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

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