Mycolic acids of Mycobacterium aurum. Structure and biogenetic implications

Mycobacterium aurum (type strain) was analyzed for its mycolate content. Three types of mycolates were identified: di-unsaturated, oxo and dicarboxy mycolates, each type being constituted by two subtypes. The acid released by pyrolysis was identified as docosanoic acid. By use of mass spectrometry and oxidation techniques, the structures of these six subtypes of mycolates were elucidated. They contain Z and E double bonds, the latter having an adjacent methyl branch. No cyclopropane ring was observed. All the methyl branches occurring in these mycolates derived from methionine, the methyl-branched chiral center adjacent to the double bond having an R configuration. The structures of the major di-unsaturated mycolates suggest that they cannot be the precursors of oxo mycolates. The amount of long-chain secondary alcohol (2-octadecanol) obtained from the whole cells was found to be much greater than that expected from hydrolysis of wax ester mycolate (ester of 2-octadecanol and dicarboxy-mycolic acid). Further investigations showed that 2-octadecanol was also present in triacylglycerols, esterifying the omega-carboxyl group of long-chain fatty acids structurally related to dicarboxy-mycolates.     

Mycolic acid metabolic filiation and location in Mycobacterium aurum and Mycobacterium phlei

Synchronous cultures of Mycobacterium aurum were used to prove a close relationship between cellular division and active synthesis of mycolic acids (characteristic long-chain 3-hydroxyacids, branched at position 2), confirming previous proposals. Mycolic acid biosynthesis was studied in two species (Mycobacterium phlei and M. aurum) each producing three types of mycolic acids: di-unsatured mycolates, oxomycolates and wax-ester mycolates (ester of dicarboxymycolic acid and 2-icosanol or 2-octadecanol). It was shown that unsaturated mycolates and oxomycolic acids were not directly related, whereas a metabolic filiation was confirmed between oxomycolate and wax ester mycolate: the latter derived from the former by a Baeyer-Villiger oxidation step, as has been proposed on the basis of structural considerations. By observing the labelling of the different mycolate pools in the cell, i.e. the organic-solvent-extractable fraction (essentially containing esters of trehalose and of glycerol) and the cell residue (assumed to be the cell-wall polymers), it was clear that oxomycolates and unsaturated mycolates appeared first in the extractable lipids, then in the wall-linked mycolates while wax-ester mycolates appeared first as wall-linked derivatives. Thus, it is proposed that mycolates could follow separate routes involving differently located enzymes to reach their complex forms either in extractable lipids or in the wall-linked arabino-galactan.     

THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO-NIGRAL PATHWAY

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices. Unilateral transection of the striato-nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1-¹⁴C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U-¹⁴C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U-¹⁴C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side. Incubation of slices from substantia nigra with β-mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.     

The regulation of glyceride synthesis in isolated white-fat cells. The effects of acetate, pyruvate, lactate, palmitate, electron-acceptors, uncoupling agents and oligomycin

1. The incorporation of 5mm-[U-(14)C]glucose into glyceride fatty acids by fat cells from normal rats incubated in the presence of 20munits of insulin/ml was increased by acetate, pyruvate, palmitate, NNN'N'-tetramethyl-p-phenylenediamine, phenazine methosulphate, dinitrophenol, tetrachlorotrifluoromethyl benzimidazole and oligomycin. Lactate did not stimulate glucose incorporation into fatty acids. The effects of these agents were concentration-dependent. 2. In the presence of 5mm-glucose+insulin, [U-(14)C]acetate, [U-(14)C]pyruvate and [U-(14)C]lactate were incorporated into fatty acids in a concentration-dependent manner, thereby further increasing the total rate of fatty acid synthesis. 3. NNN'N'-tetramethyl-p-phenylenediamine decreased the incorporation of [U-(14)C]pyruvate into fatty acids in normal cells and increased the incorporation of [U-(14)C]lactate into fatty acids. 4. In fact cells from 72h-starved rats the stimulatory effects of NNN'N'-tetramethyl-p-phenylenediamine upon glucose and lactate incorporation into fatty acids were totally and partially abolished respectively whereas the stimulatory effects of acetate upon glucose incorporation were retained. 5. Combinations of the optimum concentrations of the substances that stimulate glucose incorporation into fatty acids were tested and compared. The effects of acetate+NNN'N'-tetramethyl-p-phenylenediamine and acetate+palmitate upon normal cells were additive. The effects of NNN'N'-tetramethyl-p-phenylenediamine+palmitate were not additive. It was found that total fatty acid synthesis in the presence of glucose was most effectively increased by raising the concentration of pyruvate in the incubation system. 6. The significance of these results in supporting the proposal that fatty acid synthesis from glucose in adipose tissue is a ;self-limiting process' is discussed.     

