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-gamma-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 microM and 10 microM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-gamma-S was comparable. 100 microM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 microM 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-gamma-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 microM GDP-beta-S or 5 mM EDTA rapidly reversed the activation due to GTP-gamma-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-beta-S or removal of Mg2+.
Litosch
I
I
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism*; D008274:Magnesium; D010738:Type C Phospholipases / Q000378:metabolism*
D006165:Guanylyl Imidodiphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010738:Type C Phospholipases; D008274:Magnesium; D002118:Calcium