The coenzyme A requirement of mammalian fatty acid synthetase: evidence for involvement in the elongation of acyl-enzyme thioesters

We have confirmed that coenzyme A is required for rat fatty acid synthetase activity (T. C. Linn, M. J. Stark, and P. A. Srere, 1980, J. Biol. Chem.255, 1388–1392). When rat liver or mammary gland fatty acid synthetase was assayed in the presence of a CoA-scavenging system such as ATP citrate lyase, almost complete inhibition of fatty acid synthesis was observed. The inhibition was reversed by addition of CoA or pantetheine, but not by addition of N-acetylcysteamine or other thiols. In the absence of CoA, the rate of elongation of acyl moieties on both native fatty acid synthetase and fatty acid synthetase lacking the chain-terminating thioesterase I component (trypsinized fatty acid synthetase) was reduced 100-fold. All of the palmitate synthesized slowly by the CoA-depleted native multienzyme was released, by the thioesterase I component, as the free fatty acid; only shorter-chainlength acyl moieties remained bound to the enzyme. The acyl-S-multienzyme thioesters formed by the trypsinized fatty acid synthetase in the absence of CoA contained saturated moieties of chain length C₆-C₁₆; addition of CoA promoted elongation of the acyl-S-multienzyme thioesters without release from the enzyme. The transfer of acetyl and malonyl moieties from CoA to the multienzyme, the reduction of S-acetoacetyl-N-acetylcysteamine and S-crotonyl-N-acetylcysteamine, and the dehydration of S-β-hydroxybutyryl-N-acetylcysteamine, reactions catalyzed by the fatty acid synthetase, were not dependent on the presence of CoA. The hydrolysis of acyl-S-multienzyme catalyzed by thioesterase I, the resident chain-terminating component of the fatty acid synthetase, and thioesterase II, a monofunctional mammary gland chain-terminating enzyme, was also independent of CoA availability as was hydrolysis of an acyl-S-pantetheine pentapeptide isolated from the multienzyme. On the basis of these observations we conclude that CoA is required for the elongation of acyl moieties on the fatty acid synthetase but not for their release from the multienzyme.     

Cerulenin resistance in a cerulenin-producing fungus. Isolation of cerulenin insensitive fatty acid synthetase

Cerulenin, an antifungal antibiotic isolated from a culture filtrate of Cephalosporium caerulens, is a potent inhibitor of fatty acid synthetase systems. This antibiotic specifically blocks the activity of β-ketoacyl thioester synthetase (condensing enzyme). The mechanism of the resistance of C. caerulens to cerulenin was investigated. The rate of growth in medium containing up to 100 gmg/ml cerulenin was as rapid as that in cerulenin-free medium. At a cerulenin concentration of 300 μg/ml, the rate of growth was still more than half that of the control. The addition of cerulenin (200 μg/ml) to a culture of growing cells has almost no effect on the incorporation of [${}^{14}\text{C}$]acetate into cellular lipids. Fatty acid synthetase was purified from C. caerulens to homogeneity. Properties of this fatty acid synthetase were almost the same as those of yeast fatty acid synthetase except for the sensitivity to cerulenin. C. caerulens synthetase is much less sensitive to cerulenin than fatty acid synthetases from other sources. These findings suggested that the insensitivity of C. caerulens fatty acid synthetase plays an important role in the cerulenin resistance of this fungus.     

Fatty acid synthetase assay employing bicyclic diones as substrates.

A rapid and convenient spectrophotometric assay for fatty acid synthetase is presented. It is based on the NADPH-dependent reduction of compounds such as cis-decalin-1,4-dione by the β-ketoacyl-ACP reductase component of the fatty acid synthetase complex. The assay has applications where rapid, semiquantitative data are desired.     

Occurrence of apoAcyl carrier protein and an S-alkylation-resistant form of holoAcyl carrier protein in Escherichia coli.

The 4′-phosphopantetheine prosthetic group of holoacyl carrier protein (holoACP) in Escherichia coli turns over independently of the apoprotein, due to the activities of holoACP hydrolase and holoACP synthetase. There is no measurable pool of apoACP in pantothenate-supplemented cells of a pantothenate-requiring mutant, but extended incubation on deficient medium, with exhaustion of cellular coenzyme A (CoA), leads to slow accumulation of the apoprotein. It is concluded that, although the activities of the synthetase and hydrolase are about equal in crude extracts, in the cells an excess synthetase activity maintains ACP completely as holoACP unless cells are artifically depleted of CoA, the donor of the 4′-phosphopantetheine group. About 20% of the holoACP in normal cells was designated as “holoACP esters,” being resistant to S-alkylation unless first treated with neutral hydroxylamine; this proportion increased to about 80% in pantothenate starvation. A preliminary attempt to identify acyl portions from this material was unsuccessful. The proportion of this material was not elevated in other strains under conditions which show feedback inhibition of fatty acid biosynthesis in vivo.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

S-Adenosylmethionine,cyclopropane fatty acid synthase,and the production of lactobacillic acid in Lactobacillus plantarum

S-Adenosylmethionine (AdoMet) levels in Lactobacillus plantarum were found to increase concomitantly with the production of membrane cyclopropane fatty acids under normal growth conditions. This increase in AdoMet did not occur when the pH of the culture medium (initially pH 6.5) was not allowed to fall (pH 4 or lower) during growth. When the culture medium was maintained at pH 6.5, cyclopropane fatty acid synthesis also remained low. While the activity of cyclopropane fatty acid synthase is increased as the pH decreases, the activity of AdoMet synthetase is largely unaffected by the variation of pH of the culture medium. The production of cyclopropane fatty acids is also dependent upon continued protein synthesis; in the presence of chloramphenicol cyclopropane fatty acid synthase activity is decreased, resulting in a lowered production of cyclopropane fatty acids. A dramatic increase in AdoMet levels occurs in the presence of chloramphenicol. It is proposed that AdoMet levels, in conjunction with cyclopropane fatty acid synthase activities, regulate cyclopropane fatty acid synthesis in L. plantarum.     

Subcellular Sites Involved in Lipid Synthesis in Saccharomyces cerevisiae

When the crude ribosomal fraction of Saccharomyces cerevisiae was separated into "light" and "heavy" fractions, fatty acid synthetase was concentrated in the former, whereas acetyl-Coenzyme A synthetase, fatty acid "desaturase," and squalene oxidocyclase were found in the latter. The "desaturase" sedimented with the ribosomal material and was not solubilized by low concentrations of sodium deoxycholate (DOC). The other two systems found in the "heavy" fraction sedimented with the membranes, but, upon solubilization of the membranes by DOC, these enzyme systems remained as particles.     

Differential effects of Mg2+ on various hydrolytic and synthetic activities of multifunctional glucose-6-phosphatase

The adaptive synthesis of fatty acid synthetase in the livers of rats fed a fat-free diet following 48 hr of fasting has been studied using immunochemical methods. The development of fatty acid synthetase activity during adaptive synthesis occurs about 3 hr following feeding, whereas the synthesis of material precipitable by anti-fatty acid synthetase serum, as judged by the incorporation of ³H-labeled amino acids into the immunoprecipitate, commenced within 1 hr. Extracts of liver of rats fed a fat-free diet for 1–3 hr following fasting contain increasing amounts of material which competes with purified fatty acid synthetase for antibody binding sites, even though they have no fatty acid synthetase activity. This suggests the presence of enzymatically inactive precursors of fatty acid synthetase in the liver extracts. The incorporation of [¹⁴C]pantothenate into fatty acid synthetase during adaptive synthesis follows the same pattern as the development of enzyme activity, indicating that these enzymatically inactive precursors of fatty acid synthetase may represent an apoenzyme which is converted to the enzymatically active holoenzyme by the incorporation of the 4′-phosphopantetheine prosthetic group. The subcellular site of synthesis of fatty acid synthetase was shown to be in the pool of polysomes that are not membrane bound, rather than in the rough endoplasmic reticulum.     

