Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes
1976; Elsevier BV; Volume: 452; Issue: 2 Linguagem: Inglês
10.1016/0005-2744(76)90206-0
ISSN1878-1454
Autores Tópico(s)Ion channel regulation and function
ResumoDetermination of specific GTPase (EC 3.6.1.—) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 μM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5′(β,γ-imino)triphosphate(App(NH)p) and suppression of the transfer of γ-32P from GTP to ADP with an ATP regeneration system. Under these conditions catecholamines 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 GTPase, 3–7; and residual non-specific NTPase, 3–5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the β-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 μM, 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-ethylmale imide (MalNEt) at 0°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 μM. These GTPase activities were inhibited by ITP but not by CTP and UTP.
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