PURIFICATION AND PROPERTIES OF A PHOSPHATIDIC ACID-PREFERRING PHOSPHOLIPASE A(1) FROM BOVINE TESTIS - EXAMINATION OF THE MOLECULAR-BASIS OFITS ACTIVATION

We recently identified a cytosolic phospholipase A(1) activity in bovi ne brain and testis that preferentially hydrolyzes phosphatidic acid s ubstrates. We also showed that the enzyme displays sigmoidal kinetics toward phosphatidic acid substrates in a Triton X-100 mixed micelle as say system (Higgs, H. N., and Glomset, J. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9574-9578), In the present work we purified the bovi ne testis enzyme 14,000 fold and used a combination of size exclusion chromatography, labeling with the phospholipase A inhibitor, methyl ar achidonyl fluorophosphonate, and SDS-polyacrylamide gel electrophoresi s to provide evidence that it is a homotetramer of 110-kDa subunits. S tudies of the molecular basis of the enzyme reaction in Triton micelle s revealed that (a) a nonhydrolyzable sn-1-alkyl-2-oleoyl analogue of phosphatidic acid activated the enzyme 30-fold in a sigmoidal fashion (Hill coefficient 3.2, EC(50) 4 mol %) without substantially affecting its preference for specific diacyl phosphoglyceride substrates, (b) t he activator promoted tight binding of the enzyme to micelles, and (c) the enzyme's activity toward unsaturated phosphatidic acid substrates was affected by the location and nature of the fatty acyl chain doubl e bonds.