Purification and characterization of Ca2+-dependent phospholipases A(2) from rat kidney

Three phospholipase A(2) (PLA(2)) activities were identified in rat kidney, In the particulate fraction a PLA(2) activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group II PLA(2). This PLA(2) 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 PLA(2) from rat liver mitochondria, platelet, and spleen. The cytosolic fraction of rat kidney contained at least two PLA(2) 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 PLA(2), exhibited a preference for arachidonic acid at the sn-2 position of glycerophospholipids, and was already optimally active at submillimolar Ca2+ concentrations. The cytosolic PLA(2) activity that did not bind to the anion-exchange column was purified by gel filtration, immunoaffinity chromatography using immobilized polyclonal antibodies to group I PLA(2), and C18 reversed-phase chromatography. Immunological properties and N-terminal sequence analysis identified this enzyme as rat group I PLA(2). Rat glomerular mesangial cells contained only group II and high molecular mass PLA(2) enzymes. (C) 1996 Academic Press, Inc.