title: Demonstration of similar calcium dependencies by mammalian high and low molecular mass phospholipase A2.
abstract: The in vitro Ca2+ dependencies of arachidonyl (AA)-selective high molecular mass phospholipase A2 (HMM, 85 kDa-PLA2) and human low molecular mass (LMM-Type II, 14 kDa)-PLA2 were compared. When the LMM-PLA2 and HMM-PLA2 enzymes were examined for hydrolysis against [3H]AA Escherichia coli in an ethyleneglycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA)-free buffer system, neither enzyme demonstrated activity below 10 microM free Ca2+. Beyond 11 microM Ca2+ both enzyme activities increased steadily exhibiting 50% of maximal activity at 0.1 and 1.0 mM, respectively. Using EGTA-regulated free Ca2+ buffers, both enzymes responded in a biphasic manner, achieving 50% of the maximum response by 0.5 microM Ca2+, stabilizing up to 0.1 mM, then further increasing with exposure to millimolar Ca2+ concentrations. Replacement of [3H]AA-labeled phosphatidylethanolamine vesicles for [3H]AA E. coli or using Tris-HCl buffer instead of HEPES buffer did not alter these findings significantly. The presence of EGTA had a pronounced concentration-dependent effect on the activity of both the HMM- and LMM-PLA2 enzymes but only in the range of 0 to 100 microM free Ca2+. EGTA (EC50 approximately 200 microM) reduced the concentration of Ca2+ required by PLA2 to achieve 50% of maximal acylhydrolysis. In contrast, the Type I bovine pancreatic PLA2 required millimolar Ca2+ concentrations to elicit 50% of the maximal response in both EGTA-free or EGTA-containing systems, which is concordant with its extracellular role as a digestive enzyme. These data suggest that the LMM-Type II PLA2 and HMM-PLA2 are both activated at submicromolar, intracellularly relevant, Ca2+ concentrations and therefore have the ability to contribute to cellular lipid metabolism.
authors.affiliation: SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.
authors.forename: L A
authors.initials: LA
authors.lastname: Marshall
authors.forename: A
authors.initials: A
authors.lastname: McCarte-Roshak
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002021:Buffers; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D002417:Cattle; D006224:Cricetinae; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010741:Phospholipases A / Q000187:drug effects* / Q000378:metabolism; D054467:Phospholipases A2; D014316:Tritium
chemical_list: D002021:Buffers; D007527:Isoenzymes; D014316:Tritium; D016718:Arachidonic Acid; D004533:Egtazic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium