BOVINE RETINAL PIGMENT EPITHELIUM CONTAINS NOVEL TYPES OF PHOSPHOLIPASE A2

We have recently demonstrated the presence of phospholipase A 2 (PLA 2 ) activity in cells from bovine retinal pigment epithelium (RPE) [Jacob et al. (1996) J. Biol. Chem. 271 , 19209-19218]. We report here our results on the characterization of this RPE-PLA 2 activity. We show that RPE probably contains two types of PLA 2 enzyme, as indicated by the results obtained with different PLA 2 -active fractions eluted from cation-exchange columns and treated with Ca 2+ /EGTA, dithiothreitol, p -bromophenacyl bromide or heat. These results, in addition to those from PLA 2 assays using different substrates, also suggest that RPE-PLA 2 enzymes are different from the well-known secretory, cytoplasmic and Ca 2+ -independent forms. Sequential extraction of RPE with (1) isotonic, (2) hypertonic and (3) detergent-containing PBS argues for the presence of weakly membrane-associated enzymes. Control experiments using ‘back and forth’ TLC allowed us to discriminate between PLA 2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA 2 enzymes. These results, together with those obtained by treating RPE homogenates with H 2 SO 4 , guanosine 5ʹ-[γ-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA 2 enzymes. We conclude that RPE contains novel types of PLA 2 that are different from the secretory, cytoplasmic and Ca 2+ -independent forms.