An extracellular lipopolysaccharide-phospholipid-protein complex produced by Escherichia coli grown under lysinelimiting conditions

Lysine limitation during growth of the lysine-requiring mutant of Escherichia coli 12408 resulted in the excretion of a complex containing 60% of lipopolysaccharide, 26% of extractable phospholipid and 11% of protein. The complex was obtained from the culture filtrate in yields of about 0·5g./l. by precipitation with chloroform or gel filtration; some purification steps are described. The greater part of the phospholipid consisted of phosphatidylethanolamine, which contained four main fatty acids; two monoenoic acids and a cyclopropane acid were esterified mainly in the β-position, and a saturated acid was located mainly in the γ-position. The protein component was relatively insoluble and contained an excess of acidic over basic amino acids and little cystine. The lipopolysaccharide resembled in composition the intracellular lipopolysaccharides from rough strains of E. coli. Both protein and lipopolysaccharide constituents of the complex were serologically active; the complex was less toxic than the purified lipopolysaccharide. In the electron microscope the complex showed a mixture of particles of various sizes and shapes. Rods and hollow spheroids (diameter 12–200mμ) were most common and resembled the particles previously found surrounding cells actively excreting the complex. The chloroform-precipitated material showed a tubular lamellar structure. Soluble lipopolysaccharide prepared from the complex also consisted of hollow spheres and rods.     

Biosynthesis of adenosyl-d-methionine and adenosyl-2-methylmethionine by Candida utilis

Supplementation of the culture medium of Candida utilis with d-methionine or 2-methyl-dl-methionine leads to intracellular synthesis of S-adenosyl-d-methionine and S-adenosyl-2-methylmethionine. The identity of the sulfonium compounds was established by tracer technique, chromatography, acid hydrolysis, and examination of the released methionine and 2-methylmethionine. In addition to the expected sulfur amino acid component, both adenosine sulfonium fractions contained S-adenosyl-l-methionine. This is explained by transmethylation of S-adenosyl-d-methionine and of S-adenosyl-2-methyl-methionine with endogenous l-homocysteine; the resulting l-methionine reacts with ATP to form S-adenosyl-l-methionine. Experiments with purified cell-free preparations of S-adenosylmethionine synthetase (EC 2.5.1.6) from C. utilis confirmed the reaction of ATP with d-methionine or 2-methyl-dl-methionine.     

Fat Metabolism in Higher Plants XXXVI: Long Chain Fatty Acid Synthesis in Germinating Peas

A low lipid, high starch containing tissue, namely cotyledons of germinating pea seedlings was examined for its capacity to synthesize fatty acid. Intact tissue slices readily incorporate acetate-¹⁴C into fatty acids from C₁₆ to C₂₄. Although crude homogenates synthesize primarily 16:0 and 18:0 from malonyl CoA, subsequent fractionation into a 10,000g pellet, a 10⁵g pellet and supernatant (soluble synthetase) revealed that the 10⁵g pellet readily synthesizes C₁₆ to C₂₈ fatty acids whereas the 10,000g and the supernatant synthesize primarily C₁₆ and C₁₈. All systems require acyl carrier protein (ACP), TPNH, DPNH if malonyl CoA is the substrate and ACP, Mg²⁺, CO₂, ATP, TPNH, and DPNH if acetyl CoA is the substrate. The cotyledons of germinating pea seedlings appear to have a soluble synthetase and 10,000g particles for the synthesis of C₁₆ and C₁₈ fatty acid, and 10⁵g particles which specifically synthesize the very long chain fatty acid from malonyl CoA, presumably via malonyl ACP.     

