SEARCH · PubMed Health

Results for “PHOSPHOLIPASE”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources

PubMed1995

The coupling of pertussis toxin-sensitive G proteins to phospholipase A2 and adenylyl cyclase in CHO cells expressing bovine rhodopsin.

The regulation of phospholipase A2 by G protein-coupled receptors is examined in CHO cells which normally express the purinergic receptor and have been transfected with bovine rhodopsin. The purinergic receptor has been reported to activate both phospholipase C and phospholipase A2 in this cell line. In contrast, bovine rhodopsin by itself is not able to activate phospholipase A2. However, the photoreceptor does potentiate purinergic receptor-mediated phospholipase A2 activation in a light-dependent manner. Both the purinergic receptor stimulation of phospholipase A2 and the enhanced activity mediated by rhodopsin are completely pertussis toxin-sensitive, suggesting the regulation of phospholipase A2 by a member of the Gi family of G proteins. Both of these receptors also inhibit adenylyl cyclase activity. Rhodopsin-mediated inhibition of adenylyl cyclase is pertussis toxin-sensitive, whereas inhibition by the purinergic receptor is calcium-sensitive but not pertussis toxin-sensitive. These results suggest (1) that rhodopsin is similar to other receptors that normally couple to Gi when expressed in cultured cells and (2) that regulation of adenylyl cyclase and PLA2 in CHO cells by rhodopsin and the purinergic receptor occur via distinct pathways.

Adenosine Triphosphate↗

PubMed1993

Crystal structure of a porcine pancreatic phospholipase A2 mutant. A large conformational change caused by the F63V point mutation.

The highly homologous bovine and porcine pancreatic phospholipase A2 (85% amino acid residue identity) show a large conformational difference in the loop from residue 59 to 71. In bovine phospholipase A2 residues 59 to 66 adopt an alpha-helix conformation, while residues 67 to 71 are in a surface loop. Residues 59 to 66 in the porcine enzyme have a random coil conformation, and residues 67 to 71 form a short 3(10)-helix. It has been suggested that most probably this conformational difference is caused by the substitution Val63 (bovine) to Phe63 (porcine) in the otherwise invariant loop 59 to 70. To test this hypothesis, a mutant porcine phospholipase A2 was constructed in which residue Phe63 was replaced by a Val. The activity of this F63V mutant towards aggregated substrates was about half the activity of wild-type porcine phospholipase A2, but significantly different from that of the bovine enzyme. The affinity for zwitterionic interfaces was found to be intermediate between porcine and bovine phospholipase. The mutation did not have any effect on the stability of the enzyme towards denaturation by guanidine.HCl. The F63V mutant was crystallized in space group P2(1)2(1)2(1) with cell dimensions a = 79.88 A, b = 65.23 A, c = 52.62 A, with two molecules per asymmetric unit. Its three-dimensional structure was solved by molecular replacement methods, and refined to a crystallographic R-factor of 17.6% for all data between 10 and 2.2 A resolution. In one molecule the 58 to 71 loop is in very weak density, suggesting a high degree of disorder or flexibility. The conformation of the same loop in the other molecule could be determined unambiguously. It shows a conformation which resembles more that of bovine phospholipase A2 than that of porcine phospholipase. It is concluded that indeed the single F63V substitution causes a dramatic conformational change.

Amino Acid Sequence↗

PubMed1990

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and oligomers of prostaglandin B1.

