Determination of the turn-off reaction for the epinephrine-inhibited human platelet adenylate cyclase.
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 cyclic AMP formation upon addition of the alpha2-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 degrees C were obtained for the epinephrine-inhibited platelet enzyme. Cholera toxin, which does not inhibit the epinephrine-induced GTPase stimulation in platelet membranes, did not affect this turn-off reaction. In contrast, the turn-off rate of the prostaglandin-E1-stimulated human platelet adenylate cyclase, measured with GDP beta S, was reduced from about 9 min-1 to 2 min-1 at 25 degrees C by pretreatment of the membranes with cholera toxin, which inhibits the prostaglandin-E1-stimulated GTPase activity. The data strongly suggest that 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 that cholera toxin affects only the stimulatory site. The findings furthermore suggest that the activity states of these two regulatory sites control the activity of the adenylate cyclase.
D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000097:blood; D001792:Blood Platelets / Q000201:enzymology*; D055598:Chemical Phenomena; D002621:Chemistry; D000242:Cyclic AMP / Q000097:blood; D004837:Epinephrine / Q000494:pharmacology*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology
D000067956:Adenylyl Cyclase Inhibitors; D011942:Receptors, Adrenergic, alpha; D013873:Thionucleotides; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D004837:Epinephrine