DIRECT LABELING OF HORMONE-SENSITIVE PHOSPHOINOSITIDES BY A PLASMA-MEMBRANE-ASSOCIATED PTDINS SYNTHASE IN TURKEY ERYTHROCYTES

We have previously characterized phosphatidylinositol (PtdIns) synthas e and PtdIns/myo-inositol-exchange enzyme activities in ghost membrane s prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Va ziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that Pt dIns synthase activity is relatively enriched in plasma-membrane prepa rations of turkey erythrocytes and that inositol phospholipids labelle d by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated ph ospholipase C (PLC), which is present also in ghost preparations. Spec ific-radioactivity measurements of [H-3]PtdIns from ghosts labelled to equilibrium under conditions favouring [H-3]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly a ccess approx. 14 % of the total erythrocyte ghost PtdIns. Approx. 16 % of the [H-3]PtdIns labelled by the PtdIns synthase reaction can be ph osphorylated to polyphosphoinositides, which are then hydrolysed by th e receptor- and G-protein-stimulated PLC. Since the mass of PtdIns dec lines to a similar extent as [H-3]PtdIns during stimulation in the pre sence of guanine nucleotides and ATP, it is evident that both the labe lled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma mem branes were also labelled by [H-3]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities res pectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-229811, but the labelled lipids were sensit ive to purinergic-receptor-stimulated hydrolysis in reconstitution ass ays using partially purified turkey erythrocyte PLC. The results stron gly suggest that at least a portion of the PtdIns synthase in turkey e rythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.