The deacylation of phosphatidylinositol (PI) in rat brain was studied in vitro. Using 1-acyl, 2-[1-14C-oleoyl] sn-glycerol-3-phosphoinositol and [U-14C]phosphatidylinositol as substrates, a release of 14C-free fatty acid was found when incubations were conducted with the PI labelled in the 2 position, both in the 12 000 – 106 000 g pellet (microsomal) and in the 106 000 g supernate prepared from rat brain homogenate. With the 106 000 g pellet the deacylation activity was linear with time up to 15 min and was directly proportional to the amount of protein added. Two pH optima were observed, one in the region of pH 7.5 which constituted the major activity and the other in the region of pH 6.0. The apparent Km for the enzyme activity at pH 7.5 was found to be 6.2 × 10−4 M and the Vmax was 1.24 nmol of [14C]oleic acid released per minute per milligram of protein. The Ca2+ ion stimulated the activity maximally at 5 mM while other divalent cations such as Mg2+, Mn2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Ni2+, Pb2+, and Zn2+ either partially or completely inhibited the activity. The nonionic detergent, Triton X-100, stimulated the deacylation more than twofold at a concentration of 0.01%. The sulfydryl reagents, p-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide showed partial inhibition of the reaction which was reversed by the addition of dithiothreitol. The deacylation activity in the 106 000 g supernate from rat brain was found to be directly proportional to the amount of protein added, and to the time (up to 15 min). A pH optimum was observed in the region of pH 6.0. Substrate concentration studies showed that the apparent Km was 5.0 × 10−4 M and the Vmax was 3.93 nmol of [14C]oleic acid released per minute per milligram of protein. "lyso-PI," diacylglycerol, and fatty acid were formed at pH 6.0 and pH 7.5. The data obtained indicate that from 54 (pH 7.5) to 70% (pH 6.0) of the altered PI is due to phospholipase A2 activity, 24 (pH 6.0) to 28% (pH 7.5) is due to phospholipase "C-like" activity, and from 2 (pH 6.0) to 22% (pH 7.5) may be due to phospholipase A1 activity. These results provide evidence for the deacylation component of a deacylation–reacylation cycle for the generation of specific molecular species of PI.
Studies on fatty acid oxidation. 8. The effects of fatty acids on metabolism of rat-brain cortex in vitro
