Synthesis of multimethyl-branched fatty acids by avian and mammalian fatty acid synthetase and its regulation by malonyl-CoA decarboxylase in the uropygial gland

ARCHIVESOF BIOCHEMISTRY AND BIOPHYSICS Vol. 186, No. 1, February, pp. 152-163, 1978

Synthesis Mammalian

of Multimethyl-Branched Fatty Acid Synthetase Decarboxylase

Fatty Acids by Avian and

and Its Regulation

in the Uropygial

J. S. BUCKNER,2 P. E. KOLA!lTUKUDY,3 Department

of Agricultural

AND

by Malonyl-CoA

Gland’ LINDA ROGERS

Chemistry and the Program in Biochemistry and Biophysics, University, Pullman, Washington 99164

Washington

State

Received August 15, 1977; revised October 31, 1977 Fatty acid synthetase, partially purified by gel filtration with Sepharose 4B from goose liver, showed the same relative rate of incorporation of methylmalonyl-CoA (compared to malonyl-CoA) as that observed with the purified fatty acid synthetase from the uropygial gland. In the presence of acetyl-CoA, methylmalonyl-CoA was incorporated mainly into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8,10-pentamethyldodecanoic acid by the enzyme from both sources. Methylmalonyl-CoA was a competitive inhibitor with respect to malonyl-CoA for the enzyme from the gland just as previously observed for fatty acid synthetase from other animals. Furthermore, rabbit antiserum prepared against the gland enzyme cross-reacted with the liver enzyme, and Ouchterlony double-diffusion analyses showed complete fusion of the immunoprecipitant lines. The antiserum inhibited both the synthesis of n-fatty acids and branched fatty acids catalyzed by the synthetase from both liver and the uropygial gland. These results suggest that the synthetases from the two tissues are identical and that branched and n-fatty acids are synthesized by the same enzyme. Immunological examination of the 105,OOOg supernatant prepared from a variety of organs from the goose showed that only the uropygial gland contained a protein which cross-reacted with the antiserum prepared against malonyl-CoA decarboxylase purified from the gland. Thus, it is concluded that the reason for the synthesis of multimethyl-branched fatty acids by the fatty acid synthetase in the gland is that in this organ the tissuespecific and substrate-specific decarboxylase makes only methylmalonyl-CoA available to the synthetase. Fatty acid synthetase, partially purified from the mammary gland and the liver of rats, also catalyzed incorporation of [methyZ-14C1methylmalonyl-CoA into 2,4,6,8_tetramethyldecanoic acid and 2,4,6&tetramethylundecanoic acid with acetyl-CoA and propionyl-CoA, respectively, as the primers. Evidence is also presented that fatty acids containing straight and branched regions can be generated by the fatty acid synthetase from the rat and goose, from methylmalonyl-CoA in the presence of malonyl-CoA or other precursors of n-fatty acids. These results provide support for the hypothesis that, under the pathological conditions which result in accumulation of methylmalonyl-Cob, abnormal branched acids can be generated by the fatty acid synthetase.

Fatty acid synthetase of animals catalyzes the synthesis of mainly n-C,, fatty 1This is Scientific Paper No. 4886, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, Washington 99164. This work was supported in part by Grant GM18278from the Institute of General Medical Sciences of the U.S. Public Health Service. 2 Resent address: Metabolism and Radiation Laboratory, Agricultural Research Service, U.S.

acids in most tissues. Sebaceous glands, on the other hand, produce methylbranched acids (1) and uropygial glands of many birds generate ‘multiple methylbranched fatty acids as the major products (2). We have previously demonstrated that Department of Agriculture, Fargo, North Dakota 58102. s To whom all correspondence should be sent.

152 0003-9861/78/1861-0152$02.00/O Copyright0 1978 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

methylmalonyl-CoA is the precursor of the branched acids in cell-free preparations (3). A highly purified fatty acid synthetase preparation from the uropygial gland of the goose generated 2,4,6&tetramethyldecanoic acid from acetyl-CoA and methylmalonyl-CoA, while 2,4,6,&tetramethylundecanoic acid was produced when acetyl-CoA was replaced by propionyl-CoA (4). The same enzyme preparation catalyzed the synthesis of n-C,, acid from acetyl-CoA and malonyl-CoA at least two orders of magnitude faster than the rate of synthesis of the multimethyl-branched fatty acids, and the properties of this highly purified synthetase were very similar to those of the fatty acid synthetases of other animals (4). However, since the two multi-branched acids generated in uitro are also known to be the major fatty acids generated in vivo (3, 5, 6), it appeared that the fatty acid synthetase preparation did contain the enzyme responsible for the in uiuo synthesis of the branched acids. Since the fatty synthetase preparation appears to be homogeneous, these results suggested that fatty acid synthetase has the inherent capacity to catalyze the formation of multimethyl-branched acids from methylmalonyl-CoA. If such is the case if would appear likely that, in diseases involving defects in the methylmalonyl-CoA mutase reaction (7-W methyl-branched acids might be generated by the fatty acid synthetase. In this paper we present evidence which strongly suggests that the fatty acid synthetase which generates the multimethylbranched fatty acids in the uropygial gland of the goose is identical to that which generates n-acids in other tissues. The inherent ability of animal fatty acid synthetase to generate methyl-branched acids from methylmalonyl-CoA is also demonstrated with fatty acid synthetase preparations from the liver and the mammary gland of the rat. EXPERIMENTAL

PROCEDURES

Materials. White domestic geese were purchased from Richard’s Goose Hatchery, Othello, Washington, and were maintained on a low-energy breeder ration. Malonyl-CoA, acetyl-CoA, NADPH, dithio-

FATTY ACIDS

153

erythritol, Sepharose 4B, glucose8-phosphate, glucose-6-phosphate dehydrogenase, and bovine serum albumin were purchased from Sigma Chemical Co. Agar and complete and incomplete adjuvant (Freund) were purchased from Difco Laboratories. [methyZ-14C]Methylmalonyl-CoA (54 Ci/mol) and Omnifluor were purchased from New England Nuclear Corp. Isolation

and purification

of fatty acid synthetuse.

Excised uropygial glands and excised liver tissue of geese were homogenized in 100 mM phosphate buffer, pH 7.6, containing 250 mM sucrose, 1 mM MgClz, and 1 mM dithioerythritol, and the 105,OOOg supematant was obtained by ultracentrifugation as described before (3). Excised mammary glands and liver tissue of lactating rats (12-21 days postpartum) were homogenized and centrifuged as described above for the tissues of the goose. In a similar manner, 105,OOOgsupernatants were obtained from heart, brain, lung, pancreas, spleen, and adipose tissues of the goose. The fatty acid synthetase was partially purified by gel filtration with Sepharose 4B (4) from the 105,OOOgsupematants prepared from the uropygial gland and liver of the goose and the mammary gland and liver of the rat. Sepharose 4B columns (2.4 x 90 cm) were equilibrated with 100 mM phosphate buffer, pH 7.6, containing 0.5 mM dithioerythritol. The 105,OOOgsupematant (2-3 ml) was placed on top of the column and eluted with the same buffer at a flow rate of 0.4 ml/min. The absorbance of the column effluent was monitored at 280 nm with an Isco Model UA-5 monitor, and 6.0-ml fractions were collected. Enzyme assays. The rates of malonyl-CoA incorporation into fatty acids by the partially purified fatty acid synthetase preparations were determined spectrophotometrically by measuring the initial rates of NADPH oxidation at 340 nm for 5 min at 30°C (4). Reaction mixtures contained 15 pmol of phosphate buffer, pH 7.6, 0.01 pmol of acetyl-CoA, 0.02 pmol of malonyl-CoA, and enzyme in a total volume of 0.2 ml. The rate of methylmalonyl-CoA incorporation into fatty acids was determined by a radiochemical assay. Standard reaction mixtures, containing 36 pmol of phosphate buffer, pH 7.6, 0.35 Fmol of dithioerythritol, 0.18 pmol of NADPH, 0.57 pmol of glucose-g-phosphate, 1 unit of glucose-6-phosphate dehydrogenase, 0.02 pmol of acetyl-CoA, 0.063 pmol of [methylJ4C]methlmalonyl-CoA (2.8 Cilmol) and enzyme in a total volume of 0.4 ml, were incubated for 30 min at 30°C. Reactions were terminated by the addition of 0.1 ml of 45% NaOH. The alkaline reaction mixtures were heated at 100°C for 20 min and acidified with 6 N HCl, and the labeled fatty acids were extracted with chloroform and purified by thin-layer chromatography as described below.

154 Protein

BUCKNER,

KOLATTUKUDY,

was determined by the method of Lowry et bovine serum albumin as standard. Specific activities were expressed as nanomoles of malonyl-CoA or methylmalonyl-CoA incorporated per minute per milligram of protein. The incorporation of malonyl-CoA and methylmalonyl-CoA into fatty acids was determined using incubation times and protein concentrations which were in the linear range. Chromatography. Thin-layer chromatography was performed with silica gel G on 20 x 20-cm plates which were activated for 12 h at 100°C. Free fatty acids and fatty acid butyl esters were purified with hexane:ethyl ether:formic acid (4O:lO:l) as the developing solvent. Radio gas-liquid chromatography was performed with a Perkin-Elmer Model 801 gas chromatograph equipped with a flame ionization detector and an ei?luent splitter, attached to a Barber-Coleman radioactivity monitor. Analysis of fatty acid butyl esters was done on a coiled stainless steel column (0.6 x 300 cm) packed with 5% OV-1 on 80-100 mesh Gas-Chrome Q with an argon carrier gas flow of 80 ml/min. The column temperature was programmed from 160 to 240°C with a 2Wmin rate. Authentic samples were used as standards for thinlayer and gas chromatography. Determination of radioaztivity. Radioactivity on thin-layer chromatograms was monitored with a Berthold thin-layer scanner. Solutions containing 14C were assayed for radioactivity, after mixing with 15 ml of 30% ethanol in toluene containing 4 g/ liter of Omnifluor, by liquid scintillation spectrometry with a Packard Model 3003 Tri-Carb scintillation spectrometer. An internal standard of [14Cltoluene was used to determine the counting efficiency, which was usually 74%. Preparation of derivatives. The butyl esters of fatty acids were prepared by refluxing the fatty acids with 14% boron trifluoride in n-butanol for 30 min. The reaction was stopped by the addition of an equal volume of H,O. The fatty acid butyl esters were recovered by extraction with chloroform. The isolated fatty acid butyl esters were purified by thinlayer chromatography as described above. Preparation of antiserum. About 5 mg of purified fatty acid synthetase from goose uropygial glands was dissolved in 0.5 ml of 0.9% NaCl and mixed with 0.5 ml of complete Freund’s adjuvant, and the mixture was emulsified by ultrasonic treatment with the needle probe of a Biosonik III. This emulsion was injected subcutaneously into a rabbit at multiple sites. After 2 weeks, the same rabbit was injected with an additional 5 mg of protein, emulsified with incomplete Freund’s adjuvant. Two weeks aRer the second injection, the rabbit was bled from the ear and the antiserum was prepared and stored at -20°C. Antiserum against malonyl-CoA decarboxylase

al. (13) with

AND

ROGERS

was prepared by immunizing rabbits with puriiied malonyl-CoA decarboxylase as described before (14). Zmmunodij&sion. Double-diffusion analyses were performed according to the method of Ouchterlony (15) on 1% agar in 0.9% NaCl contained in petri dishes. After diffusion was complete, nonagglutinated protein was removed from the agar by repeated washings with 0.9% NaCl followed by distilled HzO. The agglutinated protein bands were stained with a solution of 0.5% amido black, 5% HgCl, in 5% acetic acid for 30 min and the excess dye was removed from the agar with 7.5% acetic acid. Immunodiffision was usually performed with undiluted antiserum placed in the 20-~1 center well and protein samples placed in each of six equally spaced outer wells. Protein concentrations of 14-20 mglml were used in immunodiffusion analyses for the 105,OOOg supernatants of brain, heart, lung, spleen, pancreas, liver, and adipose tissues of the goose. RESULTS

AND

DISCUSSION

Since cell-free preparations from the uropygial gland of goose were shown to catalyze the incorporation of methylmalonyl-CoA into branched fatty acids and malonyl-CoA into n-fatty acids (3, 41, it is possible that the glandular tissue of goose contains either two different synthetases (one for methylmalonyl-Cob incorporation and another for malonyl-CoA) or a single enzyme which can synthesize either branched or n-fatty acids. The predominance of multimethyl-branched fatty acids exclusively in the glandular wax suggested that perhaps the gland contained a synthetase specific for methylmalonyl-CoA. If such is the case, liver, a tissue which contains little multi-branched fatty acids, should not contain such an enzyme. Initial experiments with equal amounts of protein from the 105,OOOgsupernatants showed that methylmalonylCoA was incorporated into fatty acids only by the glandular extract and not by the liver extract (3). However, these results are misleading since the two preparations most likely contained different amounts of synthetase. It has been shown that fatty acid synthetase constitutes an unusually large portion of the total soluble protein of the goose uropygial gland (4), whereas liver tissue would not be expected to contain such an abundance of this enzyme. To overcome this problem, fatty acid syn-

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

thetase, obtained from liver extracts, was partially purified by gel filtration chromatography with Sepharose 4B, and the resulting elution profile of proteins from the column is shown in Fig. 1. Spectrophotometric assays for fatty acid synthetase revealed that the peak of enzymatic activity was associated with the leading shoulder on the second major protein peak eluted from the column. After pooling the fractions, the partially purified fatty acid synthetase (3-4 mg/ml) was concentrated by ultrafiltration to approximately 20 mg/ ml. Repetition of the gel filtration step gave a preparation in which fatty acid synthetase (13 S) was the dominant protein as indicated by analytical ultracentrifugation (Fig. 1). The specific activity of this preparation for the incorporation of malonyl-CoA approached 40% of that obtained with similar preparations from the uropygial gland. Therefore, it became possible to use comparable units of enzyme from the two sources to measure their ability to synthesize branched fatty acids. The partially purified enzyme from the liver did indeed catalyze the incorporation of [methyZ-~4C]methylmalonyl-CoA into fatty acids, and the specific activity for this incorporation was about half of that observed with the enzyme preparation from the gland. Thus, the ratios of the specific activities for malonyl-CoA incorporation to those for methylmalonyl-CoA

FATTY

ACIDS

155

incorporation were quite similar for the two enzyme preparations (Table I). These results strongly suggest that the specificity of the gland enzyme is not responsible for the production of multimethyl-branched fatty acids in the gland and that the fatty acid synthetase in the gland is probably quite similar if not identical to that in the liver. If such is the case, the enzyme from the liver might catalyze the synthesis of the same multimethyl-branched fatty acids as those formed by the gland from methylmalonyl-CoA. Radio gas-liquid chromatographic analysis showed that the partially purified liver enzyme synthesizes 2,4,6,8-tetramethyldecanoic acid from acetyl-CoA and methylmalonyl-CoA (Fig. 2). This multimethyl-branched acid is also the dominant acid synthesized by the gland enzyme in vitro (3, 4) as well as in uiuo (3, 5, 6). Smaller amounts of 2,4,6,8,10-pentamethyldodecanoic acid were also produced by both enzymes. The only difference between the two preparations was that the liver enzyme generated a small amount of a longer acid. Thus, the liver enzyme is capable of synthesizing the same multi-branched acids in vitro as those generated by the gland enzyme in vitro and in uiuo. Therefore, it appears that the substrate availability must be the reason for the fact that, in uiuo, the liver generates n-fatty acids, whereas the gland produces mainly multimethyl-

FIG. 1. Sepharose 4B gel filtration of the 105,OOOg supernatant obtained from goose liver homogenates. The column (2.3 x 90 cm) was eluted with 100 mM phosphate buffer, pH 7.6, containing 0.5 mM dithioerythritol at a flow rate of 0.4 ml/min. Absorbance at 280 nm (-1 was monitored and 50 ~1 of each fraction was assayed spectrophotometrically for fatty acid synthetase activity (O-01 as described under Experimental Procedures. The inset shows the sedimentation velocity pattern of the fatty acid synthetase fraction isolated by repeated Sepharose 4B gel filtration. The analytical ultracentrifugation was done as described before (41.

156

BUCKNER, TABLE

KOLATTUKUDY.

