title: Purification and properties of a phosphatidic acid-preferring phospholipase A1 from bovine testis. Examination of the molecular basis of its activation.
abstract: We recently identified a cytosolic phospholipase A1 activity in bovine brain and testis that preferentially hydrolyzes phosphatidic acid substrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in Triton X-100 mixed micelle assay system (Higgs, H.N., and Glomset J.A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9574-9578). In the present work we purified the bovine testis enzyme 14,000-fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl arachidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresis to provide evidence that it is a homotetramer of 110-kDa subunits. Studies of the molecular basis of the enzyme reaction in Triton micelles revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl-analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC50 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) the activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain double bonds.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry, University of Washington, Seattle 98195-7370, USA.
authors.forename: H N
authors.initials: HN
authors.lastname: Higgs
authors.forename: J A
authors.initials: JA
authors.lastname: Glomset
mesh_terms: D000818:Animals; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006868:Hydrolysis; D008297:Male; D008823:Micelles; D010712:Phosphatidic Acids / Q000378:metabolism*; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054466:Phospholipases A1; D013737:Testis / Q000201:enzymology*
chemical_list: D008823:Micelles; D010712:Phosphatidic Acids; D010741:Phospholipases A; D054466:Phospholipases A1