Phosphatidylmonomethylethanolamine Research Articles

Progesterone modulation of transmembrane helix-helix interactions between the alpha-subunit of Na/K-ATPase and phospholipid N-methyltransferase in the oocyte plasma membrane.

BackgroundProgesterone binding to the surface of the amphibian oocyte initiates the meiotic divisions. Our previous studies with Rana pipiens oocytes indicate that progesterone binds to a plasma membrane site within the external loop between the M1 and M2 helices of the α-subunit of Na/K-ATPase, triggering a cascade of lipid second messengers and the release of the block at meiotic prophase. We have characterized this site, using a low affinity ouabain binding isoform of the α1-subunit.ResultsPreparations of isolated plasma membranes from Rana oocytes demonstrate that physiological levels of progesterone (or the non-metabolizable progestin R5020) successively activate phosphatidylethanolamine-N-methyltransferase (PE-NMT) and sphingomyelin synthase within seconds. Inhibition of PE-NMT blocks the progesterone induction of meiosis in intact oocytes, whereas its initial product, phosphatidylmonomethylethanolamine (PME), can itself initiate meiosis in the presence of the inhibitor. Published X-ray crystallographic data on Na/K-ATPase, computer-generated 3D projections, heptad repeat analysis and hydrophobic cluster analysis of the transmembrane helices predict that hydrophobic residues L, V, V, I, F and Y of helix M2 of the α1-subunit interact with F, L, G, L, L and F, respectively, of helix M3 of PE-NMT.ConclusionWe propose that progesterone binding to the first external loop of the α1-subunit facilitates specific helix-helix interactions between integral membrane proteins to up-regulate PE-NMT, and, that successive interactions between two or more integral plasma membrane proteins induce the signaling cascades which result in completion of the meiotic divisions.

The first product of phospholipid N-methylation, phosphatidylmonomethylethanolamine, is a lipid mediator for progesterone action at the amphibian oocyte plasma membrane.

Progesterone has been shown to act at plasma membrane receptors on the amphibian oocyte to trigger a cascade of changes in membrane phospholipids and to initiate the G(2)/M transition of the first meiotic division. The earliest event (0-1 min) is the transient N-methylation of phosphatidylethanolamine (PE) to form phosphatidylmonomethylethanolamine (PME), demonstrated using [(3)H]glycerol to prelabel oocyte plasma membrane PE. [(3)H]Glycerol-labeled PME rises 10-fold within the 1-2 min after exposure to progesterone and accounts for conversion of about 50% of the [3H]Glycerol-labeled PE. [(3)H]PME levels slowly decline over the following 10-30 min. [(3)H] or [(14)C] labeled fatty acid experiments showed that newly formed PME is enriched in linoleic or palmitic, but not in arachidonic acid, indicating that specific PE pools undergo progesterone-induced N-methylation. Two plasma membrane changes: activation of serine protease, and Ca(2+) release from the oocyte surface coincide with PME formation; both are prevented by pretreatment of oocytes with the N-methylation inhibitor, 2-methylaminoethane. Media containing PME micelles release both protease and Ca(2+) from intact oocytes within the first 1-2 min. The immediate downstream metabolites of PME, PDE and PC, do not induce serine protease activity or Ca(2+) release. We conclude that progesterone initially activates N-methyltransferase in the oocyte plasma membrane, and that the first product, PME, is responsible for activation of serine protease in the plasma membrane and the release of Ca(2+) from the oocyte surface.

Progesterone-induced phospholipid N-methylation and sphingomyelin synthesis in the amphibian oocyte plasma membrane: a second source of the 1,2-diacylglycerol second messenger associated with the G 2/M transition