I

INCORPORATION OF MALONYL-COA AND METHYLMALONYL-COA INTO FATTY ACIDS BY FATTY ACID SYNTHETASES FROM THE UROPYCIAL GLAND AND LIVER OF GOOSE AND THE MAMMARY GLAND AND LIVER OF RAT’ Sp+fic acSpecificactivity Enzyme source

(nmol incorporated/minlmg)

tiwXt;;Ftio

Malo-

nylCoA

MethylmalonylCoA

(methyl malonylCoA/malonyl-CoA)

Goose Uropygial gland Liver

541 192

0.6 0.3

1.1 1.5

Rat Mammary gland Liver Mammary glandb

238 86 94

2.1 0.4 0.23

8.8 4.6 2.4

o Amounts of protein added to the reaction mixtures for spectrophotometric assays with malonylCoA were as follows: uropygial gland and liver of goose, 0.4 and 1.9 pg, respectively; mammary gland and liver of rat, 0.8 and 2.1 pg, respectively. Amounts of protein added to the reaction mixtures for radiochemical assays with [methyPClmethylmalonyl-CoA were as follows: uropygial gland and liver of goose, 200 and 370 pg, respectively; mammary gland and liver of rat, 180 and 590 rg, respectively. Other conditions for the spectrophotometric and radiochemical assays and the isolation of products are described under Experimental Procedures. b 237 pg of protein used.

branched fatty acids. In rats fatty acid synthetase from the mammary gland has been reported to be identical to that from the liver (16). Comparison of all of the properties of the synthetase from the goose liver with those of the enzyme from the uropygial gland indicates that the enzymes from the two tissues were quite similar, although the in uiuo products of the enzyme are quite different in the two tissues. To test further whether the synthetases from the two tissues are identical, Ouchterlony double-diffusion analyses were performed. The enzyme from the goose liver cross-reacted with the antibody against the synthetase from the gland and complete fusion of the immunoprecipitant lines suggested that the synthetases from the two tissues are immunologically identical (Fig. 3). Fur-

AND

ROGERS

thermore, the enzymatic activity of the liver synthetase was inhibited by the antiserum prepared against the enzyme isolated from the gland. The increasing degree of inhibition of the liver enzyme caused by increasing amounts of the antiserum (prepared against the gland enzyme) was very similar to that observed with the synthetase from the gland. Incorinto poration of methylmalonyl-CoA branched fatty acids by the synthetase from both the uropygial gland and the liver of the goose was also inhibited by the antiserum prepared against the synthetase isolated from the uropygial gland. All of these results very strongly suggest that the fatty acid synthetase in the uro-

l--r--l I 0

IO

20

30

TIME (min) FIG. 2. Radio gas-liquid chromatograms of fatty acids (as butyl esters) generated from [methyl14Clmethylmalonyl-CoA and acetyl-CoA by the purified fatty acid synthetases from goose liver (L) and uropygial glands (G). The bottom tracing shows the flame ionization detector response obtained from a mixture of authentic n-fatty acid butyl esters CC,,C,,), and the other smooth tracing shows the flame ionization detector response from the butyl esters of the naturally occurring branched acids from the uropygial gland. The components labeled a, b, and c have been identified as 2,4,6,8-tetramethyldecanoic acid, 2,4,6,8-tetramethylundecanoic acid, and 2,4,6,8,10-pentamethyldodecanoic acid, respectively, by mass spectrometry (3). The procedures for the recovery of fatty acids and the preparation of butyl ester derivatives and radio gas-liquid chromatography conditions are described under Experimental Procedures.

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

FIG. 3. Immuno-double-diffusion analysis (Ouchterlony) of the purified fatty acid synthetases from goose liver and goose uropygial glands. The center well contained undiluted antiserum obtained from rabbits injected with purified fatty acid synthetase isolated from the goose uropygial gland. Alternate outer wells marked L contained partially puritied fatty acid synthetase (15 mg/ml) from the goose liver, whereas the other three contained purified synthetase from the uropygial gland (3 mg/ml). Immunodiffusion conditions are described under Experimental Procedures.

pygial gland is identical to that in the liver and that the same fatty acid synthetase catalyzes the formation of n-fatty acids from malonyl-CoA and the methylbranched fatty acids from methylmalonylCoA. The demonstration that the fatty acid synthetase from the goose synthesizes 2,4,6,&%tetramethyldecanoic acid in vitro, from acetyl-CoA and methylmalonyl-CoA, provided an explanation for the observation that the uropygial gland produces this multimethyl-branched acid in uiuo. The problem of low specific activity observed for the incorporation of methylmalonyl-CoA is probably offset by the large amount of synthetase (one-sixth to one-third of the total soluble protein) present in the gland, and there is a possibility that some factor present in the gland activates the enzyme in Go. However, the multibranched acid is generated when methylmalonyl-CoA is the only available substrate and the synthetase in vitro shows a high degree of preference for the

