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PubMed1982

Phospholipase A2 activity in carrageenin-induced inflammatory tissue of rats.

Phospholipase A2 activity was detected in 7-day-old carrageenin-induced inflammatory tissue of rats using a synthetic substrate. 1-acyl-2-[3H]arachidonyl-phosphatidylcholine. The inflammatory tissue was homogenized in saline containing 1 M KCl, and the 105,000 g supernatant fraction was placed on a Sephadex G-100 column. The partially purified phospholipase A2 had a pH optimum at 6-7 and was Ca2+ dependent. p-Bromophenacyl bromide was strongly inhibitory to the partially purified phospholipase A2 (IC50 = 1.44 x 10(-5) M). A moderate inhibition was observed with indomethacin. Cycloheximide and dexamethasone, which inhibit prostaglandin production in inflammatory tissue, exerted no direct inhibitory action on the phospholipase A2. There were no direct inhibitory effects of quinacrine, bradykinin, or actinomycin D. The cell-free supernatant fraction of the inflammatory exudate of 7-day-old carrageenin-induced granulation tissue was found to have no phospholipase A2 activity.

Animals↗
PubMed1986

Mechanism of phospholipase A2-induced conduction block in bullfrog sciatic nerve. II. Biochemistry.

The biochemical changes associated with conduction block following exogenous application of purified phospholipase A2 to bullfrog sciatic nerves were investigated. Nerves were treated with concentrations of phospholipase A2 needed to produce at least a 50% decrease in the compound action potential after a 2-2 1/2 h incubation. This phospholipase A2-induced conduction block was associated with lipid hydrolysis, depletion of high energy phosphates and decreases in [3H]saxitoxin binding. Forty-two percent of the nerve phosphatidylcholine and 45% of the nerve phosphatidylethanolamine were hydrolyzed. [3H]Saxitoxin binding was decreased by 85% in association with conduction block attained with phospholipase A2 incubation. There were significant decrements in adenosine triphosphate levels (-58%) and in phosphocreatine levels (-78%), but no difference in the specific activities of these phosphate compounds. It is concluded that a number of mechanisms can account for the conduction failure resulting from phospholipase A2, including disruption of sodium channels needed for propagation of regenerative nerve impulses and the depletion of high energy phosphates needed to maintain ionic gradients.

Adenine Nucleotides↗
PubMed1988

Diacylglycerol stimulates phospholipase A2 from Swiss 3T3 fibroblasts.

We recently demonstrated that diacylglycerol induced arachidonate release and prostaglandin E2 synthesis in 3T3 fibroblasts, and greatly augmented prostaglandin E2 synthesis in response to submaximal and maximal concentrations of bradykinin. We have now partially purified a phospholipase A2 from the cells. When phosphatidyl[3H]choline was used as substrate, several diacylglycerols augmented phospholipase A2 activity. Diacylglycerol was effective at concentrations as low as 30 nM. Protein kinase C inhibition did not affect diacylglycerol's stimulation of phospholipase A2. Diacylglycerol did not alter the calcium requirement for phospholipase A2 or its pH optimum. The present study demonstrates that the effect of diacylglycerol to augment arachidonate metabolism is at the level of phospholipase A2, itself.

Animals↗
PubMed1990

Exacerbation of rat adjuvant arthritis by intradermal injection of purified mammalian 14-kDa group II phospholipase A2.

Appreciable phospholipase A2 activity was detected in a hind paw homogenate from rats with adjuvant arthritis or carrageenan-induced edema. The activity was neutralized by treatment with antibody raised against rat platelet secretory phospholipase A2, indicating that 14-kDa group II phospholipase A2 was induced in the hind paw during the process of inflammation. Injection of purified rat platelet phospholipase A2 into the hind paw of rats with adjuvant-induced arthritis resulted in exacerbation of edema in a dose-dependent manner, whereas no effect was observed on either normal rats or animals with carrageenan-induced edema under the same experimental conditions. These results suggest the importance of mammalian group II phospholipase A2 in the pathogenesis of some types of inflammation.

Animals↗
PubMed1985

Effect of phospholipase A2 on Ca2+-stimulated adenosine triphosphatase activity in microsomal fraction of rat submandibular gland.