In vitro synthesis and O acetylation of peptidoglycan by permeabilized cells of Proteus mirabilis.

The synthesis and O acetylation in vitro of peptidoglycan by Proteus mirabilis was studied in microorganisms made permeable to specifically radiolabelled nucleotide precursors by treatment with either diethyl ether or toluene. Optimum synthesis occurred with cells permeabilized by 1% (vol/vol) toluene in 30 mM MgCl2 in in vitro experiments with 50 mM Tris-HCl buffer (pH 6.80). Acetate recovered by mild base hydrolysis from sodium dodecyl sulfate-insoluble peptidoglycan synthesized in the presence of UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was found to be radioactive. Radioactivity was not retained by peptidoglycan synthesized when UDP-[acetyl-1-14C]N-acetyl-D-glucosamine was replaced with both unlabelled nucleotide and either [acetyl-3H]N-acetyl-D-glucosamine or [glucosamine-1,6-3H]N-acetyl-D-glucosamine. In addition, no radioactive acetate was detected in the mild base hydrolysates of peptidoglycan synthesized in vitro with UDP-[glucosamine-6-3H]N-acetyl-D-glucosamine as the radiolabel. Chasing UDP-[acetyl-1-14C]N-acetyl-D-glucosamine with unlabelled material served to increase the yield of O-linked [14C]acetate, whereas penicillin G blocked both peptidoglycan synthesis and [14C]acetate transfer. These results support the hypothesis that the base-labile O-linked acetate is derived directly from N-acetylglucosamine incorporated into insoluble peptidoglycan via N----O transacetylation and not from the catabolism of the supplemented peptidoglycan precursors followed by subsequent reactivation of acetate.     

Localisation and characterization of the fatty acid synthesizing system in cells of Glycine max (soubean) suspension cultures

In course of a study of fatty acid synthetase in higher plants, non-green cell suspension cultures of Glycine max (soybean) served as model tissues. For the first time, a fatty acid synthesizing system was characterized in cell cultures of higher plants and was found to be solely located in proplastids of the cells. Optimum activity of the fatty acid synthesizing system in proplastids was observed between pH 8.0 and 8.2; with [1-14C]acetate as substrate, cofactors required were CoA, ATP, Mn2+, Mg2+, HCO3-, NADH and NADPH. The system was more sensitive towards NADH than NADHP. [1-14C]Acetate,[2-14C]-malonate and [3-14C]pyruvate served as precursors for fatty acids, indicating the presence of pyruvate dehydrogenase activity in proplastids. In disrupted proplastids, [2-14C]malonylCoA was a better precursor than [1-14C]acetylCoA. After incubation of proplastids with [2-14C]malonate, a small shift, from palmitic acid to higher homologs, of label incorporated was observed, as compared to incorporation of label from [1-14C]acetate and [3-14C]pyruvate. Under the conditions of the experiment, only small amounts of polyunsaturated fatty acids, the main fatty acid components of this organelle, were synthesized. In respect to fatty acid synthesis, the non-green cell suspension culture resembles photosynthetic leaf tissue.     

Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase

Allicin is shown to be a specific inhibitor of the acetyl-CoA synthetases from plants, yeast and mammals. The bacterial acetyl-CoA-forming system, consisting of acetate kinase and phosphotransacetylase, was inhibited too. Non-specific interaction with sulfhydryl-groups could be excluded in experiments with dithioerythritol and p-hydroxymercuribenzoate. Binding of allicin to the enzyme is non-covalent and reversible. [14C]-Acetate incorporation into fatty acids of isolated plastids was inhibited by allicin with an I50-value lower than 10 microM. Other enzymes of the fatty acid synthesis sequence were not affected, as was shown using precursors other than acetate.     

Lipogenesis and cholesterogenesis in the liver of rats after cholesterol loading

Intensity of fatty acids and separate classes of lipids synthesis was studied in vitro in the liver of white rats at loading by cholesterol in the dose of 300 mg/kg once a day during 30 days by incubation of organ homogenate with [6-(14)C] glucose, [2-(14)C] lysine, [1-(14)C] palmitic acid with following determination of radioactivity of fatty acids, phospholipids, cholesterol, acylglycerols radioactivity was investigated. The inhibition of fatty acids and separate classes of lipids synthesis in vitro in the liver of white rats at loading by cholesterol at the use of [6-(14)C] of glucose and [2-(14)C] lysine, as predecessors of fatty acids and lipids and stimulation of lipids synthesis at the use of [1-(14)C] palmitic acid as the predecessor was established. The loading of white rats by cholesterol results in its synthesis inhibition in the liver during incubation of its homogenates with [6-(14)C] glucose and does not influence the cholesterol synthesis during incubation of homogenates with [2-(14)C] lysine and [1-(14)C] palmitic acid. Thus synthesis of fatty acids and their use in the phospholipids and acylglycerols synthesis in the liver of white rats with hypercholesterolemia sharply decreases during incubation of their homogenates with [6-(14)C] glucose and [2-(14)C] lysine, and the synthesis of cholesterol, phospholipids and acylglycerols - increases during incubation with [1-(14)C] palmitic acid.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Requirement of glucose for mycolic acid biosynthetic activity localized in the cell wall of Bacterionema matruchotii

When the localization of mycolic acid biosynthetic activity was examined with Bacterionema matruchotii cells disrupted by the ultrasonic vibration method, activity was detected only in the cell wall fraction, not in the inner membrane nor in the 78,000g supernatant. Either the supernatant or sugar was absolutely required for the incorporation of [14C]palmitate into mycolic acids. Among sugars examined, glucose was most effective, with maltose being second. Unexpectedly, trehalose was inert. As to substrate, the present system utilized free palmitic acid rather than palmitoyl-CoA. The reaction products from palmitate and glucose were glucose mycolate and trehalose monomycolate, in which the label from [14C]palmitate or [14C]glucose was incorporated. Glucose palmitate was also formed. Addition of trehalose resulted in a shift from glucose mycolate to trehalose monomycolate. These data clearly indicate that sugars play an important role in the synthesis of mycolic acids from free fatty acids.     

The Substrate Utilization and Concentration of 14C Photosynthates in Citronella under Fe Deficiency

Changes in the utilization pattern of primary substrate, viz. [U-14C] acetate, 14CO2 and [U-14C] saccharose, and the contents of 14C fixation products in photosynthetic metabolites (sugars, amino acids, and organic acids) were determined in Fe-deficient citronella in relation to the essential oil accumulation. There was an overall decrease in photosynthetic efficiency of the Fe-deficient plants as evidenced by lower levels of incorporation into the sugar fraction and essential oil after 14CO2 had been supplied. When acetate and saccharose were fed to the Fe-deficient plants, despite a higher incorporation of label into sugars, amino acids, and organic acids, there was a lower incorporation of these metabolites into essential oils than in control plants. Thus, the availability of precursors and the translocation to a site of synthesis/accumulation, severely affected by Fe deficiency, is equally important for the essential oil biosynthesis in citronella.     

Tetraenoic and pentaenoic mycolic acids from Mycobacterium thamnopheos. Structure, taxonomic and biosynthetic implications

On the basis of the analysis of mycolates, the type strain of Mycobacterium thamnopheos has been considered as a member of the genus Nocardia. In a comparative study conducted on mycobacterial species we found that M. thamnopheos synthesized two types of mycolate having the same mobilities on thin-layer chromatography as those of mycobacteria, but different from nocardomycolates. Mass spectrometry analyzes showed that the major series of both types consisted of polyunsaturated mycolic acids, ranging from C72 to C78 with four or five double bonds. On pyrolytic mass spectrometry or gas chromatography, the least polar mycolates released mainly monounsaturated C22 esters whereas the other type yielded saturated C20 and C22 esters. These results suggested that M. thamnopheos might be more related to the Aurantiaca taxon than to mycobacteria and Nocardia. The permanganate-periodate oxidation products of esters obtained by pyrolysis of the least polar mycolates showed that they contained docosen-4-oic and docosen-6-oic acids. Both types of mycolate esters yielded the same set of long-chain meroaldehydes on pyrolysis. These meroaldehydes were significantly distinct from those of mycobacterial mycolates in the location of the double bonds. After hydrogenation of the double bond located in the alkyl-branched chain, the two types of mycolates had the same mobility on thin-layer chromatography, indicating that the difference of migration was due to the additional double bond found in the least polar mycolates. Based on stereochemical data, the relative configuration of both mycolates was found to be threo, like that established for all mycolates studied so far.     

Studies on the biosynthesis of iturin, an antibiotic of Bacillus subtilis, and a lipopeptide containing beta-hydroxy fatty acids

The biosynthesis of iturin, an antibiotic containing a beta-amino fatty acid, was studied by incubating Bacillus subtilis in the presence of various 14C-labelled precursors. Sodium acetate or palmitic acid were incorporated into the beta-amino acids of iturin. Among the alpha-amino acids (asparagine, glutamine, serine, proline and tyrosine) in the peptidic part of iturin, asparagine appears to be the best precursor. In the presence of sodium [14C]acetate or [14C]asparagine, there was a synthesis of radioactive compound (compound X) before the synthesis of radioactive iturin. Compound X contained asparagine and/or aspartic acid, glutamine and/or glutamic acid and beta-hydroxy fatty acids.     

The crucial role of trehalose and structurally related oligosaccharides in the biosynthesis and transfer of mycolic acids in Corynebacterineae

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.     

Propionate inhibits hepatocyte lipid synthesis

Oat bran lowers serum cholesterol in animals and humans. Propionate, a short-chain fatty acid produced by colonic bacterial fermentation of soluble fiber, is a potential mediator of this action. We tested the effect of propionate on hepatocyte lipid synthesis in rats using [1-14C]acetate, 3H2O, and [2-14C]mevalonate as precursors. Propionate produced a statistically significant inhibition of cholesterol biosynthesis from [1-14C]acetate at a concentration of 1.0 mM and from 3H2O and [2-14C]mevalonate at concentrations of 2.5 mM. Propionate also produced a significant inhibition of fatty acid biosynthesis at concentrations of 2.5 mM using [1-14C]acetate as a precursor. The demonstration of propionate-mediated inhibition of cholesterol and fatty acid biosynthesis at these concentrations suggests that propionate may inhibit cholesterol and fatty acid biosynthesis in vivo and may mediate in part the hypolipidemic effects of soluble dietary fiber. Further studies are needed to clarify this action of propionate and to establish the exact mechanisms by which the inhibition occurs.     

Radiolabelling Studies on the Lipid Metabolism in the Marine Brown Alga Dictyopteris membranacea

The lipid metabolism of the marine brown alga D. membranaceawas investigated using [2–¹⁴C]acetate, [1–¹⁴C]myristate,[l–^I4C]oleate and [l–¹⁴C]arachidonate as precursors.On incubation with [2–¹⁴C]acetate, 18:1 and 16:0 werethe main products formed by de novo synthesis and incorporatedinto polar lipids. With all the exogenous substrates used, DGTAwas strongly labelled and the subsequent rapid turnover of radioactivitysuggested a key role for this lipid in the redistribution ofacyl chains and most likely also in the biosynthesis of theeukaryotic galacto-lipids produced in the absence of PC. Inthe glycolipids a continuous accumulation of radioactivity wasobserved with all the substrates used. The labelling kineticsof molecular species of MGDG suggested the desaturation of 18:1to 18:4 and of 20:4 (n-6) to 20:5 (n–3) acids on thislipid. Both PG and PE were primary acceptors of de novo synthesizedfatty acids and exogenous [l–¹⁴C]oleate, but no evidenceexists for a further processing of acyl chains on these lipids.TAG, although strongly labelled with all exogenous [l–¹⁴CJacids,was not labelled when [2–¹⁴C]acetate was used as a precursorindicating the flux of endogenous fatty acids to be differentof that of exogenously supplied fatty acids. (Received November 4, 1997; Accepted February 23, 1998)     

