM MgCl,; 5 rnM ATP, a 55-75% (NH&SO, precipitate of liver supernatant protein (hydrolase fraction) obtained from 48-h refed, 12-h fasted pigeons, 4 mg of protein; and 0.2 M potassium phosphate buffer, pH 7.0. Samples were incubated at 32°C for 20 min and frozen overnight, and then the proteins were separated by affinity chromatography as described in the footnote to Table III.
tigenicity. Analysis of acyl carrier protein from the nonenzymatically active protein for /3-alanine showed the absence of this component, suggesting that the apofatty acid synthetase lacks the 4’-phosphopan-
APO- AND
HOLOFATTY
16
FIG. 6. The dependence (2) specific activity of fatty milligram of protein (0-O); tase on the time of refeeding DEAE-cellulose step of the
24
ACID
SYNTHETASES
589
52 40 I
of (1) total fatty acid synthetase from two pigeon livers (O-O); acid synthetase in nanomoles of palm&ate formed per minute per and (3) incorporation of [Wlpantetheine into fatty acid synthebirds after a 48-h fast (A- - -A). Enzyme was purified through the standard procedure (10
FIG. 7. The dependence of the quantities of apoand holoenzymes on the time of refeeding. Apofatty acid synthetase protein in milligrams (+O); holofatty acid synthetase protein in milligrams (O---O); fatty acid synthetase activity in nanomoles of palmitate formed per minute per milligram of holofatty acid synthetase (A-A); fatty acid synthetase activity in nanomoles of palmitate formed per minute per milligram of apofatty acid synthetase (A--A).
tetheine prosthetic group. This conclusion was supported by the isolation of apoacyl carrier protein. Tables III and IV present data showing that a system exists in the supernatant solution of pigeon liver homogenate which is capable of effecting the interconversion of apo- and holofatty acid synthetases. The apo- to holoenzyme conversion occurs in the presence of CoA and ATP. In this reaction, a 4’-phosphopantetheine group is
transferred from CoA to the apofatty acid synthetase with the formation of the enzymatically active fatty acid synthetase complex. Presumably, this is the mechanism by which enzymatically inactive apoenzyme is converted to holoenzyme during the synthesis of the enzymatically active fatty acid synthetase complex in the liver cell. The conversion of holo- to apoenzyme was effected in the presence of Mg2+ and ATP with a pigeon liver supernatant fraction containing 4’-phosphopantetheine hydrolase activity. As can be seen from Table IV the amount of holo- converted to apofatty acid synthetase is small, but the activity of the remaining holoenzyme is decreased fivefold from the starting materia14 It is of interest to note that the activities of the 4’-phosphopantetheine transferase and hydrolase vary with the nutritional state of the pigeon. For 4’-phosphopantetheine transferase activity, the birds were refed for 12 h after a 48-h fast; no activity was detected in fasted pigeons. The 4’-phosphopantetheine hydrolase ac4 This decrease in activity has been found to be due to a kinase present in the enzyme fraction containing the hydrolase. This result has suggested to us the possibility that phosphorylation-dephosphorylation may also play a role in regulating the activity of the fatty acid synthetase complex (22).
KIM
ET AL.