The effect of phospholipase A2 on the Ca2+-ATPase (EC 3.6.1.3) activity in the microsomal fraction of rat submandibular gland was kinetically studied in vitro. The Ca2+-ATPase activity was significantly increased by the treatment with phospholipase A2 in the presence of bovine serum albumin as a scavenger for hydrolyzed products. When the microsomal fraction was incubated with phospholipase A2 in the absence of bovine serum albumin, the Ca2+-ATPase activity was not altered. The Vmax and Km values for both ATP and Ca2+ were increased by the phospholipase A2 treatment, respectively. These results indicated that the activation of Ca2+-ATPase by the phospholipase A2 treatment is due to the increase of Vmax.

Animals↗
PubMed1987

Characterization of the structure and function of three phospholipases A2 from the venom of Agkistrodon halys pallas.

Three monomeric phospholipases A2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air-water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

Amino Acid Sequence↗
PubMed1987

In vivo synergy of cardiotoxin and phospholipase A2 from the elapid snake Naja mossambica mossambica.

The lethality of elapid snake venoms is due to toxic polypeptides which are alpha-neurotoxins, phospholipases A2 and cardiotoxins. In contrast to alpha-neurotoxins, less is known about the mode of action of phospholipases A2 and cardiotoxins at a cellular and molecular level, although it has been demonstrated that the hemolytic effect of cardiotoxin and its action on muscle can be accelerated in vitro by phospholipase A2. Here we show that when mice are injected i.v. with phospholipase A2, a significant decrease in survival time is observed if a sub-LD50 amount of cardiotoxin is injected simultaneously. Furthermore, the survival time is drastically reduced when phospholipase A2 (2.3 LD50) is injected first, followed 15 min later by doses of cardiotoxin as small as 0.18 LD50. These results strongly suggest a synergy between the two polypeptides.

Animals↗
PubMed1987

Molecular weight variations in the diversity of phospholipase A2 forms in reptile venoms.

Reptile venoms exhibit a wide diversity of phospholipase A2 forms when examined by electrophoretic and chromatographic techniques which separate on the basis of net charge. In principle, diversity in charge among the enzyme forms could result from two types of structural modifications: (i) pretranslational modifications, such as differences in amino acid sequences; (ii) post-translational modifications, such as partial proteolysis or hydrolysis of amide functions of asparagine and glutamine residues. Some types of modifications alter both charge and molecular weight. A radiolabeling technique has been developed which allows detection of phospholipase A2 enzymes after electrophoretic separation on the basis of molecular weight in polyacrylamide gels containing sodium dodecylsulfate. Venom preparations were radiolabeled by incubation with p-dodecylphenacylbromide, which was prepared radiolabeled to high specific activity with tritium. The labeling agent was dispersed in phosphatidylcholine liposomes and incubated with venom preparations under conditions of optimal enzyme activity. The preparations were then extracted with ether and hexane to remove substrate and unbound label and subjected to polyacrylamide gel electrophoresis under standard conditions. Protein-bound radiolabel was detected by fluorography and autoradiography. Patterns of labeled species obtained by gel electrophoresis of selected venoms using separation on the basis of charge were similar to the patterns of phospholipase A2 variants obtained under the same electrophoretic conditions using enzymatic detection in phosphatidylcholine-containing gels. Examination of a variety of snake and lizard venoms using this technique revealed the presence of multiple molecular weight forms of labeled enzymes, but the extent of diversity was less than that observed with the same venoms using separation of phospholipase electrophoretic variants on the basis of charge. The results are consistent with diversity in reptile venom phospholipase A2 forms being derived from differences in both molecular weight and charge.

Animals↗
PubMed1989

Isolation of an acidic phospholipase A2 from the venom of Agkistrodon acutus (five pace snake) and its effect on platelet aggregation.

A phospholipase A2 from the venom of the snake Agkistrodon acutus was purified by immunoaffinity chromatography and fast protein liquid chromatography (FPLC) as a single band by PAGE and SDS-PAGE. The estimated mol.wt was 16,400 by SDS-PAGE and 16,900 by gel filtration and the isoelectric point was 4.9. The ten N-terminal amino acid residues are homologous to those of the acidic phospholipases A2 from other crotalid venoms. The purified enzyme showed a potent inhibitory effect on platelet aggregation induced by ADP, collagen and sodium arachidonate in human platelet-rich plasma. The platelet aggregation by these inducers was completely suppressed when the concentration of the venom phospholipase A2 was 10-100 micrograms/ml. However, at 20 micrograms/ml, platelet aggregation could be elicited in washed human platelet suspension. Aspirin (28 micrograms/ml), an inhibitor of cyclooxygenase, inhibited the aggregating effect of the phospholipase A2. It is proposed that the stimulatory mechanism of the phospholipase A2 might be due to the liberation of arachidonic acid from phospholipids in the membrane of platelets and the formation of thromboxane A2.

