Purification and characterization of a soluble phospholipase A2 from guinea pig lung.
Guinea pig lung cytosolic phospholipase A2 was purified to near homogeneity by chromatography on a phosphocellulose column, followed by Q-Sepharose, S-Sepharose, gel filtration chromatography and reverse-phase HPLC. The purified enzyme exhibited an apparent molecular weight of 16,700 by SDS-polyacrylamide gel electrophoresis. Active enzyme eluted from the gel at an apparent molecular weight of 16,700. The purified enzyme exhibited a pH optimum of 9.0 and was calcium-dependent. Guinea pig lung phospholipase A2 hydrolyzed phosphatidylcholine and phosphatidylethanolamine equally well. Substrates containing unsaturated fatty acids in the sn-2 position were hydrolyzed preferentially to those containing saturated fatty acids. Anionic detergents stimulated enzyme activity while nonionic detergents inhibited the enzyme. Disulfide reducing agents dithiothreitol, glutathione and 2-mercaptoethanol modestly stimulated enzyme activity. The sulfhydryl aklylating agent n-ethylmaleimide had no effect on enzyme activity and only high concentrations of p-hydroxymercuribenzoic acid inhibited enzyme activity. The histidine modifying agent, bromophenacyl bromide did not inhibit guinea pig lung phospholipase A2 under conditions in which Crotalus adamanteus phospholipase A2 was inhibited 80%. Manoalide inhibited guinea pig lung phospholipase A2 in a concentration-dependent manner (IC50 = 2 microM). Antibodies prepared against porcine pancreatic phospholipase A2 specifically immunoprecipitated guinea pig lung phospholipase A2 suggesting that the major phospholipase A2 in guinea pig lung cytosol is immunologically related to pancreatic phospholipase A2 in agreement with the biochemical properties of the enzyme.
Bennett
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CF
Department of Molecular Pharmacology, Smith Kline and French Laboratories, King of Prussia, PA.
McCarte
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A
Crooke
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D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008970:Molecular Weight; D010741:Phospholipases A / Q000302:isolation & purification* / Q000378:metabolism; D054467:Phospholipases A2; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D003902:Detergents; D010741:Phospholipases A; D054467:Phospholipases A2