Reconstitution of Catecholamine-Stimulated Guanosinetriphosphatase Activity

β-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 (G_s) 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 (P_i) in response to β-adrenergic agonists. The initial rate of isoproterenol-stimulated hydrolysis is approximately 1 mol of GTP hydrolyzed·min⁻¹·mol⁻¹ of G_s. 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]GTPγS) to G_s in the vesicles. Activity in the absence of agonist, or in the presence of agonist plus a β-adrenergic antagonist, is 8–25% of the hormone-stimulated activity. Guanosinetriphosphatase (GTPase) is not saturated at 10 µM GTP, and the response to GTP is formally consistent either with the existence of multiple K_m's or of a separate stimulatory site for GTP. The GTPase activity of G_s 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 G_s, although [³⁵S]-GTP-γS binding is increased by Lubrol solubilization.