Characterization of acidic Ca2+-independent phospholipase A2 of bovine lung

An acidic Ca2+-independent phospholipase A(2) (aiPLA(2)) has been isolated previously from rat lung and a human cDNA has been described. This study applied the method to larger scale isolation of the native protein from the bovine lung. A polyclonal antibody was generated to a 15 amino acid synthetic peptide based on a conserved rat/human sequence. This antibody recognized a single protein band with an estimated molecular mass of approximate to 29 kDa in a soluble fraction obtained from bovine lung homogenate. A 29 kDa protein that reacted with the aiPLA(2) antipeptide antibody was detected in fractions containing aiPLA(2) activity on sequential column chromatographies. The partially purified enzyme showed 176-fold increase over the homogenate in Ca2+-independent pendent PLA(2) activity at pH 4. Activity was maximal with phosphatidylcholine substrate and was significantly less with phosphatidylethanolamine and anionic phospholipids. The enzyme had no acyl group preference in phosphatidylcholine and showed no preference for oxidized substrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiPLA(2) activity was inhibited by a transition state phospholipid analog (MJ33, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine protease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacyl bromide, and ATP. Analysis of N-terminal amino acid sequence for the 29 kDa protein demonstrated its high homology to human 26 kDa aiPLA(2). These was no significant change in molecular mass of the protein following treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA(2) immunoreactivity with lamellar body, lysosomal, and cytosolic fractions. These results indicate isolation from bovine lung of a 29 kDa acidic Ca2+-independent phospholipase A(2) homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholine. (C) 1998 Elsevier Science Inc. All rights reserved.