The effects of progesterone and GTPγS on phospholipid N-methylation and sphingomyelin synthesis were studied in plasma-vitelline membranes isolated from amphibian ( Rana pipiens) oocytes. Plasma-vitelline membranes were preincubated with S- adenosyl- l [methyl- 3 H] methionine for w min at 20°C and total phospholipids extracted at 0, 15, 30 and 60 s after addition of progesterone and/or GTPγS. Progesterone levels (3 μM) that induce meiosis in the intact oocyte stimulated [ 3H-methyl]incorporation into phosphatidylmonomethylethanolamine (PME) 9–10-folrd over the first 6 s with smaller increases in phosphatidyldimethylethanolamine (PDE) and phosphatidylcholine (PC). [ methyl- 3H] labeling of sphingomyelin (SM) rises after 30 s, approaching that of [ methyl- 3H]PME by 60 s. 17β-Estradiol, a noninducer of meiosis, was inactive. When oocytes were prelabeled with [ 3H]palmitic acid, it was found that a fall in [ 3H]ceramide coincides with the transient increase in [ 3H]SM, indicating that the end product of N-methylation (PC) undergoes a transfer reaction with ceramide to form SM and 1,2-DG. GTPγS levels previously reported to stimulate PC-specific phospholipase C activity in oocyte plasma membranes (5 μM) also stimulated both [ methyl- 3H]PME and [ methyl- 3H]SM formation. An inhibitor of phospholipid N-methylation, 2-(methyl-amino)ethanol, blocked stimulation of [ methyl- 3H]SM synthesis by both progesterone and GTPγS as well as induction of meiosis by progesterone. Progesterone thus acts at the oocyte plasma membrane to stimulate PE N-methyltransferase and SM synthase. The finding that GTPγS mimics progesterone suggests that N-methyltransferase is mediated by G-protein(s). The transient increase in 1,2-DG which we had previously reported to occur within 1–2 min following progesterone stimulation of the Rana oocyte appears to arise from PC by two different pathways: SM synthesis and hydrolysis of PC by phospholipase C.

Choline deficiency in cultured adrenal medullary cells: effect on phosphatidylcholine biosynthesis.

The effect of choline deficiency on the composition and biosynthesis of the major membrane phospholipids was examined in adrenal medullary cells maintained in suspension cultures. The amount and proportions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in these cells were not affected by the removal of choline from the culture media. However, the rate of biosynthesis of choline at the phosphatide level by the stepwise methylation of PE increased twofold within 24 h after choline was removed from the culture media, while ethanolamine incorporation into PE was increased by 50%. In contrast, the rate of incorporation of labeled choline into PC, presumably via CDP-choline, was virtually identical in cells that had been preincubated in the presence or absence of 1 mM choline. These results demonstrate that cultured cells of neural origin are capable of compensating for lack of exogenous choline by forming choline at the phosphatide level through the sequential methylation of PE. The hypolipidemic drug, DH-990, when added to the culture media, inhibited conversion of phosphatidylmonomethylethanolamine (PME) to PC, but had no effect on the N-methylation of PE. This differential effect indicates that the initial N-methylation of PE is catalyzed by an enzyme that is distinguishable from the enzyme(s) catalyzing the conversion of PME to PC.

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.

Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties

In the yeast Saccharomyces cerevisiae, two membrane-associated enzymes catalyze the three-step methylation of Phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Phosphatidylethanolamine methyltransferase (PEMT) catalyzes the first methylation reactions (PE → phosphatidylmonomethylethanolamine (PMME)) and phospholipid methyltransferase (PLMT) catalyzes the second two methylation reactions (PMME → phosphatidyldimethylethanolamine (PDME) → PC). Using gene disruption mutants of the S. cerevisiae OPI3 and CHO2 genes, we independently studied the enzymological properties of microsome-associated PEMT and PLMT, respectively. The enzymological properties of the enzymes differed with respect to their pH optima, cofactor requirements and thermal lability. For the PEMT reactions, the apparent K m values for PE and S-Adenosylmethionine (AdoMet) were 57 μM and 110 μM, respectively. For the PLMT reactions, the apparent K m values for PMME and PDME were 380 μM and 180 μM, respectively. The apparent K m values for AdoMet were 54 μM and 59 μM with PMME and PDME as substrates, respectively. S-Adenosylhomocysteine ( AdoHcy) was a competitive inhibitor of PEMT ( K i = 12 μM) and PLMT ( K) i = 57 μM and K i = 54 μM for PMME and PDME, respectively) with respect to AdoMet. AdoHcy was a noncompetitive inhibitor of PEMT ( K) i , = 160 μM) and PLMT K i = 320 μM and K i = 120 μM) with respect to PE and PMME and PDME, respectively.

Modification of phospholipid polar head group with monomethylethanolamine and dimethylethanolamine decreases cholesteryl ester and triacylglycerol synthesis in cultured human fibroblasts

Modification of the phospholipid polar head group was achieved by supplementation of the growth medium of cultured human fibroblasts with the choline analogues monomethylethanolamine (ME) or dimethylethanolamine (DE) at a concentration of 80-200 micrograms/mL for 48 hr. The maximum concentration of phosphatidylmonomethylethanolamine (PME) or phosphatidyldimethylethanolamine (PDE) reached without affecting the phospholipid/protein ratio was about 45% of total phospholipids. Incorporation of oleic acid into cholesteryl esters and triacylglycerols was markedly inhibited after supplementation with ME or DE, and accounted for 60% and 40% of controls, respectively, at 200 micrograms/mL, whereas incorporation into phospholipids was not affected. AcylCoA:cholesterol acyltransferase (ACAT) and diacylglycerol acyltransferase (DGAT) activities measured on cell-free extracts appeared to be decreased also by phospholipid polar head group modification, whereas the overall phospholipid acyltransferase activity remained unchanged. The intracellular content of cholesteryl esters and triacylglycerols, determined by the isotopic equilibrium method with radioactive cholesterol and glycerol, was found to be diminished to 50-60% and 40-50% of controls, respectively, after supplementation with the choline analogues. The study showed that modification of the phospholipid polar head group affects the activity of membrane-bound enzymes involved in the metabolism of neutral lipids.

Effect of toluene on rat synaptosomal phospholipid methylation and membrane fluidity

This study investigated the effects of toluene (1 g/kg, 1 hr, i.p.) on rat synaptosomal phospholipid methylation (PLM), phospholipid composition, and membrane fluidity. Toluene significantly decreased basal PLM (35%) in studies using [ 3H]methionine ([ 3H]Met) as the methyl donor; this was reflected by similar decreases in phosphatidylmonomethylethanolamine (PME) (30%). No effects were observed in either PLM reactions that used [ 3H]adenosylmethionine ([ 3H]AdoMet) as methyl donor, or AdoMet synthetase, suggesting that toluene preferentially affects PLM reactions that derive methyl groups from [ 3H]Met. Also, toluene decreased synaptosomal phosphatidylethanolamine (PE) (24%), the initial substrate for PLM, and the addition of PE back to PE-depleted synaptosomes restored methyltransferase activity. Agonist-stimulated PLM using norepinephrine (NE) demonstrated that agonist-receptor coupling returned PLM to control values in synaptosomes from toluene-treated rats. NE-stimulated PLM was also blocked by propranolol (PRO), suggesting a role for toluene in receptor-mediated events. Membrane fluidity studies demonstrated that in vivo administration of toluene increased the outer synaptosomal membrane fluidity, whereas in vitro administration of toluene had no effect. Our observations support a positive relationship between increased PLM activity and increased outer, not core, membrane fluidity. These data demonstrate that specific toluene-phospholipid interactions occur in synaptosomes, resulting in altered membrane composition, function and fluidity.

Head Group Specificity in the Requirement of Phosphatidylcholine Biosynthesis for Very Low Density Lipoprotein Secretion from Cultured Hepatocytes

We have demonstrated that hepatic very low density lipoprotein (VLDL) secretion requires active phosphatidylcholine (PC) synthesis via either the CDP-choline pathway or phosphatidylethanolamine (PE) methylation pathway (Yao, Z., and Vance, D.E. (1988) J. Biol. Chem. 263, 2998-3004). In the present work, the head group specificity of phospholipid synthesis required for lipoprotein secretion was investigated in cultured hepatocytes isolated from choline-deficient rats. When N-monomethylethanolamine (0.1 mM) or N,N-dimethylethanolamine (0.1 mM) was added to the culture medium, the cells synthesized correspondingly phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). However, the synthesis of PDME could correct the impaired VLDL secretion only to a limited extent, whereas the synthesis of PMME inhibited VLDL secretion. Although dimethylethanolamine did not promote VLDL secretion as well as choline, dimethylethanolamine altered the increased triacylglycerol synthesis in the choline-deficient cells as effectively as choline. Supplementation of the culture medium with ethanolamine (0.1 mM) had little effect on cellular PE or PC levels, nor was normal VLDL secretion resumed. However, the amounts of cellular PC and PE were both decreased when the medium was supplemented with N-monomethylethanolamine or N,N-dimethylethanolamine. These results suggest that the choline head group moiety of PC is specifically required for normal VLDL secretion and cannot be replaced with ethanolamine, monomethylethanolamine, or dimethylethanolamine. In addition, the impaired VLDL secretion from the choline-deficient hepatocytes could also be corrected by supplementation of betaine (0.2 mM) and homocysteine (0.2 mM), indicating the utilization of a methyl group from betaine for PC formation via methylation of PE.

