Properties and purification of an arachidonoyl-hydrolyzing phospholipase A 2 from a macrophage cell line, RAW 264.7

Abstract

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1- O-alkyl-2-arachidonoylglycerophosphocholine (1- O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A 2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A 2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A 2 that hydrolyzed 1-O- hexadecyl-2-[ 3H] arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (l-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A 2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A 2 eluted between molecular weight markers of 67000 and 150000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 μmol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A 2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.