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PubMed1986

Contamination of commercial preparations of xanthine oxidase by a Ca2+-dependent phospholipase A2.

Using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate, we demonstrate that many, but not all, commercial preparations of xanthine oxidase contain phospholipase A2 activity as a contaminant. Phospholipase A2 activity (64.3-545.6 nmol phospholipid hydrolyzed per min per mg protein) was optimal in the neutral to alkaline pH range, was Ca2+-dependent, and was unaffected by the addition of xanthine. Phospholipase A2 activity was totally inhibited by 1.0 mM EDTA while radical production by xanthine plus xanthine oxidase was unaffected by EDTA. Even chromatographically purified xanthine oxidase (Sigma Grade III) contained substantial phospholipase A2 activity (64.3 nmol/min per mg). Since the preparation of xanthine oxidase employs proteolytic digestion of milk or buttermilk by pancreatin, an extract of pancreas which is an organ rich in phospholipase A2 activity, we speculate that the contaminant phospholipase A2 is introduced by this treatment. Because xanthine oxidase is used extensively to study free radical-induced cell injury and membrane phospholipid alterations, the presence of a potent extracellular phospholipase A2 may have influenced previously published reports and such studies in the future should be interpreted with care.

Calcium↗

PubMed1985

Some aspects of rat platelet and serum phospholipase A2 activities.

Rat platelet lysate contained appreciable phospholipase A2 activity. In agreement with literature data this enzymatic activity eluted in the void volume of a Sephadex G-100 column. When the void volume peak was chromatographed over a Matrex gel blue A column, part of the phospholipase A2 activity ran through, whereas the remainder was bound to the gel. The latter activity could be eluted with buffers containing a high salt concentration. In contrast, phospholipase A2 activity solubilized from rat platelet lysates by treatment with high salt eluted from Sephadex G-100 columns with an apparent molecular weight of 10-15 kDa. This solubilized enzyme completely bound to Matrex gel blue A and, in the presence of Ca2+ also to an alkylphosphocholine-AH Sepharose affinity column. No indications were obtained for the presence of inactive phospholipase A2 and activator proteins in rat platelet lysates as described by Etienne, J., Grüber, A. and Polonovski, J. ((1980) Biochim. Biophys. Acta 619, 693-698; (1982) Biochemie 64, 377-380). Phospholipase A2 activity, both the associated form in platelet lysate and the monomeric form as eluted from Sephadex G-100 was slightly inhibited by trifluoperazine but calmodulin exerted no stimulation. Likewise, phospholipase A2 activity from rat serum eluted in the void volume of a Sephadex G-100 column. Rather than indicating the presence of high molecular weight forms of the enzyme, this is apparently caused by association with lipids or other proteins, in that chromatography in the presence of high salt revealed a molecular weight similar to that found for solubilized platelet phospholipase A2 activity.

Animals↗

PubMed1986

Vitamin E inhibits platelet phospholipase A2.

One of the most important functions of phospholipase A2 is the release of arachidonic acid from membrane phospholipids for the synthesis of biologically active eicosanoids. We have demonstrated in our laboratory that vitamin E inhibits platelet phospholipase A2 in a dose-dependent manner. Rats fed a 100 ppm or a 1000 ppm vitamin E diet exhibit diminished phospholipase A2 activity compared to those fed a vitamin E-free diet. Addition of vitamin E to a sonicated platelet suspension resulted in further suppression of the phospholipase A2 activity in all groups of rats. In order to gain insight into the mechanism of vitamin E inhibition of platelet phospholipase A2, we partially purified this enzyme by gel filtration chromatography. Enzyme activity was localized in the soluble supernatant fraction of a high-speed spin. This partially purified rat platelet phospholipase A2 had an absolute requirement for Ca2+ and was inhibited by various forms of tocopherol. Tocol inhibited the enzyme to a greater extent than either D- or DL-alpha-tocopherol, while there was little or no effect from DL-alpha-tocopherol acetate. These results emphasize the importance of the hydroxyl moiety on the chromanol of the vitamin E molecule for its inhibitory action, compared to that of the methyl groups which are absent in tocol. This inhibitory action of vitamin E on platelet phospholipase A2 suggests a crucial function for vitamin E in regulating arachidonate release from the membrane phospholipids and its subsequent metabolism.

Animals↗

PubMed1986

Partial characterization of rat heart cytosolic phospholipase A1 and demonstration of essential sulfhydryl groups.

The cytosol of rat heart has been previously shown to contain phospholipase A activity in substantial amounts. This paper describes the isolation and partial purification of rat heart cytosolic phospholipase A. After homogenization of rat heart followed by centrifugation to remove membraneous material, the phospholipase A activity was isolated by ammonium sulfate precipitation and further purified by gel permeation chromatography with Sephadex G-100 in the presence of 5 mM taurodeoxycholate. Two peaks were isolated: a minor peak at the void volume and major peak corresponding to a molecular weight of 45,000. The molecular weight observed in HPLC gel permeation chromatography experiments was also Mr 45,000 and was not significantly affected by the nature of the detergent used. Phospholipase A was purified 77-fold over the crude cytosol. Further purification could not be attained due to lability of the phospholipase A activity. The enzyme is a phospholipase of the A1 type which does not require Ca2+ and lacks lipase or transacylase activity. It is unusual for the phospholipases A described to date, since it is inhibited by thiol reagents and is protected by beta-mercaptoethanol, suggesting the presence of essential sulfhydryl residues.

Animals↗

PubMed1987

Relation between binding and the action of phospholipases A2 on Escherichia coli exposed to the bactericidal/permeability-increasing protein of neutrophils.

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis.

Antimicrobial Cationic Peptides↗

PubMed1988

Hydrolysis of 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine, a common precursor of platelet-activating factor and eicosanoids, by human platelet phospholipase A2.

The metabolism of platelet-activating factor (PAF) and arachidonic acid is linked through the common intermediate 1-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine (alkylarachidonoyl-GPC). Hydrolysis of alkylarachidonoyl-GPC by phospholipase A2 may initiate the biosynthesis of both PAF and eicosanoids, since alkyllyso-GPC is formed for acetylation to PAF and arachidonic acid is liberated for conversion to biologically active metabolites. In order to elucidate the regulation and functional role of human platelet phospholipase A2 in the pathway leading to the formation of both classes of lipid mediators, we have characterized its action upon alkylarachidonoyl-GPC. Human platelet phospholipase A2 was solubilized and then partially purified in the presence of n-octyl-beta-D-glucopyranoside (octyl glucoside). Hexadecylarachidonoyl-GPC was prepared biosynthetically using platelet sonicates, purified by two-step high-performance liquid chromatography (HPLC) and suspended in buffer by sonication. Our results indicate that deacylation of alkylarachidonoyl-GPC by platelet phospholipase A2 has an absolute requirement for Ca2+. It occurs at submicromolar concentrations of free Ca2+ and exhibits a biphasic Ca2+-dependence with activity plateaus at 10 microM and 2 mM. Phospholipase A2-mediated hydrolysis of alkylarachidonoyl-GPC is increased 2-fold by albumin and is enhanced 5-fold if 1,2-dioleoylglycerol is incorporated into the substrate dispersion. The substrate dependence and specificity of platelet phospholipase A2 for 1-alkyl- vs. 1-acyl-linked subclasses of arachidonic acid containing phosphatidylcholine was examined with 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3-phosphocholine (hexadecylarachidonoyl-GPC) and 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (palmitoylarachidonoyl-GPC). We found that the substrates were deacylated equivalently. We conclude that, in stimulated platelets, in the presence of increased levels of cytoplasmic free Ca2+ and newly generated diacylglycerol, alkylarachidonoyl-GPC may be rapidly hydrolyzed by phospholipase A2 and may serve as a precursor of both PAF and eicosanoids.

Albumins↗

PubMed1993

Lysosomal phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase: specificity for the sn-1 fatty acid of the donor and co-purification with phospholipase A1.

Positional specificities in donor and acceptor phospholipids of the lysosomal phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase have been determined. Comparison of the transfer of labelled fatty acid from sn-1 [14C]acyl and sn-2 [14C]acylphosphatidylcholines by extracts of rat liver lysosomes revealed that fatty acids in the sn-1 position were exclusively transferred. Degradation of the acylphosphatidylglycerol product by Rhizopus arrhizus lipase, highly specific for fatty acids esterified to sn-1 or sn-3 positions, indicated that sn-1 or sn-3 rather than sn-2 positions had been acylated. Assays of phospholipase A1, phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase, the conversion of lysophosphatidylglycerol to bis(monoacylglycero)phosphate and phospholipase A2 were performed at various steps in the purification of lysosomal phospholipase A1. After the penultimate step of chromatofocusing, there was a 1086-fold increase of phospholipase A1 specific activity over the homogenate and this was accompanied by a 11 998-fold increase of phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase specific activity. A second preparation carried through to the final step of gel-filtration retained a similar ratio of acyltransferase activity. On the other hand, specific activities of phospholipase A2 and of the conversion of lysophosphatidylglycerol to bis(monoacylglycero)phosphate increased to the step where enzyme was solubilized from lysosomes, but were lost from later steps. These findings suggest that phosphatidylcholine: bis(monoacylglycero)phosphate acyltransferase is catalyzed by lysosomal phospholipase A1. The site of acylation in the bis(monoacylglycero)phosphate acceptor appears to be either sn-1 or sn-3. Since the lysosomal extracts did not catalyze the transacylation of phosphatidylglycerol, we conclude that the formation of acylphosphatidylglycerol in lysosomes requires the sequential acylation of lysophosphatidylglycerol to form bis(monoacylglycero)phosphate by an unidentified enzymatic mechanism followed by a transacylation of bis(monoacylglycero)phosphate in either sn-1 or sn-3 position to form acylphosphatidylglycerol which is catalyzed by phospholipase A1.

