A Receptor and G-protein-regulated Polyphosphoinositide-specific Phospholipase C from Turkey Erythrocytes II. Pzy-PURINERGIC RECEPTOR AND G-PROTEIN-MEDIATED REGULATION OF THE PURIFIED ENZYME RECONSTITUTED WITH TURKEY ERYTHROCYTE GHOSTS*

The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a PZupurinergic receptor that employs an unidentified Gprotein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264,6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstution of the enzyme using [‘Hlinositol prelabeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to Pzu-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased Pzy-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 rg of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. I&, values for adenosine 5’-0-(2-thiodiphosphate), guanosine 5’-3-0-(thio)triphosphate, and AlF,activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.