MEMBRANE-BINDING OF PHOSPHOLIPASES C-BETA(1) AND C-BETA(2) IS INDEPENDENT OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE AND THE ALPHA-SUBUNIT AND BETA-GAMMA-SUBUNIT OF G-PROTEINS

We have measured the membrane binding affinities of purified phosphati dylinositol-specific phospholipases C-beta(1) and C-beta(2) to membran es of varying lipid composition using fluorescence methods. Our studie s show that these proteins bind with affinities of 10(-5)-10(-4) M, wi th a small dependence on lipid type. Binding was relatively insensitiv e to the presence of phosphatidylinositol-specific phospholipases C-be ta s' major physiological substrate, phosphatidylinositiol 4,5-bisphos phate, as well as the presence of Ca2+, which is required for activity . The presence of purified GTP gamma S-activated alpha(11) subunits of heterotrimeric guanine nucleotide binding proteins (G proteins) did n ot alter the membrane binding affinity of phosphatidylinositol-specifi c phospholipases C-beta(1), even though all is a potent activator of t his protein. Similarly, the presence of purified beta gamma subunits o f G proteins did not alter the membrane association of phosphatidylino sitol specific phospholipases C-beta(2) even though these subunits str ongly activate this isoform. These results argue against a recruitment model for PLC-beta activation by G proteins, negatively charged lipid s, Ca2+, or substrate, and suggest that activation occurs through asso ciation of the membrane-bound species.