Fusao Hirata

Identification and properties of methyltransferases that synthesize phosphatidylcholine in rat brain synaptosomes.

Abstract: Rat brain was found to enzymatically methylate phospholipids to form phosphatidylcholine with S‐adenosyl‐l‐methionine serving as the methyl donor. Methyltransferase activity was localized in the microsomes and synaptosomes. In synaptosomes, at least two enzymes were found to be involved in the formation of phosphatidylcholine. The first methyltransferase which catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl‐N‐monomethylethanolamine was found to have a pH optimum of 7.5, a low Km for 5‐adenosyl‐l‐methionine and a partial requirement for Mg2. Methyltransferase I is tightly bound to membranes. The second methyltransferase (II) catalyzes the successive methylations of phosphatidyl‐N‐monomethylethanolamine to phosphatidyl‐N, N‐dimethylethanolamine and then to phosphatidylcholine. In contrast to methyltransferase I, methyltransferase II has a pH optimum of 10.5, a high apparent Km for S‐adenosyl‐l‐methionine and no requirement for Mg2. Methyltransferase II is easily solubilized by sonication. The highest specific activity for both enzymes was found in the synaptosomal plasma membrane.

Phospholipid methylation affects immunoglobulin E-mediated histamine and arachidonic acid release in rat leukemic basophils

Abstract Antigenic stimulation of rat basophilic leukemia cells sensitized with immunoglobulin E causes the release of histamine as well as arachidonic acid and its metabolites. The release of these substances is preceded by an increase in phospholipid methylation. Inhibition of phospholipid methylation is correlated to the inhibition of histamine release. Inhibition of methylation also reduces arachidonate release. Phospholipid methylation appears to be associated with both histamine secretion and the release of arachidonate and its metabolites.

IgE-mediated histamine release in rat basophilic leukemia cells: Receptor activation, phospholipid methylation, Ca2+ flux, and release of arachidonic acid

Abstract Stimulation of IgE receptors on rat basophilic leukemia cells causes a transient rise and fall of methylated phopholipids, Ca 2+ influx, and release of arachidonic acid previously incorporated into phosphatidylcholine and liberation of histamine. Inhibition of phospholipid methylation by methyltransferase inhibitors, 3-deazaadenosine and homocysteine thiolactone, almost completely blocks the influx of Ca 2+ , and release of arachidonic acid and histamine. Stimulation of immunoglobulin E receptors by antigen releases only [ 14 C]arachidonic acid but not [ 14 C]linoleic acid, [ 14 C]oleic acid and [ 14 C]stearic acid, all of which were previously incorporated into phospholipids. [ 14 C]Arachidonate was found to be incorporated mainly into phosphatidylcholine. The phosphatidycholine rich in arachidonate appeared to be synthesized to a considerable extent by the transmethylation pathway. These findings suggest that in rat basophilic leukemia cells, immunoglobulin E receptors, phospholipid methyltransferases, Ca 2+ ion channel, and phospholipase(s) that cause release of arachidonic acid and the discharge of histamine are associated.

Phospholipase activation in the IgE-mediated and Ca2+ ionophore A23187-induced release of histamine from rat basophilic leukemia cells

Abstract The importance of phospholipase(s) activation in the IgE-mediated and ionophoreinduced histamine release from the rat basophilic leukemia cell line has been examined. The activation of phospholipase(s) as measured by [ 14 C]arachidonic acid release and the release of histamine both required Ca 2+ and were temporally parallel. Inhibition of phospholipase(s) activity by the inhibitors mepacrine and α-parabromoacetophenone also correlated with the inhibition of histamine release. [ 14 C]Arachidonic acid released by the phospholipase(s) was mainly metabolized to prostaglandin D 2 . The inhibition of the cyclooxygenase pathway by indomethacin did not affect histamine release. 5,8,11,14-Eicosatetraynoic acid inhibited both histamine and [ 14 C]arachidonic acid release suggesting an effect not only on the cyclooxygenase and lipoxygenase pathways but also on the phospholipase(s). These results suggest that activation of phospholipase appears to be necessary for histamine release in the rat bosophilic leukemia cells.

Evidence for two methyltransferases involved in the conversion of phosphatidylethanolamine to phosphatidylcholine in the rat liver

Abstract The stepwise methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) occurs in the rat liver. The properties of two methyltransferases that are involved in this stepwise methylation of PE to PC in the rat liver are reported. Rat microsomal membranes were used as the source of the enzymes and S -adenosyl- l -[methyl- 3 H]methionine as the methyl donor. The first methyltransferase converted PE to phosphatidyl- N -methylethanolamine (PME), and the second methyltransferase converted PME to PC. The products were characterized by thin-layer chromatography and high-pressure liquid chromatography. After incubation of rat liver microsomes with the methyl donor, three peaks corresponding to PME, dimethyl-PME (PMME), and PC were found and quantified. The first methyltransferase had low K m (0.83 μ m ), pH optimum of 8, and was activated by Mg 2+ . The second methyltransferase had a high K m (~67 μ m ) and a pH optimum of 10. The proportion of the first methyltransferase in the microsomal membranes was increased by repeated washings in hypotonic medium containing EDTA. When the microsomal membranes were subjected to repeated mild sonication and centrifugation at 105,000 g a fraction of the second methyltransferase was solubilized (i.e., appeared in the nonparticulate fraction). The solubilized enzyme utilized dipalmitoyl-PME and -PMME as substrates. Both enzymes were also present in mitochondrial and nuclear membranes with highest specific activities occurring in the microsomal membranes.

Protein methylesterase and leukocyte chemotaxis.

Abstract The involvement of protein carboxylmethylesterase in leukotaxis has been studied. The enzyme is localized in the cytosolic fraction of neutrophils. In intact cells the presence of a chemoattractant induces a rapid stimulation of enzymatic activity. An half-maximal stimulation of this activity is induced by a concentration (3 n M ) of peptide attractant that is close to that (1 n M ) producing an half-maximal chemotactic response. In addition, the presence of a specific antagonist of peptide attractants lowers the stimulated esterase response of neutrophils to these chemotactic factors. The role of another enzyme, protein carboxylmethylase, has been re-examined. In the initial stages (1 min) of chemotactic stimulation, a highly variable increase in the activity of this enzyme occurs, while over a longer period (30 min) there is an apparent decrease. The results are consistent with a role for an increased turnover of protein methylesters in leukotaxis. The reversible neutralization of charge that would accompany this turnover process may contribute to alterations of membrane events that occur in the earliest stages of leukotaxis.

Phospholipid methyltransferase asymmetry in synaptosomal membranes

The sequential methylation of phosphatidylethanolamine to form phosphatidylcholine is carried out by two methyltransferases in rat brain synaptosomes. The first enzyme methylates phosphatidylethanolamine to form phosphatidylmonomethylethanolamine. The second enzyme methylates the monomethylated phospholipid two additional times, forming phosphatidylcholine. Experiments comparing the rate of methylation between intact and lysed synaptosomes indicate that synaptosomes accumulateS-adenosyl-l-methionine and that the first methylation takes place on the cytoplasmic side of the membrane. Studies comparing trypsin digestion of proteins in intact and lysed synaptosomes indicate that the first enzyme is localized on the cytoplasmic side of the membrane and the second enzyme faces the external surface. Phospholipase C hydrolyzed phosphatidylcholine formed by methylation, suggesting its localization in the external layer of the phospholipid bilayer. A mechanism for an enzyme-mediated flip-flop of phospholipids from the cytoplasmic side to the outer surface of the synaptosomal plasma membrane is presented.

The relationship between phospholipid methylation and calcium influx in murine lymphocytes stimulated with native and modified Con A

Native Con A and two chemical derivatives, divalent dimeric Con A and monovalent dimeric Con A. induced a transient increase of phospholipid methylation, Ca2+ influx, and also increased DNA synthesis in murine lymphocytes. For each of the individual mitogens, the dose-response curves for these three activities were very similar. However, there were major differences between the dose-response curves for Con A and each of its two chemical derivatives. On the other hand, the time course of phospholipid methylation for each lectin reached a maximum at about 10 min after the addition of lectin, and then gradually decreased to control levels. In like manner, Ca2+ influx reached its maximum at approximately 5 min. The lectin-stimulated increase in phospholipid methylation occurred in calcium-free medium, while the inhibitor of phospholipid methylation, 3-deaza-SIBA, also suppressed the increased calcium influx. This suggests that the Ca2+ influx might be regulated by early phospholipid methylation. Further, in the absence of calcium, the methylated phospholipids do not undergo Con A-accelerated breakdown by phospholipase A2. This suggests that the increased influx of calcium is necessary for the activation of phospholipase A2, an enzyme that hydrolyses methylated phospholipids to yield arachidonic acid and lysolecithin. Blocking any of these biochemical steps also blocked subsequent DNA synthesis, suggesting that the pathway may be required for the activation of lymphocytes.

ROLE FOR METHYLATION IN LEUKOCYTE CHEMOTAXIS

Key words: Chemotaxis, leukocyte, methylation, protein carboxyl groups, membrane phospholipids

Molecular Mechanisms of the Modulation of Phospholipid Metabolism by Glucocorticoids

Glucocorticoids are hormones from the adrenal cortex and have diverse effects on the metabolisms of purines, fats, amino acids, and carbohydrates in a variety of tissues and organs. The actions of glucocorticoids have been well documented with respect to the capacity of organisms to resist environmental changes and noxious stimuli, especially stress (Axelrod and Reissin, 1984). These hormones are reported generally to act in concert with a variety of other hormones in a permissive manner. Many hormones, neurotransmitters, and drugs cause release of arachidonic acid, a precursor of leukotrienes and prostaglandins, from target tissues or organs, and glucocorticoids suppress such receptor-mediated release of arachidonic acid (Kuehl and Egan, 1980). Since prostaglandins and leukotrienes are now believed to be inflammatory mediators, the antiinflammatory activity, a major action, of glucocorticoids has been proposed to be associated with inhibition of this arachidonic acid release (Kuehl and Egan, 1980). In the present chapter, I describe the molecular mechanism of actions of glucocorticoids whereby these hormones block the release of arachidonic acid stimulated by hormones, neurotransmitters, and drugs.