PURIFICATION OF A 110-KDA PHOSPHOINOSITIDE PHOSPHOLIPASE-C THAT IS ACTIVATED BY G-PROTEIN BETA-GAMMA-SUBUNITS

We report the purification from bovine brain cytosol of a 110-kDa phosphoinositide-specific phospholipase C (PLC-110) that was markedly stimulated by G-protein betagamma-subunits. The enzyme was purified approximately 2000-fold with a yield of 4%. On the basis of size and immunological cross-reactivity, PLC-110 was distinct from 150-kDa PLC-beta1, 145-kDa PLC-gamma1, and 85-kDa PLC-delta1. An antiserum to a peptide corresponding to a conserved PLC Y domain sequence cross-reacted with PLC-110. PLC-110 was also recognized by two antisera selective for NH2-terminal and internal sequences in PLC-beta3, but not by a third peptide antiserum to the COOH terminus of this enzyme, suggesting that PLC-110 is related to PLC-beta3. Reconstitution of purified PLC-110 with betagamma-subunits produced greater than 100-fold activation, indicating that no other proteins were required. Half-maximal activation was observed at approximately 60 nM betagamma and full activation at approximately 500 nM betagamma. PLC-110 maximally hydrolyzed phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate at 1 muM Ca2+, but showed no activity toward phosphatidylinositol at Ca2+ concentrations up to 1 mM. Concentrations of purified guanosine 5'-O-(3-thiotriphosphate)-liganded alpha(q) that fully activated PLC-beta1 failed to stimulate PLC-110. This observation indicates that the site at which betagamma interacts with PLC-110 is distinct from that at which alpha(q) regulates the activity of PLC-beta isozymes.