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

Abstract

The time course of activation and deactivation and the degree of activation at steady state [ E _a ] / [ E _tot ] of adenylate cyclase, in semi-purified rat pancreatic plasma membranes, were compatible with a simple two-state model with three rate constants, so that [ E _a ] / [ E _tot ] = k ₊₁ /( k ₊₁ + k ₂ + 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 k ₂ was unaltered at 7 min ^-1 , so that [ E _a ] / [ E _tot ] increased 15-fold, from 4 to 61%. A similar activation was obtained after cholera toxin pretreatment but by a different mechanism. The toxin pretreatment exerted a major inhibitory effect on the value of k ₂ and on the corresponding GTPase activity. A pretreatment at the high cholera toxin concentration (30 pg/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.