Amino Acids↗
PubMed1990

Purification and amino acid sequence of basic protein I, a lysine-49-phospholipase A2 with low activity, from the venom of Trimeresurus flavoviridis (Habu snake).

A basic protein (pI 10.2), named basic protein I, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative of the protein and its peptides produced by chemical (cyanogen bromide and formic acid) and enzymatic (chymotrypsin, Achromobacter protease I, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was similar in sequence to phospholipases A2 from the venoms of crotalid and viperid snakes. A most striking feature of this protein is that aspartic acid at the 49th position common in phospholipases A2 is replaced by lysine. When the protein acted on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine, oleic acid was preferentially released, indicating that the protein has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was 1.5% that of T. flavoviridis phospholipase A2 isolated previously. The fact that both affinity to Ca2+ and reactivity to p-bromophenacyl bromide of basic protein I are approximately one order of magnitude lower than those of T. flavoviridis phospholipase A2 might explain the low activity of basic protein I.

Amino Acid Sequence↗
PubMed1990

Purification and properties of several phospholipases A2 from the venom of Australian king brown snake (Pseudechis australis).

Thirteen isoenzymes of phospholipases A2 were purified from the venom of Australian king brown snake, Pseudechis australis. They (except phospholipase A2 Pa-9C) showed normal properties of snake venom phospholipases A2; the apparent mol. wts were about 13,000, the optimum pH values were around 8, calcium ion was indispensable for the enzymatic activity and the optimum calcium ion concentrations were more than 5 mM. Phospholipase A2 Pa-9C had a lag period at the initial stage of the enzymatic reaction. The enzymatic activities determined by the titration method using 1,2-dipalmitoylglycerophosphocholine as a substrate at 37 degrees C were 10,500 units/mg for Pa-1G and 75 units/mg for Pa-13. The lethal activities measured by i.v. injections in mice were 0.09 micrograms/g body wt for Pa-5 and 6.8 micrograms/g body wt for Pa-13. The lethal activity correlates with the enzymatic activity (correlation coefficient of 0.92), and both activities showed no relationship to the basicity of the enzyme. Pa-1G is the first acidic phospholipase A2 (pI = 6.4) with high neurotoxicity (0.13 micrograms/g body wt).

Animals↗
PubMed1982

Inhibition of phospholipase A2 activity by Fluosol, an artificial blood substitute.

This paper describes the effect of Fluosol on phospholipase A2 activity both in vitro and in vivo. This compound inhibited both postheparin plasma and hepatic phospholipase A2 activities. Furthermore, in Fluosol transfused rats the postheparin plasma phospholipase A2 activity was 41% below that observed in control animals. The inhibition of phospholipase A2 activity was due to the micellar forms of pluoronic, F-68, a detergent present in Fluosol. It is postulated that the inhibition of phospholipase A2 activity by Fluosol might interfere with the release of arachidonic acid, a substrate used in biosynthesis of prostaglandins.

Animals↗
PubMed1989

Raman and infrared studies on the conformation of porcine pancreatic and Crotalus durissus terrificus phospholipases A2.

Raman and infrared spectroscopies were used to investigate conformational features of Crotalus durissus terrificus and porcine pancreatic phospholipases A2, as well as the proenzyme of the latter. The results indicate that conformational changes occur for the phospholipase molecules as a consequence of different experimental conditions such as change of physical state, presence of certain ionic species and interaction with a model substrate analog. Amorphous and crystalline solid phospholipase present discrepant conformational features. Conformational transitions were detected for the pancreatic zymogen----phospholipase A2 transformation and different secondary contents were observed for a toxic and a nontoxic form of the phospholipase molecule. All those structural changes have been shown to involve primarily the architecture of the polypeptide backbone rather than the conformation of amino acid residue side-chains. Disulfide bridges have shown consistently a gauche-gauche-gauche geometry which has not been disturbed by any of the experimental conditions employed. The external occurrence of tryptophan residues has been a common feature for the systems assayed, as well as the predominant localization of tyrosine residues in hydrophilic environment, probably at the molecular surface.

Animals↗
PubMed1982

Role of phospholipase A2 and prostaglandin E in growth and differentiation of myeloid leukemic cells.