Inhibition of human phospholipases A2 by cis-unsaturated fatty acids and their oxidative metabolites and/or polymers was studied using partially purified human phospholipases A2 and [1-14C]oleate labelled, autoclaved E. coli as substrate. As previously reported for other phospholipases A2, oleic and arachidonic acids inhibited human synovial fluid phospholipase A2 with IC50s of 15 and 30 microM respectively. Air oxidation of arachidonic acid or hydroxylation of oleic acid (12-hydroxy-oleate) substantially relieved that inhibition. Similarly, the enzymatically oxidatized metabolite of arachidonate, prostaglandin B1 (PGB1), did not inhibit enzymatic activity. However, prostaglandin Bx (PGBx), an oligomer (n = 6) of PGB1, was a potent inhibitor of Ca(++)-dependent, neutral-active phospholipase A2 activities. Enzymatic activity in acid extracts from human neutrophils, platelets, sperm, plasma, synovial fluid, endometrium, degenerative disc, and snake venom was inhibited by PGBx with IC50s ranging from 0.5-7.0 microM. Inhibition was independent of substrate phospholipid concentration over a 24-fold range (5-120 microM) and PGBx quenched the tryptophan fluorescence of snake venom phospholipase A2 in a dose-dependent manner. Agonist-induced (A23187) release of arachidonic acid from prelabelled human neutrophils and cultured human endothelial cells was also inhibited by PGBx with IC50s of 3 and 20 microM, respectively. These results illustrate that oxidative reactions of cis-unsaturated fatty acids relieve their natural inhibitory activity, and polymerization of an inactive fatty acid metabolite yields a potent inhibitor of in vitro and in situ phospholipase A2 activity.

Arachidonic Acid↗

PubMed1992

Characteristics and possible functions of mast cell phospholipases A2.

Phospholipase A2 activity in lysates of mast cells and their related cells [mouse bone marrow-derived IL-3 dependent mast cells (BMMC), rat connective tissue mast cells (CTMC), and rat mastocytoma RBL-2H3 cells] was measured using phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) as exogenous substrates. Both BMMC and RBL cells showed rather high phospholipase A2 activity, whereas CTMC showed only weak activity. These cells contained at least three types of phospholipase A2. Type 1 enzyme showed no appreciable affinity to heparin, and preferentially hydrolyzed either PC or PE, both of which have an arachidonic acid at the sn-2 position. The activity was absorbed by monoclonal antibody against rabbit platelet cytosolic 85-kDa phospholipase A2. Type 2 enzyme had an affinity to heparin, and was completely inhibited by anti-rat platelet 14-kDa secretory phospholipase A2. This enzyme could be expressed as an "ecto-type" enzyme on the cell surface and might be secreted from cells when mast cells are activated. Type 3 enzyme also had an affinity to heparin, but was separated from type 2 enzyme on reverse-phase HPLC. This enzyme did not interact with anti-14-kDa secretory enzyme antibody. Purified type 3 enzyme (30-kDa) specifically hydrolyzed PS. p-Bromophenacylbromide inhibited all types of phospholipase A2, whereas mepacrine inhibited type 2 and type 3 enzymes, but not type 1 enzyme. Type 2 enzyme was also inhibited by the specific antibody, complement degradation product, and a small-molecular-weight inhibitor. Histamine release was inhibited by all these inhibitors, whereas PGD2 production was inhibited only by p-bromophenacylbromide. Possible roles for these phospholipases A2 in mast cell function are proposed.

Animals↗

PubMed1996

Cloning of a phospholipase C-delta 1 of rabbit skeletal muscle.

The phospholipase C isoform responsible for the increase in the total myoplasmic inositol 1,4,5-trisphosphate concentration during tetanic contraction of isolated skeletal muscle and its mechanism of activation is not known. We have cloned and sequenced a phospholipase C cDNA of rabbit skeletal muscle coding for a protein of 745 amino acids with a molecular mass of 84,440 kDa. The deduced amino acid sequence exhibits the phospholipase C-specific domains X and Y which according to current knowledge very likely represent the catalytic centre of the enzyme. An overall sequence homology of 88% to the phospholipase C-delta 1 of rat brain suggests that the encoded protein represents a phospholipase C-delta 1 isoform of rabbit skeletal muscle. Northern blot analysis shows, that this phospholipase C-delta is dominantly expressed in skeletal muscle, less strongly in smooth muscle (uterus) and lung and weakly in heart, kidney and brain. In the N-terminal part of the primary structure a consensus sequence for a canonical EF-hand Ca2+ binding domain can be identified together with a short positively charged motif which recently has been suggested to be essential for the binding of phosphatidylinositol 4,5-bisphosphate. If these two domains which are unique for phospholipase C-delta are sufficient in establishing a mechanism for the activation of the enzyme, inositol 1,4,5-trisphosphate formation in skeletal muscle could be the consequence of an increase in myoplasmic Ca2+.

