Mechanism of adenylate cyclase activation by cholera toxin: Inhibition of GTP hydrolysis at the regulatory site

Abstract

Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-siimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity coincided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD.It is propsed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site,is an essential turn-off mechanism, The results have shown that the toxin concurrently inhibits the GTPase activity and enhances the GTP-dependent adenylate cyclase activity. Furthermore, the effects of GTP on the adenylate cyclase in preparations treated with cholera toxin resembled those seen with the hydrolysis-resistant analog Gpp(NH)p in untreated preparations. We therefore propose that cholera toxin activates the adenylate cyclase via an inhibition of the turn-off GTPase reaction. ['y-32P]GTP was prepared by the method of Glynn and Chappell (14). MATERIALS AND METHODSTurkey Erythrocyte Membranes. These were prepared by digestion of the nuclei with DNase, as described for frog erythrocytes (15), with the following modifications: the pH of the Tris buffer used for lysis and for washes of the membranes was 7.9 at 230 and 2-mercaptoethanol (2 mM) was used instead of dithiothreitol. The membranes were stored in liquid nitrogen and thawed on the day of the experiment.Toxin Treatment. Adenylate cyclase in membrane preparations from turkey erthrocytes was activated with cholera toxin (120 ,g/ml) that had been incubated for 15 min at 370 in turkey erythrocyte cytosol containing 0.6 mM dithiothreitol.t The cytosol was prepared from packed erythrocytes lysed by freezing in liquid nitrogen, followed by thawing at 370, susAbbreviations: Gpp(NH)p, guanosine 5'-(f,-y-imino)triphosphate; App(NH)p, adenosine 5'-(,By-imino)triphosphate; GTPase, guanosinetriphosphatase; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. * To whom correspondence should be addressed.

Cholera toxin promotes the generation of semi-mature porcine monocyte-derived dendritic cells that are unable to stimulate T cells

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