Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes.
Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.
D000262:Adenylyl Cyclases / Q000097:blood; D000818:Animals; D002395:Catecholamines / Q000494:pharmacology*; D002396:Catechols / Q000494:pharmacology; D004298:Dopamine / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D020558:GTP Phosphohydrolases / Q000097:blood*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009638:Norepinephrine / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000097:blood*; D011433:Propranolol / Q000494:pharmacology; D014422:Turkeys
D002395:Catecholamines; D002396:Catechols; D010656:Phenylephrine; D011433:Propranolol; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases; D007545:Isoproterenol; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine