Figure 7
Quantification of [γ32-P] GTP bound to cyclase complex A: Quantification [32yP- GTP] in cyclase(s) complex at t = 0. Membranes (50 μg protein) were incubated with [γ32-P] GTP and isoproterenol, isoproterenol + baclofen or baclofen (each 200 nM) in presence of EDTA (1 mM) for 20 min at 20°C. The membranes were then placed in ice and solubilized, and the cyclase(s) complex was isolated by Forskolin-agarose. The radioactivity in the isolated 'pull down' was measured by filtration on nitrocellulose membrane. Bars represent the mean +/-SE of 3 experiments. Control experiments with only membrane, CTx or PTx treated membrane are also shown. Lane 1: control membrane, lane 2: iso, lane 3: bac, lane 4: iso + bac, lane 5: iso + bac + CTx, lane 6: CTx, lane 7: PTx B: Quantification [32yP-GTP] in cyclase(s) complex at t = 5 min. [γ32-P] GTP bound to cyclase(s) complex was measured 5 min after a large excess of GTP was added. Membranes were incubated as indicated above, placed in ice and complemented with Mg (5 mM) for 5 min, then solubilized in the presence of NaF (100 μM). The cyclase(s) complex was isolated as above. The radioactivity in the isolated 'pull down' was measured by filtration on nitrocellulose membrane. Bars represent the mean +/-SE of 3 experiments. * p < 0.01 and ** p < 0.005 Lane 1: control membrane, lane 2: iso, lane 3: bac, lane 4: iso + bac, lane 5: iso + bac + CTx, lane 6: CTx, lane 7: PTx