Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Enrichment of fungi and degradation of styrene in biofilters

Summary Experiments were set up in order to enrich styrene-degrading fungi in biofilters under conditions representative for industrial off-gas treatment. From the support materials tested, polyurethane and perlite proved to be most suitable for enrichment of styrene-degrading fungi. The biofilter with perlite completely degraded styrene when amounts ranging between 290 and 675 mg/m in the influent gas were present. An elimination capacity of at least 70 g styrene per m3 filter bed per hour was calculated.     

Long-term effects of cis and trans monounsaturated (18:1) and saturated (16:0) fatty acids on the synthesis and secretion of apolipoprotein A-I- and apolipoprotein B-containing lipoproteins in HepG2 cells

The objective of this study was to compare the long-term effects of oleic (cis 18:1), elaidic (trans 18:1), and palmitic (16:0) acids on hepatic lipoprotein production, using HepG2 cells as an experimental model. The net accumulation in the medium of apolipoprotein A-I (apoA-I) was not significantly altered by fatty acids, whereas that of apoB was increased with oleic and elaidic acids. Oleic acid, and to a lesser extent elaidic and palmitic acids, increased the mass of triglycerides in the medium and the incorporation of [(3)H]glycerol into secreted triglycerides. The incorporation of [(14)C]acetate into cellular and secreted total cholesterol was stimulated by 96% and 83%, respectively, with elaidic acid but was not significantly modified by oleic or palmitic acid. Relative to oleic acid, the secretion of (14)C-labeled phospholipids and triglycerides was decreased 28% to 31% with elaidic and palmitic acids whereas that of free cholesterol and cholesteryl esters was enhanced 93% and 73%, respectively, with elaidic acid but remained unchanged with palmitic acid. Compared with oleic acid, elaidic acid stimulated the secretion of very low density lipoprotein cholesterol (VLDL-Chol), low density lipoprotein cholesterol (LDL-Chol), and high density lipoprotein cholesterol (HDL-Chol) by 43%, 70%, and 34%, respectively, whereas palmitic acid decreased VLDL-Chol but had no significant effect on LDL-Chol and HDL-Chol. The ratios of total cholesterol to HDL-Chol were 3.17, 3.60, and 3.25 with oleic, elaidic, and palmitic acids, respectively; the corresponding ratios of LDL-Chol to HDL-Chol were 0.87, 1.10, and 0.93, respectively. Compared with oleic and palmitic acids, the LDL and HDL particles secreted in the presence of elaidic acid contained higher levels of free cholesterol and cholesteryl esters and a lower content of phospholipids. The phospholipid-to-total cholesterol ratios of HDL were 1.05, 0.40, and 0.76 with oleic, elaidic, and palmitic acids, respectively.Our results indicate that in comparison with cis monounsaturated and saturated fatty acids, trans fatty acids have more adverse effects on the concentration and composition of lipoproteins secreted by HepG2 cells.     

The effects of topical application of various fatty acids on pheromone and glandular lipid biosynthesis in the moth Heliothis virescens

Binary mixtures of deuterium-labeled palmitic acid and an excess of different fatty acids were applied to the sex pheromone gland of female Heliothis virescens and the effects on the terminal steps of pheromone biosynthesis, including incorporation of fatty acids into the glandular lipids, observed. Relative to labeled palmitic acid applied alone, application of all the binary mixtures resulted in decreased levels of the labeled pheromone component, (Z)-11-hexadecenyl acetate (Z11-16:OAc), but there was generally no decrease in the amounts of labeled pheromone precursor, (Z)-11-hexadecenoate, nor labeled palmitate in the glandular lipids. These data suggest that the excess of fatty acid in the gland inhibits Delta11-desaturation. However, in the case of excess myristoleic acid, the amount of labeled (Z)-11-hexadecenoate increased significantly, suggesting that this acid inhibited fatty acid reduction. Dose-response tests with certain of the fatty acids were consistent with the above interpretations and further indicated that the gland had a high capacity for rapidly activating and incorporating excess fatty acids into the glandular lipids. Finally, application of the various fatty acids resulted in increased levels of these acids in the gland and, in the cases of myristoleic, palmitoleic and myristic acids, it also resulted in increased levels of the corresponding aldehydes, which had previously been detected in the gland of female H. virescens. This suggests that the fatty acid reductase in H. virescens is not highly specific for the major component, and that the final ratio of pheromone components is determined in part by the availability of their corresponding fatty acids in the gland.     

