Phosphatidic acid and polyphosphoinositide formation in a broken cell preparation from rat brain: Effects of different incubation conditions.

The formation of phosphatidic acid (PA) and polyphosphoinositides in rat brains in vitro was studied under hypotonic conditions by incubating a lysed crude synaptosomal fraction with [?-(32)P]ATP for 10 s. Only phosphatidic acid, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) were labelled. The formation of PIP(2) decreased with increasing preincubation times. PIP and PA formation, however, were not affected. The rate of phosphorylation of PIP, PIP(2) and PA was linear with incubation time for at least 10 s, indicating that the ATP/enzyme ratio was sufficiently high. The maximal incorporation of phosphate into the three phospholipids was found at 30 degrees C. Ten millimolar Mg(2+) and pH 6.5 were the best compromise for measuring the incorporation of phosphate into PIP, PIP(2) and PA simultaneously. A sodium acetate-magnesium acetate buffer system gave optimal kinase activities. The results obtained show that, with minor adjustments of the incubation conditions, it is possible to measure simultaneously the formation of PIP, PIP(2) and PA.