Ligand requirements for the relaxation of adenylate cyclase from activated and inhibited states

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Abstract
The turkey erythrocyte adenylate cyclase system binds tightly the inhibitory nucleotide GDP, and a pretreatment step with isoproterenol and GMP is required to restore activation. Under identical pretreatment condition, the release of labeled nucleotide is complete within 1 min whereas the restoration of activation by Gpp(NH)p requires 15 min. A study of the ligand requirements of the slow step shows the following: The role of GMP is that of an obligatory allosteric regulator. Cholera toxin modification of the system abolishes the requirement for GMP with a considerable enhancement in the reaction rate. GMP is without effect on the relaxation process with the activator Gpp(NH)p as the resident nucleotide. In sharp contrast, EDTA (without effect in a GDP-occupied complex) markedly potentiates alterations from the Gpp(NH)p-occupied state. Formation of a GDP/guanosine 5''-O-(2-thiodiphosphate) (GDP.beta.S) hybrid leads to the suppression of both F- and Gpp(NH)p activation. F- activation is restored by isoproterenol alone, while GMP is still required to restore Gpp(NH)p activation. Covalent modification or nucleotide analog occupancy of the regulatory complex can probably modify the allosteric role for GMP, with consequences for the rate of the slow step.