Metabolism of bis(monoacylglycero)phosphate in macrophages

To further elucidate the role of bis(monoacylglycero)phosphate in lysosomes, its metabolism was assessed by incubation of intact and disrupted macrophages in the presence of labeled lipid precursors. In rabbit pulmonary macrophages bis(monoacylglycero)P accounted for 17.9% and acylphosphatidylglycerol for 2.6% of phospholipid phosphorus. Major fatty acids in bis(monoacylglycero)P were oleic (47%), linoleic (29%), and arachidonic (6.4%); those in acylphosphatidylglycerol were of similar distribution except for a high content of palmitic acid (20%). When homogenates of rabbit pulmonary and peritoneal macrophages, rat pulmonary macrophages, and human blood leukocytes were incubated with sn[(14)C]glycerol-3-phosphate and CDP-diacylglycerol at pH 7.4, there was labeling of bis(monoacylglycero)P and acylphosphatidylglycerol that correlated with content of bis(monoacylglycero)P. When intact rabbit pulmonary macrophages were incubated for 60 min with [(3)H]glucose and [(32)P]orthophosphate, small amounts of label appeared in bis(monoacylglycero)P and only traces in acylphosphatidylglycerol. In contrast, incubation of intact cells with the (14)C-labeled fatty acid precursors palmitic, oleic, and arachidonic acids resulted in much greater labeling of the two lipids. Labeling of phospholipids was greatest with arachidonate as precursor and least with palmitate; after 60 min, labeling of bis(monoacylglycero)P with arachidonate was 10- and 50-fold greater than with oleate and palmitate, respectively, and was exceeded only by that of phosphatidylcholine. Calculated ratios of labeling of fatty acid to P, particularly those for arachidonate, were much greater for bis(monoacylglycero)P and for acylphosphatidylglycerol than for other phospholipids. This suggests a uniquely high turnover of fatty acids in bis(monoacylglycero)P and acylphosphatidylglycerol and thus a more specific role for these compounds in metabolism of complex lipids in the lysosome.-Huterer, S., and J. Wherrett. Metabolism of bis(monoacylglycero)phosphate in macrophages.     

Uptake and metabolism of circulating chylomicron triglyceride by rabbit aorta

To determine if chylomicron triglycerides are taken up and metabolized by the arterial wall, rabbit abdominal aortas were perfused in situ for various times up to 2 hr with blood-buffer containing isotopically labeled substrates. Labeled chylomicrons were obtained by feeding [(3)H]palmitic acid or [(3)H]glyceryl trioleate to rats and rabbits with cannulated thoracic ducts. After aortic perfusion with these chylomicrons, more than 85% of aortic lipid ester radioactivity was in triglyceride; when labeled glycerol or palmitic acid was perfused, most aortic ester lipid radioactivity was in diglycerides and phospholipids. This indicated that, during perfusion with chylomicrons, intact triglyceride molecules were taken up by aorta. The rate of triglyceride fatty acid uptake by the inner avascular segment approached maximal values at low concentrations of perfusate triglyceride fatty acids (2 mm), whereas uptake in the outer capillary perfused segment increased with increasing triglyceride fatty acid concentration (0.4-25 mm). By double-radioisotope techniques it was shown that aortic free fatty acid was derived from both perfusate free fatty acids and from hydrolysis of lipoprotein glycerides within the aortic wall. Uptake of chylomicron triglyceride by perfused aorta was independent of triglyceride hydrolysis, which was quantitatively small.     

Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus

The modification of viral glycoproteins through the covalent attachment of fatty acids was studied in baby hamster kidney (BHK) cells infected with Semliki Forest virus (SFV). Comparative pulse-chase experiments with [3H]palmitic acid and [35S]methionine revealed that a precursor polypeptide, designated p62, of the structural SFV glycoprotein and E1 serve as the primary acceptors of acyl chains. Acylation of p62 occurs immediately prior to its proteolytical cleavage to E2 and E3 emphasizing the post-translational and specific nature of this hydrophobic modification. To trace the acyl donor(s) for protein acylation the covalent attachment of fatty acids to p62 was studied after extremely short labeling periods with [3H]palmitic acid and correlated to the metabolism of the exogenous tritiated fatty acid. The shortest possible labeling time, a 10 s pulse with [3H]palmitic acid, was sufficient to acylate SFV p62. Analysis of the labeled lipids extracted from the same cells revealed that palmitoyl-CoA and phosphatidic acid showed the highest specific radioactivity among the tritiated lipid species. Out of these lipid species palmitoyl-CoA was identified as the functional acyl donor lipid in a cell-free system for the acylation of polypeptides.     

The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus.

[3H]Myristic and [3H]palmitic acid were compared as tracers for the fatty acylation of cellular lipids and viral glycoproteins in chicken embryo cells infected with fowl plague and Semliki Forest virus (SFV). Both of these substrates are incorporated into glycerolipids to a similar extent, whereas sphingolipids show much higher levels of palmitate than myristate after a 20 h labeling period. Both fatty acid species were found to be subject to metabolic conversions into longer chain fatty acids yielding 11.7% C16:0 from [3H]myristic and 11.8% C18:0 from [3H]palmitic acid. The reverse, a metabolic shortening of the exogenous acyl-chains yielding, for instance, significant levels of myristic acid from palmitic acid was not observed. Out of the various [3H]fatty acids present after in vivo labeling with [3H]myristic acid (C14:0) the elongated acyl-species arising from metabolic conversion (e.g., C16:0; C18:0) are preferred over myristic acid in the acylation of SFV E1 and E2 and of the influenza viral hemagglutinin (HA2). During acylation of exogenous E1 from SFV in vitro incorporation of palmitic acid from palmitoyl CoA exceeds that of myristic acid from myristoyl CoA by a factor of 37. This indicates that specificity for the incorporation of fatty acids into viral membrane proteins occurs at the level of the polypeptide acyltransferase(s).     

Myristyl and palmityl acylation of the insulin receptor

The presence of covalently bound fatty acids in the insulin receptor has been explored in cultured human (IM-9) lymphocytes. Both alpha (Mr = 135,000) and beta (Mr = 95,000) subunits of the receptor incorporate [3H]myristic and [3H]palmitic acids in a covalent form. The effects of alkali and hydroxylamine on the labeled subunits indicate the existence of two different kinds of fatty acid linkage to the protein with chemical stabilities compatible with amide and ester bonds. The alpha subunit contains only amide-linked fatty acid while the beta subunit has both amide- and ester-linked fatty acids. Analysis by high performance liquid chromatography after acid hydrolysis of the [3H]myristate- and [3H]palmitate-labeled subunits demonstrates the fatty acid nature of the label. Furthermore, both [3H]myristic and [3H]palmitic acids are found attached to the receptor subunits regardless of which fatty acid was used for labeling. The incorporation of fatty acids into the insulin receptor is dependent on protein synthesis and is also detectable in the Mr = 190,000 proreceptor form. Fatty acylation is a newly identified post-translational modification of the insulin receptor which may have an important role in its interaction with the membrane and/or its biological function.     