A chloroplast-associated fatty acid synthetase system in Euglena

Fatty acid synthetase activity in etiolated Euglena gracilis strain Z is independent of added ACP and associated with a high-molecular-weight complex of the type found in yeast. Cells grown in the dark and then greened by illumination in a resting medium develop a second enzyme system which is dependent on added ACP and generally resembles the corresponding E. coli and plant enzymes. Cycloheximide has no effect on the appearance of the ACP-dependent fatty acid synthetase in greening cells whereas chloramphenicol causes complete inhibition at concentrations which decrease chlorophyll synthesis by 66%. An induction of the ACP-dependent fatty acid synthetase in the absence of chloroplast development occurs on exposure of dark-grown cells to doses of ultraviolet light which selectively affect proplastid nucleoprotein. This enzyme induction by ultraviolet light is inhibited by chloramphenicol. The protein synthesis machinery of the chloroplast appears to be responsible, either directly or indirectly, for the appearance of the ACP-dependent fatty acid synthetase of Euglena.     

A high-performance liquid chromatographic method for the enantiomeric analysis of the enzymatically synthesized coenzyme A ester of 2-tetradecylglycidic acid

The enantiomeric composition of an enzymatically synthesized sample of the coenzyme A ester of 2-tetradecylglycidic acid (TDGA-CoA) was determined by the use of high-performance liquid chromatography with a chiral stationary phase. The stationary phase was commercially available and consisted of (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to aminopropyl silica gel. Analysis was performed using the phenacyl derivative of 2-tetradecylglycidic acid (TDGA), obtained by mild hydrolysis of the TDGA-CoA followed by reaction of the extracted TDGA with phenacyl chloride. Chromatography showed the enantiomeric purity of TDGA-CoA, synthesized in a rat liver microsomal enzyme mixture over a 2-h period, to be a 15.6:1 ratio of the R:S enantiomers (88% ee). The result demonstrates the steroselectivity of the long-chain fatty acid-coenzyme A synthetase for chiral fatty acid epoxide, TDGA.     

Isolation and characterization of a tryptic fragment containing the thioesterase segment of fatty acid synthetase from the uropygial gland of goose.

Trypsin treatment of purified fatty acid synthetase from the uropygial gland of goose released a 33,000 molecular weight peptide from the 270,000 molecular weight synthease. A combination of ammonium sulfate precipitation, Sephadex G-100 gel filtration, anion-exchange chromatography with QAE-Sephadex, and cation-exchange chromatography with cellulose phosphate gave rise to the first homogeneous preparation of the 33,000 molecular weight fragment containing fatty acyl-CoA thioesterase activity. Amino acid composition of this peptide was quite similar to that of the intact fatty acid synthetase except for a lower valine content; a partial specific volume of 0.734 was calculated for the thioesterase fragment. The pH optimum for the thioesterase was near 7.5 and the enzyme showed a high degree of preference for CoA esters of fatty acids with 16 or more carbon atoms. Palmitoyl-CoA inhibited the enzyme and therefore the rate of hydrolysis was not proportional to the amount of protein at low concentrations. Inclusion of bovine serum albumin in the reaction mixture prevented this inhibition. Disregarding the substrate inhibition, an apparent K_m of 5 × 10^?5m and a V of 340 nmol/min/mg were calculated. The thioesterase was inhibited by active serine-directed reagents such as phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate as well as by SH-directed reagents as p-chloromercuribenzoate and N-ethylmaleimide. The isolated thioesterase fragment generated antibodies in rabbits and the antithioesterase inhibited the enzymatic activity of fatty acid synthetase. The antithioesterase showed immunoprecipitant lines with fatty acid synthetase from the uropygial gland and the synthetase from the liver of goose. Anti-fatty acid synthetase prepared against the enzyme from the gland cross-reacted with the thioesterase segment. Even though the synthetase from the uropygial gland synthesizes multimethyl-branched fatty acids in vivo, the thioesterase segment of this synthetase appears to be quite similar to that isolated from the rat.     

