The deacylation of phosphatidylinositol by rat brain preparations

Abstract

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.

Degradation of Arachidonoyl‐Labeled Phosphatidylinositols by Brain Synaptosomes

“…Phospholipase A activity is known to be activated by bile salts (Gatt, 1968;Cooper and Webster, 1970;Webster, 1973;, but the DG lipases (at pH 7-8) described by Cabot and Gatt (1976) are inhibited by bile salts as well as by Triton X-100. The results from this study are similar to those obtained by Shum et al (1979), although they incubated labeled diacyl-GPI with brain fractions in the presence of calcium and in the absence of bile salts. In their study, active deacylation of diacyl-GPI by phospholipase A (mainly A2) was observed, along with hydrolysis to give DG (Shum et al, 1979).…”

“…The results from this study are similar to those obtained by Shum et al (1979), although they incubated labeled diacyl-GPI with brain fractions in the presence of calcium and in the absence of bile salts. In their study, active deacylation of diacyl-GPI by phospholipase A (mainly A2) was observed, along with hydrolysis to give DG (Shum et al, 1979).…”

“…Since unsaturated fatty acids are released from membrane phospholipids through the action of phospholipase A, it is possible that these two hydrolytic processes are interrelated by some unknown mechanism yet to be investigated. We believe that the effect of calcium may be an important aspect for consideration, as this divalent cation could stimulate both phospholipase A2and diacyl-GPI-specific phospholipase C activities in brain and other tissues in the absence of detergent (Michell, 1975;Shum et al, 1979). Experiments are being conducted in our laboratory to explore these possibilities further.…”

Activities of enzymes metabolizing phospholipids in rat cerebral ischemia

“…Subcellular fractionation was carried out essentially by the method of Shum et al (1979). The brains were homogenized with 9 vol of ice-cold 0.25M sucrose.…”

Changes in lipid metabolites and enzymes in rat brain due to ischemia and recirculation

“…Subcellular fractions were prepared by the method of Shum et al (1979), and protein was determined by the method of Lowry et al (1951). The procedures for enzyme assays were described in detail in our previous communication (Hirashima et al, 1989).…”