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PubMed1979

Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study.

Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.

Alanine↗

PubMed1982

Release of lysosomal phospholipase A1 and A2 into cytosol and rapid turnover of newly-formed lysophosphatidylcholine in FL cells during fusion-from-within induced by measles virus.

The subcellular distribution of the phospholipase activities and metabolism of lysophosphatidylcholine in cultured human cell line (FL cells) during "fusion-from-within" induced by measles virus were studied. During cell fusion, fairly high activities of phospholipase A1 and A2, the optimal pHs of which were acidic, appeared in the cytosol. These phospholipases were confirmed to be of lysosomal origin by the following facts: (1) the properties of these phospholipases were the same as those of lysosomal phospholipase A1 and A2; (2) a decrease in the total activity of phospholipases A1, A2 and acid phosphatase in the lysosomal fraction of the infected cells coincided with an increase in their activity in the cytosol. The release of lysosomal phospholipase A1 and A2 into the cytosol of the infected cells appeared to be related to the extent of cell fusion-from-within. Phospholipase A1 and A2 released to the cytosol hydrolyzed the cell membrane-bound phospholipids to form lysophospholipids. 1-[1-14C]Palmitoyl-GPC, incorporated into the cells from the culture medium, was rapidly converted into phosphatidylcholine, triacylglycerol and phosphatidylethanolamine in the cells during fusion-from-within, but the radioactive lysophosphatidylcholine and fatty acids were hardly detectable, indicating a rapid turnover of cellular lysophosphatidylcholine during cell fusion. The subcellular lysophospholipid acyl-hydrolase activities of the infected cells were higher than those of normal cells. Possible relation between the membrane fusion induced by measles virus and the phospholipid metabolism in the infected cells were discussed.

Cell Fusion↗

PubMed1980

Correlation of enzymatic activity and anticoagulant properties of phospholipase A2.

Some highly purified phospholipases A from the venom of viperidae, crotalidae and elapidae were found to hve anticoagulant properties. All phospholipases which exhibited anticoagulant properties are characterized by a high isoelectric point, but not all strongly basic phospholipases are anticoagulant. Anticoagulant phospholipases hydrolyse highly packed monomolecular films of phospholipids without any lag time while non-anticoagulant phospholipases present considerable induction times indicative of a low penetrating power. When the ester linkages in the procoagulant lipids were replaced by the non-hydrolysable ether bonds, the mixture retained its clotting ability even in the presence of phospholipases, thus suggesting that anticoagulant phospholipases prevent clot formation by hydrolysis of phospholipids. This was confirmed by chemical modification of phospholipases, viz. alkylation of the active-centre histidine with 1-bromo-octan-2-one. This modification yielded proteins which had lost their anticoagulant properties but which retained a high affinity for phospholipids.

Animals↗

PubMed1989

Release of phospholipase A2 activity from rat vascular smooth muscle cells mediated by cAMP.

Primary cultures of smooth muscle cells (SMC) derived from rat aorta release a phospholipase A2 activity into the culture medium. Phospholipase A2 activity was determined with [1-14C]oleate-labelled Escherichia coli as substrate. The enzyme has a neutral pH optimum and the activity is critically dependent on the free calcium concentration, with significant activity in the micromolar range of free calcium. Treatment of SMC with the beta agonist salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cAMP concentration, increase the release of phospholipase A2 activity in a dose-dependent manner. Likewise, the addition of the membrane-permeable cAMP analogues, (Sp)-adenosine 3',5'-[thio]phosphate and N6,O-2'-dibutyryladenosine 3',5'-phosphate, enhance the release of phospholipase A2 activity from SMC in a dose-dependent manner. There is a lag period of about 4 h before a significant secretion of phospholipase A2 can be detected under basal, as well as under stimulated conditions. The forskolin analogue 1,9-dideoxyforskolin, which is inactive as a stimulator of adenylate cyclase, has no effect on phospholipase A2 secretion. Likewise, the potent vasoconstrictive peptide angiotensin II activates inositol phospholipid turnover in SMC, but has no effect on phospholipase A2 release. Pretreatment of SMC with actinomycin D or cycloheximide completely suppresses basal and cAMP-stimulated secretion of phospholipase A2 activity, thus demonstrating that transcription and protein synthesis are necessary for enzyme release.

Albuterol↗

PubMed1991

Phospholipase activity of Mycobacterium leprae harvested from experimentally infected armadillo tissue.

Three types of phospholipase activity--phospholipase A1, A2, and lysophospholipase--were detected in Mycobacterium leprae harvested from armadillo tissue at about 25% of the specific activity found in a slowly growing mycobacterium, Mycobacterium microti, which was grown in medium to optimize its phospholipase activity. The highest activity found was lysophospholipase, which released fatty acid from 2-lyso-phosphatidylcholine. Phospholipase activity was detected by using phosphatidylcholine and phosphatidylethanolamine. Differences in relative activities with these three types of substrate distinguished phospholipase activity in M. leprae extracts from armadillo liver extracts. Furthermore, retention of activity in M. leprae after NaOH treatment showed that the activity associated with M. leprae was not host derived. The specific activity of phospholipase was 20 times higher in extracts of M. leprae than in intact M. leprae organisms. Diazotization, a treatment which abolishes activities of surface enzymes exposed to the environment by the formation of covalent azide bonds with exposed amino groups, did not affect M. leprae's phospholipase activity, with one exception: release of arachidonic acid from phosphatidylcholine, which was partially inhibited. Phenolic glycolipid I, the major excreted amphipathic lipid of M. leprae, inhibited phospholipase activity, including release of arachidonic acid, for both M. leprae- and armadillo-derived activity.

Animals↗

PubMed1986

Effect of various blockers of arachidonic acid metabolism on release of beta-endorphin- and adrenocorticotropin-like immunoreactivity induced by phospholipase A2 from rat adenohypophysis in vitro.

Anterior pituitary quarters were incubated in vitro and the release of beta-endorphin-like (beta-End-IR) and adrenocorticotropin-like immunoreactivity (ACTH-IR) was determined. The effect of phospholipase A2 as well as the effect of various compounds known to influence arachidonic acid metabolism under certain conditions were examined. Phospholipase A2 increased the release of beta-End-IR and ACTH-IR. This effect was reversible, concentration-dependent (1-400 ng/ml) and inhibited in calcium-free medium and in the presence of CoCl2 (5 mM) or phospholipase A2 inhibitors (p-bromophenacylbromide, 21 microM; mepacrine, 1 mM). The phospholipase A2-induced beta-End-IR release was accompanied by the release of prostaglandin E2. Inhibition of cyclooxygenase activity by indomethacin (14 or 140 microM) did not change beta-End-IR release induced by phospholipase A2 (5 ng/ml). The effects of blockers of lipoxygenase (nordihydroguaiaretic acid, NDGA; AA861) or lipoxygenase plus cyclooxygenase (BW755C; eicosatetraynoic acid, ETYA) on phospholipase A2-induced release of beta-End-IR were diverse. BW755C (up to 250 microM) and AA861 (up to 100 microM) produced no effect. However, NDGA or ETYA inhibited phospholipase A2-induced beta-End-IR release. NDGA (100 microM) produced a maximum inhibition by about 40% (p less than 0.05), whereas ETYA (100 microM) produced a maximum inhibition by about 85% (p less than 0.001). These data are consistent with the view that phospholipase A2 releases endogenous arachidonic acid which is transformed into products which stimulate ACTH and beta-endorphin release from the corticotrophs; the metabolizing enzyme (possibly a lipoxygenase or epoxygenase) is sensitive to NDGA and especially to ETYA.

Acetophenones↗

PubMed1993

Tumor necrosis factor-alpha potentiates phospholipase A2-stimulated release and metabolism of arachidonic acid in cultured intestinal epithelial cells (INT 407).

Tumor necrosis factor-alpha (TNF-alpha), a known pro-inflammatory cytokine, has been suggested to play a role in the pathogenesis of inflammatory bowel disease (IBD) by mediating damage to the intestinal epithelial cells. The present study demonstrates that TNF-alpha potentiates release and metabolism of 14C-labeled arachidonic acid (14C-AA) in cultured intestinal epithelial cells (INT 407). Although TNF-alpha on its own was but a weak stimulator of cellular 14C-AA turnover, it significantly potentiated the release of 14C-AA and 14C-labeled prostaglandin E2(14C-PGE2) after stimulation with three known phospholipase A2 activators: phospholipase. C from Clostridium perfringens, the calcium ionophore A23187, and the phorbol ester 4-beta-phorbol-12-myristate-13-acetate (PMA). The phospholipase A2 inhibitor quinacrine significantly reduced both AA and PGE2 release after combined stimulation with phospholipase C and TNF-alpha. In contrast to its effect on the AA turnover, TNF-alpha did not affect the phospholipase C-stimulated production of platelet-activating factor (PAF-acether). Taken together, these findings indicate that a) TNF-alpha potentiates phospholipase A2-stimulated AA release from cultured intestinal epithelial cells; b) TNF-alpha may stimulate phospholipase A2-dependent AA release without affecting the formation of PAF-acether and c) pretreatment with TNF-alpha potentiates the formation of PGE2 after stimulation with phospholipase A2 activators. In summary, the present investigation points to the possibility that TNF-alpha may stimulate intestinal epithelial cells to produce biologically active AA metabolites and that this stimulation may be modulated by components of the intestinal luminal content, like bacterial toxins.

Arachidonic Acid↗

PubMed1990

A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7.

Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis.

Animals↗

PubMed1990

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture.

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.

Adipose Tissue↗

PubMed1985

Immunoreactive phospholipase A2 in serum in acute pancreatitis and pancreatic cancer.

Immunoreactive phospholipase A2 (EC 3.1.1.4) was measured by a new sensitive time-resolved fluoroimmunoassay in the serum of 58 healthy subjects and 103 patients with acute pancreatitis. Patients with acute pancreatitis were grouped according to the etiology and clinical severity of the disease. The mean phospholipase A2 concentration in the reference (healthy) group was 5.5 (SD 1.9) micrograms/L. In acute pancreatitis the mean phospholipase A2 concentration was increased on the first day after hospital admission in all groups, and returned to normal somewhat more slowly than did serum amylase, especially in the patients with severe alcoholic pancreatitis. In this latter group the mean concentration of serum phospholipase A2 on the first day was 42.6 (SD 29.5) micrograms/L. In patients with pancreatic cancer, serum phospholipase A2 was 29.2 (SD 21.3) micrograms/L. The phospholipase A2 and amylase values were closely associated in all groups. The clinical sensitivities were 90.9% for severe alcoholic pancreatitis and 87.5% for pancreatic cancer. Immunochemical determination of phospholipase A2 in serum provides fast and specific detection of injury to pancreatic acinar cells. In addition to the early diagnosis of acute pancreatitis, follow-up determinations of phospholipase A2 seem to be useful in differentiating between mild and severe forms of pancreatitis.

Acute Disease↗

PubMed1989

Immunoaffinity purification, partial sequence, and subcellular localization of rat liver phospholipase A2.

Monoclonal antibodies against rat liver mitochondrial phospholipase A2 were used to develop a rapid immunoaffinity chromatography for enzyme purification. The purified enzyme showed a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the N-terminal 24 amino acids was determined. This part of the sequence showed only 25% homology with that of rat pancreatic phospholipase A2 but was 96% identical to that of rat platelet and rat spleen membrane-associated phospholipase A2. These enzymes are distinguished from pancreatic phospholipases A2 by the absence of Cys-11. In rat liver phospholipase A2 activity has been reported in various subcellular fractions. All of these require Ca2+ and have a pH optimum in the alkaline region, but little is known about the structural relationship and quantitative distribution of these enzymes. We have investigated these points after solubilization of the phospholipase A2 activity from total homogenates and crude subcellular fractions by extraction with 1 M potassium chloride. Essentially all of the homogenate activity could be solubilized by this procedure indicating that the enzymes occurred in soluble or peripherally membrane-associated form. Gel filtration and immunological cross-reactivity studies indicated that phospholipases A2 solubilized from membrane fractions shared a common epitope with the mitochondrial enzyme. The quantitative distribution of the immunopurified enzyme activity among subcellular fractions followed closely that of the mitochondrial marker cytochrome c oxidase. Rat liver cytosol contained additional Ca2+-dependent and -independent phospholipase activities.

Amino Acid Sequence↗

PubMed1987

Inhibition of phospholipase A2 by "lipocortins" and calpactins. An effect of binding to substrate phospholipids.

The "lipocortins" are a group of proteins that have been reported to inhibit phospholipase A2 by direct interaction with enzyme. Two proteins which have been identified as lipocortin on the basis of inhibition of phospholipase A2 activity have recently been cloned and sequenced. These have been shown to be identical to the calpactins, which are membrane cytoskeletal proteins serving as major substrates of the tyrosine protein kinases. We have now found that two forms of calpactin (I and II) inhibit porcine pancreatic phospholipase A2 in an assay using Escherichia coli cells or extracted phospholipid vesicles as substrate, but only when the substrate concentration is very low. Both calpactins, as well as another, 73-kDa inhibitory protein, were found to bind purified phospholipids and E. coli cell membranes directly. Kinetic studies show that the inhibition of phospholipase A2 by calpactin can be overcome by high phospholipid substrate concentrations, whether E. coli cells or isolated phospholipid vesicles are used. For example, in the presence of 5 X 10(-10) M phospholipase A2 and 1 X 10(-7) M calpactin, the inhibition decreases from 100 to 0% as phospholipid in vesicles is raised from 2 to 8 microM. The evidence reported here strongly suggests that in vitro inhibition of phospholipase A2 by lipocortin is due to sequestering of the phospholipid substrate by the inhibitor protein, rather than a direct interaction with the phospholipase. These results raise questions about the physiological significance of the inhibition of phospholipases by calpactins.

Animals↗

PubMed1986

Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets.

Data in the previous paper suggest that epinephrine can mobilize a small pool of arachidonic acid via an enzymatic pathway distinct from phospholipase C and that this pathway is blocked by perturbations that block Na+/H+ exchange. The present studies demonstrate that epinephrine and ADP stimulate a phosphatidylinositol-hydrolyzing phospholipase A2 activity in human platelets. This occurs even when measurable phospholipase C activation, platelet secretion, and secondary aggregation are blocked with the thromboxane A2 receptor antagonist SQ29548. Furthermore, perturbants of Na+/H+ exchange diminish lysophosphatidylinositol production in response to epinephrine, ADP, and thrombin, but not to the Ca2+ ionophore A23187. Artificial alkalinization of the platelet interior with methylamine reverses the effect of the Na+/H+ antiporter inhibitor, ethylisopropylamiloride, on thrombin-stimulated lysolipid production, suggesting that the alkalinization of the platelet interior which would occur secondary to activation of Na+/H+ exchange might play an important role in phospholipase A2 activation. In addition, treatment of platelets with methylamine increases the sensitivity of phospholipase A2 to activation by the Ca2+ ionophore A23187, suggesting that changes in pH and Ca2+ may regulate phospholipase A2 activity synergistically. Finally, epinephrine causes a prompt decrease in platelet-chlortetracyclin fluorescence even in the presence of cyclooxygenase inhibitors, suggesting that epinephrine is able to mobilize membrane-bound Ca2+ independent of phospholipase C activation. Taken together, the data suggest that epinephrine-provoked stimulation of phospholipase A2 activity may occur as a result of Ca2+ mobilization and a concomitant intraplatelet alkalinization resulting from accelerated Na+/H+ exchange.

Adenosine Diphosphate↗

PubMed1988

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line.

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.

Acetonitriles↗

PubMed1987

Effect of prolonged physical exercise on muscular phospholipase A2 activity in rats.

Prolonged muscular exercise stimulates glucose uptake by the working muscles themselves. The mechanism of this phenomenon is at present unclear. It has been proposed that the kallikrein-kinin-prostaglandin system plays a role in the physiological regulation of muscular glucose metabolism during exercise. Since bradykinin can stimulate phospholipase A2, a key enzymatic step in prostaglandin synthesis, phospholipase A2 activity was assayed in rats at rest and in rats compelled to swim for 60 minutes. The physiological significance of an increase in muscular phospholipase A2 activity is not clear. Since bradykinin can stimulate both muscular glucose uptake and phospholipase A2 activity, it is possible that the increased activity of this enzyme is involved in the exercise-induced increase of muscular glucose uptake. Phospholipase A2 activity was strongly increased in the exercising rat muscles. A small but significant increase in phospholipase A2 activity was observed in the heart, whereas no variation in activity was demonstrated in either the kidney or the liver of exercising rats. These findings strongly indicate that prolonged exercise increases muscular phospholipase A2 activity only in the muscle and heart. This phenomenon appears to be strongly related to muscular contraction, since other stress situations such as cold exposure did not modify phospholipase A2 activity. Our data are in agreement with the hypothesis of a possible involvement of prostaglandins in the priming action of insulin on glucose uptake during muscular work.

Animals↗

PubMed1983

The interaction of dialkyl ether lecithins with phospholipase A2 (Naja naja naja).

Dialkyl lecithins are nonhydrolyzable substrate analogues for phospholipase A2 (Naja naja naja). Short chain dialkyl lecithins (which form monomers and micelles), and long chain ether-linked lecithins (which form bilayers or can be solubilized in detergent micelles) have been used to study phospholipase A2/phospholipid interactions. The results of gel filtration, 1H and 31P NMR spectroscopy, kinetic assays and UV difference spectroscopy show that binding of the ether-linked lecithins is independent of metal ion in pure lecithin systems (monomer, micelle, or bilayer), but requires Ca2+ for tight binding when Triton X-100 is used to solubilize the lecithin in mixed micelles. The apparent KD for dihexadecyl phosphatidylcholine is 2 mM in sonicated vesicles, but is increased to 6 mM in Triton-mixed micelles in the presence of Ca2+. When phospholipase A2 binds monomeric and micellar dialkyl lecithin, the lecithin phosphate resonance is broadened considerably, indicating that head group motion is restricted. Ca2+ enhances this interaction with the head group in these pure lipid systems. In similar 31P NMR experiments with dihexadecyl phosphatidylcholine solubilized in Triton X-100 mixed micelles, phospholipase A2 does not cause comparable broadening of the phosphate resonance, even in the presence of Ca2+ where gel filtration shows binding occurs. The kinetic effect of the ether-linked lipid inhibitors on phospholipase activity toward ester-linked lecithins is also modulated by the presence of Triton X-100. Ether-linked lecithins are much more potent inhibitors in two-component (dialkyl- diacyl-) lecithin micelles and bilayers than in complex mixed micelles with Triton X-100. Detergent-induced changes in the characteristics of the micellar interface are implicated in this inhibition phenomenon. The interaction of phospholipase A2 with sonicated vesicles of dihexadecyl phosphatidylcholine has also been examined by 1H and 31P NMR spectroscopy. When phospholipase A2 binds to vesicles, it promotes loss of encapsulated lanthanide ions, and appears to accelerate transbilayer phospholipid "flip-flop" rates. These results are discussed in terms of membrane asymmetry studies using phospholipase A2.

Calcium↗

PubMed1982

Phospholipase A2-induced rat mast cell secretion. Role of arachidonic acid metabolites.

Highly purified porcine phospholipase A2 induced noncytotoxic rat cell degranulation, as indicated by release of histamine without release of the cytoplasmic marker lactate dehydrogenase. Ultrastructural studies using transmission, scanning, and freeze fracture electron microscopic techniques indicated that phospholipase A2-induced degranulation was comparable to that caused by other mast cell secretagogues. Secretory changes noted were fusion of perigranular and plasma membranes, formation of vacuoles containing less electron-dense granules, and exocytosis of the altered granules through pores in the plasma membrane, without alteration in other intracellular organelles. The earliest consistent feature of the exocytotic process (within 1 minute) was the formation of plasma membrane bulges overlying cytoplasmic granules, with depletion of intramembranous particles from the bulges and a reduction in surface microridges. Phospholipase A2-induced mast cell degranulation was blocked by the phospholipase inhibitor 4-bromophenacyl bromide and by eicosa-5,8,11,14-tetraynoic acid (ETYA) but not by indomethacin. Since ETYA inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism and indomethacin only the cyclooxygenase pathway, these findings are compatible with the mediation of phospholipase A2-induced mast cell degranulation by a lipoxygenase product of the released arachidonic acid ETYA, however, may inhibit phospholipase activity directly and thus affect degranulation by phospholipase A2 in this way. These studies indicate that phospholipase A2 can induce mast cell degranulation and provides evidence that is compatible with, but not proof of, mediation of this process by a lipoxygenase product of arachidonic acid metabolism.

Animals↗

PubMed1993

Reconstitution of thromboxane A2 receptor-stimulated phosphoinositide hydrolysis in isolated platelet membranes: involvement of phosphoinositide-specific phospholipase C-beta and GTP-binding protein Gq.

Activation of human platelets by the arachidonic acid metabolite thromboxane A2 and the thromboxane A2 mimic U46619 is mediated through phosphoinositide-specific phospholipase C-catalysed hydrolysis of phosphoinositides. We have established conditions to reconstitute U46619-stimulated phosphoinositide breakdown by addition of guanine nucleotides and soluble platelet phospholipase C activities to isolated 32P-labelled membranes. Receptor-activated phosphoinositide hydrolysis was observed in the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[S]) or GTP plus U46619. Phosphoinositide hydrolysis was dependent on both GTP and U46619, with half-maximal stimulation observed at 5 microM and 500 nM respectively. Phospholipase C isoenzymes beta, gamma 1, gamma 2 and delta were purified from platelet cytosol and their ability to reconstitute GTP[S]-dependent and GTP/U46619-dependent phosphoinositide hydrolysis determined. Phospholipase C-beta and -delta, but not phospholipase C-gamma 1 or -gamma 2, catalysed phosphoinositide breakdown in the presence of GTP[S]. In contrast, only phospholipase C-beta was able to reconstitute GTP-dependent U46619-induced hydrolysis. The participation of GTP-regulatory proteins in the reconstitution of GTP[S]- and GTP/U46619-induced phosphoinositide hydrolysis was examined using antibodies to the C-terminals of the alpha-subunits of three of the heterotrimeric GTP-binding proteins expressed in human platelets Gq, Gi2 and Gi3. Anti-Gq antibody, but not anti-Gi2 or Gi3 antibody, inhibited both GTP[S]- and GTP/U46619-dependent reconstitution of phosphoinositide hydrolysis with phospholipase C-beta. In contrast GTP[S]-stimulated hydrolysis by phospholipase C-delta was not inhibited by any of the G-protein antibodies. These results show the functional specificity of GTP-binding proteins and phospholipase C isoenzymes in mediating agonist-induced phosphoinositide hydrolysis in human platelets.

Blood Platelets↗