β Subunit copurifies with GppNHp-activated adenylyl cyclase

Previous kinetic studies (Tolkovsky, A. M., Braun, S., and Levitzki, A. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 213-222) and biochemical studies (Arad, H., Rosenbusch, J., and Levitzki, A. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6579-6583) from our laboratory suggest that G_s or α_s remain associated with the catalytic subunit of adenylyl cyclase (C) throughout the activation cycle of adenylyl cyclase by hormone receptors. In this study we have purified GppNHp-activated bovine brain adenylyl cyclase over 3000-fold under mild solution conditions. We demonstrate that although the enzyme is permanently activated it retains the β subunit when bound to a forskolin-agarose affinity column as long as it is not exposed to high salt concentrations. The stoichiometry of α_s to β to C is close to unity, suggesting that βγ subunits do not dissociate from G_s upon its activation. The complex γβα_s(GppNHp) · C dissociates partially when migrating on a Superose 12 fast protein liquid chromatography molecular-seiving column. This partial dissociation probably results from the relatively diluted state of the enzyme at a high degree of purity. Prolonged ultracentrifugation of the complex also causes partial dissociation of the βγ subunits from α_s(GppNHp) · C. The apparent contradiction between the results reported here and the observation that βγ subunits inhibit cyclase activity when added to platelet membranes (Katada, T., Bokoch, G. M., Northrup, J. K., Ui, M., and Gilman, A. G. (1984a) J. Biol. Chem. 259, 3568-3577) is discussed. We suggest an alternative model to account for this inhibitory effect of added βγ subunits.