Purification and characterization of Ca(2+)-dependent phospholipases A2 from rat kidney.

Three phospholipase A2 (PLA2) activities were identified in rat kidney. In the particulate fraction a PLA2 activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group II PLA2. This PLA2 was partially solubilized and purified to near homogeneity. The amino acid sequence at the N-terminus of the purified enzyme was identical to that of the 14-kDa rat group II PLA2 from rat liver mitochondria, platelet, and spleen. The cytosolic fraction of rat kidney contained at least two PLA2 activities which could be separated on a Mono Q column. Upon gel filtration the activity that eluted from the anion-exchange column in the salt gradient behaved as a high molecular mass PLA2, exhibited a preference for arachidonic acid at the sn-2 position of glycerophospholipids, and was already optimally active at submillimolar Ca2+ concentrations. The cytosolic PLA2 activity that did not bind to the anion-exchange column was purified by gel filtration, immunoaffinity chromatography using immobilized polyclonal antibodies to group I PLA2, and C18 reversed-phase chromatography. Immunological properties and N-terminal sequence analysis identified this enzyme as rat group I PLA2. Rat glomerular mesangial cells contained only group II and high molecular mass PLA2 enzymes.