Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.
The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but 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 three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.
Groupe de recherche en énergie et information biomoléculaires, Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Canada.
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Van Themsche
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Jacob
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Salesse
D015153:Blotting, Western; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism
D005227:Fatty Acids; D010741:Phospholipases A; D054467:Phospholipases A2