Amino Acid Sequence↗

PubMed1994

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗

PubMed2000

Level of circulating phospholipase A2 in prediction of the prognosis of patients with suspected myocardial infarction.

OBJECTIVES: Atherosclerotic lesions result from inflammatory-proliferative responses of the endothelium and smooth muscle of the arterial wall. Poor prognosis of acute myocardial infarction (AMI) patients has been associated with elevated levels of acute phase proteins including C-reactive protein. We investigated the significance of circulating phospholipase A2 in the long-term prognosis of suspected AMI patients. METHODS: The concentration of phospholipase A2 was measured by an immunoassay in sera of 100 suspected AMI patients. Admission phospholipase A2 95 % fractile outliers were excluded to eliminate the effect of acute infectious diseases. The total and atherosclerotic mortalities were followed for a 4-year period. RESULTS: The most powerful prognostic limit for both admission (p = 0.02, RR = 2.6 and 95% CI = 1.2 to 5.6) and maximal (p = 0.06, RR = 2.4 and 95% CI = 0.96 to 5.9) phospholipase A2 groups was > or =8 microg/l. The admission phospholipase A2 level had an independent prognostic significance for atherosclerotic mortality (p = 0.04, RR = 2.4 and CI = 1.02 to 5.8) in multivariate analysis with CK-MB and age. CONCLUSIONS: The elevated serum phospholipase A2 level at admission is an independent predictor of long-term atherosclerotic mortality in patients with suspected AMI. The prognostic significance of phospholipase A2 weakens during hospitalisation concomitant to the onset of the acute inflammatory response to myocardial injury.

Aged↗

PubMed1998

Duodenal secretion of phospholipase A2, amylase, and bicarbonate in chronic pancreatitis.

Phospholipase A2 has been suggested to be involved in the pathogenesis and pathophysiology of acute pancreatitis. We determined phospholipase A2 and amylase activities in duodenal juice collected during a secretin test from 30 consecutive patients who were suspected to have chronic pancreatitis or biliary disease. The patients underwent endoscopic retrograde cholangiopancreatography (ERCP) the following day. In the 8 patients with ERCP findings of advanced chronic pancreatitis, the mean outputs of phospholipase A2, amylase, and bicarbonate were reduced by 74%, 72%, and 60% compared to the respective values in the 13 (control) patients without a diagnosis of any pancreatic disorder or jaundice. In the 3 patients with recurrent pancreatitis but normal ERCP findings and in the 6 patients with jaundice the output values were not significantly reduced compared to those in the patients without any pancreatic disorder or jaundice. The outputs of amylase and phospholipase A2 were not significantly interrelated, whereas the outputs of phospholipase A2 and bicarbonate correlated well. Receiver characteristic (ROC) curves confirmed the high specificity and sensitivity of phospholipase A2 or bicarbonate output in patients with ERCP findings of advanced chronic pancreatitis compared to those with no changes in pancreatic ducts, with similar probability values of 0.880 +/- 0.111 (SEM), compared to the respective lower value of amylase, 0.676 +/- 0.118. Phospholipase A2 and bicarbonate output proved of equal value as markers of chronic pancreatitis and were superior to amylase output in the secretin test.

Acute Disease↗

PubMed1981

Studies on (K+ + H+)-ATPase. IV. Effects of phospholipase C treatment.

(1) The total phospholipid content of a gradient purified (K+ + H+)-ATPase preparation from pig gastric mucosa is 105 mumol per 100 mg protein, and consists of 29% sphingomyelin, 29% phosphatidylcholine, 28% phosphatidylethanolamine, 10% phosphatidylserine and 4% phosphatidylinositol. The cholesterol content corresponds to 50 mumol per 100 mg protein. (2) Treatment with phospholipase C (from Clostridium welchii and Bacillus cereus) results in an immediate decrease of the phosphate content. Up to 50% of the phospholipids are hydrolyzed by each phospholipase C preparation alone, without further hydrolysis by increased phospholipase concentration or prolonged incubation time. Combined treatment with the two phospholipase C preparations, sequentially or simultaneously, hydrolyzes up to 65% of the phospholipids. (3) The (K+ + H+)-ATPase and K+ stimulated p-nitrophenylphosphatase activities are decreased proportionally with the total phospholipid content, indicating that these enzyme activities are dependent on phospholipids. (4) Phospholipase C treatment does not change optimal pH, Km value for ATP and temperature dependence of the gastric (K+ + H+)-ATPase, but slightly decreases the Ka value for K+. (5) Phospholipase C treatment lowers the AdoPP[NH]P binding and phosphorylation capacities, suggesting that inactivation occurs primarily on the substrate binding level. (6) Most of the results can be understood by assuming that hydrolysis of the phospholipids by phospholipase C leads to aggregation of the membrane protein molecules and complete inactivation of the aggregated ATPase molecules.

4-Nitrophenylphosphatase↗

PubMed1991

Chronic ethanol ingestion modifies liver microsomal phosphatidylserine inducing resistance to hydrolysis by exogenous phospholipase A2.

Chronic ethanol ingestion leads to the acquisition of a tolerance to membrane lipid disordering, a lowered partition coefficient to hydrophobic compounds and a resistance to the hydrolysis of the phospholipids by exogenous phospholipase A2. Anionic phospholipids have been implicated as being responsible for the resistance to lipid disordering and a number of modifications to these phospholipids are known to occur as a result of chronic ethanol-ingestion. In this study the basis of the resistance to phospholipase A2 in hepatic microsomes was investigated. It was found that chronic ethanol-induced modifications to each of the major phospholipid classes was responsible to some extent for the resistance to phospholipase A2, however, PS was particularly potent considering it is a compositionally minor constituent. The effect was interpreted as a reduced ability to activate the phospholipase A2 since PS acts as an essential activator of phospholipase A2 (along with PI). Fatty acid analysis revealed that the chronic ethanol-treatment resulted in a elevated level of docosahexaenoate with a parallel reduction in arachidonate in phosphatidylserine. Lipid packing and organization is important in the regulating the level of exogenous phospholipase A2 activity but the activity was not found to correlate with lipid order of different phosphatidylserine species. It is concluded that subtle differences in the molecular species arrangement or disposition around the enzyme may be responsible for the altered phospholipase A2 interaction with the membrane induced by chronic ethanol-treatment. One implication of this study is that other anionic phospholipid dependent membrane proteins, of which there are many known examples, may also be modified as a result of chronic ethanol-ingestion.

Alcoholism↗

PubMed1984

Studies on enzymes related to diacylglycerol production in activated platelets. I. Phosphatidylinositol-specific phospholipase C: further characterization using a simple method for determination of activity.

A simple method of determination of phosphatidylinositol-specific phospholipase C activity in soluble platelet extracts has been devised. It is based on the use of a total lipid extract from rat liver microsomes incubated with [3H]inositol in the presence of MnCl2. Phosphatidylinositol hydrolysis can thus be detected by determining hydrosoluble radioactivity formed upon incubation with enzyme fractions. Owing to the presence of other phospholipids in the assay system, phospholipase C was inhibited. However, activity was restored by sodium deoxycholate (0.1%, w/v). Optimal conditions also included calcium (1-10 mM) and a pH between 5 and 7, allowing the detection of phospholipase C without the need for purifying the substrate. Using this simplified procedure, platelet phospholipase C was submitted to preparative electrofocusing and to gel filtration chromatography on Sephacryl S-200. Phospholipase C focused in one single peak at pH 6.1. An Mr of 86 000 was found upon gel chromatography of a crude extract, against 68 000 when phospholipase C had been previously purified by electrofocusing. These data indicate that phospholipase C might be associated with lipids or with an Mr 20 000 protein, the significance of which is discussed.

Blood Platelets↗

PubMed1984

Nonspecific inhibition of enzymes by p-bromophenacyl bromide. Inhibition of human platelet phospholipase C and modification of sulfhydryl groups.