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

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-pentamethyl-dodecanoic 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 105,000g 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 tissue-specific 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 [methyl-¹⁴C]methylmalonyl-CoA into 2,4,6,8-tetramethyldecanoic acid and 2,4,6,8-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-CoA, abnormal branched acids can be generated by the fatty acid synthetase.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

An Acyl-CoA Synthetase in Mycobacterium tuberculosis Involved in Triacylglycerol Accumulation during Dormancy

Latent infection with dormant Mycobacterium tuberculosis is one of the major reasons behind the emergence of drug-resistant strains of the pathogen worldwide. In its dormant state, the pathogen accumulates lipid droplets containing triacylglycerol synthesized from fatty acids derived from host lipids. In this study, we show that Rv1206 (FACL6), which is annotated as an acyl-CoA synthetase and resembles eukaryotic fatty acid transport proteins, is able to stimulate fatty acid uptake in E. coli cells. We show that purified FACL6 displays acyl-coenzyme A synthetase activity with a preference towards oleic acid, which is one of the predominant fatty acids in host lipids. Our results indicate that the expression of FACL6 protein in Mycobacterium tuberculosis is significantly increased during in vitro dormancy. The facl6-deficient Mycobacterium tuberculosis mutant displayed a diminished ability to synthesize acyl-coenzyme A in cell-free extracts. Furthermore, during in vitro dormancy, the mutant synthesized lower levels of intracellular triacylglycerol from exogenous fatty acids. Complementation partially restored the lost function. Our results suggest that FACL6 modulates triacylglycerol accumulation as the pathogen enters dormancy by activating fatty acids.     

Control of fatty acid synthetase mRNA levels in rat liver by insulin, glucagon, and dibutyl cyclic AMP

The quantity of translatable fatty acid synthetase mRNA in liver of rats subjected to different hormonal states was determined with a rabbit reticulocyte lysate cell-free translation system. Both membrane-free polysomal and total cellular poly (A)-containing RNA were translated. The level of translatable fatty acid synthetase mRNA was 11-fold or more lower in livers of diabetic rats than in similar animals treated with insulin. In contrast, both glucagon and dibutyl cyclic AMP caused a 3-fold reduction over controls in the amount of translatable fatty acid synthetase mRNA in livers of animals refed a fat-free diet for 12 hr. These changes are consistent with the previously reported alterations in the relative rates of fatty acid synthetase synthesis measured in vivo. This suggests that the changes in the amount of fatty acid synthetase that occur in liver in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.     

Adaptive synthesis of fatty acid synthetase and acetyl-CoA carboxylase by isolated rat liver cells.

Hepatocytes were isolated at specified times from livers of diabetic and insulin-treated diabetic rats during the course of a 48-h refeeding of a fat-free diet to previously fasted rats. The rates of synthesis of fatty acid synthetase and acetyl-CoA carboxylase in the isolated cells were determined as a function of time of refeeding by a 2-h incubation with l-[U-¹⁴C]leucine. Immunochemical methods were employed to determine the amount of radioactivity in the fatty acid synthetase and acetyl-CoA carboxylase proteins. The amount of radioactivity in the fatty acid synthetase synthesized by the isolated cells was also determined following enzyme purification of the enzyme to homogeneity. Enzyme activities of the fatty acid synthetase and acetyl-CoA carboxylase in the cells were measured by standard procedures. The results show that isolated liver cells obtained from insulintreated diabetic rats retain the capacity to synthesize fatty acid synthetase and acetyl-CoA carboxylase. The rate of synthesis of the fatty acid synthetase in the isolated cells was similar to the rate found in normal refed animals in in vivo experiments [Craig et al. (1972) Arch. Biochem. Biophys. 152, 619–630; Lakshmanan et al. (1972) Proc. Nat. Acad. Sci. USA69, 3516–3519]. In addition the relative rate of synthesis of fatty acid synthetase was stimulated greater than 20-fold in the diabetic animals treated with insulin. Immunochemical assays, when compared with enzyme activities, indicated the presence of an immunologically reactive, but enzymatically inactive, form or “apoenzyme” for both the fatty acid synthetase and acetyl-CoA carboxylase. The synthesis of these immunoreactive and enzymatically inactive species of protein, as well as the synthesis of the “holoenzyme” forms of both enzymes, requires insulin.     

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-¹⁴C]18:0)-CoA was synthesized from [1-¹⁴C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-¹⁴C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-¹⁴C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [¹⁴C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.