Figure 6
Determination of the fractional occupancy and hydrolysis of GTP in the catalytic site of Gαs complexed with adenylyl cyclase(s) A: Fractional occupancy of GTP in cyclase(s) associated Gs . Comparative kinetics of GTP hydrolysis at 0°C. Membranes (50 μg protein per assay) were incubated with [γ32-P] GTP (1 μM, 10 000 cpm/pmol) and isoproterenol, isoproterenol + baclofen or baclofen (each 200 nM) in the presence of EDTA (1 mM) for 20 min at 20°C. The samples were placed on ice and complemented with Mg (5 mM) and an excess of cold GTP (1 mM) for 10 min, then the membranes were solubilized in presence of NaF (100 μM) and a 'pull down' experiment using Forskolin-agarose was carried out as above. The radioactivity in the isolated 'pull down' was measured by filtration on nitrocellulose membranes and recorded as a percentage (+/-SE) of the radioactivity measured in parallel experiment using membranes treated with cholera toxin (CTx). Bars represent the mean +/-SE of 5 experiments. * represents values of iso+bac significantly different from iso or bac (P < 0.01) B: Single turnover GTP hydrolysis of cyclase(s) associated Gs at 0°C. Membranes (50 μg protein per assay) were incubated with iso, iso+bac or bac (each 200 nM) and with [γ32-P] GTP (1 μM, 10 000 cpm/pmol) and EDTA (1 mM) for 20 min at 20°C. The GTPase reaction was initiated by the addition of MgCl2 (5 mM) and an excess of cold GTP (1 mM) in ice. At various time points, NaF (100 μM) was added and membranes were immediately solubilized and incubated with Forskolin-agarose. Radioactivity in the 'pull down' complex attached to the Forskolin-agarose was counted as above and reported as percentage of the radioactivity at t = 0* P < 0.05 and ** P < 0.01