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PubMed1991

Calcium is sufficient but not necessary for activation of sheep platelet cytosolic phospholipase A2.

In this study we demonstrate that: (1) although the major phospholipase A2 present in sheep platelets is activated by calcium ions, it can effectively catalyze hydrolysis of the sn-2 ester linkage in phospholipids in the absence of calcium; (2) expression of calcium-independent phospholipase A2 activity can be induced by NaCl utilizing purified (but not crude) cytosolic enzyme; and (3) calcium-independent phospholipase A2 activity is regulated by a reconstitutable cytosolic protein. Collectively, these results underscore the fundamental catalytic differences between extracellular and intracellular calcium-dependent phospholipases A2 and demonstrate that calcium is sufficient, but not necessary, for the activation of this class of intracellular phospholipases A2.

Animals↗
PubMed1993

Correlation between phospholipase A2 activity and intra-Golgi protein transport reconstituted in a cell-free system.

A wide variety of phospholipase A2 inhibitors blocks intra-Golgi protein transport reconstituted in a cell-free system. Phospholipase A2 activity detectable under the protein transport assay conditions is actually inhibited by the inhibitors. There is a good correlation between the inhibition of protein transport and that of phospholipase A2 activity. Prolactin secretion from GH3 cells is also blocked by a membrane-permeable phospholipase A2 inhibitor, suggesting the physiological relevance to inhibition of protein transport in vitro by phospholipase A2 inhibitors.

Acetylglucosamine↗
PubMed1993

Monoclonal antibodies against rat liver mitochondrial phospholipase A2: epitope analysis and application in western blotting.

1. Eight cell-lines producing monoclonal antibodies, raised against rat liver mitochondrial phospholipase A2, were investigated with respect to epitope-recognition. It was shown that all antibodies tested were directed to an identical epitope. 2. This epitope is a conformational one, since treatment of phospholipase A2 with the reducing agent dithiothreitol lowered the antibody binding significantly. 3. To increase sensitivity, Western blot analyses have to be performed on protein samples lacking dithiothreitol or beta-mercaptoethanol. The conditions described in this report allow the detection of the phospholipase A2 in rat liver homogenates. 4. When rat liver mitochondrial phospholipase A2 was purified by Ultrogel AcA 54 gel filtration, a nearly homogeneous protein preparation was obtained, as judged by SDS-PAGE. Western blot analysis of this preparation, however, clearly indicated the phospholipase A2 to correspond to a hardly visible protein band.

Animals↗
PubMed1991

Detection and characterization of extracellular phospholipase A2 in pleural effusion of patients with tuberculosis.

Extracellular phospholipase A2 activity has been identified in pleural fluid of patients with tuberculosis. This enzyme is a calcium requiring protein and has a pH optimum of 10.0. The enzyme was inhibited by the active site-directed histidine reagent, rho-bromophenacyl bromide. Ionic and non-ionic detergents, or the sulfhydryl reagent dithiothreitol, caused loss of enzyme activity. When substrate specificity was tested using 2-[1-14C]linoleoyl phospholipids as substrates, phosphatidyl-ethanolamine was the best substrate, followed by phosphatidylserine and phosphatidylcholine. This phospholipase A2 showed high affinity for heparin, and was recognized by a monoclonal antibody raised against phospholipase A2 from human synovial fluid. These findings suggest that an extracellular phospholipase A2, which may belong to the 14K group II phospholipase A2 family, exists in the pleural fluid of patients with tuberculosis.

Acetophenones↗
PubMed1985

A rapid method for the purification of monomeric and/or dimeric phospholipases A2 in crotalid snake venoms.

We have developed a simple two-step procedure for the separation of monomeric (14,000 mol. wt) and dimeric (28,000 mol. wt) phospholipases A2 from the venoms of Crotalidae family snakes. All venom phospholipases A2 studied thus far exist as monomers under acidic conditions and are chromatographed as such on a column of G-50 Sephadex (superfine) equilibrated in 5% acetic acid. Separation of dimeric phospholipases A2 from any monomeric enzyme(s) in pools of enzyme thus obtained is achieved by chromatography on a second column of G-50 Sephadex (superfine) identical to the first but developed in 1% ammonium bicarbonate. This method has been applied to an investigation of the prevalence of monomeric and/or dimeric enzymes in venoms of the Crotalidae family. The distribution of monomeric and dimeric phospholipases correlates well with phylogeny. In the more primitive Crotalidae genera, such as Trimeresurus and Agkistrodon, monomeric phospholipases A2 are predominant. In the more highly evolved Crotalus genus, only dimeric enzymes are found.

Animals↗
PubMed1986

Structure-function relationships of phospholipases. I: Prediction of presynaptic neurotoxicity.

The hydropathic indexes of 24 phospholipases have been calculated from their amino acid sequences. The presynaptic neurotoxic potential of venom phospholipases can be predicted by the use of hydropathy profiles. The presynaptically acting phospholipases have a distinct hydrophobic region around the residues 80-110 which is probably involved in the interaction with the presynaptic membranes. This region is present as a separate helix in the tertiary structure of phospholipases. Such a hydrophobic region is absent in non-neurotoxic phospholipases.

Amino Acid Sequence↗
PubMed1992

An electrophoretic study on phospholipase A2 isoenzymes in the venoms of Central American crotaline snakes.

The number and isoelectric points of phospholipase A2 isoenzymes were studied in the venoms of 12 Central American crotaline snakes of the genera Bothrops, Crotalus, Lachesis and Agkistrodon. The study was carried out by using a methodology based on electrophoretic separation of venoms, transfer to nitrocellulose and detection of activity of the bands by an indirect hemolytic assay in agarose-erythrocyte-egg yolk gels. All venoms tested had indirect hemolytic activity, although they varied in the number and isoelectric point of their phospholipases A2. Most venoms had predominantly acidic isoenzymes, with the exception of A. bilineatus which had mainly basic isoenzymes and B. schlegelii which had both acidic and basic isoenzymes. Analysis of interindividual variability in B. asper venom demonstrated that two phospholipase A2 isoenzymes are present in some venoms but absent in others. Polyvalent antivenom was effective in neutralizing phospholipase A2 activity of the 12 venoms tested, when venoms and antivenom were incubated in the fluid phase. This work demonstrates a conspicuous interspecific variability in the number and isoelectric points of phospholipases A2 present in Central American crotaline snake venoms.

Animals↗
PubMed1995

Binding proteins on synaptic membranes for crotoxin and taipoxin, two phospholipases A2 with neurotoxicity.

Crotoxin and taipoxin are both neurotoxic phospholipases A2 capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been considered to be necessary but not sufficient for the blockade. Since many phospholipases A2 with comparable or even higher enzymatic activity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation of this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the isolated synaptic membrane fraction from guinea-pig brain, whereas specific binding is not detected with the nontoxic pancreatic phospholipase A2. Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polypeptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all, other toxic phospholipases A2 also appear to be ligands for the three polypeptides. We now report studies on partial purification of these polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-binding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca(2+)-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the Na(+)-dependent uptake of norepinephrine, dopamine and serotonin by the synaptosomes. In contrast, choline uptake is not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Activation of phospholipase A2 on lipid bilayers.

So far, three phospholipases A2 that display activation kinetics during the time course of hydrolysis of DPPC LUV have been found to undergo a fluorescence change coincident with the activation: the monomer (AppD49) and the dimer enzymes from A. piscivorus piscivorus and the dimer enzyme from C. atrox. The porcine pancreatic enzyme produces similar time courses of hydrolysis but does not display a concurrent fluorescence change. It is assumed that other phospholipases A2 will behave similarly in terms of the hydrolysis reaction. Which enzymes respond with a similar change in intrinsic fluorescence during the time course may well depend on the position of tryptophan residues and the amino acid sequence. Even though a given phospholipase A2 may not change its fluorescent properties on activation, the simultaneous monitoring of the hydrolysis reaction and the fluorescence of probes of the bilayer structure can be done with any phospholipase A2. A variety of probes exist which are sensitive to slightly different membrane properties and could be used as described here for TMA-DPH. For example, 1,3-dipyrenylpropane is sensitive to the apparent microviscosity of the bilayer is terms of the ability of molecules to translationally diffuse in the membrane. 6-Palmitoyl-2-[[2-(trimethylammonio)ethyl]methylamino]naphthalene chloride is sensitive to the ability of a molecule to rotate in the bilayer and displays large changes in its steady-state fluorescence as the anisotropy of the bilayer changes. 6-Propionyl-2-(dimethylamino)naphthalene is sensitive to the polarity and degree of hydration of its environment. Finally, a compound titled NK-529 has recently been introduced that apparently monitors the lateral phase separation of fatty acids in the bilayer. The fact that activation of phospholipase A2 can be monitored during the time course of hydrolysis of DPPCLUV makes this system an excellent choice for studying the mechanisms of activation and possible effects of various activators and inhibitors. The experimental system described here provides a way to determine whether such regulators exert their effects through alterations of the properties of the membrane and/or the enzyme. Importantly, this system allows one to seek temporal correlations of the various events in the process.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1994

The expression of phospholipase A2 and lipocortins (annexins) I, II and V in human fetal membranes and placenta in association with labour.

We have studied the expression of the cellular, type two phospholipase A2 and lipocortins (annexins) I, II and V in human amnion, chorion-decidua and placenta using northern analysis. We found no difference in the expression of phospholipase A2 or lipocortin V in tissues obtained before or after labour. Lipocortin I expression was found to decrease in amnion and placenta but increase in chorion decidua with the onset of labour, while expression of lipocortin II increased only in amnion. These results support the hypothesis that the increased phospholipase A2 activity in the fetal membranes and placenta which is associated with labour is not due to increased phospholipase A2 gene expression, but that post translational control of phospholipase A2 activity may be mediated through changes in lipocortin I expression.

Annexin A1↗
PubMed1995

Plasmalogens, phospholipases A2 and signal transduction.

Several lines of evidence indicate that the breakdown of plasmalogens in neural membranes during neurodegenerative diseases is a receptor-mediated process catalyzed by a plasmalogen-selective phospholipase A2. This enzyme has recently been purified from bovine brain. It does not require Ca2+ and is localized in cytosol. It has a molecular mass of 39 kDa and is strongly inhibited by glycosaminoglycans, with the pattern of inhibition being heparan sulfate > hyaluronic acid > chondroitin sulfate > heparin. This plasmalogen-selective phospholipase A2 is also inhibited by gangliosides and sialoglycoproteins. Substrate specificity and the effects of metal ions, detergents and inhibitors suggest that this phospholipase A2 is different from the well-known 85 kDa Ca(2+)-dependent cytosolic phospholipase A2 that has recently been cloned and is not plasmalogen-selective. The plasmalogen-selective phospholipase A2 may be regulated by glycosaminoglycans and sialoglycoconjugates and may be involved in the regulation of K+ channels. This enzyme, which plays a major role in the release of fatty acids during ischemic injury and reperfusion, shows promise as a major target for drug therapy.

Animals↗
PubMed1992

Release of carrot plasma membrane-associated phosphatidylinositol kinase by phospholipase A2 and activation by a 70 kDa protein.

Plasma membranes were isolated from carrot (Daucus carota L.) cells grown in suspension culture and treated with phospholipase A2 from snake or bee venom for 10 min. As a result of this treatment, phosphatidylinositol kinase activity was recovered in the soluble fraction. There was no detectable diacylglycerol kinase or phosphatidylinositol monophosphate kinase activity released from the membranes after the phospholipase A2 treatment. Treating the plasma membranes with phospholipase C or D did not release PI kinase activity. The phospholipase A2-released PI kinase was activated over 2-fold by a heat stable, soluble 70 kDa protein. The partially purified 70 kDa activator increases the Vmax but does not affect the Km of the phospholipase A2-released PI kinase.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Inhibition of phospholipase A2 and insulin secretion in pancreatic islets.