This study demonstrates that p-bromophenacyl bromide irreversibly inhibits, in a time- and dose-dependent manner, yeast alcohol dehydrogenase, bovine pancreatic alpha-chymotrypsin, human platelet phosphatidylinositol (PI)-specific phospholipase C, in addition to the neutral-active and calcium-dependent phospholipase A2 of human platelets. The PI-specific phospholipase C has maximal activity at pH 5,5 is calcium-dependent, and is strongly inhibited by sulfhydryl reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and methylmethane thiosulfonate . Increasing concentrations of DTNB produced concomitant inhibition of phospholipase C activity and titration of sulfhydryl groups. In contrast, human platelet phospholipase A2 activity was unaffected by concentrations of DTNB that titrated sulfhydryl groups, and completely inhibited PI-specific phospholipase C activity. Treatment of cysteine with p-bromophenacyl bromide resulted in modification of the amino acid as demonstrated by paper chromatography, and loss of titratable sulfhydryl groups. These data show that p-bromophenacyl bromide inhibits a wide spectrum of enzymatic activities including PI-specific phospholipase C. This reagent modifies amino acid residues other than active-site histidines and therefore has a broader reactivity than previously considered. Thus, it should not be used as a selective inhibitor of enzymes in crude cellular experiments.

Acetophenones↗

PubMed1988

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells.

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.

Animals↗

PubMed1991

Subcellular localization of rat gastric phospholipase A2.

In the present study, we have performed experiments to gain some insight into the subcellular localization and biochemical properties of gastric mucosal phospholipase A2. After classical subcellular fractionation of whole glandular stomach mucosa, we found that gastric phospholipase A2 was essentially enriched in the 105,000 x g pellet that contains microsomes and plasma membranes. Except for the cytosol, all the subcellular fractions exhibited similar phospholipase A2 activity (i.e., optimum of pH, calcium dependence, apparent Km and positional specificity). The high-speed pellet was further characterized by ultracentrifugation on a sucrose gradient. Data showed that the sedimentation profile of phospholipase A2 was quite similar to those of plasma membrane markers and more specifically to an apical membrane marker. These results, taken together, showed that a gastric phospholipase A2 is distributed among the various subcellular fractions (as a result of cross-contamination) together with the membrane fraction on which it is associated. It is proposed that this fraction is the apical plasma membrane which would be the main site of phospholipase A2 action for arachidonic acid release. Lysophospholipase showed the same sedimentation profile as phospholipase A2, whereas acyl CoA-lysophosphatidylcholine: acyltransferase mainly sedimented with heavy microsomes. The substrate specificity of the enzyme was assessed by endogenous hydrolysis of gastric mucosal phospholipids. We were able to show that the enzyme acts at nearly the same rate on two major gastric membrane phospholipids, namely phosphatidylcholine and phosphatidylethanolamine.

Animals↗

PubMed1994

Quantification of the interaction between lysolecithin and phospholipase A2.

The rate of hydrolysis of phosphatidylcholine bilayers by phospholipase A2 may be either enhanced or inhibited by the presence of lysolecithin depending on the experimental conditions examined. To further understand the relationship of lysolecithin to phospholipase A2 activity, the binding of lysolecithin to phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus was examined by fluorescence spectroscopy. The tryptophan emission intensity of the enzyme was enhanced by 70% upon addition of lysolecithin. The binding isotherm for lysolecithin to the phospholipase A2 estimated from the fluorescence change was biphasic, with a clear break in the curve occurring at the critical micelle concentration of the lysolecithin. Several observations suggested that the phospholipase A2 was capable of hydrolyzing the lysolecithin although at a rate far below that of phospholipid hydrolysis. These experiments were repeated using several other species of phospholipase A2, and the results were found to be general among the enzymes except the lys-49 isozyme from A. p. piscivorus which displayed neither the dependence on the critical micelle concentration for binding nor the ability to hydrolyze lysolecithin. These results were used as the basis for a quantitative analysis of enzyme fluorescence changes that occur during the time course of phospholipid hydrolysis and of the mechanism whereby lysolecithin inhibits the hydrolysis of phosphatidylcholine bilayers by phospholipase A2.

Agkistrodon↗

PubMed1991

Eicosanoid generation from antigen-primed mast cells by extracellular mammalian 14-kDa group II phospholipase A2.

The extracellular form of 14-kDa group II phospholipase A2 has been found to accumulate at various types of inflammatory sites. In the present paper, we have studied the possible role of the extracellular 14-kDa group II phospholipase A2 in the process of prostaglandin production in activated rat mast cells. When mast cells obtained from the peritoneal cavity of rats were sensitized with IgE, challenged with antigen and then exposed to extracellular 14-kDa group II phospholipase A2, appreciable release of prostaglandin D2 was observed. Generation of prostaglandin D2 was dependent on the concentration of the phospholipase A2 as well as that of the antigen, while no appreciable prostaglandin D2 generation was observed with cells in the absence of the antigen. No histamine release was observed under the same conditions. Phosphatidylcholine in mast cell membranes was appreciably hydrolyzed to liberate free arachidonic acid when mast cells were incubated with 14-kDa group II phospholipase A2 added exogenously in the presence of the antigen. Both the generation of prostaglandin D2 and the release of arachidonic acid were retarded by inhibitors specific to 14-kDa group II phospholipase A2. Thus, 14-kDa group II phospholipase A2 may function in the process of inflammation by acting on IgE-antigen-primed mast cells, which are not fully activated, to generate eicosanoids.

Animals↗

PubMed1991

Inhibition of pancreatic phospholipase A2 activity by uteroglobin and antiflammin peptides: possible mechanism of action.

We investigated the possible mechanism of inhibition of porcine pancreatic phospholipase A2 in vitro by rabbit uteroglobin and by the antiflammin peptides. We optimized the conditions of phospholipase A2 assay using a deoxycholate-phosphatidylcholine mixed micellar substrate and established the activity of these inhibitors under optimized conditions. The results of fluorescence studies and crosslinking experiments indicate that the inhibitors interact with the enzyme in solution and affect the increase in intrinsic fluorescence of phospholipase A2 observed upon interaction with a mixed micellar substrate. In addition, we identified a sequence similarity between the antiflammin peptides, the putative active region of uteroglobin and a region in pancreatic phospholipase A2. This region of phospholipase A2 has been previously identified as being involved in the regulation of dimerization of this enzyme, and is conserved in the pancreatic-type enzymes. Taken together, these observations suggest that uteroglobin and antiflammins interact with porcine pancreatic phospholipase A2 and this may, at least in part, explain the enzyme inhibitory effect of these molecules observed in vitro. One possible mechanism of this effect may be an interference with the dimerization process of phospholipase A2 which is associated with interfacial activation.

Amino Acid Sequence↗

PubMed1991

Immunochemical cross-reactivity of two phospholipase A2 neurotoxins, agkistrodotoxin and crotoxin.

Polyclonal rabbit antisera were raised against the phospholipase A2 neurotoxin agkistrodotoxin (AGTX) from Agkistrodon blomhoffii brevicaudus venom and against the phospholipase A2 subunit (component-B, CB) of crotoxin from Crotalus durissus terrificus venom. Anti-AGTX antibodies cross-reacted strongly with crotoxin and crotoxin-like molecules and more weakly with other phospholipases A2 from the venoms of Viperidae and Crotalidae. On the other hand, anti-CB antibodies cross-reacted with AGTX, and also recognized ammodytoxin A and the phospholipase A2 from Vipera berus venom, but not other phospholipases A2 from Crotalidae and Viperidae. Anti-AGTX and anti-CB antibodies were able to inhibit the phospholipase A2 activity and to neutralize the lethal potency of the homologous and heterologous toxins (AGTX or crotoxin). Immunoaffinity chromatography columns were used to isolate anti-AGTX antibodies which recognized CB (91% of the total anti-AGTX antibodies), and anti-CB antibodies which recognized AGTX (52% of the total anti-CB antibodies). Immunochemical investigations performed with each type of antibody indicated that the majority of AGTX antigenic determinants are present on crotoxin component-B and on phospholipases A2 from Viperidae venoms, and that some of these determinants are involved in the neutralization of lethal potency and in the inhibition of enzymatic activity of AGTX and crotoxin.

Animals↗