1-Acylglycerol-3-Phosphate O-Acyltransferase↗

PubMed1992

Initiator-promoter coupling of phospholipases D and A2 in platelets upon cholesterol incorporation.

Since phospholipases exist within a membrane lipid environment, it is not unreasonable to assume that cholesterol capable of changing the lipid environment can effect the coupling relationship among the signal transducing components. Our previous study showed that the 'molecular switch' through which membrane cholesterol modulates cyclic nucleotide levels and Na+/H+ exchange within human platelets is phospholipase A2. We demonstrate here that membrane cholesterol initiates the activation of phosphatidyl choline phospholipase D and phosphatidic acid thus generated promotes the activation of its phospholipase A2 in the presence of extraplatelet calcium. More important, inhibition of phospholipase D by zinc blocks the activation of phosphatidic acid phospholipase A2 in platelets upon cholesterol incorporation. Our result led us to postulate that membrane cholesterol induced initiation promotion coupling of phospholipases D and A2 in human platelets may be responsible for the hypersensitized state of platelets in hypercholesterolemic patients.

Blood Platelets↗

PubMed1982

Effects of beta-bungarotoxin and phospholipase A2 from Naja naja atra snake venom on ATPase activities of synaptic membranes from rat cerebral cortex.

Non-neurotoxic phospholipase A2 of Formosan cobra venom possessed higher hydrolytic activity on phosphatidylcholine vesicles and also had higher inhibitory action on Na+-K+-ATpase and Mg2+-ATPase of the rat synaptic membrane than neurotoxic beta-bungarotoxin of Formosan Krait Venom. Na+-K+-ATPase was more susceptible than Mg2+-ATPase to the inhibitory action of toxins, especially in the presence of Triton X-100. The inhibition of ATPases by toxins followed the Michaelis-Menton equation. It is interesting that various phospholipids and ions influenced phospholipase A2 and beta-bungarotoxin inhibition of ATPases. Sphingomyelin antagonized phospholipase A4 more profoundly than beta-bungarotoxin, while egg lecithin had the reverse effect. Both phosphatidylethanolamine and phosphatidylserine protected Na+-K+-ATPase from the inhibitory action of phospholipase A2 but not that of beta-bungarotoxin. High K+ (30 mM) did not affect, while Ca2+ (0.2 mM) decreased, the inhibitory action of phospholipase A2 on Na+-K+-ATPase; in contrast, high K+ reversed, and Ca2+ increased, that of beta-bungarotoxin. These findings imply that phospholipase A2 and beta-bungarotoxin may have different substrate specificities and prefer different conformational states of the membrane for binding. This may explain, at least in part, why beta-bungarotoxin is neurotoxic, while phospholipase A2 is not.

Adenosine Triphosphatases↗

PubMed1991

Chemotactic peptide stimulation of arachidonic acid release in HL60 cells, an interaction between G protein and phospholipase C mediated signal transduction.

The mechanism of phospholipase A2 activation by chemotactic peptide was investigated in human promyelocytic HL60 cells. N-Formyl-methionyl-leucyl-phenylalanine (fMetLeuPhe) and the non-hydrolyzable GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) induced arachidonic acid release in permeabilized and metabolically inhibited HL60 cells, a preparation in which calcium was buffered and inositol phospholipid hydrolysis was inhibited. Inositol phosphate generation and arachidonic acid were shown to be temporally dissociated. These results suggest that receptor-dependent phospholipase C activity is not required for fMetLeuPhe to induce arachidonic acid release. However, fMetLeuPhe effects were highly calcium-dependent and inhibition of phospholipase C reduced fMetLeuPhe stimulation of arachidonic acid release even in the permeabilized cell preparation. We conclude that although phospholipase A2 activation is linked to the fMetLeuPhe receptor independent of phospholipase C, actions of phospholipase C to mobilize calcium and release diacylglycerol may be important to phospholipase A2 activation in the intact cell.

