Determining steps in the regulatory GTPase cycle of rat pancreatic adenylate cyclase

The time course of activation and deactivation and the degree of activation at steady state [Ea]/[Etot] of adenylate cyclase, in semi-purified rate pancreatic plasma membranes, were compatible with a simple two-state model with three rate constants, so that [Ea]/[Etot] = k+1/(k+1 + k2 + k-1). The hormone CCK-8 increased k+1 with GTP in a dose-dependent manner, from 0.2 to 10.9 min-1; k-1 increased from 0.01 to 0.3 min-1, i.e. in proportion, but k2 was unaltered at 7 min-1, so that [Ea]/[Etot] increased 15-fold, from 4 to 61%. A similar activation was obtained after cholera toxin pretreatment by a different mechanism. The toxin pretreatment exerted a major inhibitory effect on the value of k2 and on the corresponding GTPase activity. A pretreatment at the high cholera toxin concentration (10 micrograms/ml) exerted two additional effects that became evident when p[NH]ppG rather than GTP was used as activating nucleotide: (a) a relatively large increase in k-1 from an unmeasurably low control value to 0.3 min-1, and (b) a four-fold increase in the p[NH]ppG activation rate, k+1. This contrasted with the action of CCK-8, which increased k-1 and k+1 in proportion.