Purification and properties of phospholipase A1 from bovine brain.

Phospholipase A, (PLA,) was isolated from a soluble fraction of bovine brain.The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and heparin-Sepharose CL-GB column chromatography.Mono Q, Sephacryl 5-300, and Mono S high resolution column chromatography in the presence of the detergent 3-[(3-cholamidopropy1)dimethylammonio]-l-propanesulfonic acid (10 m) and glycerol (lo%, v/v) was required to further separate the enzyme from contaminating material.The purified PLA, eluted from the Sephacryl S-3OOHR column in a volume corresponding to a molecular mass of 365 kDa and migrated as two bands (M, = 112,000 and 95,000) when separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.Chromatofocusing, hydroxylapatite, and lectin affinity column chromatography and nondenaturing polyacrylamide gel electrophoresis were unsuccessful in separating the two electrophoretic bands, implying a close association or similarity.The purified enzyme was stable in solutions containing detergent and glycerol and was insensitive to metal chelators, dithiothreitol, phenylmethylsulfonyl fluoride, and diisopropyl fluorophosphate, but was inactivated by heat (60 "C) and ZnClz.At pH 7.5, the purified enzyme showed highest specific activity, 23.8 pmoYmin-mg, when l-palmitoyl-2-[1-'4C]arachidonoyl-phosphatidylethanolamine was the substrate.The rate of catalysis was optimal at a pH of 9.0 and could be enhanced 2-fold by Ca2+, M e , and S$+, but not Mn".The enzyme catalyzed the specific hydrolysis of acyl groups from the sn-1 position of a broad range of phospholipid substrates, including lysophospholipids, and accounts for most of the soluble phospholipase A, activity of bovine brain.Phospholipids are integral components of biological membranes and harbor within their structures a variety of bioactive agents (1, 2).One such agent is arachidonic acid which is mainly found in the sn-2 position, and hydrolysis of arachidonoyl-substituted phospholipids occurs preferentially during membrane phospholipid turnover (3-5).Arachidonic acid may modulate the activity of protein kinase C and other proteins ( 6 4 , but its most important role is to be oxygenated to eicosanoids (9-ll), which invoke a variety of physiological responses (11).Eicosanoid production is generally limited by the availability of free arachidonic acid (9,12), hence lipases which mobilize arachidonic acid are of considerable interest.Multiple pathways contribute to the formation of free arachi-* The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.