Purification and properties of phospholipase A1 from bovine brain.
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, and Mono S high resolution column chromatography in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (10 mM) and glycerol (10%, v/v) was required to further separate the enzyme from contaminating material. The purified PLA1 eluted from the Sephacryl S-300HR column in a volume corresponding to a molecular mass of 365 kDa and migrated as two bands (M(r) = 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 degrees C) and ZnCl2. At pH 7.5, the purified enzyme showed highest specific activity, 23.8 mumol/min-mg, when 1-palmitoyl-2-[1-14C]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+, Mg2+, and Sr2+, but not Mn2+. 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 A1 activity of bovine brain.
Pete
M J
MJ
Howard Hughes Medical Institute, Vanderbilt University School of Medicine Nashville, Tennessee 37232-0295.
Ross
A H
AH
Exton
J H
JH
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002793:Cholic Acids / Q000494:pharmacology; D002845:Chromatography / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D010741:Phospholipases A / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D054466:Phospholipases A1; D010743:Phospholipids / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013379:Substrate Specificity
D002793:Cholic Acids; D010743:Phospholipids; D012492:Salts; D010741:Phospholipases A; D054466:Phospholipases A1; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate