Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

The sensitivity of different phospholipase A2 (PLA2)-active fractions elute d from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigme nt epithelial (hRPE) cells probably contain two different intracellular PLA 2 enzymes. Control experiments using "back-and-forth" thin-layer chromatogr aphy confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experi ments where no cross-reactivity was observed between the hRPE cytosolic PLA 2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) an d cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PL A2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s . We also report an additional hRPE-PLA2 enzyme that is secreted and that e xhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is char acteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted w eakly with an antiserum directed against porcine pancreatic group I sPLA2 b ut strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are th ree distinct PLA2 enzymes in cultured hRPE cells, including two novel intra cellular PLA2s and a 22-kDa secreted sPLA2 enzyme.