Characterization of high affinity GTPase activity correlated to beta-adrenergic receptor stimulation of adenylyl cyclase in rat parotid membranes.
beta-Adrenergic receptor stimulation of adenylyl cyclase involves the activation of a GTP-binding regulatory protein (G-protein, termed here Gs). Inactivation of this G-protein is associated with the hydrolysis of bound GTP by an intrinsic high affinity GTPase activity. In the present study, we have characterized the GTPase activity in a Gs-enriched rat parotid gland membrane fraction. Two GTPase activities were resolved; a high affinity GTPase activity displaying Michaelis-Menten kinetics with increasing concentrations of GTP, and a low affinity GTPase activity which increased linearly with GTP concentrations up to 10 mM. The beta-adrenergic agonist isoproterenol (10 microM) increased the Vmax of the high affinity GTPase component approx. 50% from 90 to 140 pmol/mg protein per min, but did not change its Km value (approximately 450 nM). Isoproterenol also stimulated adenylyl cyclase activity in parotid membranes both in the absence or presence of GTP. In the presence of a non-hydrolyzable GTP analogue, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), isoproterenol increased cAMP formation to the same extent as that observed with AlF-4. Cholera toxin treatment of parotid membranes led to the ADP-ribosylation of two proteins (approximately 45 and 51 kDa). Cholera toxin also specifically decreased the high affinity GTPase activity in membranes and increased cAMP formation induced by GTP in the absence or the presence of isoproterenol. These data demonstrate that the high affinity GTPase characterized here is the 'turn-off' step for the adenylyl cyclase activation seen following beta-adrenergic stimulation of rat parotid glands.
Hiramatsu
Y
Y
Clinical Investigations and Patient Care Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, MD 20892.
Ambudkar
I S
IS
Baum
B J
BJ
D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002462:Cell Membrane / Q000201:enzymology; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010306:Parotid Gland / Q000201:enzymology*; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction
D011943:Receptors, Adrenergic, beta; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D011433:Propranolol; D000242:Cyclic AMP; D020558:GTP Phosphohydrolases; D000262:Adenylyl Cyclases; D007545:Isoproterenol