Sex pheromone biosynthetic pathway in Spodoptera littoralis and its activation by a neurohormone

Deuterium-labeled fatty acids have been used to elucidate the sex pheromone biosynthetic pathway in Spodoptera littoralis. Label from palmitic acid was incorporated during the scotophase into all the pheromone acetates and their corresponding fatty acyl intermediates. (Z,E)-9,11-tetradecadienyl acetate, the major component of the pheromone blend, is synthesized from palmitic acid via tetradecanoic acid, which, by the action of a specific (E)-11 desaturase and subsequently a (Z)-9 desaturase, is converted into (Z,E)-9,11-tetradecadienoate. By further reduction and acetylation, this compound leads to the dienne acetate. Deuterated precursors applied to the pheromone gland during the photophase were also incorporated into the pheromone. The percentage of labeled (Z,E)-9,11-tetradecadienyl acetate relative to natural compound was significantly higher during the light period. Label incorporation from different intermediates into the pheromone was stimulated by injection of brain-subesophageal ganglion extract during the photophase. The influence of the pheromone biosynthesis-activating neuropeptide on the biosynthetic pathway is discussed.     

Acylation of cellular proteins with endogenously synthesized fatty acids

A number of cellular proteins contain covalently bound fatty acids. Previous studies have identified myristic acid and palmitic acid covalently linked to protein, the former usually attached to proteins by an amide linkage and the latter by ester or thio ester linkages. While in a few instances specific proteins have been isolated from cells and their fatty acid composition has been determined, the most frequent approach to the identification of protein-linked fatty acids is to biosynthetically label proteins with fatty acids added to intact cells. This procedure introduces possible bias in that only a selected fraction of proteins may be labeled, and it is not known whether the radioactive fatty acid linked to the protein is identical with that which is attached to the protein when the fatty acid is derived from endogenous sources. We have examined the distribution of protein-bound fatty acid following labeling with [3H]acetate, a general precursor of all fatty acids, using BC3H1 cells (a mouse muscle cell line) and A431 cells (a human epidermoid carcinoma). Myristate, palmitate, and stearate account for essentially all of the fatty acids linked to protein following labeling with [3H]acetate, but at least 30% of the protein-bound palmitate in these cells was present in amide linkage. In BC3H1 cells, exogenous palmitate becomes covalently bound to protein such that less than 10% of the fatty acid is present in amide linkage. These data are compatible with multiple protein acylating activities specific for acceptor protein fatty acid chain length and linkage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Docosahexaenoic,arachidonic, palmitic,and oleic acids are differentially esterified into phospholipids of frog retina

Docosahexaenoic acid (22:6n-3) is highly enriched in the retina. To determine if retinal cells take up and metabolize fatty acids in a specific manner, retinas from Rana pipiens were incubated for 3 h with an equimolar mixture of tritiated 22:6n-3, arachidonic acid (20:4n-6), palmitic acid, and oleic acid. The radiolabeling of retinal lipids was determined and compared to the endogenous fatty acid content of the lipids. The results showed that in most, but not all, cases, the relative labeling with the four precursor fatty acids was similar to their relative abundance in each glycerolipid. Thus, during retinal glycerolipid synthesis, either through de novo or acyl exchange reactions, fatty acids are incorporated in proportions reflecting their steady-state mass levels. Since other studies with labeled glycerol have shown greater differences between early labeling patterns and molecular species mass, the final incorporation we report may be due primarily to acyl exchange reactions.     

Assay of the concentration and (13)C isotopic enrichment of propionyl-CoA,methylmalonyl-CoA,and succinyl-CoA by gas chromatography-mass spectrometry

We developed gas chromatography-mass spectrometry assays for the concentration and mass isotopomer distribution of propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA in tissues. The assays involves perchloric acid extraction of the tissue, spiking the extract with [(2)H(5)]propionyl-CoA and [(2)H(4)]succinyl-CoA internal standards, and isolation of short-chain acyl-CoA fraction on an oligonucleotide purification cartridge. Propionyl-CoA is reacted with sarcosine and the formed N-propionylsarcosine is assayed as its pentafluorobenzyl derivative. Methylmalonyl-CoA and succinyl-CoA are hydrolyzed and the corresponding acids assayed as tert-butyl dimethylsilyl derivatives. The assay was applied to a study of [U-(13)C(3)]propionate metabolism in perfused rat livers. While propionyl-CoA is only M3 labeled, succinyl-CoA is M3, M2, and M1 labeled because of isotopic exchanges in the citric acid cycle. Methylmalonyl-CoA is M3 and M2 labeled, reflecting reversal of S-methylmalonyl-CoA mutase. Thus, our assays allow measuring the turnover of the coenzyme A derivatives involved in anaplerosis of the citric acid cycle via precursors of propionyl-CoA, i.e., propionate, odd-chain fatty acids, isoleucine, threonine, and valine.     

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.     

Characterisation of hexane-degrading microorganisms in a biofilter by stable isotope-based fatty acid analysis, FISH and cultivation

The hexane-degrading bacterial community of a biofilter was characterised by a combination of stable isotope-based phospholipid fatty acid analyses, fluorescence in situ hybridisation and cultivation. About 70 bacterial strains were isolated from a full-scale biofilter used for treatment of hexane containing waste gas of an oil mill. The isolation approach led to 16 bacterial groups, which were identified as members of the Alpha-, Beta- and Gammaproteobacteria, Actinobacteria and Firmicutes. Three groups showed good growth on hexane as the sole source of carbon. These groups were allocated to the genera Gordonia and Sphingomonas and to the Nevskia-branch of the Gammaproteobacteria. Actively degrading populations in the filter material were characterised by incubation of filter material samples with deuterated hexane and subsequent phospholipid fatty acid analysis. Significant labelling of the fatty acids 16:1 cis10, 18:1 cis9 and 18:0 10methyl affiliated the hexane-degrading activity of the biofilter with the isolates of the genus Gordonia. In vitro growth on hexane and in situ labelling of characteristic fatty acids confirmed the central role of these organisms in the hexane degradation within the full-scale biofilter.     

Influence of chain length and unsaturation on the effects of fatty acids on phosphoglyceride biosynthesis in isolated rat and pig hepatocytes.

Hepatocytes isolated from rat or pig by collagenase perfusion were incubated with [3H]glcyerol and different albumin-bount fatty acids. Among C22 fatty acids docosahexaenoic acid stimulated phosphatidylethanolamine synthesis in rat hepatocytes most effectively. Addition of docosahexaenoic acid plus either palmitic or stearic acid resulted almost in the same stimulation whereas combinations of this acid with lauric or myristic acid had no effect. Lauric acid and myristic acid alone inhibited phosphatidylethanolamine synthesis. The chain length specificity for monoenoic fatty acids was similar, the hexadecenoic and octadecenoic acids (both cis and trans) being most stimulatory. The addition of 0.2 mM ethanolamine markedly stimulated phosphatidylethanolamine synthesis, but most effects of fatty acids were similar in its presence or absence.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

omega-Oxidation of fatty acids and the acetylation p-aminobenzoic acid.

p-Aminobenzoic acid was fed to normal and alloxan-induced diabetic rats injected with [omega-14C]labeled and [2-14C]labeled fatty acids. The p-acetamidobenzoic acid that was excreted was hydrolyzed to yield acetate which was degraded. The distribution of 14C in the acetates formed when an [omega-14C]labeled fatty acid was injected was similar to that when a [2-14C]labeled fatty acid was injected. This contrasts with the finding that in acetates from 2-acetamido-4-phenylbutyric acid excreted when 2-amino-4-phenylbutyric acid was fed, there was a difference in the distributions of 14C, a difference attributable to omega-oxidation of the fatty acid. Acetylation of p-aminobenzoic acid is then concluded to occur in a different cellular environment than that of 2-amino-4-phenylbutyric acid, one in which omega-oxidation is not functional. When 2-amino-4-phenylbutyric acid was fed and [6-14C]palmitic acid injected, rather than [16-14C]palmitic acid, the distribution of 14C in acetate was the same as when [2-14C]palmitic acid was injected. This indicates that the dicarboxylic acid formed on omega-oxidation of palmitic acid does not undergo beta-oxidation to form succinyl-CoA. Thus, glucose is not formed via omega-oxidation of long-chain fatty acid.     

Palmitoylation of membrane proteins inSpiroplasma floricola

Covalent modification ofSpiroplasma floricola membrane proteins by fatty acids was determined by in vivo labeling of the cells with radioactive fatty acids followed by separation on one-dimensional SDS-polyacrylamide gels and visualization by autoradiography. Approximately 25 different proteins were found to be labeled with [³H]-palmitate, whereas almost none were labeled with [³H]-oleate. The radioactivity could not be removed from the palmitoylated membrane proteins by boiling in SDS or by exhaustive extraction with chloroform-methanol (21). Nevertheless, treating the palmitoylated proteins with a 0.1N KOH solution removed approximately 70% of the bound [³H]-palmitate. The major protein-bound fatty acid species were identified, following their release from the protein by chemical cleavage, as palmitic acid and stearic acid (83% and 7.5%, respectively).     

Molecular species analysis of 1,2-diacylglycerol released in response to progesterone binding to the amphibian oocyte plasma membrane

Progesterone, the physiological inducer of amphibian meiosis, acts within minutes at plasma membrane receptors of the Rana pipiens oocyte to release 1,2-diacylglycerol (DAG) from plasma and intracellular membranes. High-performance liquid chromatography (HPLC) analysis of lipid extracts of uninduced oocytes indicates the presence of at least three classes of DAG with a total DAG content of about 150 micromol/kg wet weight. Within 3-5 min after exposure to progesterone, there was a differential increase in all three DAG classes with a twofold increase in total DAG by 10 min. The fatty acid composition of the DAGs in uninduced and progesterone-stimulated oocytes was compared using thin layer chromatographic analysis of lipid extracts from oocytes double-labeled with [14C] or [3H]glycerol and [14C] or [3H]fatty acids. The ratio of labeled fatty acid/labeled glycerol was measured in phosphatidylcholine (PC), phosphatidylinositol (PI) and DAG. The linoleic (18:2) or arachidonic (20:4) acid/glycerol ratios in basal DAG were low compared to that in PC or PI. In contrast, the myristic (14:0), palmitic (16:0) or oleic (18:1) acid/glycerol ratios in basal DAG were relatively high compared to the ratio in PC and PI. A transient increase in both linoleic and palmitic acid labeling of DAG occurred within the first 1-2 min in progesterone-treated oocytes, followed by a return to or below the basal level. Arachidonic and myristic acid labeling of DAG fall within the first minute after progesterone treatment, followed by a sustained rise over the next 10 min. The [3H]oleic acid/[14C]glycerol ratio of DAG does not change significantly following exposure to progesterone. Pretreatment with a phospholipid N-methylation inhibitor (2-methylaminoethane) precluded the rise in linoleic and palmitic acid-rich DAG, whereas pretreatment with a diglyceride kinase inhibitor (D102) produced a sustained elevation of linoleic and palmitic acid-rich DAG. These results indicate that the DAG released in response to progesterone is composed of multiple new molecular species of DAG and that both the palmitate and linolate-rich forms are rapidly phosphorylated to form phosphatidic acid (PA). The newly formed DAG species differ from the basal DAG species and reflect sequential activation of sphingomyelin (SM) synthase, PC-specific phospholipase D (PLD) and PI-specific phospholipase C in response to progesterone, which we have described previously.