A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from Turkey erythrocytes: II. P_2Y-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 P_2Y-purinergic receptor that employs an unidentified G-protein 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 [³H]inositol 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 P_2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P_2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 μg 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. K_0.5 values for adenosine 5′-O-(2-thiodiphosphate), guanosine 5′-3-O-(thio)triphosphate, and AlF₄^- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.