THE GPPNHP-ACTIVATED ADENYLYL CYCLASE COMPLEX FROM TURKEY ERYTHROCYTE-MEMBRANES CAN BE ISOLATED WITH ITS BETA-GAMMA SUBUNITS

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[beta,gamma-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the beta-gamma subunits copurify with the cyclase. The stoichiometry of the cyclase to the alpha subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (alpha(s)) to the beta subunit is close to unity, demonstrating that the beta-gamma subunits do not dissociate from the G(s) . cyclase complex (G(s), guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the beta-gamma subunits could be separated from the alpha(s) . cyclase complex. Previously reported results on bovine brain cyclase also showed that the G(s) . cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999-10004]. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that beta-gamma subunits are not released from G(s) upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine-nucleotide-binding regulatory protein cannot be via shifting the alpha(s) to beta-gamma equilibrium as is commonly believed, and an alternate hypothesis is proposed.