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.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002766:Cholecystokinin / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000502:physiology; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D051381:Rats
D006150:Guanine Nucleotides; D002766:Cholecystokinin; D002772:Cholera Toxin; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases