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 Gs.cyclase complex (Gs, 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 Gs.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, 9999-10,004]. 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 Gs 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.
Bar-Sinai
A
A
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel.
Marbach
I
I
Shorr
R G
RG
Levitzki
A
A
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D004789:Enzyme Activation / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D046911:Macromolecular Substances; D012995:Solubility; D013329:Structure-Activity Relationship; D014422:Turkeys
D046911:Macromolecular Substances; D006165:Guanylyl Imidodiphosphate; D000262:Adenylyl Cyclases