CHARACTERIZATION OF ACIDIC CA2-INDEPENDENT PHOSPHOLIPASE A(2) OF BOVINE LUNG()

An acidic Ca2+-independent phospholipase A(2) (aiPLA(2)) has been isol ated previously from rat lung and a human cDNA has been described. Thi s study applied the method to larger scale isolation of the native pro tein 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 mole cular mass of approximate to 29 kDa in a soluble fraction obtained fro m 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 purifie d enzyme showed 176-fold increase over the homogenate in Ca2+-independ ent pendent PLA(2) activity at pH 4. Activity was maximal with phospha tidylcholine substrate and was significantly less with phosphatidyleth anolamine and anionic phospholipids. The enzyme had no acyl group pref erence in phosphatidylcholine and showed no preference for oxidized su bstrate, but activity was less with 1-O-alkyl phosphatidylcholine. aiP LA(2) activity was inhibited by a transition state phospholipid analog (MJ33, cyl-3-trifluoroethylglycero-sn-2-phosphomethanol), serine prot ease inhibitors, and the anti-peptide antibody but was insensitive to arachidonoyl trifluoromethyl ketone, bromoenol lactone, p-bromophenacy l 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 fo llowing treatment with endoglycosidase F. Western blot of subcellular fractions from rat lung indicated aiPLA(2) immunoreactivity with lamel lar body, lysosomal, and cytosolic fractions. These results indicate i solation from bovine lung of a 29 kDa acidic Ca2+-independent phosphol ipase A(2) homologue of the rat and human enzyme and provide evidence for specificity in the metabolism of lung surfactant phosphatidylcholi ne. (C) 1998 Elsevier Science Inc. All rights reserved.