Mechanism of pertussis toxin action on the adenylate cyclase system. Inhibition of the turn-on reaction of the inhibitory regulatory site.

Abstract

The activation reaction of the inhibitory guanine nucleotide-binding regulatory site of the adenylate cyclase system was studied in membranes of rat adipocytes, S49 lymphoma wild-type cells and their cyc- variants, pretreated without and with the Bordetella pertussis toxin, islet-activating protein (IAP), by measuring the kinetics of adenylate cyclase inhibition by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The IAP treatment, which caused a loss of GTP and hormone-induced adenylate cyclase inhibition, did not prevent enzyme inhibition by the stable GTP analogue. However, in either cell type studied, pretreated with IAP, the lag phase of GTP[S] inhibitory action was largely increased by about fivefold compared to control membranes. Similar to the controls, the lag phase of GTP[S] inhibition of adenylate cyclase in membranes of IAP-pretreated cells was shortened in the presence of an inhibitory hormone. Furthermore, the lag phase of inhibition by GTP[S] was decreased with increasing concentrations of Mg2+. The data indicate that the pertussis toxin does not principally prevent an interaction of the inhibitory guanine nucleotide regulatory site of the adenylate cyclase system with either the catalytic moiety or an inhibitory hormone receptor. The data, furthermore, suggest that the toxin inhibits the activation reaction (turn-on reaction) of the inhibitory coupling component. This inhibition, which may take place at a Mg2+-binding site, can account for the observed functional loss of GTP and hormone-induced adenylate cyclase inhibition after IAP treatment.