Determination of the Turn‐Off Reaction for the Epinephrine‐Inhibited Human Platelet Adenylate Cyclase

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Abstract
Epinephrine inhibits human platelet adenylate cyclase by an .alpha.2-adrenoceptor-mediated and GTP-dependent process. The turn-off reaction for this epinephrine-inhibited enzyme was studied by measuring the rate of cAMP formation upon addition of the .alpha.2-adrenoceptor antagonist, yohimbine, or upon addition of an excess of the stable GDP analog, guanosine 5''-O-(2-thiodiphosphate) (GDP.beta.S), which competitively inhibited the action of GTP in the epinephrine-induced inhibition. The decay of the inhibited state of the adenylate cyclase was used to calculate the rate constant of the turn-off reaction. With both methods, almost identical koff values of 0.6-0.7 min-1 at 25.degree. C were obtained for the epinephrine-inhibted platelet enzyme. Cholera toxin, which does not inhibit the epinephrine-induced GTPase stimulation in platelet membranes, did not affect this turn-off reaction. The turn-off rate of the prostaglandin[PG]-E1-stimulated human platelet adenylate cyclase, measured with GDP.beta.S, was reduced from about 9 to 2 min-1 at 25.degree. C by pretreatment of the membranes with cholera toxin, which inhibits the PGE1-stimulated GTPase activity. Evidently, the guanine nucleotide regulatory site, mediating epinephrine-induced adenylate cyclase inhibition, is activated and inactivated by similar mechanisms as is the site mediating adenylate cyclase stimulation, and cholera toxin affects only the stimulatory site. The activity states of these 2 regulatory sites may control the activity of the adenylate cyclase.