Studies on enzymes related to diacylglycerol production in activated platelets. II. Subcellular distribution, enzymatic properties and positional specificity of diacylglycerol- and monoacylglycerol-lipases.
The subcellular distribution of diacylglycerol- and monoacylglycerol-lipases has been studied in human platelets. Using a fractionation procedure on Percoll gradient (Perret, B., Chap, H. and Douste-Blazy, L. (1979) Biochim. Biophys. Acta 556, 434-446), the enzyme activity displayed the same profile as that of [3H]concanavalin A, a plasma membrane marker. This result was confirmed with highly purified platelet plasma membranes prepared by adsorption onto polyethylenimine-bonded polyacrylamide beads (Kinoshita, T., Nachman, R.L. and Minick, R. (1979) J. Cell Biol. 82, 688-696). Studies with isolated membranes or crude homogenate revealed that the enzyme requires calcium or magnesium and displays an optimal pH of 6.2, showing that it is able to hydrolyse diacylglycerol under conditions where phosphatidylinositol-specific phospholipase C is fully active. Using diacylglycerol labelled in the 1- or 2-position, it was found that the two fatty acids are released at the same rate, which is supported by the lack of monoacylglycerol accumulation and by the observation that monoacylglycerol is hydrolysed at a 20-fold faster rate than diacylglycerol. Increasing concentrations of Mg-ATP promote the conversion of diacylglycerol into phosphatidic acid by diacylglycerol kinase, but only high concentrations become inhibitory for diacylglycerol lipase. These results are discussed in the light of our former hypothesis that arachidonic acid release from platelet phospholipids might occur through the sequential action of a phosphatidylinositol-specific phospholipase C coupled to a diacylglycerol lipase (Mauco, G., Chap, H., Simon, M.F. and Douste-Blazy, L. (1978) Biochimie 60, 553-561). The possible role of this enzyme in the regulation of the activity of protein kinase C is also emphasized.
D001792:Blood Platelets / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000097:blood*; D002413:Cations, Divalent; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D002499:Centrifugation, Density Gradient; D004075:Diglycerides / Q000097:blood*; D005989:Glycerides / Q000097:blood*; D006801:Humans; D007700:Kinetics; D008071:Lipoprotein Lipase / Q000097:blood* / Q000302:isolation & purification; D008994:Monoacylglycerol Lipases / Q000097:blood* / Q000302:isolation & purification; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D002413:Cations, Divalent; D004075:Diglycerides; D005989:Glycerides; D002265:Carboxylic Ester Hydrolases; D008994:Monoacylglycerol Lipases; D008071:Lipoprotein Lipase