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

Abstract

Treatment of turkey erythrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both 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 and 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. Continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5''-(.beta.,.gamma.-imino)triphosphate [Gpp(NH)p]. The toxin-treated adenylate cyclase was maximally activated, in the presence of isoproterenol, by GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only 1/5 of the activity achieved with hormone plus Gpp(NH)p. The toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for an extended period (half-time of 3 min) upon subsequent addition of the .beta.-adrenergic blocker, propranolol. The native enzyme was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.