Arachidonic acid may be an important mediator of insulin secretion since (1) glucose activates phospholipase A2 thus increasing endogenous unesterified levels of arachidonic acid, (2) arachidonic acid mobilizes Ca2+ from the islet endoplasmic reticulum and (3) arachidonic acid has been proposed to regulate voltage-dependent Ca2+ channels in the beta-cell. We have used the phospholipase A2 inhibitor, (p-amylcinnamoyl)anthranilic acid (ACA), to determine whether phospholipase A2 activation is required for glucose-induced insulin secretion. ACA inhibited in a dose-dependent manner glucose-induced insulin secretion, as well as glyceraldehyde and alpha-ketoisocaproic acid-induced insulin secretion. ACA also totally abolished glucose-induced arachidonate accumulation but did not affect phospholipase C suggesting that it was specific for phospholipase A2. Furthermore, ACA did not inhibit glucose oxidation. These observations suggest that glucose-induced arachidonate increase is essential for insulin secretion.

Animals↗
PubMed1992

Anionic currents of chick sensory neurons are affected by a phospholipase A2 purified from the venom of the taipan snake.

A neurotoxic phospholipase A2 was purified from the venom of the taipan snake Oxyuranus scutellatus scutellatus by three consecutive chromatographic steps on ion exchange resins, followed by an affinity column prepared with a phosphatidylcholine derivative attached to Sepharose. The phospholipase was shown to be of type A2 (specific activity of 85 units/mg protein), and an apparent molecular weight of 16,000. Amino acid analysis shows the presence of approx. 150 residues with the N-terminal amino acid sequence: NLAQFGFMIRCANGGSRSALDYADYGC, different from all the phospholipases described until now. This enzyme is lethal to experimental mice (LD50 = 10 micrograms/20 g mouse weight) and affects ionic currents in chick (Gallus domesticus) dorsal root ganglion cells, measured by the whole-cell clamp technique. In symmetrical external/internal ionic solutions, after suppression of Na+, K+ and Ca2+ currents, external application of phospholipase at a low concentration (30 nM) was shown to increase the baseline current in a reversible manner. The augmented response was voltage-dependent and the effect was much greater for negative currents. In the presence of a salt gradient across the membrane (out 40 mM NaCl/in 140 mM CsCl), the current reversal potential revealed a shift in the positive direction typically due to Cl- ion flux through the membrane. External application of a 50 microM concentration of picrotoxin caused a reversible reduction of the phospholipase-induced chloride current. Moreover, no appreciable current block was detected after addition of 50 microM DIDS.

Amino Acid Sequence↗
PubMed1995

Hydrogen peroxide activation of cytosolic phospholipase A2 in vascular smooth muscle cells.

We have reported previously that hydrogen peroxide induces arachidonic acid release from prelabeled vascular smooth muscle cells. Here, we studied the effect of hydrogen peroxide on the phosphorylation of cytosolic phospholipase A2 in these cells. Hydrogen peroxide induced a rapid, time-dependent increase in the phosphorylation of cytosolic phospholipase A2. Hydrogen peroxide also increased arachidonic acid release from prelabeled cells in a time-dependent manner similar to that of phosphorylation of cytosolic phospholipase A2. Protein kinase C depletion significantly inhibited the hydrogen peroxide-stimulated cytosolic phospholipase A2 phosphorylation and arachidonic acid release. Hydrogen peroxide caused a time-dependent increase in mitogen activated protein kinase activity. Taken together, these findings suggest that cytosolic phospholipase A2 may, at least in part, contribute to arachidonic acid release induced by hydrogen peroxide and this effect appears to be mediated by protein kinase C and mitogen activated protein kinase.

Animals↗
PubMed1996

Transfection of C3H10T1/2 cells with the Harvey-ras oncogene reduces cytosolic phospholipase A2 function.

Several lines of evidence suggest that phospholipase A2 may play a role in the activated ras-mediated transformation process. In the present study, phospholipase A2 activity and expression were assessed in a murine fibroblast cell line (C3H10T1/2 cells) that was stably transfected with the Harvey ras oncogene, a cellular model used for studying multistage carcinogenesis. Reduced levels of fatty acids were released from the ras-transfected cells compared to untransfected controls. The in vitro phospholipase A2 activity apparent in the C3H10T1/2 showed preference for sn-2 arachidonyl phosphatidylcholine compared to dipalmitoyl phosphatidylcholine. The activity, as well as the cytosolic phospholipase A2 immunoreactive protein, was reduced by 50% in the ras-transfected cells compared to control cells. These results suggest that the cytosolic phospholipase A2 is the predominant form of this enzyme family expressed in C3H10T1/2 cells and that the activity and protein amount is reduced by 50% in these cells when stably transfected with the Harvey ras oncogene.

Animals↗
PubMed1995

Effect of berbamine on cytosolic phospholipase A2 activation in rabbit platelets.

The effect of berbamine, a biscoclaurine alkaloid, on cytosolic phospholipase A2 activation in rabbit platelets was investigated. Berbamine inhibited arachidonic acid liberation induced by thrombin but not that by ionomycin. The alkaloid did not affect thrombin-stimulated Ca2+ mobilization. Ca(2+)-dependent translocation of cytosolic phospholipase A2 to membranes, or the activity of partially purified cytosolic phospholipase A2. Furthermore, berbamine had no effect on the thrombin-elicited increase in cytosolic phospholipase A2 activity. However, berbamine suppressed arachidonic acid liberation in platelets stimulated with GTP-binding protein activators. Although incubation of platelet membranes with a GTP analogue decreased the islet-activating protein-catalyzed ADP-ribosylation of an approximately 40 kDa protein in the membranes, pretreatment of the membranes with berbamine did not influence the decrease in ADP-ribosylation. These results suggest that berbamine may impair GTP-binding protein-mediated activation of cytosolic phospholipase A2, probably without influencing the enzyme translocation to membranes or the increase in the enzyme activity, and thus may cause the suppression of thrombin-induced arachidonic acid liberation.

Alkaloids↗
PubMed2004

Chronic carbamazepine selectively downregulates cytosolic phospholipase A2 expression and cyclooxygenase activity in rat brain.

BACKGROUND: Carbamazepine is a mood stabilizer used as monotherapy or as an adjunct to lithium in the treatment of acute mania or the prophylaxis of bipolar disorder. Based on evidence that lithium and valproate, other mood stabilizers, reduce brain arachidonic acid turnover and its conversion via cyclooxygenase to prostaglandin E(2) in rat brain, one possibility is that carbamazepine also targets the arachidonic acid cascade. METHODS: To test this hypothesis, carbamazepine was administered to rats by intraperitoneal injection at a daily dose of 25 mg/kg for 30 days. RESULTS: Carbamazepine decreased brain phospholipase A(2) activity and cytosolic phospholipase A(2) protein and messenger RNA levels without changing significantly protein and activity levels of calcium-independent phospholipase A(2) or secretory phospholipase A(2). Cyclooxygenase activity was decreased in carbamazepine-treated rats without any change in cyclooxygenase-1 or cyclooxygenase-2 protein levels. Brain prostaglandin E(2) concentration also was reduced. The protein levels of other arachidonic acid metabolizing enzymes, 5-lipoxygenase and cytochrome P450 epoxygenase, were not significantly changed nor was the brain concentration of the 5-lipoxygenase product leukotriene B(4). CONCLUSIONS: Carbamazepine downregulates cytosolic phospholipase A(2)-mediated release of arachidonic acid and its subsequent conversion to prostaglandin E(2) by cyclooxygenase. These effects may contribute to its therapeutic actions in bipolar disorder.

Analgesics, Non-Narcotic↗