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We have investigated the effects of different lipids on the activity of a phosphatidylinositol (PI)-specific phospholipase C isolated from sheep seminal vesicular glands.Dispersions of PI in the absence of detergent are hydrolyzed at 1-3 pmol/min/mg of protein, a rate only 3-10% of that obtained when optimal concentrations of sodium deoxycholate are present.When hydrolysis of PI in microsomes from mouse L-cells is measured, only 0.01 pmol of PI is hydrolyzed/min/mg of protein.Lipid dispersions prepared from extracts of microsomes are also poor substrates.We added varying amounts of other phospholipids to PI dispersions to explore the effect on PI hydrolysis.Phosphatidylserine (PS) and phosphatidylethanolamine (PE) had only modest effects on PI hydrolysis, but phosphatidylcholine (PC) was markedly inhibitory.Diglycerides stimulated PI hydrolysis in dispersions by about 10-fold.Using small unilamellar vesicles containing PI and PE (1:0.4),we found 50 pmol of PI hydrolyzed/min/mg of protein.Incorporation of PC in increasing amounts into these vesicles inhibited PI hydrolysis by 75% at 1 PC/PI, and at 4 PC/PI, no hydrolysis occurred.PC incorporated into vesicles separate from those containing PI was not inhibitory.Incorporation of PS into vesicles containing PI and PC overcame the inhibition of PI hydrolysis in a calcium ion-and PS-dependent manner.PI hydrolysis at 3 m M Ca2' using vesicles containing PI/PC/PS/PE in proportions of 1:4:2:0.4 was 2.5 pmol/min/mg of protein, compared to undetectable hydrolysis in similar vesicles without PS.Addition of diglyceride further increased the rate of hydroIysis by 2.5-fold.We conclude that PC inhibits PI hydrolysis by interacting with the substrate.The inhibition is overcome by negatively charged lipids and high concentrations of calcium ions.Diglyceride, a product of PI hydrolysis, further stimulates PI breakdown.Since negatively charged lipids are concentrated on the inner leaflet of most cell membranes, we postulate that calcium fluxes resulting from stimulation of cell surface receptors may initiate PI breakdown which is further accelerated as diglyceride accumulates.The physiological significance of agonist-stimulated PI'
Hofmann et al. (Wed,) studied this question.