Phospholipase A2 activity and prostaglandin E synthesis have been studied in different clones of myeloid leukemic cells, which differ in their competence to be induced to differentiate by the macrophage and granulocyte differentiation-inducing protein or the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). Clones that could be induced to differentiate by this protein showed a higher basal phospholipase A2 activity than clones that could not be induced to differentiate by this protein inducer. Cell competence to be induced to differentiate by TPA did not show this correlation, and the clone with the least ability to respond to TPA showed the lowest number of binding sites for [20-3H]phorbol 12,13-dibutyrate. Differentiation induced by the protein was accompanied by a 7-14-fold increase in prostaglandin E synthesis, whereas differentiation induced by TPA did not show this increase. Externally added prostaglandin E1 did not induce differentiation but inhibited cell proliferation and the degree of inhibition in the different clones was related to the basal phospholipase A2 activity. The results indicate that increase of prostaglandin E synthesis was not an essential pre-requisite for differentiation, that prostaglandin E seems to be involved in the inhibition of cell proliferation in association with phospholipase A2, and that the differentiation-inducing protein and TPA can induce differentiation by different pathways. The amount of basal phospholipase A2 activity was also related to previously found differences in the ability of the clones to develop desensitization to beta-adrenergic hormones or prostaglandin E1.

Animals↗
PubMed1987

Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells.

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.

Actins↗
PubMed1987

Stimulation of luteinizing hormone release by melittin and phospholipase A2 in rat pituitary cells.

Gonadotropin release in rat pituitary monolayer cultures was stimulated by phospholipase A2, as well as by its activator melittin. A dose-dependent stimulation of luteinizing hormone secretion by melittin was observed in a dose range of 10(-8) to 10(-4) M. A higher dose (1 mM) melittin had a sub-optimal effect. The stimulatory action of melittin was calcium-dependent and blocked by phospholipase A2 inhibitors, chloroquine and quinacrine. Similar to melittin, phospholipase A2 enhanced the effect of LH release in a dose range of 0.1-100 units/ml. The effect of this enzyme was also calcium-dependent with optimal calcium concentrations at 1.5 mM, as obtained also for melittin. In superfusion experiments, the stimulatory action of melittin and phospholipase A2 was reproducible in their effects on LH release in gonadotrophs. In addition, melittin (10(-7) M) stimulated LH and 3H-arachidonic acid efflux in superfused pituicytes following prelabelling with radiolabelled arachidonate. These data suggest that phospholipase A2, which releases arachidonic acid from phospholipids, may participate in controlling gonadotropin secretion in gonadotrophs, since arachidonic acid and its metabolites have previously been found to enhance gonadotropin release.

Animals↗
PubMed1988

The measurement of phospholipase A2 activity in human myometrium: physiological and pathological implications.

Phospholipase A2 activity was measured in human myometrium obtained at hysterectomy in a group of 41 patients using a double isotope ratio assay based on the liberation of [14-C] oleic acid from 1-palmitoyl-2-[14-C] oleoyl phosphatidylcholine. The enzyme was shown to be calcium independent and to have an optimum pH of 7. There was no significant difference (Mann Whitney U test) in myometrial phospholipase A2 activity between proliferative and secretory phases of the menstrual cycle (ranges: 3.88-30.8 and 0.47-25.85 nmol/mg protein per h respectively) but there was a significant (P less than 0.01) increase in activity in myometrium from uteri with fibroids (median 11.33, range 2.18-30.88 nmol/mg protein per h) compared to those without fibroids (median 6.94, range 0.31-25.85 nmol/mg protein per h). Myometrial phospholipase A2 activity was significantly lower (P less than 0.001) in the 33-40 age group (median 4.71, range 0.31-6.94) compared to the 41-50 age group (median 11.35, range 2.18-30.88 nmol/mg protein per h). In the 51-55 age group phospholipase A2 activity (median 8.71, range 2.5-17.71 nmol/mg protein per h) was not significantly different from that of the other two groups. The increase in activity in the 41-50 age group was not due to the increased incidence of uterine fibroids. These findings suggest that myometrial phospholipase A2 may be important in the pathophysiology of the uterus.

Adult↗
PubMed1992

Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro.

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled less than 10% of the pregnenolone metabolites. No estrogens or 5 alpha-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3 beta-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-beta to dienone but did not affect 17-hydroxylase or 'andien-beta-synthetase'. However, when the C-17, 20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.

Aldehyde-Lyases↗