The GppNHp‐activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its βγ subunits

Abstract

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5′‐[β,γ‐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 βγ subunits copurify with the cyclase. The stoichiometry of the cyclase to the α subunit of the stimulatory guanosine‐nucleotide‐binding regulatory protein (α_s) to the β subunit is close to unity, demonstrating that the βγ 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 β_i subunits could be separated from the α_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 βγ 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 α_s to βγ equilibrium as is commonly believed, and an alternate hypothesis is proposed.