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PubMed1986

Solubilization and properties of Ca2+-dependent human platelet phospholipase A2.

Using a sonicated dispersion of radiolabeled 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine as substrate, we found that phospholipase A2 activity of human platelets was enhanced 2.4-fold by albumin (1 mg/ml). The enzyme was recovered predominantly in the cytosolic fraction of platelets with less than a third of its activity being associated with the membrane fraction. In the presence of 24 mM n-octyl-beta-D-glucopyranoside (octylglucoside) phospholipase A2 was effectively (more than 90%) extracted from platelet lysates without solubilization of platelet membranes. Ion exchange chromatography of the soluble enzyme yielded a phospholipase A2 of unchanged total activity and great stability. This phospholipase A2 was active only in the presence of divalent cations (Ca2+ greater than Sr2+ greater than Mg2+ = 0), required albumin for optimal activity and exhibited exclusive positional specificity for the acyl ester bond at the 2-position of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine. Indomethacin (500 microM), mepacrine (500 microM) and N-ethylmaleimide (4 mM) inhibited the phospholipase A2 by 69, 62 and 19%, respectively. The results are discussed in the light of previous findings on human platelet phospholipase A2.

Albumins↗

PubMed1987

Modulation by cytosol and commercial proteins of acid-active phospholipase A2 from adrenal medulla.

Expression of soluble, acid-active phospholipase A2 from a granule-enriched fraction of bovine adrenal medulla is modulated by adrenal medullary cytosol in an ionic strength- and pH-dependent manner. At 150 nM Na+, cytosol is inhibitory in a dose-dependent manner while at 14 and 50 mM Na+ inhibition is preceded by stimulation. The extent of inhibition increases with increasing Na+ while the extent of stimulation increases with decreasing Na+. Effects are seen primarily below pH 5. Bovine liver cytosol behaves similarly, and soluble, acid-active phospholipases A2 from bovine and rat liver are also modulated by cytosol. The predominant regulatory component has a molecular weight of 67 000 as determined by gel permeation chromatography. Serum albumin and other commercially available proteins affect expression of acid-active phospholipase A2 in a manner similar to cytosol. Acid-active phospholipase A2 activity is also present in cytosol, it is enhanced substantially upon precipitation of cytosolic proteins, and is subsequently suppressed by added protein. Modulatory effects of cytosol result from its interaction with substrate, not enzyme, but are not due to dilution of substrate by cytosolic phospholipids. The study demonstrates that proteins modulate expression of acid-active phospholipases A2 by interacting with substrate in an ionic strength- and pH-dependent fashion. Such interactions may be important in the in vivo regulation of acid-active phospholipases A2.

Adrenal Medulla↗

PubMed1988

In vitro inhibition of lysosomal phospholipase A1 of rat lung by amiodarone and desethylamiodarone.

Amiodarone causes phospholipid storage in the lysosomes of various types of lung cell in animals and man. It has been proposed that this is due to its ability to inhibit lysosomal phospholipase A. To investigate this further, a crude lysosomal fraction from rat lung was prepared and phospholipase A was isolated and its positional specificity was determined. Analysis of the products formed after incubation with 2-[1-14C]oleoylphosphatidylcholine showed that only phospholipase A1 activity is present. This soluble preparation of lung lysosomal phospholipase A1 was used to study inhibition by amiodarone and desethylamiodarone, in vitro. Both were extremely potent inhibitors of the lung acid phospholipase A1. To evaluate the levels of amiodarone in lung lysosomes, rats were treated with the agent for 3 days and the combined mitochondrial/lysosomal fraction of lung tissue was prepared by differential centrifugation. This fraction had been shown previously to be highly enriched in amiodarone. Purified mitochondria and lysosomes were isolated from the combined mitochondrial/lysosomal fraction with Percoll gradients and analyzed for their drug content by HPLC. Amiodarone and desethylamiodarone were present in roughly equal amounts, relative to protein, in mitochondria and lysosomes, respectively. Amiodarone appears to differ from other cationic amphiphilic drugs which cause lipidosis because the latter are more highly lysosomotropic. Although amiodarone does not appear to be highly lysosomotropic in lung, it causes lysosomal phospholipid storage because of its ability to concentrate in lung and because it inhibits lysosomal phospholipase A to a much greater extent than other cationic amphiphiles such as diethylaminoethoxyhexestrol, chloroquine and chlorphentermine.

