FATTY-ACID AND PHOSPHOLIPID SELECTIVITY OF DIFFERENT PBOSPHOLIPASE A(2) ENZYMES STUDIED BY USING A MAMMALIAN MEMBRANE AS SUBSTRATE

Previous studies using phospholipid mixed vesicles have demonstrated t hat several types of phospholipase A(2) (PLA(2)) enzymes exhibit diffe rent selectivity for fatty acids at the sn-2 position, for the type of chemical bond at the sn-1 position or for the phosphobase moiety at t he sn-3 position of phospholipids. In the present study, we have utili zed natural mammalian membranes from U937 monocytes to determine wheth er two purified 14 kDa PLA(2) isoenzymes (Type I, Type II) and a parti ally purified 110 kDa PLA(2) exhibit substrate selectivity for certain fatty acids or phospholipids. In these studies, arachidonic acid (AA) release from membranes was measured under conditions where the remode lling of AA mediated by CoA-independent transacylase (CoA-IT) activity has been eliminated. In agreement with the mixed-vesicle models, AA w as the major unsaturated fatty acid hydrolysed from membranes by the 1 10 kDa PLA(2), suggesting that this PLA(2) is selective in releasing A A from natural membranes. By contrast, Type I and Type II PLA(2)s were less selective in releasing AA from phospholipids and released a vari ety of unsaturated fatty acids at molar ratios that were proportional to the ratios of these fatty acids in U937 microsomal membranes. Exami nation of AA release from phospholipid classes indicated that all thre e enzymes released AA from the major AA-containing phospholipid classe s (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinos itol) of U937 membranes. The 110 kDa PLA(2) released AA from phospholi pid subclasses in ratios that were proportional to the AA content with in phospholipid classes and subclasses of U937 membranes. These data s uggested that the 110 kDa PLA(2) shows no preference either for the sn -1 linkage or for the sn-3 phosphobase moiety of phospholipids. By con trast, Type I and Type II PLA(2)s preferentially released AA from etha nolamine-containing phospholipids and appeared to prefer the 1-acyl-li nked subclass. Taken together, these data indicate that the 110 kDa PL A(2) selectively releases AA from U937 membranes, whereas Type I and T ype II PLA(2) release a variety of unsaturated fatty acids. Furthermor e, the 110 kDa PLA(2) releases the same molar ratios of AA from all ma jor phospholipid subclasses, whereas Type I and Type II PLA(2)s show s ome specificity for phosphatidylethanolamine when these enzymes are in cubated with a complex mammalian membrane substrate.