FATTY ACIDS

157

synthesis of n-fatty acids. The presence of an extremely active and substrate-specific malonyl-CoA decarboxylase in this gland has been suggested to be responsible for making methylmalonyl-CoA the chief substrate available to the fatty acid synthetase in uiuo (17, 18), and this decarboxylase has been purified to homogeneity (14). If this malonyl-CoA decarboxylase plays such a regulatory role this enzyme must be present specifically in this gland. To test this possibility, we prepared rabbit antiserum against the purified malonylCoA decarboxylase and examined extracts from a variety of organs of the goose for an immunologically cross-reacting material. We concentrated the 105,OOOgsupernatant from all of these tissues and conducted Ouchterlony double-diffision analyses at different protein concentrations. Uropygial gland was the only organ in which immunologically cross-reacting material was found. Thus, this enzyme appears to be a tissue-specific protein in its occurrence. To determine whether malonyl-CoA decarboxylase is present in very small quantities in other tissues, the goose liver was thoroughly examined. The 105,OOOg supernatant from the liver was subjected to gel filtration on a Sepharose 4B column and fractions were examined for malonyl-CoA decarboxylase activity. Very low activity was detected in fractions which had an elution volume similar to that observed for the enzyme from the uropygial gland (14). Upon concentration, the specific activity of the liver preparation was 1.2 nmol/min/mg, whereas the corresponding fraction from the gland of the same animal gave a value of 918 nmol/min/mg. Similarly, the total units of activity in the liver were also only a small (2.0.1%) fraction of the units found in the uropygial gland. To determine whether the small amount of malonyl-CoA decarboxylase activity found in the liver is due to the same enzyme as that purified from the gland, the antiserum prepared against the enzyme from the gland was used against the malonyl-CoA decarboxylase fraction obtained from the gel filtration step indicated above. Since no cross-reactivity

158

BUCKNER,

KOLATTUKUDY,

could be observed with Ouchterlony double-diffusion analyses this fraction was concentrated, and at high protein concentration immunoprecipitant lines were observed in spite of the complications caused by the very high protein concentration (Fig. 4). The complete fusion of the precipitant lines strongly suggested that the low amount of malonyl-CoA decarboxylase in the liver is identical to that found in the gland. Low levels of malonyl-CoA decarboxylase activity have been observed in a variety of animal tissues (19-22). However, since this enzyme has not been purified from any source other than the uropygial gland (13), it is not possible to determine whether the enzyme in the uropygial gland is similar to those present in other animal tissues. The results discussed above and in previous communications (14, 17, 18) show that a decarboxylase, which is specific for malonyl-CoA, is present in relatively large amounts (~1% of the protein) specifically in the uropygial glands of the goose. The amount of this enzyme present in the gland would be quite adequate to prevent the availability of malonyl-CoA to the fatty acid synthetase, assuming that the in vitro rates of acetyl-CoA carboxylase and malonyl-CoA decarboxylase accurately reflect the activities of these enzymes in viuo (18). Since acetyl-CoA carboxylase of the uropygial gland catalyzes the formation of methylmalonyl-CoA from propionyl-CoA at nearly the same rate as the formation of malonyl-CoA (171, this gland would be expected to contain acetyl-CoA and methylmalonyl-CoA as the major substrates for the fatty acid synthetase. With these substrates the fatty acid synthetase from the gland is known to generate 2,4,6,&tetramethyldecanoic acid as the major product (4), and this acid is known to be the major acid of the uropygial gland wax in uiuo (3, 5, 6). Thus, in this gland a single new enzyme, namely, malonyl-CoA decarboxylase, causes the synthesis of unusual multimethyl-branched acids using the same carboxylase and fatty acid synthetase as those found in other animal tissues.

AND ROGERS

FIG. 4. Ouchterlony double-diffusion analysis of the purified malonyl-CoA decarboxylase from the uropygial gland and partially purified decarboxylase from the liver of the goose. The center well contained rabbit antiserum prepared against puritied decarboxylase from the gland. Outer wells 1, 3, and 5 contained 20, 10, and 2 mg/ml of a malonylCoA decarboxylase preparation, partially purified by Sepharose 4B gel filtration from the 105,OOOg supernatant prepared from goose liver homogenate. Wells 2, 4, and 6 contained purified malonyl-CoA decarboxylase (2 mg/ml) from the uropygial gland.

Methylmalonyl-CoA is known to be a competitive inhibitor with respect to malonyl-CoA for fatty acid synthetase from other animal tissues (23). In view of the fact that fatty acid synthetase in the uropygial gland uses methylmalonyl-CoA as the substrate to generate multimethylbranched fatty acids in uiuo, it was necessary to determine whether methylmalonyl-CoA is a competitive inhibitor of this enzyme. Methylmalonyl-CoA inhibited the incorporation of malonyl-CoA, and double-reciprocal plots (l/u vs l/s) of malonyl-CoA incorporation at four different concentrations of methylmalonyl-CoA showed linear relationships. All lines intercepted at the same point on the u-axis as expected from a classical competitive inhibitor. The plot of the slope vs concentration of methylmalonyl-CoA was linear (Fig. 51, and from this plot a Ki of 1.6 x 10m5M was calculated, which is about onethird of the K, (5 x 10e5 M) for malonylCoA. Thus, fatty acid synthetase from the uropygial gland is quite similar in this respect to synthetases from other animals, although the in uiuo product of the enzyme is different. If methylmalonyl-CoA

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

FIG. 5. Determination of the Ki of methylmalonyl-CoA for the incorporation of malonyl-CoA into n-fatty acids by the purified fatty acid synthetase from the goose uropygial gland. The slope of the double-reciprocal (l/u vs l/s) plots is plotted against the concentration of the inhibitor.

simply substitutes for malonyl-CoA, the latter substrate might be a competitive inhibitor for the synthesis of multimethylbranched fatty acids. To test this possibility the effect of unlabeled malonyl-CoA on the incorporation of [3-14ClmethylmalonylCoA into fatty acids was determined. Surprisingly, low concentrations of malonylCoA stimulated the incorporation of methylmalonyl-CoA into fatty acids. For example, with a subsaturating concentration (25 PM) of methylmalonyl-CoA, 8 PM malonyl-CoA caused a 40% stimulation of incorporation of methylmalonyl-Cob. This stimulation is probably due to the formation of aliphatic chains in which both malonyl and methylmalonyl moieties have been incorporated (mixed chain). Since the rate of synthesis of branched chains is very low compared to that of straight chains, a mixed chain might be expected to be formed at intermediate rates. If such is the case, the composition of the fatty acids generated from [3J4C]methylmalonyl-CoA in the presence of unlabeled malonyl-CoA should be altered to reflect the malonyl-CoA incorporation. Radio gas-liquid chromatographic analyses showed that increasing concentrations of unlabeled malonyl-CoA increased the proportion of longer fatty acids generated from [3-14Clmethylmalonyl-CoA (Fig. 6). From their retention times, it was obvious that these longer acids were branched,

159

FATTY ACIDS

but they were not homologues of 2,4,6,8tetramethyldecanoic acid, representing incorporation of additional C, units. Thus, it appears quite clear that these longer labeled acids represent mixed chains. Since all of the known properties of the fatty acid synthetase from the uropygial glands are extremely similar to those of fatty acid synthetase from other animals (41,it appeared probable that the synthesis of multimethyl-branched fatty acids is an innate property of fatty acid synthetase of animals. To test this possibility, fatty acid synthetase was partially purified from the 105,OOOgsupernatant from rat mammary

0

30

lo TIME Ch%

FIG. 6. Radio gas-liquid chromatograms of fatty acids (as butyl esters) generated from [methylW]methylmalonyl-CoA and acetyl-CoA in the presence of malonyl-CoA by purified fatty synthetase isolated from goose uropygial glands. Each reaction containing 16 nmol of [methylmixture, lClmethylmalonyl-CoA and 315 pg of protein, was incubated with the other components (see Experimental Procedures) and malonyl-CoA at the concentrations indicated for 60 min at 30°C; other experimental details are described in the text. The arrows indicate the retention times of the n-acids indicated.

160

BUCKNER,

KOLATTUKUDY.

glands by gel filtration using a Sepharose 4B column (Fig. 7). The elution profile of the protein showed four peaks, and fatty acid synthetase activity coincided with the second peak eluted from the column. Analytical ultracentrifugation showed one major component (13 S) and few minor components, A repetition of the gel filtration step gave a preparation purified to near homogeneity as indicated by analytical ultracentrifugation (Fig. 7). By a similar procedure, partially purified fatty acid synthetase was isolated from a 105,OOOg supernatant prepared from rat liver. In this case, the degree of purification of enzyme was less than that obtained from the mammary gland. The partially purified enzymes from both mammary gland and the liver of the rat incorporated [methyZ-14C]methylmalonyl-CoA into fatty acids. The specific activity for the enzyme from mammary gland was much higher than that obtained with the liver enzyme (Table I). The specific activity obtained with malonyl-CoA as the substrate for the mammary gland enzyme was also greater than the value obtained with the liver enzyme. With the enzyme from either source, as well as with the synthetase from the uropygial gland and the liver of the goose, the rate of methylmalonyl-CoA incorporation was several orders of magnitude less than the rate of synthesis of nfatty acids from malonyl-CoA. However, the relative rate of methylmalonyl-CoA

AND ROGERS

incorporation compared to that of malonylCoA was greater with the enzyme preparations from the rat than with the enzyme from the goose (Table I), and the values showed considerable variation, particularly in the case of the enzyme preparation from the rat tissues. A probable explanation for this difference is given in a succeeding paragraph. To determine the nature of the fatty acids generated by the fatty acid synthetase of rat from methylmalonyl-CoA, the labeled fatty acids derived from [methyl14Clmethylmalonyl-CoA were subjected to radio gas-liquid chromatographic analyses. To avoid the possibility of losses due to volatility, butyl esters were used for these analyses. The major product derived from methylmalonyl-CoA with acetyl-CoA as the primer was 2,4,6,8tetramethyldecanoic acid (Fig. 8a, peak A), but a significant amount of the higher homologue, 2,4,6,8,10+entamethyldodecanoic acid (peak C) was also formed. The higher and lower homologues (representing incorporation of 6 and 3 C3 units, respectively) were detectable, but only in very small quantities. Small amounts of 2,4,6,8-tetramethylundecanoic acid and smaller amounts of 2,4,6,8,10-pentamethyltridecanoic acid were also found. These acids are obviously generated from propionyl-CoA (as primer), which might have been generated from methylmalonyl-CoA. To test whether propionyl-CoA can in fact func-

FRACTION NUMBER FIG. 7. Sepharose 4B gel filtration of the 105,OOOg supernatant prepared from the mammary gland homogenates of lactating rata. The experimental procedures used for the recovery of purified fatty acid synthetase are the same as those described in Fig. 1, with the exception that 10 ~1 of each fraction was assayed for fatty acid synthetase activity (O-O). The inset shows a sedimentation velocity pattern of the fatty acid synthetase fraction isolated by repeated Sepharose 4B filtration. The analytical ultracentrifugation was done as described before (4).

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

0

IO

161

FATTY ACIDS

20 TIME (min)

30

40

FIG. 8. (a) Radio gas-liquid chromatograms of fatty acids (as butyl esters) generated from [methyl-Wlmethylmalonyl-CoA, with acetyl-CoA (A) or propionyl-CoA (P) as the starter, by the partially purified fatty acid synthetase from rat mammary glands. A, 2,4,6,8-tetramethyldecanoic acid; B, 2,4,6,8-tetramethylundecanoic acid; C, 2,4,6,8,10pentamethyldodecanoic acid, D, 2,4,6,7,10pentamethyltridecanoic acid. Identification was by comparison of the retention times with those of naturally occurring fatty acids previously identified by mass spectrometry (3). In the top tracing the unmarked component is tentatively identified from its retention time to be 2,4,6,-trimethylnonanoic acid. (b) Radio gas-liquid chromatogram of fatty acids (as butyl esters) generated from [m.ethyZ-‘4C]methylmalonyl-CoA and acetyl-CoA by a partially purified fatty acid synthetase preparation from the mammary gland (M) and liver (L) of a lactating rat. The bottom tracing shows the flame ionization detector response from a coinjected mixture of authentic n-fatty acid butyl esters (&,-C&J and the butyl esters of the fatty acids from the goose uropygial gland. In both cases, procedures for the recovery of fatty acids, preparation of butyl ester derivatives, and radio gas-liquid chromatography conditions are as described under Experimental Procedures. A, 2,4,6,8-tetramethyldecanoic acid from the gland lipid.

tion as the primer, unlabeled propionylCoA was added instead of acetyl-CoA into a reaction mixture containing [methyl14C]methylmalonyl-CoA. Radio gas-liquid chromatographic analyses of the products showed that the major products were 2,4,6&tetramethylundecanoic acid and a smaller amount of what appears to be (from the retention time) 2,4,6-trimethylnonanoic acid (Fig. 8a). This trimethyl acid is a substantial product only when propionyl-CoA is the primer, although the corresponding acid generated from acetylCoA (2,4,6-trimethyloctanoic acid) was detectable. In any case it is quite obvious that fatty acid synthetase from rat, which does not generate significant amounts of multimethyl-branched acids in uiuo, can

also synthesize the same multimethylbranched acids as those generated by the uropygial gland of the goose in uivo and by the synthetase isolated from the gland. With the synthetase from the rat the relative rates of synthesis of branched and n-fatty acids (ratio shown in Table I), as well as the nature of the products generated from methylmalonyl-Cob, showed considerable variation. For example, with preparations which allowed the higher relative rates of incorporation of methylmalonyl-CoA, the major labeled fatty acids showed retention times greater than those generated by the enzyme from the goose (Fig. 8b). The retention times of these acids did not coincide with those of n-fatty acids and bromination did not affect the

162

BUCKNER.

KOLATTUKUDY.

of radioactivity distribution. pattern Therefore, it is obvious that these acids are branched, but the retention times indicated that the major acids are not simply higher homologues of 2,4,6,8-tetramethyldecanoic acid representing incorporation of additional C, units. The most probable explanation appears to be that the acids generated by this enzyme preparation from the rat contain both straight portions and branches; similar 14C distribution patterns suggesting the formation of mixed chains have been previously observed in crude preparations of rat liver fatty acid synthetase (24). The enzyme isolated from the rat tissue contains either short n-acids or their precursors and they participate in the synthesis of fatty acids from methylmalonyl-CoA, giving rise to a mixed population of acids. The fact that the addition of malonyl-CoA stimulates incorporation of methylmalonyl-CoA by the goose enzyme to give a mixed population of acids (see above) supports this conclusion. Furthermore, the observation that the rate of incorporation of methylmalonyl-CoA is higher for the enzyme preparation from the rat than those from the goose is also consistent with the explanation indicated above. Among the fatty acid synthetase preparations from the rat, those which showed a low relative incorporation of methylmalonyl-CoA, somewhat comparable to those observed with the enzyme preparation from the goose (Table I), gave the fully branched acids (methyl branch on each of the alternate carbon atoms). On the other hand, preparations which showed higher relative rates of incorporation of methylmalonyl-CoA gave mixed chains. The physiological factors which give rise to these variations are unknown. The size of the branched and mixed chains generated by the fatty acid synthetase preparations also shed some light on the chain termination process. The fatty acid synthetase preparations from both the rat and the goose generate n-C,, acid as the major product from malonyl-CoA, and the chain-terminating thioesterase segment, isolated from the synthetase from rat (25) and goose (261, shows specificity for thioesters of n-C,, and longer acids.

AND

ROGERS

On the other hand, when the aliphatic chains are built solely from methylmalonyl-CoA, tetramethyldecanoic acid is the major product. In this case, since the condensation rate is very low, the relative rate of thioesterase competes effectively with condensation. When aliphatic chains containing straight and branched portions are formed the condensation rate is presumably higher than that obtained with fully branched chains (as indicated by the higher relative rates of incorporation of methylmalonyl-CoA in the presence of malonyl-CoA and with some enzyme preparations from the rat shown in Table 1). Under these conditions the products formed fall between n-C,, acid and the tetramethyldecanoic acid with respect to gas chromatographic retention time and hydrophobicity. Presumably, as the chains contain more straight regions further condensation effectively competes against the thioesterase, resulting in larger products. The limiting case is n-C,, acid, in which case the thioester is rapidly hydrolyzed by the thioesterase segment of the synthetase. Thus, the notion that the specificity of the thioesterase determines the size of the product (27) and the more detailed concept, that the relative rates of condensation vs chain termination determine the size of the product (28), are further illustrated by the synthesis of n- and branched acids observed in the present investigation. The results presented in this paper strongly suggest that fatty acid synthetase from animals is capable of generating tetramethyl and pentamethyl fatty acids when methylmalonyl-CoA is the sole substrate available, whereas a mixed population of acids containing both straight and branched portions would result when both malonyl-CoA and methylmalonyl-CoA are available. Thus, under the pathological conditions, which result in defects in the methylmalonyl-CoA isomerase reaction (7-11, 291, this intermediate could accumulate. For example, under B,, deliciency, elevated levels of methylmalonylCoA have been observed (30). Under such conditions methylmalonyl-CoA could participate in fatty acid synthesis, as demon-

ENZYMATIC

SYNTHESIS

OF MULTIMETHYL-BRANCHED

strated in the present paper, and the branched acids thus generated would be expected to generate functionally defective membranes and therefore play a role in pathogenesis. Thus, the results presented in this paper provide a reasonable biochemical explanation for the finding that methyl-branched acids are detected in the lipids under conditions which do not permit a normal functioning of the methylmalonyl-CoA isomerase (31, 32). REFERENCES 1. DOWNING, D. T. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, P. E., ed.), pp. 18-42, Elsevier, New York. 2. JACOB, J. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, P. E., ed.), pp. 94-141, Elsevier, New York. 3. BIJCKNER, J. S., AND KOLATTIJKIJDY, P. E. (1975) Biochemistry 14, 1774-1782. 4. BUCKNEB, J. S., AND KOLATTVKVDY, P. E. (1976) Biochemistry 15, 1948-1957. 5. MURRAY, K. E. (1962). Aust. J. Chem. 15, 510520. 6. ODHAM, G. (1963)Ark. Kern. 21, 379-393. PFOC. Nat. Acad. Sci. USA 63, 191-197. 7. ANDO, T., RA~MV~~EN, K., NYHAM, W. L., DONNELL, G. N., AND BARNES, N. D. (1971) J.

Pediat. 73, 827-832. 9. PANT, S. S., ABBVRY, A. K., AND RICHAIWBON, E. P. (1968) Acta Neural. Stand. Suppl. 35, 6-36. 9. OBEBHOLZER, V. G., LEVIN, B., BVFLGE~~, E. S., AND YOUNG, W. F. (1967) Arch. Dis. Child 42, 492-504. 10. STOKKE, O., ELDJARN, L., NORVM, K. R., STEENJOHNSON, J., AND HALVOR~EN, S. (1967) &and. J. C&n. Lab. Znvest. 20, 313-328. 11. MAHONEY, M. J., HART, A. C., STEEN, V. D., AND ROSENBERG, L. E. (1975) Proc. Nat. Acad.

Sci. USA 72, 2799-2803. 12. MORROW, G., BARNESS, L. A., CARDINALE, G. J., ABELES, R. H., AND FLAKB, J. G. (1969) Proc. Nat. Acad. Sci. USA 63, 191-197. 13. LOWRY, 0. H., R~SEBROVGW, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275.

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ACIDS

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14. BVCKNER, J. S., KOLATTVKVDY, P. E., AND PovLOSE, A. J. (1976) Arch. B&hem. Biaphys. 177, 539-551. 15. OVCHTERLONY, 0. (1953) Acta Pathol. Microbial. Stand. 32, 231-240. 16. SMITH, S. (1973) Arch. Biochem. Biophys. 156, 751-758. 17. BVCKNER, J. S., AND KOLATTVKVDY, P. E. (1975) Biochemistry 14, 1768-1773. 18. BVCKNER, J. S., AND KOLATTVKVDY, P. E. (1976) in Chemistry and Biochemistry of Natural Waxes (Kolattukudy, P. E., ed.), pp. 148-184, Elsevier, New York. 19. NAKADA, H. I., WOLFE, J. B., AND WICK, A. N. (1957) J. Biol. Chem. 226, 145-152. 20. LANDRISCINA, C., GNONI, G. V., AND QVAGLIARIELU), E. (1971) Eur. J. B&hem. 19, 573580. 21. BOONE, S. C., AND WAKIL, S. J. (1970) Biochemistry 9, 1470-1479. 22. KOEPPE, A. H., MITZEN, E. J., AND AMMOVRI, A. A. (1974) Biochemistry 13,3589-3595. 23. FORWARD, S. A., AND G~MPERTZ, D. (1970) En-

zymologia 39, 379-390. 24. CARDINALE, G. J., CARTY, T. J., AND ABELBE, R. H. (1970) J. Biol. Chem. 245, 3771-3775. 25. SMITH, S., AGRADI, E., LIBERTINI, L. AND DILEEPAN, K. N. (1976) Proc. Nat. Acad. Sci. USA 73, 1184-1188. 26. BEWRD, C. J., KOLA~VHVDY, P. E., AND ROGERS, L. (1978) Arch. Biochem. Biophys. 186. 139-151. 27. BARNES, E. M., AND WAKIL, S. J. (1968) J. Bioz.

Chem. 243, 2955-2962. 28. SUMP&R, M., OEBTERHELT, D., RIEPERTINGER, C., AND LYNEN, F. (1969) EUF. J. Biochem. 10, 377-387. 29. MORROW, G., MAHONEY, M. J., MATHEWS, C., AND LEBOWITZ, J. (1975) Pediut. Res. 9, 641644. 30. FRENKEL, E. P., KITCHENS, R. L., HEREIH, L. B., AND FRENKEL, R. (1974) J. Biol. Chem. 249, 6984-6991. 31. GARTON, G. A., STAID, J. R., SMITH, A., AND SIDDONS, R. C. (1975) Lipids 10, 855-857. 32. KISHIMOT~, Y., WILLIAMS, M., MOSER, H. W., HIGNITE, C., AND BIEMANN, K. (1973) J. Lipid Res. 14, 69-77.