The effect of excess K+ on two different tricarboxylate cycles in rat brain cortex slices

The authors compared, in rat brain cortex slices, the oxidation of labelled glucose and acetate and the conversion of these precursors into amino acids during incubation in control salt-glucose medium and in medium with 47 mM K+, with the aim of determining with which of the two determinable tricarboxylate cycles raised oxygen consumption is associated in the presence of excess K+. Under the experimental conditions it was found that from U-[14C]-glucose more than double the amount of [14C]-CO2 was formed and that the rate of [14C] incorportation into the amino acids was likewise roughly doubled. This is indicative of activation of processes in the tricarboxylate cycle associated with the large glutamate pool. Incorporation from 1-[14C]-acetate into the total amino acids was not affected. Specific activity in glutamate and asparate was more than doubled, while glutamine specific activity fell to less than half. [14C]-CO2 production fell to 65%. This shows that the tricarboxylate cycle associated with the small glutamate pool, which is probably localized in the glia cells, did not participate in raised oxygen consumption in the presence of excess K+.     

Different elongation pathways in the biosynthesis of acyl groups of trichome exudate sugar esters from various solanaceous plants

Two common pathways are known for elongation of aliphatic acids via acetate in biological organisms: the fatty acid synthase (FAS) and the alpha-ketoacid elongation (alphaKAE) pathways. The alphaKAE route is utilized in many biosynthetic pathways, including the tricarboxylic acid cycle, leucine biosynthesis, and in formation of coenzyme B, glucosinolates, alpha-ketoadipate, sugar-ester acyl acids, short-chain alcohols of yeast and Clostridium species, 2-amino-4-methylhex-4-enoic acid, and l-gamma-phenyl butyrine. In the FAS route, both carbons from acetyl-acyl carrier protein are retained per elongation cycle, while in the alphaKAE route only one carbon from acetyl-coenzyme A is retained. Available evidence indicates that different members of the family Solanaceae may use one or the other of these elongation mechanisms in the synthesis of acyl groups of trichome-exuded sugar esters. In both, precursors for elongation are derived from branched-chain amino acid metabolism. Here we compared radiolabeling patterns in sugar-ester acyl groups from trichomes (the specific tissue in which sugar esters are synthesized) of the tobaccos, Nicotiana benthamiana, N. gossei, N. glutinosa, of Petunia x hybrida cv. Falcon Red & White, and Datura metel, and epidermal peels of Lycopsersicon pennellii after their synthesis from [2-(14)C]-, [1-(14)C]- and [1,2-(14)C]acetate. Recovered acyl acids were purified and then degraded to determine label distribution between the carboxyl termini and the remainder of the molecules. Six- and 20-h incubations were studied, and membrane fatty acids were monitored as internal controls for FAS-mediated elongation. Results are consistent with participation of alphaKAE in synthesis of sugar-ester acyl groups of tobaccos and petunia, but apparently FAS is utilized in the formation of these groups in L. pennellii and D. metel.     

METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

A new radiochemical method for determination of pyruvate dehydrogenase complex and acetyl-coenzyme A synthetase

A radiochemical method for assaying pyruvate dehydrogenase complex and acetyl-coenzyme A synthetase is described, using [2-14C]pyruvate and [1-14C]acetate, respectively, as radiolabeled precursors. The assay is based on nonenzymatic O-acylation of excess dithioerythritol (DTE) by enzymatically formed acetyl-coenzyme A. [1-14C]Acetyl-DTE is easily extracted from the incubation mixture by organic solvents and separated from the unreacted labeled substrates.