ADP-ribosylation of G(s) promotes the dissociation of its α and β subunits

We have utilized purified reactants and cofactors to examine the form of the stimulatory guanine nucleotide-binding regulatory component (G(s)) of adenylate cyclase that serves as a substrate for ADP-ribosylation by cholera toxin; we have also investigated some of the consequences of that covalent modification. Activation of G(s) with nonhydrolyzable analogs of GTP, which causes dissociation of its subunits, completely inhibits the toxin-catalyzed covalent modification. However, this effect cannot be explained by subunit dissociation, since activation of G(s) by fluoride is not inhibitory and ADP ribosylation of the α (45,000-Da) subunit of G(s) proceeds equally well in the presence and absence of the β (35,000-Da) subunit. ADP-ribosylation of the α subunit of G(s) decreases its apparent affinity for the β subunit; however, the affinity of α and ADP-ribosyl-α for GTP appear to be approximately the same. ADP-ribosylation of G(s) thus promotes the dissociation of its α and β subunits. This effect may account for or contribute to the activation of adenylate cyclase by cholera toxin.