Diacylglycerol kinase and lipase activities in rat brain subcellular fractions

Phosphatidic acid synthesis via diacylglycerol kinase and free fatty acid release via diacylglycerol lipase were investigated in rat brain subcellular fractions using membrane-bound [I-(14)C]arachidonoyl-diacylglycerol as substrate. Labeled diacylglycerol was generated by incubating brain membranes containing [I-(14)C]arachidonoyl-phosphatidylinositols in the presence of deoxycholate and Ca(2+). Incubation of the prelabeled synaptosomes enriched in [1-(14)C]arachidonoyl-diacylglycerols or incubation of brain subcellular fractions with heat-treated prelabeled membranes resulted in the release of free fatty acids from the diacylglycerols. When incubations were carried out in the presence of ATP, MgCl(2) and NaF, both free fatty acid release and conversion of diacylglycerols to phosphatidic acids were observed. The conversion of diacylglycerols to phosphatidate or their hydrolysis to free fatty acids were linear with time for at least 15 min. In three brain subcellular fractions examined, diacylglycerol kinase activity indicated a pH maximum of 7.4. The free fatty acid release was enhanced slightly by Ca(2+) (1 mM), but Ca(2+) (0.5-4 mM) in the presence of Mg(2+) (10 mM) was inhibitory to the diacylglycerol kinase reaction. Phosphatidate formation was also inhibited by an excessive amount of deoxycholate added to the incubation mixture. Among the brain subcellular fractions, diacylglycerol kinase was more active in synaptic vesicles and cytosol than in the microsomal fraction, whereas diacylglycerol lipase activity was higher in the cytosol fraction than in the membrane fractions. Upon washing the membranes by centrifugation, a substantial portion of the diacylglycerol kinase activity was removed after the first washing, whereas the diacylglycerol lipase activity remained essentially unchanged. The metabolic role of arachidonoyl-diacylglycerols in brain membranes in relation to the biosynthesis of phosphatidate and the release of arachidomic acid is discussed.