GTP-binding proteins in membranes and the control of adenylate cyclase activity.

摘要: To identify the GTP-binding site of catecholamine-stimulated adenylate cyclase (EC 4.6.1.1) from pigeon erythrocyte membranes photoreactive GTP derivatives have been synthesized. One them, P”-(4-azidoanilido)-PI-5’-guanosine triphosphate, proved to be a potent activator particulate and soluble which binds with high affinity (K,,ss 3.3 x lo-’ M) competes effectively guanyl-5’yl imidodiphosphate (Gpp(NH)p) for same binding site. Photoactivation [“‘PIPJ-(4-azidoanilidoj-PI-5’-GTP-labeled resulted in covalent incorporation label into four major proteins M, = 86,000, 52,000, 42,000, 23,000, whereas Lubrol PX-solubilized only 42,000 23,000 were covalently labeled, although preparations stimulated by guanylnucleotides about extent as membranous preparations. The bulk nucleotide sites, >95%, could separated without loss activity centrifugation solubilized through sucrose density gradient, most protein remained associated activity. Detergent-solubilized inactivated on contact GTP-Sepharose matrix. Inactivation was due dissociation complex two fractions, one contained guanylnucleotide-binding sites. Reactivation occurred recombination fraction released Gpp(NH)p or not adsorbed GTP-Sepharose. Furthermore, reactivated rabbit myocardial depleted proteins. Reconstitution experiments fractions obtained isoproterenol-treated suggested that characteristic synergistic amplification hormone action is mediated via guanyl protein. Guanylnucleotide-binding also