Amiodarone↗

PubMed1988

Calcium-independent phospholipase A2 in rat tissue cytosols.

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.

Animals↗

PubMed1989

Oligomers of prostaglandin B1 inhibit in vitro phospholipase A2 activity.

Oligomers of prostaglandin B1 inhibited phospholipase A2 extracted from human neutrophils in a dose-dependent manner (IC50 = 5 microM), while the monomer was not inhibitory at concentrations of 10 microM or less. The inhibitory activity of PGB1 oligomers increased with increasing polymer size; PGB dimer had approximately one-half the maximal inhibitory activity of PGBx, while a trimer was almost as inhibitory as a tetramer and PGBx (n = 6). PGBx as an oil or as a water-soluble sodium-salt-inhibited Ca2(+)-dependent phospholipase A2 from snake venom, bovine pancreas, human neutrophil and platelet, human synovial fluid, and human sperm with IC50 values ranging from 0.5-7.5 microM. Inhibition was independent of added Ca2+ and was independent of substrate phospholipid concentration. Interaction of purified snake venom phospholipase A2 (Naja mocambique) with PGBx resulted in dose-dependent quenching of the enzyme's tryptophan fluorescence; 50% quench was noted with a molar ratio of PGBx/enzyme of 1.5. Inhibition of phospholipase A2 activity by PGBx was relieved in a dose-dependent manner by either defatted or untreated bovine serum albumin. PGBx is a potent in vitro inhibitor of a wide spectrum of phospholipases A2, and as illustrated in the accompanying paper, has profound inhibitory effects on arachidonic acid mobilization in human neutrophils and vascular endothelial cells. Modulation of cellular and extracellular phospholipases A2, and the bioactive transmitters generated by this catalytic event, may be a basic mechanism by which oligomers of prostaglandin B1 exert their reported membrane-protective effects.

Blood Platelets↗

PubMed1990

Studies on the acyl-chain selectivity of cellular phospholipases A2.

The selective release of arachidonate, as opposed to monoenoic and dienoic fatty acids, after stimulation of cells has suggested the involvement of arachidonate-selective phospholipases A2. Supportive evidence for the existence of such enzymes has also come from in vitro experiments. We have studied the acyl-chain selectivity of phospholipase A2 preparations obtained from human polymorphonuclear leukocytes, human platelets and rat platelets using sn-2-[14C]oleoylphosphatidylcholine and sn-2-[3H]arachidonoylphosphatidylcholine either as single substrates or in doubly labeled mixtures. In either case, no evidence for acyl-chain selectivity was observed for human PMN and rat platelet phospholipase A2. Additional experiments with human PMN homogenates and derived extracts yielded no indication for the selective loss of an arachidonate-selective phospholipase A2. Results with human platelet cytosol were highly suggestive for the presence of an arachidonoyl-selective phospholipase A2 when separate phosphatidylcholine species were assayed. This apparent selectivity was progressively lost when the substrates were mixed or embedded in a membrane of 1-palmitoyl-2-linoleoylphosphatidylcholine. The implications for occurrence of arachidonate-selective phospholipase A2 are discussed.

Animals↗

PubMed1983

Suppression by phospholipase A2 inhibitors of secretion of catecholamines from isolated adrenal medullary cells by suppression of cellular calcium uptake.

The involvement of phospholipase A2 in the secretion of catecholamines and cellular uptake of 45Ca2+ was investigated in isolated bovine adrenal medullary cells. In these cells, stimulation of cholinergic receptors by carbamylcholine causes the activation of receptor-linked Ca-channels and influx of Ca2+ is known to trigger the secretory process. Phospholipase A2 inhibitors, such as quinacrine, chloroquine, quinine and p-bromophenacyl bromide, all inhibited the secretion of catecholamines evoked by carbamylcholine in a dose-dependent manner. These phospholipase A2 inhibitors also inhibited the cellular uptake of 45Ca2+ evoked by carbamylcholine with similar dose-response curves to those for inhibition of catecholamine secretion. The inhibition by phospholipase A2 inhibitors was found to be distinct from inhibition by d-tubocurarine which competitively blocks acetylcholine receptors, and from inhibition by diltiazem which acts as a Ca-antagonist at Ca-channels. Phospholipase A2 inhibitors seem to suppress the secretion of catecholamines by interfering with the linkage between acetylcholine receptors and Ca-channels by the membrane effects including the inhibition of endogenous phospholipase A2 activity of the adrenal medullary cells.

Acetophenones↗

PubMed1989

Synthetic peptide from lipocortin I has no phospholipase A2 inhibitory activity.

Two anti-inflammatory peptides corresponding to a high amino acid similarity region between lipocortins were synthesized and tested on their ability to inhibit porcine pancreatic phospholipase A2. Kinetic assays using monomeric and aggregated phospholipids did not reveal any phospholipase A2 inhibitory activity. The peptides did not inhibit phospholipase A2 activity on monolayers of negatively charged substrate and did not prevent phospholipase A2 action on mixed micelles of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine and sodiumdeoxycholate. Ultraviolet difference spectroscopy did not show binding of the peptides to phospholipase A2. Therefore we conclude that these anti-inflammatory peptides do not inhibit pancreatic phospholipase A2 in vitro, in contrast to the results recently published [(1988) Nature 335, 726-730].

Amino Acid Sequence↗

PubMed1988

Crystal structure of bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide.

Bovine pancreatic phospholipase A2 covalently inhibited by p-bromo-phenacyl-bromide was crystallized from 50% (v/v) 2-methyl-2,4-pentanediol. The space group was P3(1)21 with cell dimensions a = b = 46.73 A and c = 102.5 A (1 A = 0.1 nm). Diffraction data were collected by oscillation photography from one single crystal of dimensions 0.2 mm x 0.2 mm x 0.2 mm. The crystal structure was determined to a resolution of 2.5 A by crystallographic refinement of a starting model, which consisted of native bovine pancreatic phospholipase A2 positioned and oriented in the P3(1)21 cell as in the bovine pro-phospholipase A2. The crystallographic R-factor decreased from 0.378 to 0.197 after 70 refinement cycles. For the greater part the three-dimensional structure was very similar to that of native phospholipase. The inhibitor group shows up clearly. However, as in solution, there is no calcium ion bound any more in the active site, and this causes a significant conformational change in the loop from residue 59 to 73. This loop is remote from the calcium binding site. Interestingly, this is the same loop that also shows different conformations in other phospholipase A2 molecules. The inhibitor molecule has hydrophobic interactions with Phe5 and Cys45. Rational design of specific and potent inhibitors of phospholipase A2 catalysis is discussed on the basis of the present three-dimensional structure.

Acetophenones↗

PubMed1983

The role of phospholipase C in platelet responses.

Degradation of inositides induced by phospholipase C in activated platelets leads to the formation of 1,2-diacylglycerol (1,2-DG) and its phosphorylated product, phosphatidic acid (PA). We have studied the relationship between activation of phospholipase C and the appearance of specific platelet responses, such as phosphorylation of proteins, shape change, release reaction and aggregation induced by different stimuli such as thrombin, platelet-activating factor, collagen, arachidonic acid (AA) and dihomogamma linolenic acid. A low degree of platelet activation induces only shape change which is associated with partial activation of phospholipase C (formation of phosphatidic acid), and phosphorylation of both a 40K molecular weight protein (protein kinase C activation) and a 20K molecular weight protein (myosin light chain). A higher degree of platelet activation induces aggregation, release of serotonin and a higher level of phospholipase C and protein kinase C activities. Metabolism of AA occurs concomitantly to aggregation and serotonin release, but AA metabolites are not related to the shape change of human platelets. Platelet shape change and the initial activation of phospholipase C induced by thrombin or platelet-activating factor is independent of the metabolites derived from cyclo-oxygenase activity. Further activation of phospholipase C which occurs during platelet aggregation and release reaction is, however, partly dependent on cyclo-oxygenase metabolites.

Arachidonic Acid↗

PubMed1983

Change in membrane lipid fluidity induced by phospholipase A activation: a mechanism of endotoxic shock.

The effects of endotoxin administration on the fluidity of dog liver plasma membranes and their relationship with changes in phospholipase A2 activity were studied. Endotoxin administration decreased the fluidity of liver plasma membranes and this decrease was reversible by phosphatidylcholine. The endotoxin-induced decrease in membrane fluidity could be mimicked by digesting control liver membranes with exogenous phospholipase A2. Endotoxin administration also increased the endogenous phospholipase A2 activity. Endotoxin in vitro had no phospholipase A2-like activity but it activated the hydrolytic activity of exogenous phospholipase A2. Based on these data, it is concluded that endotoxin administration decreased the fluidity of canine liver plasma membranes by acting through activation of phospholipase A2. The decrease in membrane lipid fluidity induced by endotoxin administration may play a significant role in the development of the pathophysiology of endotoxic shock at the cellular level.

Animals↗

PubMed1986

Cardiotoxicity of Naja nigricollis phospholipase A2 is not due to alterations in prostaglandin synthesis.

The basic phospholipase A2 from Naja nigricollis snake venom is cardiotoxic, causing decreased contractility and arrhythmias at concentrations which induce low levels of phospholipid hydrolysis. Cardiac tissue has a high content of arachidonic acid at the sn-2 position of the major membrane phospholipids, thus increased prostaglandin synthesis might contribute to the cardiotoxic effects of N. nigricollis phospholipase A2. Intracellular action potentials and cardiac contractility were monitored in the isolated right ventricular wall of the rat heart exposed for 1 hr to N. nigricollis phospholipase A2, with or without indomethacin, or to arachidonic acid. The tissues were homogenized, prostaglandins extracted and the 6-keto PGF1 alpha and PGE2 content of the hearts determined. The physiologic effects and prostaglandin content of hearts treated with N. nigricollis phospholipase A2 were not altered by indomethacin nor mimicked by concentrations of arachidonic acid comparable to that present in N. nigricollis phospholipase A2-treated tissue. These results support our previous suggestion that exogenously applied N. nigricollis phospholipase A2 causes cardiotoxic effects by a mechanism that is independent of phospholipid hydrolysis.

Animals↗

PubMed1986

Effects of beta-bungarotoxin and Naja naja atra snake venom phospholipase A2 on acetylcholine release and choline uptake in synaptosomes.

Beta-bungarotoxin is a potent presynaptically acting snake venom toxin that exhibits phospholipase A2 activity. We compared the effects of beta-bungarotoxin and a less toxic snake venom phospholipase A2 on synaptosomal 3H-acetylcholine release and 3H-choline uptake. The purpose of these experiments was to study the mode by which beta-bungarotoxin inhibits 3H-acetylcholine release in this preparation. Under non-depolarizing conditions, both beta-bungarotoxin and Naja naja atra phospholipase A2 stimulated 3H-acetylcholine release from a synaptosomal fraction preloaded with 3H-choline. Beta-bungarotoxin was more potent, but less efficacious, than N. naja atra phospholipase A2. In contrast, both toxins inhibited 3H-acetylcholine release from the synaptosomal fraction incubated with 3H-choline after toxin exposure. In agreement with the results obtained by monitoring acetylcholine release, beta-bungarotoxin and N. naja atra phospholipase A2 appeared to block 3H-choline uptake into the synaptosomal fraction non-competitively. Although the toxins may cause the release of unlabeled choline from synaptosomes, the block of labeled choline uptake could not be explained by decreased specific activity of 3H-choline in the bathing medium. Therefore, beta-bungarotoxin and N. naja atra phospholipase A2 block 3H-acetylcholine release from synaptosomes indirectly by inhibiting the uptake of 3H-choline necessary for 3H-acetylcholine synthesis. In comparing these results using 3H-choline to those in the literature obtained with deuterated choline, there appears to be a difference in apparent toxin action that relates to the type of label (3H or 2H) attached to choline.

Acetylcholine↗

PubMed1991

Structure-function relationships among neurotoxic phospholipases: NN-XIII-PLA2 from Indian cobra (Naja naja naja) and VRV PL-V from Russell's viper (Vipera russelli) venoms.

Though venom phospholipases induce various pharmacological effects their mechanism of action is in some cases unclear. There may be separate pharmacological sites on the venom phospholipase molecule. In order to understand the structure-function relationships among venom phospholipases, studies on interaction of venom phospholipases with its antibodies and various alkaloids were carried out. The alkaloids aristolochic acid, ajmaline and reserpine were incapable of inhibiting the phospholipase A2 activity of NN-XIII-PLA2 but inhibited its edema inducing potency and partially inhibited the symptoms of neurotoxicity. The direct and indirect hemolytic activity remain unaffected. Polyclonal antibodies (anti PL-V Ig) to a neurotoxic PLA2 VRV PL-V neutralized the neurotoxic symptoms and lethality of VRV PL-V without affecting its in vitro phospholipase A2 activity when egg PC was used as the substrate. However, they inhibited the catalytic activity of VRV PL-V when synaptosomes were used as the substrate. Our results indicate the presence of multiple pharmacologically active sites apart from catalytic site.