tivity was detected in refasting pigeons. Roncari (6) has obtained similar results with the hydrolase from rat liver where hydrolase activity is high in fasted rats but not detectable in rats fed ad Zibitum. The separation and identification of apoand holofatty acid synthetases has suggested the possibility that these forms may also be involved in the short-term regulation of fatty acid synthetase activity in liver. Hence, assays were carried out to determine the quantities of apo- and holoenzyme in pigeon liver during a period of fasting and refeeding. The results (Fig. 7) clearly show that there is a change in the quantity of apo- and holoenzyme during refeeding. The quantity of apoenzyme first increases and then declines on refeeding fasted birds, and then it increases when the birds are subjected to a period of fasting. This change in quantities of apo- and holoenzyme reinforces the suggestion that the interconversion of these forms may represent a physiologically significant short-term mechanism of control of fatty acid synthetase activity. The separation of apofatty acid synthetase from rat liver fatty acid synthetase has also been accomplished by using the same procedure as reported above for the pigeon liver enzyme. However, the separation of apoenzyme from rat liver fatty acid synthetase has been complicated by the presence of a 7s component in association with the DEAE-cellulose-purified enzyme (15). ACKNOWLEDGMENTS We wish to thank Professor Harold Deutsch of the Department of Physiological Chemistry, University of Wisconsin, for the amino acid analyses. REFERENCES 1. EL~VSON, J., AND VAGEL~~, P. R. (1968)J. Biol. Chem. 243, 3603-3611. 2. VAGELOS, P. R., AND LARRABEE, A. R. (1967) J. Biol. Chem. 242, 1776-1781. 3. TWETO, J., LIBERATI, M., AND LARRABEE, A. R. (1971) J. Biol. Chem. 246, 2468-2471.
4. Yu, H. L., AND BURTON, D. N. (1974) Arch. Biochem. Biophys. 161, 297-305. 5. Yu, H. L., AND BURTON, D. N. (1974) Biochem. Biophys. Res. Commun. 61, 483-488. 6. &NCARI, D. A. K. (1975)Canad. J. Biochem. 53, 135-142. 7. QURESHI, A. A., KIM, M., LORNITZO, F. A., JENIK, R. A., AND PORTER, J. W. (1975) Biothem. Biophys. Res. Commun. 64, 836-844. 8. KUMAR, S., DORSEY, J. A., MUESING, R. A., AND PORTER, J. W. (1970) J. Biol. Chem. 245,47324744. 9. LARSSON, P., AND MOSBACH, H. (1971) Biotechnol. Bioeng. 13, 393-398. 10. Hsu, R. Y., WASSON, G., AND PORTER, J. W. (1965) J. Biol. Chem. 240, 3736-3746. 11. LORNITZO, F. A., QURESHI, A. A., AND PORTER, J. W. (1974) J. Biol. Chem. 249, 1654-1656. 12. LORNITZO, F. A., QURESHI, A. A., AND PORTER, J. W. (1975) J. Biol. Chem. 250, 4520-4529. 13. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 14. MURPHY, J. G., AND KIES, M. W. (1960)Biochim. Biophys. Actu 45, 382-384. 15. COLLINS, J. M., CRAIG, M. C., NEPOKROEFF, C. M., KENNAN, A. L., AND PORTER, J. W. (1971) Arch. Biochem. Biophys. 143, 343-353. 16. QURESHI, A. A., LORNITZO, F. A., Hsu, R. Y., AND PORTER, J. W. (1976)Arch. B&hem. Biophys. 177, 379-393. 17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S. (1958) Anal. Chem. 30, 1190-1206. 18. GERRITSEN, I., REHBERG, M. L., AND WAISMAN, H. A. (1965) Anal. Biochem. 11, 460-466. 19. MAJERUS, P. W., ALBERTS, A. W., AND VAGELOS, P. R. (1964) Proc. Nat. Acad. Sci. USA 51, 1231-1238. 20. ELLMAN, G. L. (1970) Arch. Biochem. Biophys. 82, 70-77. 21. HEDRICK, J. L., AND SMITH, A. J. (1968) Arch. Biochem. Biophys. 126, 155-164. 22. QURESHI, A. A., JENIK, R. A., KIM, M., LORNITZO, F. A., AND PORTER, J. W. (1975) Biothem. Biophys. Res. Commun. 66, 344-351. 23. NEPOKROEFF, C. M., LAKSHMANAN, M. R., NESS, G. C., MUESING, R. A., KLEINSEK, D. A., and PORTER, J. W. (1974)Arch. B&hem. Biophys. 162, 340-344. 24. LAKSHMANAN, M. R., NEPOKROEFF, C. M., KIM, M., AND PORTER, J. W. (1975)Arch. B&hem. Biophys. 169, 737-745. 25. TWETO, J., AND LARRABEE, M. (1972) J. Biol. Chem. 247, 4990-4904.