The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture.

Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [3H]monomethylethanolamine (MME) and [3H] dimethylethanolamine (DME). The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivative. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [3H]MME and [3H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.

Calcium-dependent phosphorylation of the amphibian oocyte plasma membrane: An early event in initiating the meiotic divisions

Plasma membranes isolated from Rana oocytes showed a 7–10 fold increase in the Ca 2+-dependent phosphorylation of endogenous protein following exposure to meiotic stimuli (progesterone, insulin) either in vivo or in-vitro . Exogenous phosphatidylmonomethylethanolamine (PME) was effective in stimulating Ca 2+-dependent membrane phosphorylation and also induced meiosis. Induction of phosphorylation was blocked by the protease inhibitor leupeptin, as are all other responses to meiotic stimuli. Phosphatidylserine was inactive when added to intact oocytes, but stimulated membrane phosphorylation nearly 15-fold when added to isolated membranes. The results indicate a link between phospholipid methylation and protein kinase C activation.

Modulation of phospholipid methylation in rabbit leukocytes by indomethacin.

Methylation of membrane phospholipids has been implicated as an early biochemical signal for the chemotactic migration of phagocytic cells into inflammatory sites. In this study, the ability of indomethacin to modulate phospholipid methylation as one of its mechanisms of antiinflammatory action was investigated. Nontoxic doses of indomethacin (10(-4) M and 10(-5) M) were found to retard the methylation of phosphatidylmonomethylethanolamine (PMME) to phosphatidyldimethylethanolamine (PDME) and phosphatidylcholine (PC) in rabbit leukocytes, suggesting inhibition of methyltransferase II activity. Indomethacin was, however, without effect on the uptake of L-[methyl 3H]methionine by rabbit leukocytes. It is suggested that the inhibition of membrane phospholipid methylation could result in the suppression of inflammatory responses such as prostaglandin and leukotriene synthesis, generation of reactive oxygen radicals, and chemotaxis.

The phospholipid- N-methyltransferase of rat brain microsomes

The phospholipid- N-methyltransferase activity of rat brain microsomes had an optimum pH of 11.0 in the absence or presence of phosphatidylethanolamine (PE) but pH 10.0 in the presence of phosphatidyl-monomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). An apparent K m for S-adenosyl methonine from 0.10 to 0.12 mM was observed with exogenous methylated phospholipids PMME or PDME. Methylated neutral lipid was the major lipid produced in the absence of the exogenous acceptors. Two exogenous phospholipids, PMME and PDME, significantly stimulated microsomal phospholipid- N-methyltransferase activity and the predicted methylated phospholipids were the major products. PE additions did not cause any stimulation of methylated lipid formation. Preincubation of particles at temperatures from 40 to 100° C resulted in a loss in the microsomal phospholipid- N-methyltransferase activity that was stimulated by PMME and PDME.

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg 2+ (10 m m) or Ca 2+ (1 m m), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 m m Mg 2+ and 1 m m Ca 2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent K m values for S-adenosyl- l-methionine were 20 and 400–500 μ m for the conversion of PE to PME and for the conversion of PME to PC they were 5 μ m and 40 μ m. S-Adenosyl- l-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [ 14C]methyl incorporation into total lipid. The apparent K i value for AdoHcy was 44.7 μ m. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approxiamtely 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A 2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A 2 in the thymocyte plasma membrane.

Chemical methylation of phosphatidylethanolamine by S-adenosylmethionine

The chemical methylation of phosphatidylethanolamine (PE) by S-adenosyl methionine (SAM) is most active when carried out at alkaline pH's. Phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine are less effective reactants. The PE present in the microsomal and myelin membrane can serve as an acceptor in this reaction. Thin layer chromatography indicates the formation of the expected products.

Norepinephrine stimulation of phospholipid methylation in rat cortical synaptosomes: Fact or artifact?

Synaptosomes from rat cerebral cortex incubated with 3H-S-adenosyl-L-methionine (3H-SAM) displayed an increase in chloroform- extractable tritium when norepinephrine was added to the reaction mixture. The products of this mixture were maximally generated from intact synaptosomes, only partially inhibited by propranolol, and not enhanced by exogenous phospholipids. Thin layer chromatographic analysis of these chloroform extracts in three solvent systems yielded large norepinephrine- stimulated peaks of radioactivity that did not consistently co-chromatograph with authentic methylated phospholipid standards: phosphatidylmonomethylethanolamine (PME), phosphatidyldimethylethanolamine (PDE), and phosphatidylcholine (PC). Further, attempts to identify these peaks of radio-activity using as standards several putative methylated products of varied chemical classes, failed to elucidate likely candidates. It appears that while norepinephrine markedly stimulates the amount of tritium extracted into the chloroform phase, careful and positive structural elucidation of formed products is required before it can be concluded that these are indeed methylated phospholipids.

