Beta 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 Gs or alpha 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 beta subunit when bound to a forskolin-agarose affinity column as long as it is not exposed to high salt concentrations. The stoichiometry of alpha s to beta to C is close to unity, suggesting that beta gamma subunits do not dissociate from Gs upon its activation. The complex gamma beta alpha 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 beta gamma subunits from alpha s (GppNHp). C. The apparent contradiction between the results reported here and the observation that beta gamma 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 beta gamma subunits.
Marbach
I
I
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Bar-Sinai
A
A
Minich
M
M
Levitzki
A
A
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002540:Cerebral Cortex / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004789:Enzyme Activation; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D012995:Solubility
D046911:Macromolecular Substances; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases