Purification and properties of phospholipase A1 from bovine brain.

摘要: Phospholipase A1 (PLA1) was isolated from a soluble fraction of bovine brain. The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and heparin-Sepharose CL-6B column chromatography. Mono Q, Sephacryl S-300, S high resolution chromatography in the presence detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (10 mM) glycerol (10%, v/v) required to further separate enzyme contaminating material. purified PLA1 eluted S-300HR volume corresponding molecular mass 365 kDa migrated as two bands (M(r) = 112,000 95,000) when separated by polyacrylamide gel electrophoresis sodium dodecyl sulfate. Chromatofocusing, hydroxylapatite, lectin affinity nondenaturing were unsuccessful separating electrophoretic bands, implying close association or similarity. stable solutions containing insensitive metal chelators, dithiothreitol, phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, but inactivated heat (60 degrees C) ZnCl2. At pH 7.5, showed highest specific activity, 23.8 mumol/min-mg, 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine substrate. rate catalysis optimal at 9.0 could be enhanced 2-fold Ca2+, Mg2+, Sr2+, not Mn2+. catalyzed hydrolysis acyl groups sn-1 position broad range phospholipid substrates, including lysophospholipids, accounts for most phospholipase activity