GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates.

摘要: Abstract The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [alpha-32P]- or [gamma-32P] GTP as substrate. binding [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S) used to quantitate total concentration Gs. 1) a saturable function GTP, with Km = 0.3 microM. MgCl2 monotonically increased activity. maximum observed turnover number about 1.5 min-1. 2) During steady-state hydrolysis, 20-40% could be trapped Gs-GDP complex 1-2% Gs-GTP. hydrolysis Gs-GTP occurred t 1/2 less than equal 5 s at 30 degrees C approximately 1 min 0 C. Hydrolysis inhibited by 1.0 mM EDTA in absence added Mg2+. 3) rate formation initial varied parallel functions concentrations (above 0.1 Mg2+). ratio accumulation constant 0.3-0.4. 4) dissociation assayable biphasic. phase accounted for 60-80% characterized min. 5) Lubrol 12A9 potently reaction parallel, inhibition product release may account hydrolysis. 6) beta subunits markedly GDP from contrast their ability stimulate S. 7) GDP, S, guanyl-5'-yl imidodiphosphate (Gpp(NH)p) competitively Gs-GDP. S Gpp(NH)p noncompetitively, displayed mixed inhibition, Pi did not inhibit. These data are interpretable terms coexistence two specific mechanistic pathways overall reaction.