Phospholipid Methyltransferase Activity Research Articles

Regulation of phospholipid synthesis in yeast

Phospholipid synthesis in the yeast Saccharomyces cerevisiae is a complex process that involves regulation by both genetic and biochemical mechanisms. The activity levels of phospholipid synthesis enzymes are controlled by gene expression (e.g., transcription) and by factors (lipids, water-soluble phospholipid precursors and products, and covalent modification of phosphorylation) that modulate catalysis. Phosphatidic acid, whose levels are controlled by the biochemical regulation of key phospholipid synthesis enzymes, plays a central role in the regulation of phospholipid synthesis gene expression.

Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

Background/Aims: Methylation of phosphatidylethanolamineto phosphatidylcholine predominantly takes place in mitochondrial-associated membrane and the endoplasmic reticulum of the liver. The transport of the phospholipids from endoplasmic reticulum to the bile canalicular membrane is via vesicular and protein transporters. In the bile canalicular membrane a flippase enzyme helps to transport phosphatidylcholine specifically to the biliary leaflet. The phosphatidylcholine then enters the bile where it accounts for about 95% of the phospholipids. We postulated that the increased proportion of phosphatidylcholine in the bile canalicular membrane and the bile compared to the transport vesicles may be due to a methyltransferase activity in the bile canalicular membrane which, using s-adenosyl methionine as the substrate, converts phosphatidylethanolamine on the cytoplasmic leaflet to phosphatidylcholine, which is transported to the biliary leaflet. The aim of our study was to demonstrate and partially characterise methyltransferase activity in the bile canalicular membrane. Methods: Organelles were obtained from hamster liver by homogenisation and separation by sucrose gradient ultracentrifugation. These, along with phosphatidylethanolamine, were incubated with radiolabelled s-adenosyl methionine. Phospholipids were separated by thin-layer chromatography and radioactivity was counted by scintigraphy. Results: We demonstrated methyltransferase activity (nmol of SAMe converted/mg of protein/h at 37°C) in the bile canalicular membrane of 0.442 (SEM 0.077, n=8), which is more than twice that found in the microsomes at 0.195 (SEM 0.013, n=8). The Km and pH optimum for the methyltransferase in the bile canalicular membrane and the microsomes were similar (Km 25 and 28 μM, respectively, pH 9.9 for both). The Vmax was different at 0.358 and 0.168 nmol of SAMe converted/mg of protein/h for the bile canalicular membrane and the microsomes, respectively. Conclusion: The presence of the methyltransferase activity in the bile canalicular membrane may be amenable to therapeutic manipulation.

Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

Demonstration and partial characterisation of phospholipid methyltransferase activity in bile canalicular membrane from hamster liver

Sperm phospholipid methyltransferase activity during preparation for exocytosis.

The present report describes experiments to evaluate phospholipid methyltransferase activity in golden hamster spermatozoa incubated under different conditions. Washed cauda epididymal sperm were incubated with taurine, in the presence or absence of epinephrine. At various times, the sperm were separated, and phospholipid methyltransferase activity measured. Also, at each time, aliquots of the sperm suspension were assayed for motility, and acrosome reactions. Some sperm incubated in the presence of taurine and epinephrine were capacitated by 3.5 h, because about 40 per cent of them can undergo the acrosome reaction 10 min after addition of the fusogen lysophosphatidylcholine. In epinephrine-free incubations the fusogen failed to stimulate acrosome reactions. On the other hand, epinephrine stimulated by twofold phospholipid methyltransferase activity from '0 time' incubated sperm, in comparison to that observed in taurine-treated cells. Enzyme activities from both taurine or epinephrine plus taurine-treated cells decreased as the incubation time of the sperm suspension increased. Kinetic properties of the sperm phospholipid methyltransferase activity were modified by the presence of taurine and epinephrine when S-adenosylmethionine was used as the substrate. These results suggest that refined molecular events occur in the sperm cell during the acquisition of fertilizing ability.

Effect of Taurine and Methionine on Sarcoplasmic Reticular Ca2+ Transport and Phospholipid Methyltransferase Activity

Summary: Perfusion of rat hearts with buffer containing 300 μM L-methionine led to a decrease in Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR) but an increase in calcium-independent Ca2+ release from junctional SR. These effects of L-methionine were not altered by exposure of the hearts to high levels of extracellular taurine, but changes in the size of the intracellular taurine pool appear to modulate calcium transport in SR through two mechanisms. First, millimolar concentrations of taurine can directly promote release of calcium from 45Ca2+-loaded junctional SR vesicles. Second, taurine serves as an inhibitor of SR phospholipid methyltransferase, an enzyme that appears to be responsible for methionine-mediated loss in Ca2+-induced Ca2+ release activity and promotion of Ca2+-independent Ca2+ release. The data imply that modulation of the intracellular taurine pool may affect cellular calcium homeostasis and myocardial contractile function. This may be important in development of the cardiomyopathy linked to taurine deficiency.

