G-PROTEIN-MEDIATED INHIBITION OF PHOSPHOLIPASE-C ACTIVITY IN A SOLUBILIZED MEMBRANE PREPARATION

In solubilized bovine brain membrane preparations AlF4- (20 muM AlCl3 plus 10 mM NaF) and 50 nM guanosine 5-O-(2-thiotriphosphate) (GTPgammaS) promoted a rapid but transient inhibition of phospholipase C (PLC) activity. Maximal inhibition was evident within 7 min of incubation, followed by reversal of inhibition. In contrast, 10 muM GTPgammaS did not induce inhibition of PLC activity but rather produced a time-dependent stimulation of PLC activity. GTPgammaS-dependent inhibition of PLC activity was concentration-dependent with half-maximal inhibition at 1 nM. Inhibition was antagonized by guanosine 5-O-(2-thiodiphosphate (GDPbetaS). Pertussis toxin delayed the onset of inhibition by GTPgammaS but did not prevent the inhibitory effect. Alpha(o)-GTPgammaS or alpha(o)-GDP had little effect on PLC activity. Alpha(i)-GTPgammaS and alpha(i)-GDP produced a 15% inhibition of PLC activity. Betagamma subunits did not inhibit basal PLC activity but did attenuate the net degree of inhibition due to GTPgammaS. Inhibition was associated with a decrease in the Ca2+ sensitivity of PLC. Preincubation of membranes with anti-PLC-beta1 antibody, but not anti-PLC-gamma1 or anti-PLC-delta1, prevented the GTPgammaS-mediated inhibition of PLC. These studies implicate PLC-beta1 as an effector system that is under negative modulation by a G protein-dependent mechanism.