Reconstitution of catecholamine-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase.
The stimulatory GTP-binding protein (Gs) of adenylate cyclase, purified from rabbit liver, and beta-adrenergic receptors, partially purified 1000-4000-fold from turkey erythrocyte plasma membranes, were coreconstituted into unilamellar phospholipid vesicles. The molar ratio of Gs to receptors in the vesicles varied from 3 to 10 in different preparations, as measured by guanosine 5'-O-(3-[35S]thiotriphosphate) [( 35S]GTP gamma S) binding to Gs and [125I]iodocyanopindolol binding to receptors. Activation of reconstituted Gs by GTP gamma S was stimulated up to 10-fold by the addition of the beta-adrenergic agonist (-)-isoproterenol. Activation was assayed functionally by reconstitution with the catalytic unit of adenylate cyclase. Because of the relative purity of this preparation, the quasi-irreversible binding of [35S]GTP gamma S could also be measured in the vesicles and was shown to parallel the functional activation of Gs under all conditions. Most of the assayable Gs in the vesicles could interact with the receptors and undergo agonist-stimulated activation. Agonist-stimulated activation and [35S]GTP gamma S binding were complete in less than 3 min, even under suboptimal conditions, and could go to completion in less than 20 s under maximal stimulation. Agonist-stimulated binding did not require appreciable free Mg2+ (less than 0.1 mM). Activation in the absence of agonist was stimulated by free Mg2+, but maximal activation took up to 10 min in the presence of 50 mM MgCl2. Reconstitution increased the stability of Gs to thermal denaturation. The addition of beta-adrenergic agonist further stabilized Gs, presumably by the formation of a stable agonist-receptor-Gs complex.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D004355:Drug Stability; D004910:Erythrocyte Membrane / Q000032:analysis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D006358:Hot Temperature; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000032:analysis; D008274:Magnesium / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011489:Protein Denaturation; D011817:Rabbits; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*; D014422:Turkeys
D008081:Liposomes; D011943:Receptors, Adrenergic, beta; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D008274:Magnesium; D007545:Isoproterenol