Effect of Taurine and Methionine on Sarcoplasmic Reticular Ca2+ Transport and Phospholipid Methyltransferase Activity

Perfusion of rat hearts with buffer containing 300 microM L-methionine led to a decrease in Ca(2+)-induced Ca2+ release in sarcoplasmic reticulum (SR) but an increase in calcium-independent Ca2+ release from junctional SR. These effects of L-methionine were not altered by exposure of the hearts to high levels of extracellular taurine, but changes in the size of the intracellular taurine pool appear to modulate calcium transport in SR through two mechanisms. First, millimolar concentrations of taurine can directly promote release of calcium from 45Ca(2+)-loaded junctional SR vesicles. Second, taurine serves as an inhibitor of SR phospholipid methyltransferase, an enzyme that appears to be responsible for methionine-mediated loss in Ca(2+)-induced Ca2+ release activity and promotion of Ca(2+)-independent Ca2+ release. The data imply that modulation of the intracellular taurine pool may affect cellular calcium homeostasis and myocardial contractile function. This may be important in development of the cardiomyopathy linked to taurine deficiency.

Phospholipid methylation decreases in human chorionic gonadotrophin-induced desensitized rat Leydig cells

Phospholipid methylation in Leydig cells from desensitized rats was studied. The incorporation of L-[methyl-3H]methionine into phospholipids in intact Leydig cells decreased when animals were injected with a single high dose of human chorionic gonadotrophin (hCG). This effect was detected on the first day after hCG injection and remained up to 12 days after treatment. The inhibition was not due to a reduced uptake of L-[methyl-3H]methionine. A decreased phospholipid methylation with unaltered phospholipid methyltransferase activity was observed on days 1, 6 and 12 after the hCG. On day 3 after hCG injection, phospholipid methyltransferase activity and phospholipid methylation in intact Leydig cells were both inhibited by 40%. Also, a minimal amount of LH free receptors and the lowest number of total receptors was observed at this time. Thus, a relationship between the reduced enzymatic activity and the maximal decrease in LH surface receptors is suggested. In addition, the decreased incorporation of L-[methyl-3H]methionine into phospholipids on days 1, 6 and 12 after hCG injection, could be associated with other cellular changes related to the desensitization process.

Phospholipid methyltransferase activity in diabetic rat fat cells: effect of isoproterenol and insulin.

The effects of isoproterenol and insulin on phospholipid methyltransferase (PLMT) activity were investigated in adipocytes from control and streptozotocin-diabetic rats. PLMT activity was assayed by measuring the rate of incorporation of 3H-methyl groups from S-adenosyl-L-[methyl-3H] methionine into phospholipids. Basal PLMT activity was higher in adipocytes from diabetic animals. Treatment of adipocytes with isoproterenol induced a concentration-dependent stimulation of PLMT activity. In control adipocytes, the maximal effect was obtained at 100 nM isoproterenol with 2.3 fold increase in PLMT activity and a half maximal effect at 25 nM. In adipocytes from diabetic rats, a lower dose of isoproterenol (10 nM), caused 1.2 fold increase with a half maximal effect at 4 nM. Addition of 100 nM insulin inhibited basal PLMT activity and the stimulatory effect of isoproterenol in both types of adipocytes. The beta-adrenergic blocking agent propranolol inhibited the stimulatory effect of isoproterenol on PLMT activity in control and diabetic adipocytes. Intracellular concentration of cAMP was higher in diabetic adipocytes but decreased to normal values after incubation in the presence of insulin.

Regulation of phosphatidylethanolamine methyltransferase and phospholipid methyltransferase by phospholipid precursors in Saccharomyces cerevisiae

Phosphatidylethanolamine methyltransferase (PEMT) and phospholipid methyltransferase (PLMT), which are encoded by the CHO2 and OPI3 genes, respectively, catalyze the three-step methylation of phosphatidylethanolamine to phosphatidylcholine in Saccharomyces cerevisiae. Regulation of PEMT and PLMT as well as CHO2 mRNA and OPI3 mRNA abundance was examined in S. cerevisiae cells supplemented with phospholipid precursors. The addition of choline to inositol-containing growth medium repressed the levels of CHO2 mRNA and OPI3 mRNA abundance in wild-type cells. The major effect on the levels of the CHO2 mRNA and OPI3 mRNA occurred in response to inositol. Regulation was also examined in cho2 and opi3 mutants, which are defective in PEMT and PLMT activities, respectively. These mutants can synthesize phosphatidylcholine when they are supplemented with choline by the CDP-choline-based pathway but they are not auxotrophic for choline. CHO2 mRNA and OPI3 mRNA were regulated by inositol plus choline in opi3 and cho2 mutants, respectively. However, there was no regulation in response to inositol when the mutants were not supplemented with choline. This analysis showed that the regulation of CHO2 mRNA and OPI3 mRNA abundance by inositol required phosphatidylcholine synthesis by the CDP-choline-based pathway. The regulation of CHO2 mRNA and OPI3 mRNA abundance generally correlated with the activities of PEMT and PLMT, respectively. CDP-diacylglycerol synthase and phosphatidylserine synthase, which are regulated by inositol in wild-type cells, were examined in the cho2 and opi3 mutants. Phosphatidylcholine synthesis was not required for the regulation of CDP-diacylglycerol synthase and phosphatidylserine synthase by inositol.

Interaction of Taurine with Methionine

Perfusion of isolated, working rat heart with buffer containing 300 microM methionine in the presence of 2.5 U/L insulin led to a 15% decrease in cardiac work and a four-fold decrease in sarcolemmal Na(+)-Ca2+ exchange activity. These effects of methionine were largely prevented by inclusion of 10 mM taurine in the buffer supplemented with methionine and insulin. Taurine also reduced the extent of 3H-methyl group incorporation from radioactive methionine into myocardial phospholipids by approximately 45%. Assays of sarcolemmal phospholipid methyltransferase activity at catalytic sites I, II, and III revealed that taurine inhibited N-methylation activity approximately 30%. The data imply that the ability of taurine to modulate myocardial contraction and calcium transport may be related to taurine-mediated inhibition of phospholipid N-methylation.

Effects of S-adenosyl- l-methionine on phospholipid methyltransferase activity changes induced by thioacetamide

Effects of S-adenosyl- l-methionine on phospholipid methyltransferase activity changes induced by thioacetamide

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.

Membrane Phospholipid Methylation is Associated with Surfactant Secretion in Rabbit Type II Alveolar Epithelial Cells

We investigated the involvement of membrane phospholipid methylation in receptor-mediated secretion of surfactant in adult rabbit type II alveolar epithelial cells (type II pneumocyte). Phospholipid methyltransferase activity was found in type II pneumocyte microsomes. Cell cultures of adult rabbit type II pneumocytes were then used to assay methyltransferase activity in the presence of the beta-adrenergic agonist, terbutaline, and the methyltransferase inhibitor, 3-deazaadenosine. Terbutaline predictably stimulated adenylate cyclase activity and surfactant secretion. It was also found to stimulate incorporation of methyl groups into phosphatidylcholine and to increase beta-adrenergic receptor availability as assayed by binding of dihydroalprenolol (DHA). Surfactant secretion, as well as adenylate cyclase activity, were stimulated by terbutaline and were inhibited by 3-deazaadenosine. 3-Deazaadenosine did not inhibit DHA binding. These results suggest that phospholipid methylation plays a role in stimulus-secretion coupling in adult rabbit type II pneumocytes.

1,2-Dimethylhydrazine-induced premalignant alterations in the S-adenosylmethionine/ S-adenosylhomocysteine ratio and membrane lipid lateral diffusion of the rat distal colon

Prior studies by our laboratory, utilizing the 1,2-dimethylhydrazine experimental model of colonic cancer, had shown that administration of this procarcinogen for 5 weeks was found to increase phospholipid methyltransferase activity and the fluidity of rat distal colonic brush-border membranes. The present studies were conducted to further explore these ‘premalignant’ colonic phenomena. Male albino rats of the Sherman strain were subcutaneously injected with dimethylhydrazine (20 mg/kg body wieght per week) or diluent for 5 weeks. Animals from each group were killed, distal colonic tissue harvested and the levels of S-adenosylmethionine, S-adenosylhomocysteine and decarboxylated S-adenosylmethionine measured by high performance liquid chromatography. The activity of methionine adenosyltransferase was also examined in these tissues. Additionally, brush-border membranes were isolated from the distal colonocytes of control and treated-animals and examined and compared with respect to their phospholipid methylation activities as well as their lipid fluidity as assessed by the rotational mobilities of the probes 1,6-diphenyl-1,3,5-hexatriene and dl-12-(9-anthroyl)stearic acid and translational mobility of the fluorophore pyrenedecanoic acid. The results of these studies demonstrated: (1) phospholipid methyltransferase activity in rat colonic plasma membranes was increased concomitantly with increases in the cellular levels of S-adenosylmethionine and the S-adenosylmethionine/ S-adenosylhomocysteine ratio in the distal colonic segment of treated-animals; and (2) the lateral diffusion of rat distal colonic brush-border membrane lipids, as assessed by the ratio of excimer/monomer fluorescence intensities of the fluorophore pyrenedecanoate, was also increased after dimethylhydrazine administration to these animals for 5 weeks.

Phospholipid methyltransferase from Drosophila melanogaster: Purification and properties

The phospholipid methyltransferase (PMTase) activity from Drosophila melanogaster has been purified ∼ 190,000-fold to give a preparation with a final sp.act. ∼4.3 μmol/min/mg protein. Gel filtration and HPLC methods show that this activity resides in a protein complex of M w = 140,000–150,000 dalton. Since the preparation gives several bands on SDS and native polyacrylamide gel electrophoresis, subunit composition has not been determined. The activity is sensitive to protein denaturing agents such as heat and proteases and shows inhibition by S-adenosyl homocysteine. Integrity of sulphydryl groups is essential for the stability of the enzyme. Zinc is a potent inhibitor, while manganese and calcium have no significant effect on activity. Micromolar concentrations of magnesium stimulate the activity, but millimolar concentrations inhibit the PMTase. There is no absolute requirement for exogenous lipid for activity, and evidence is presented that the enzyme is a lipoprotein and carries its own substrates. The incorporation of methyl groups into phosphatidylcholine and phosphatidyl- N, N-dimethylethanolamine was highest around pH 7.5. A high degree of methyl group incorporation into the monomethyl derivative also occurred at a lower pH. A Michaelis-Menten plot of methyl group incorporation into the total lipid fraction gives an average K m of 120 μM for S-adenosyl methionine. Methylation occurs on the base of the phospholipid. The ratio of the three methylated products formed is highly variable, with the monomethyl or the dimethyl products generally being the highest labelled under standard conditions. No separation of enzyme activities is observed during purification and on gel filtration a single peak is obtained, which shows all three methylating activities. Thus, while the variable ratios of the three products may indicate more than one enzyme, the single peak on gel filtration suggests these have almost identical molecular weights; it is possible they exist as a tight complex, or there is just one enzyme.

A phospho-oligosaccharide mimics the effect of insulin to inhibit isoproterenol-dependent phosphorylation of phospholipid methyltransferase in isolated adipocytes.

Addition of isoproterenol to isolated rat adipocytes prelabeled with [32P]phosphate caused an increase in the phosphorylation and activation of phospholipid methyltransferase. 32P-Labeled phospholipid methyltransferase was recovered by immunoprecipitation and gel electrophoresis. Analysis of 32P-labeled peptides revealed one site of phosphorylation regulated by isoproterenol, and analysis of phosphoamino acids demonstrated that the incorporation of [32P]phosphate was on phosphoserine. Incubation of adipocytes with isoproterenol in the presence of insulin or a phospho-oligosaccharide inhibited the phosphorylation and activation of this enzyme. The inhibitory effect of insulin on the phosphorylation of phospholipid methyltransferase was reversible, and it was mimicked by a phospho-oligosaccharide. The phospho-oligosaccharide was generated by hydrolysis of an isolated glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The insulin-like effect of this phospho-oligosaccharide on the phosphorylation of phospholipid methyltransferase was demonstrated in isolated adipocytes, and the effect was abolished by treatment of the phospho-oligosaccharide with 10% NH4OH, nitrous acid, or sodium periodate. These data suggest that in intact adipocytes the effect of insulin to inhibit the phosphorylation/activation of phospholipid methyltransferase is mediated by a phospho-oligosaccharide generated by a phosphatidylinositol-specific phospholipase C.

Hormonal regulation of phospholipid methyltransferase by 3',5'-cyclic adenosine monophosphate-dependent and independent mechanisms.

Treatment of isolated rat adipocytes with epinephrine or isoproterenol caused a time- and concentration-dependent increase in phospholipid methyltransferase (PLMT) activity that was blocked by propranolol and unaffected by phentolamine. Forskolin mimicked the stimulatory effect on PLMT, and insulin inhibited this effect. In both the absence and presence of insulin, there was a linear relationship between PLMT activity and lipolysis. PLMT activity was also increased in response to oxytocin, which does not activate adenylate cyclase in adipocytes and does not stimulate lipolysis. The effects of oxytocin were inhibited by insulin and were additive with those of isoproterenol on PLMT. These data support the hypothesis that in adipocytes, PLMT is activated by a cAMP-dependent protein kinase and a cAMP-independent mechanism, both of which can be regulated independently, and both of which are sensitive to inhibition by insulin.

Stimulation of adipocyte phospholipid methyltransferase activity by phorbol 12-myristate 13-acetate. Differential regulation of phospholipid methyltransferase and lipolysis.

The present studies demonstrate that treatment of rat adipocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) causes a dose-dependent stimulation of phospholipid methyltransferase (PLMT) activity. The stimulatory effect of PMA was not additive with that of isoprenaline or forskolin. The sensitivity of stimulated PLMT activity to inhibition by insulin, however, was decreased in the presence of PMA. The inhibitory effect of a maximal concentration of insulin on PLMT was unchanged in the presence of PMA. In contrast with the effects on PLMT, the lipolytic response of adipocytes to isoprenaline and the anti-lipolytic response to insulin were unaffected by PMA. These data suggest that PLMT is, whereas hormone-sensitive lipase is not, an intracellular target for the action of PMA. The lack of effect of PMA on lipolysis suggests that PLMT and hormone-sensitive lipase can be regulated by separate mechanisms. Furthermore, phorbol esters do not interfere in the regulatory pathway whereby insulin inhibits PMLT or lipolysis.

Protein kinase C catalyses the phosphorylation and activation of rat liver phospholipid methyltransferase

When a partially purified rat liver phospholipid methyltransferase is incubated with [gamma-32P]ATP and rat brain protein kinase C, phospholipid methyltransferase (Mr 50,000, pI 4.75) becomes phosphorylated. Phosphorylation of the enzyme showed Ca2+/lipid-dependency. Protein kinase C-dependent phosphorylation of phospholipid methyltransferase was accompanied by an approx. 2-fold activation of the enzyme activity. Activity changes and enzyme phosphorylation showed the same time course. Activation of the enzyme also showed Ca2+/lipid-dependency. Protein kinase C mediates phosphorylation of predominantly serine residues of the methyltransferase. One major peak of phosphorylation was identified by analysis of tryptic phosphopeptides by isoelectrofocusing. This peak (pI 5.2) differs from that phosphorylated by the cyclic AMP-dependent protein kinase (pI 7.2), demonstrating the specificity of phosphorylation of protein kinase C. Tryptic-peptide mapping by h.p.l.c. of the methyltransferase phosphorylated by protein kinase C revealed one major peak of radioactivity, which could be resolved into two labelled phosphopeptides by t.l.c. The significance of protein kinase C-mediated phosphorylation of phospholipid methyltransferase is discussed.

Enhancement by L-Methionine of Contractile Responses to Acetylcholine and High KCl in Uterine Segment

The contractile responses of isolated uterine segments from 17β-estradiol-3-benzoate-treated ovariectomized rats to acetylcholine (ACh) and high KCl in Ca-depleted modified Locke-Ringer solution on addition of CaCl2 were used as idicators of Ca2+ influxes through ACh receptor- and voltage-operated Ca2+ rhannels, respectively. L-Methionine (L-Met) significantly enhanced these responses. The enhancement depended on the time of treatment with L-Met and concentration of L-Met. 3-Deazaadenosine (3-DAA) plus homocysteine thiolactone (HCTL), which inhibit S-adenosylmethionine-dependent methylation, caused dose-dependent inhibition of these contractile responses to ACh and high KCl. These inhibitory effects of 3-DAA plus HCTL were significantly attenuated in the presence of L-Met. Protein carboxylmethyltransferase and phospholipid methyltransferase activities were detected in the isolated uterine segment under conditions similar to those in which the contractile responses were observed. 3-DAA plus HCTL inhibited these enzyme activities. These findings suggest that S-adenosylmethionine-dependent methylations of protein and/or phospholipid in isolated uterine segment are involved in the contractile responses to ACh and high KCl in Ca-depleted modified Locke-Ringer solution on addition of CaCl2.