Regulation of phosphatidylcholine biosynthesis in mammalian cells. III. Effects of alterations in the phospholipid compositions of Chinese hamster ovary and LM cells on the activity and distribution of CTP:phosphocholine cytidylyltransferase.

The activity and subcellular distribution of CTP:phosphocholine cytidylyltransferase in LM and Chinese hamster ovary cells in which the phospholipid composition had been altered by supplementary feeding with choline analogues were examined. Decreased levels of cellular phosphatidylcholine with corresponding increased levels of either phosphatidylethanolamine, phosphatidylmonomethylethanolamine, or phosphatidyldimethylethanolamine resulted in increased CTP:phosphocholine cytidylyltransferase activity in cell homogenates. In addition, a significantly larger fraction of the total cytidylyltransferase activity was membrane-bound in these cells. The activity of the cytidylyltransferase from cytosolic extracts of both cell types was found to be greatly increased when assayed in the presence of either phosphatidylmonomethyl ethanolamine or phosphatidyldimethylethanolamine. The lysophosphatide forms of these lipids were found to be poor activators of the cytidylyltransferase. These findings suggest that the regulation of phosphatidylcholine biosynthesis is at least partially dependent on information transfer from membranes to CTP: phosphocholine cytidylyltransferase. That is, in the presence of phosphatidylcholine-deficient membranes of cytidylyltransferase becomes activated and associated with the membranes.

Developmental regulation of phospholipid methylation in rat brain synaptosomes

Phospholipid methylation was studied in cortical synaptosomes prepared from 7 and 14 day and adult rat brain. Using varying concentrations of [ 3H] S-adenosylmethionine, Km and Vmax values were determined for the formation of [ 3H] phosphatidylmonomethylethanolamine (PME), [ 3H] phosphatidyl-dimethylethanolamine and [ 3H] phosphatidylcholine (PC). At 25°C, the Km values for the formation of all three products, significantly decreased with development. Increasing the temperature to 37°C increased the Km values in the 14 day and adult but not the 7 day preparation. The Vmax values at 25°C were highest at 7 and 14 days, depending on the product and then decreased in the adult. At 37°C, the Vmax values were highest in the 14 day preparation. The overall results are discussed in terms of the developmentally regulated decrease in both synaptic membrane PC and membrane fluidity.

Phospholipid methylation and human platelet function

Lysed human platelet preparations were shown to methylate phosphatidylethanolamine to the mono-, di- and trimethyl products. At a low concentration of S-adenosylmethionine (SAM). 2 μM, the major product formed was phosphatidylmonomethylethanolamine (PNE). There was a broad pH optimum from 9 to 11 for the formation of PNE. The combined formation of phosphatidyldimethylethanolamine (PNNE) and phosphatidylcholine (PC) showed a peak of activity at pH 8.2 followed by a broad shoulder up to pH 12. At a higher concentration of SAM (200 μM). the predominant product formed was PC; the pH dependence for formation of the combination of PNNE and PC under these conditions showed a broad peak from 9 to 11. Inhibitors of SAM-dependent methylations. S-adenosylhomocysteine (SAH) and 3-deazaadenosine (3-DZA), blocked phospholipid methylation in lysed platelets and whole cells respectively. 3-DZA, did not inhibit platelet responses to thrombin, ADP, epinephrine, collagen, arachidonic acid and the calcium ionophore A23187 as measured by serotonin release, platelet aggregation, malondialdehyde formation, clot retraction, or arachidonic acid release at high agonist concentrations, and appeared to potentiate responses at low concentrations. Prostaglandin-induced inhibition of platelet function was unaffected by 3-DZA. Thrombin stimulation of platelets failed to produce the changes in methylation of phospholipids that have been associated with receptor activation of other cell types. Phospholipid methylation is not required for intial activation of platelets but the inhibition of this methylation may activate platelet function at low levels of agonists.

Phospholipid composition and metabolism of Micrococcus denitrificans.

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of (32)P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of (32)P or acetate-1-(14)C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.