title: Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements.
abstract: The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.
authors.affiliation: Howard Hughes Medical Institute, Vanderbilt University School of Medicine, Nashville, TN 37232.
authors.forename: S J
authors.initials: SJ
authors.lastname: Taylor
authors.forename: J H
authors.initials: JH
authors.lastname: Exton
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001127:Arginine Vasopressin / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols / Q000378:metabolism; D054801:Phosphoinositide Phospholipase C; D010743:Phospholipids / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
chemical_list: D006150:Guanine Nucleotides; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D010743:Phospholipids; D001127:Arginine Vasopressin; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D008274:Magnesium; D002118:Calcium