Mechanism of activation of adenylate cyclase byVibrio cholerae enterotoxin

Abstract

The kinetics and properties of the activation of adenylate cyclase by cholera enterotoxin have been examined primarily in toad erythrocytes, but also in avian erythrocytes, rat fat cells and cultured melanoma cells. When cholera toxin is incubated with intact cells it stimulates adenylate cyclase activity, as measured in the subsequently isolated plasma membranes, according to a triphasic time course. This consists of a true lag period of about 30 min, followed by a stage of exponentially increasing adenylate cyclase activity which continues for 110 to 130 min, and finally a period of slow activation which may extend as long as 30 hr in cultured melanoma cells. The progressive activation of adenylate cyclase activity by cholera toxin is interrupted by cell lysis; continued incubation of the isolated membranes under nearly identical conditions does not lead to further activation of the enzyme. The delay in the action of the toxin is not grossly dependent of the number of toxin-receptor (G_M1 ganglioside) complexes, and is still seen upon adding a second dose of toxin to partially stimulated cells. Direct measurements indicate negligible intracellular levels of biologically active radioiodinated toxin in either a soluble or a nuclear-bound form. The effects are not prevented by Actinomycin D (20μg/ml), puromycin (30 μ/ml), cycloheximide (30 μg/ml), sodium fluoride (10mM) or sodium azide (1mM); KCN, however, almost completely prevents the action of cholera toxin. The action of the toxin is temperature dependent, occurring at very slow or negligible rates below certain critical temperatures, the values of which depend on the specific animal species. The transition for toad erythrocytes occurs at 15 to 17°C, while rat adipocytes and turkey erythrocytes demonstrate a discontinuity at 26 to 30°C. The temperature effects are evident during the lag period as well as during the exponential phase of activation. The rate of decay of the stimulated adenylate cyclase activity of cultured melanoma cells indicates a half-time of about 36hr. The rate of exponentially increasing activity and extent of enzyme activation are related to the number of bound toxin molecules according to a Langmuir adsorption isotherm and are half-maximal when about 2000 molecules of toxin are bound per cell. It is proposed that initially cholera toxin binds ineffectively, but that it is converted to an active form during the lag phase. This process may involve lateral motion of a toxin-G_M1 ganglioside complex within the plane of the membrane. The kinetics of adenylate cyclase activation are consistent with the possibility that during the exponential phase a bimolecular association is proceeding between the active form of the cholera toxin and some other membrane component. The possibility is considered that the cholera toxin molecule may bind directly to adenylate cyclase. These considerations may prove useful in understanding the possible interactions of active hormone-receptor complexes with adenylate cyclase in cell membranes.