Alkaloids↗

PubMed1982

The phospholipases of Trypanosoma brucei bloodstream forms and cultured procyclics.

Phospholipase from Trypanosoma brucei bloodstream forms was characterized and subsequently localized. The enzyme had a specific activity of 100 nmol . min-1 . mg-1 protein. The major portion (greater than 90%) was a soluble phospholipase A1 with a pH optimum around 6; the remainder, also phospholipase A1, was particle-bound and had an optimal activity around pH 5.2. Both enzymes were maximally activated by 0.2% Triton X-100 but differed in their sensitivity towards the inhibitory action of higher concentrations of this detergent and diisopropyl fluorophosphate, the particle-bound activity being more sensitive than the soluble one. Cell fractionation showed that the particle-bound, more acidic phospholipase A1 was associated with alpha-mannosidase- and acid proteinase-containing lysosomes. Cultured procyclic trypomastigotes also contained phospholipase A but its specific activity was only 15% of that of bloodstream forms. This drastic reduction in overall activity upon transformation from bloodstream to culture form was the result of a decrease in soluble phospholipase, whereas the lysosomal activity essentially remained unchanged.

Animals↗

PubMed1988

Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo.

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A2 enzyme species whose activity is regulated by Na+/H+ exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660-8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197-9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na+/H+ antiporter enables the phospholipase A2 enzyme to function at ambient or low concentrations of intraplatelet Ca2+. To test the hypothesis that the Ca2+ dependence of platelet phospholipase A2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca2+ dependence of this enzyme as a function of changes in pH (from pH 6.8-8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca2+ (EC50 1-2 microM) and plateaued above 10 microM Ca2+. Enzymatic activity measured at 4.8 microM Ca2+ was increased by raising the pH from 5.5 to 8.0 (EC50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca2+ concentrations. The interdependent regulation of phospholipase A2 activity by changes in pH and Ca2+ suggests that phospholipase A2 could serve to integrate changes in intracellular pH and available Ca2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.

Binding, Competitive↗

PubMed1989

Phospholipase C-mediated intestinal mucosal damage is ameliorated by quinacrine.

Phospholipase C from Clostridium perfringens, when injected into a closed loop of the rat small intestine in vivo, caused an increase in the activity of intraluminal N-acetyl-beta-glucosaminidase and mucosal permeability to sodium fluorescein, indicating damage to the mucosa. Phospholipase C also caused an influx of granulocytes (neutrophils) into the mucosa, as shown by the myeloperoxidase activity--a granulocyte neutrophil marker, and increased localized lipid peroxidation. Pretreatment of animals with quinacrine, a known inhibitor of phospholipase A2, prevented the increases in the luminal N-acetyl-beta-glucosaminidase activity, mucosal permeability, malondialdehyde and myeloperoxidase activity after deposition of phospholipase C in the gut lumen. It is concluded that phospholipase C might impair the function of the mucosal barrier and increase the permeability of the gut to undesirable molecules and pathogens. Part of its action may be mediated via phospholipase A2 activation since pretreatment with quinacrine afforded protection.

Animals↗

PubMed1985

Phospholipase A2 dependent release of fatty acids from peroxidized membranes.

Unilamellar vesicles (liposomes) consisting of liver phosphatidylcholine and phosphatidylethanolamine were used as model membranes and subjected to lipid peroxidation. Following peroxidation samples were treated with phospholipase A2 from snake venom and subsequently analyzed for products of lipid peroxidation and of phospholipase A2 action. A significantly increased susceptibility to phospholipase A2 was noted for liposomes subjected to peroxidation, as compared to controls, which correlated with the extent of lipid peroxidation measured by the formation of thiobarbituric acid reacting products and conjugated dienes. Low levels of peroxides were detected in control liposomes and these peroxidases were rapidly cleaved by phospholipase A2, such that nearly 40% of the total peroxide content was associated with free fatty acids after l5 min incubation. Oxidized liposomes contained over seven fold the level of lipid peroxidation products, and the were also rapidly cleaved by phospholipase A2 where over 50% were recovered as free fatty acids following l5 min of exposure to phospholipase A2. Along with this high order of removal of oxidized fatty acids, a marked hydrolysis of intact fatty acids was also observed. The extent of fatty acid release was roughly correlated with the degree of fatty acid unsaturation. A substantial increase in the release of arachidonic acid was found when peroxidized membranes were analyzed.

Fatty Acids↗