Conditions associated with the appearance of guanine nucleotide-dependent adenylate cyclase activity in turkey erythrocyte membranes

Abstract

The mechanisms involved in the activation of adenylate cyclase in turkey erythrocyte membranes may include the release of inhibitory nucleotides from the guanine nucleotide regulatory protein (N) and the subsequent formation of an active GTP-N complex. We have studied the conditions required tor the appearance of guanine nucleotide-dependent adenylate cylase activity in turkey eryhrocyte membranes in an effort to understand further the mechanisms involved in its activation. Turkey erythrocye membranes, whose adenylate cylase enzyme is usually poorly responsive to the direct actions of GTP or its analogue. Gpp(NH)p. became markedly responsive after previous exposure to isoproterenol in a sucrose-containing buffer. Without sucrose, isoproterenol alone did not lead to Gpp(NH)p responsiveness. Maximum expression of Gpp(NH)p-dependent adenyate cylase activity required prior incubation with both GMP and isoproterenol. The requirement tor sucrose when isoproterenol was used without GMP was lost when GMP was present. With or without GMP. isoproterenol induced a half-maximum effect after 5 min at 37°. The development and maintenance of Gpp(NH)p-dependent adenyate cylase activity were mediated, in part. by beta-adrenergic receptors. Following a 10-min incubation period with isoproterenol and GMP. the addition of the beta-adrenergic inhibitor, propranolol. for 4 additional min completely reversed the stimulation produced by isoproterenol and GMP. In addition, high-affinity agonist binding to beta-receptors was necessary, but not sufficient, for the development of Gpp(NH)p responsiveness.