Adenosine Triphosphate↗

PubMed1986

Contamination of commercial preparations of calmodulin by phospholipase A2.

In the course of studies of the possible regulation of cellular phospholipase A2 activities by calcium and calmodulin, it was observed that some of the commercial preparations of calmodulin contained significant phospholipase A2 activity. Six commercially available calmodulin sources were compared for the presence of contaminating phospholipase A2 activity, relative purity by SDS-gel electrophoresis, and relative biological activity in stimulating calmodulin-deficient phosphodiesterase. One of the commercial calmodulin sources contained a relatively high specific phospholipase A2 activity (1.30 +/- 0.11 nmol [1-14C]arachidonic acid released/mg protein per h) and yielded two major bands in SDS-gel electrophoresis. Two of the calmodulin sources tested were relatively free of phospholipase A2 activity, were quite pure (one band on SDS-gel) and had high biological activity in stimulating calmodulin-deficient phosphodiesterase. Thus, investigators using commercially available preparations of calmodulin should be aware of the contamination of some of these sources by phospholipase A2 activity. These findings may be of importance to investigators considering the role of calmodulin in activating a variety of calcium-dependent enzymes, including phospholipase A2.

Animals↗

PubMed1998

Mast cell degranulation induced by two phospholipase A2 homologues: dissociation between enzymatic and biological activities.

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.

Animals↗

PubMed1994

Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases.

A series of mixed-chain diacyl-PCs which contain an omega-COOH on the sn-2 chain [1-Cx-2-Cy-(COOH)-PC] and bolaform (1-Cx-2,2'-Cy-1'-Cx-PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A2 (Naja naja naja) and C (Bacillus cereus). There is very little detectable phospholipase A2 activity toward pure micellar 1-acyl-2-acyl-(omega-COOH) species. In addition, when these same omega-COOH species are present at concentrations above their CMCs, they are potent inhibitors of phospholipase A2 hydrolysis of other micellar lipids. In contrast, phospholipase C hydrolysis of the same 1-acyl-2-acyl-omega-COOH)-PC species proceeds with rates comparable to that of diheptanoyl-PC. The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C8-2,2'-C12-1'-C8-PC) display quite different kinetic results. Under limiting Ca2+ conditions (100 microM) all the available sn-2 acyl bonds of the dimer are hydrolyzed. However, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followed by much slower rate. This is consistent with only the micellar 1-acyl-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A2 hydrolysis of the dimer acting as an inhibitor of subsequent phospholipase A2 activity. Phospholipase C hydrolysis of the PC dimer and the sn-2 omega-COOH PC is rapid, with both available glycerophosphate groups cleaved at presumably the same rate. These results are discussed in terms of the unique physical properties (as measured by NMR and fluorescence experiments) of these phospholipids.

Carbon Isotopes↗

PubMed1982

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine.

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.

4-Chloro-7-nitrobenzofurazan↗

PubMed1980

Methylation of histidine-48 in pancreatic phospholipase A2. Role of histidine and calcium ion in the catalytic mechanism.

It is known that His-48 is part of the active center in pancreatic phospholipase. To further elucidate the role of histidine-48 in the active center of pancreatic phospholipase A2, we have modified the enzyme with a number of bromo ketones and methyl benzenesulfonates. Rapid methylation occurred with methyl p-nitrobenzenesulfonate. Methylated phospholipase shows total loss of enzymatic activity whereas binding of substrate and the cofactor Ca2+ remains intact. Amino acid analysis of methylated equine phospholipase showed the loss of the single molecule of histidine and the formation of one molecule of 2-amino-3-(1-methyl-5-imidazolyl)propanoic acid (1-methylhistidine). Equine phospholipase was also modified by [13C]methyl p-nitrobenzenesulfonate and the methylated enzyme was studied by 13C NMR. The results indicate that the proton on the nitrogen in position 3 of the imidazole ring is involved in a strong interaction with a buried carboxylate group, thereby hindering rotation of the imidazole ring, and that the nitrogen in position 1 is involved in catalysis. These data are in full agreement with the three-dimensional structure at 1.7-A resolution of bovine pancreatic phospholipase. A catalytic mechanism is proposed in which a water molecule which is close to the nitrogen at position 1 of the imidazole ring of the Asp-99-His-48 couple acts as the nucleophile. A comparison is made between phospholipase A2 and the serine esterases.

Animals↗

PubMed1988

Isolation, properties and amino acid sequences of a phospholipase A2 and its homologue without activity from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands.

A phospholipase A2, Laticauda colubrina phospholipase A2 II (LcPLA-II), and a phospholipase A2 homologue, Laticauda colubrina phospholipase A2 homologue I (LcPLH-I), were isolated from the venom of the yellow-lipped sea snake, Laticauda colubrina, from the Solomon Islands. LcPLA-II showed phospholipase A2 activity towards egg-yolk phosphatidylcholine (24 mumol/min per mg at optimal conditions at 37 degrees C) and lethal potency (LD50 45 micrograms/kg body wt. intravenously in mice). Both of the activities were lost by treatment with p-bromophenacyl bromide. LcPLH-I showed neither phospholipase A2 activity nor lethal potency at a dose of 4.5 mg/kg body wt. in mice. It was not modified by the treatment with p-bromophenacyl bromide. LcPLA-II and LcPLH-I bound Ca2+ at a 1:1 molar ratio with KCa values of 105 microM and 44 microM at pH 8.0 respectively. Elucidation of the amino acid sequences of these two proteins showed that each protein consisted of a single chain of 118 amino acid residues, including 14 half-cystine residues. The two sequences are different from each other at 22 residues and highly homologous to those from other sources. The essential histidine residue for the phospholipase A2 activity at position 48 is replaced by an asparagine residue in the homologue LcPLH-I. Details of the separation of the peptides obtained by proteinase digestions of LcPLA-II and LcPLA-I and the determination of their amino acid sequences are given in Supplementary Publication SUP 50145 (14 pages), which has been deposited at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1988) 249, 5.

Acetophenones↗

PubMed1982

Inhibition of kidney lysosomal phospholipases A and C by aminoglycoside antibiotics: possible mechanism of aminoglycoside toxicity.

Nephrotoxicity is an important side effect of aminoglycoside antibiotics, which are used to control infections caused by Gram-negative bacteria. Accumulation of aminoglycosides and phospholipids in the lysosomes is a prominent and early feature of aminoglycoside nephrotoxicity and is characterized histologically by the presence of numerous multilamellar bodies in kidney proximal tubule cells. Previous studies have shown that the drug-induced phospholipid fatty liver in man and animals is due to concentration of certain cationic amphiphilic drugs in lysosomes with inhibition of lysosomal phospholipases. It seemed possible that this mechanism might also explain the elevated levels of phospholipid and increased numbers of multilamellar bodies reported in the kidney cortex in aminoglycoside nephrotoxicity. In this study, subcellular localization of acid phospholipases A and C has been shown to be lysosomal in rat kidney cortex. A soluble lysosomal protein fraction was isolated and found to contain both phospholipase A and phospholipase C activity. Streptomycin did not inhibit the release of fatty acids from [3H]dioleoylphosphatidylcholine. However, amikacin, dibekacin, gentamicin, and tobramycin inhibited both phospholipase A and phospholipase C. Our results suggest that the accumulation of phospholipids in lysosomes of kidney cortex, an early and pervasive feature of acute aminoglycoside nephrotoxicity, is due to inhibition of lysosomal phospholipases.

Aminoglycosides↗

PubMed1996

Binding of human phospholipase A2 type II to proteoglycans. Differential effect of glycosaminoglycans on enzyme activity.

Phospholipase A2 acting on low density lipoproteins in the extracellular arterial intima may form proinflammatory lipid mediators. Human nonpancreatic secretory phospholipase A2 has three regions that may associate with sulfated glycosaminoglycans. The apoB-100 molecule in low density lipoproteins also has glycosaminoglycan binding regions that could mediate its retention in the arterial intima. Here we report that human nonpancreatic phospholipase A2 isolated from a transfected cell line binds to glycosaminoglycans secreted by cultured human arterial smooth muscle cells. A gel mobility shift assay showed that the affinity of phospholipase A2 for glycosaminoglycans from a heparan sulfate/chondroitin sulfate proteoglycan was higher than for chondroitin sulfate glycosaminoglycans from a larger versican-like proteoglycan. Affinity chromatography confirmed these results. All glycosaminoglycans tested, at concentrations up to 100 microM, increased the activity of phospholipase A2 toward phosphatidylcholine liposomes. Above this concentration, heparan sulfate and heparin inhibited the enzyme. Heparin and chondroitin 6-sulfate increased phospholipase A2 activity on low density lipoproteins up to 4-fold at 100 microM, whereas heparan sulfate had no effect. The results indicate that human nonpancreatic secretory phospholipase A2 interacts with proteoglycans via their glycosaminoglycan moiety and that the enzyme activity may be modulated by the association of the enzyme and its substrate to the sulfated polysaccharides.

Chondroitin Sulfates↗