G protein regulation of phospholipase C activity in a membrane-solubilized system occurs through a Mg²⁺- and time-dependent mechanism

GTP-binding proteins have been implicated to function as key transducing elements in the mechanism underlying receptor activation of a membrane-associated phospholipase C activity. In the present study, the regulation of phospholipase C activity by GTP-binding proteins has been characterized in a detergent-solubilized system derived from bovine brain membranes. Guanosine-5′-(3-O-thio)triphosphate (GTP-γ-S) and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) stimulated a dose-dependent increase in phospholipase C activity with half-maximal activation at 0.6 μM and 10 μM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-γ-S was comparable. 100 μM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 μM adenylyl-imidodiphosphate and ATP, did not stimulate phospholipase C activity, indicating that specific guanine nucleotide-dependent regulation of phospholipase C activity was preserved in the solubilized state. Gpp(NH)p or GTP-γ-S stimulation of phospholipase C activity was time-dependent and required Mg²⁺. Mg²⁺ regulated the time course for activation of phospholipase C by guanine nucleotides and the ability of guanine nucleotides to promote an increase in the Ca²⁺ sensitivity of phospholipase C. 200 μM GDP-β-S or 5 mM EDTA rapidly reversed the activation due to GTP-γ-S or Gpp(NH)p. These findings demonstrate that G protein regulation of phospholipase C activity in a bovine brain membrane- solubilized system occurs through a Mg²⁺ and time-dependent mechanism. Activation is readily reversible upon addition of excess GDP-β-S or removal of Mg²⁺.