Bovine retinal pigment epithelium contains novel types of phospholipase A2.
We have recently demonstrated the presence of phospholipase A2 (PLA2) 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-PLA2 activity. We show that RPE probably contains two types of PLA2 enzyme, as indicated by the results obtained with different PLA2-active fractions eluted from cation-exchange columns and treated with Ca2+/EGTA, dithiothreitol, p-bromophenacyl bromide or heat. These results, in addition to those from PLA2 assays using different substrates, also suggest that RPE-PLA2 enzymes are different from the well-known secretory, cytoplasmic and Ca2+-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 PLA2 and phospholipase C/diacylglycerol lipase activity and confirmed that, in our assay conditions, the release of fatty acids was indeed due to PLA2 enzymes. These results, together with those obtained by treating RPE homogenates with H2SO4, guanosine 5'-[gamma-thio]triphosphate, ATP and different protease inhibitors, permitted us to make the first characterization of these RPE-PLA2 enzymes. We conclude that RPE contains novel types of PLA2 that are different from the secretory, cytoplasmic and Ca2+-independent forms.
Jacob
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M
GREIB, Département de Chimie-Biologie, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada G9A 5H7.
Weech
P K
PK
Salesse
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D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000378:metabolism; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
D005230:Fatty Acids, Nonesterified; D007527:Isoenzymes; D010741:Phospholipases A; D008071:Lipoprotein Lipase; D054467:Phospholipases A2; D010738:Type C Phospholipases