GTP BINDING PROTEINS AND ADENYLATE CYCLASE

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Choleragen (cholera toxin), the agent responsible for the devastating diarrheal disease characteristic of cholera, causes its effects on cells by activating the hormone-sensitive adenylyl cyclase. Activation results from the ADP-ribosylation of Gsa, a guanine nucleotide-binding regulatory protein of the cyclase complex. The toxin can also ADP-ribosylate other proteins as well as simple guanidino compounds such as agmatine. ADP-ribosylation of Gs alpha and other substrates is enhanced by so-called ADP- ribosylation factors (ARFs), a family of approximately 19 kDa soluble and membrane guanine nucleotide-binding proteins that have been purified from bovine brain. In the presence of GTP, but not GDP, ARF interacts with the toxin resulting in a decrease in apparent Kms for both NAD and ADP-ribose acceptor with no significant effect on Vmax. Activation of toxin by ARF was increased approximately 4-fold by 0.003% sodium dodecyl sulfate (SDS), which had little or no effect on activity in the absence of ARF; 0.01% SDS abolished toxin activity with or without ARF. In the presence of SDS at stimulatory concentrations, ARF further increased affinities for NAD and agmatine and, in addition, increased Vmax. Stimulatory effects of ARF and SDS were observed in the presence of dithiothreitol with toxin A subunit and in the absence of dithiothreitol, with the reduced and alkylated toxin A1 peptide. Thus, it appears that ARF and SDS do not activate by facilitating release of the toxin catalytic unit. Rather, ARF interacts directly with the catalytic A1 peptide and may thus potentiate the pathogenicity of cholera toxin.