Reconstitution of catecholamine-stimulated guanosinetriphosphatase activity.
beta-Adrenergic receptors were partially purified from turkey erythrocyte membranes by alprenolol-agarose chromatography to 0.25-2 nmol/mg of protein, and the stimulatory guanosine 5'-triphosphate (GTP) binding protein of adenylate cyclase (Gs) was purified from rabbit liver. These proteins were reconstituted into phospholipid vesicles by addition of phospholipids and removal of detergent by gel filtration. This preparation hydrolyzes GTP to guanosine 5'-diphosphate (GDP) plus inorganic phosphate (Pi) in response to beta-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed min-1 X mol-1 of Gs. This low rate may be limited by the hormone-stimulated binding of substrate, since it is roughly equal to the rate of binding of the GTP analogue guanosine 5'-O-(3-[35S] thiotriphosphate) [( 35S]GTP gamma S) to Gs in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a beta-adrenergic antagonist, is 8-25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 microM GTP, and the response to GTP is formally consistent either with the existence of multiple Km's or of a separate stimulatory site for GTP. The GTPase activity of Gs in vesicles is also stimulated by 50 mM MgCl2 in the presence or absence of receptor. Significant GTPase activity is not observed with Lubrol-solubilized Gs, although [35S]-GTP gamma S binding is increased by Lubrol solubilization.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D004910:Erythrocyte Membrane / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000097:blood*; D019204:GTP-Binding Proteins; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000097:blood*; D011817:Rabbits; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D014422:Turkeys
D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol