A novel phosphatidylglycerol-selective phospholipase A(2) from macrophages

In our recent studies on the synthesis of bis(monoacylglycero)phosphate (BM P), we postulated that the first step involved a PLA(2) that cleaved the 2- acyl group from phosphatidylglycerol (PC). In the present study, a novel ly sosomal PLA2 was partially purified and characterized from RAW 264.7, macro phage like cells. Cells were homogenized and delipidated, and the PLA2 acti vity in the soluble fraction was purified by Sephacryl S100 and DEAE Sephac el. Further purification was performed using Con-A Sepharose, Phenyl Sephar ose, DEAE Sephacel, and Superdex 75 FPLC. The enzyme at this stage of purif ication showed a dominant band around 45 kDa plus several minor bands on SD S-PAGE. The molecular mass determined by Superdex 75 column FPLC was about 45 kDa. The highly purified fraction hydrolyzed at the sn-l position, imply ing that this PLA2 also has some intrinsic PLA(1) activity. This enzyme pre ferentially hydrolyzed PG, has an acidic pH optima, and does not require di valent metal ions. Comparison using PG with various acyl chains on the sn-2 position showed that oleate and linoleate were preferred relative to arach idonate. MAFP, a known cytosolic PLA2 inhibitor, strongly inhibited this PL A2 activity. MJ33, AACOCF(3), DENP, and Amiodarone also gave moderate inhib ition. The characteristics of this enzyme showed this to be a new type of P LA, and the overwhelming preference for PG as substrate suggests its physio logical role is in the biosynthesis of BMP.