Figure 2

Analysis of cholera toxin [³²γP]-ADP-ribosylated Gαs bound to cyclase A: ADP ribosylation of Gás in Forskolin agarose 'pull down' Plasma membrane (50 μg protein) was incubated with isoproterenol (10 nM) (a), isoproterenol +baclofen (10 nM each) (b), or baclofen (50 nM) (c), GTP (10 μM), MgCl₂ (1 mM) and cholera toxin plus [³²P] NAD (see 'materials and methods'). A control membrane (without drugs) is shown (d). At the indicated time membranes were placed in ice and immediately solubilized in nonionic detergent, and a 'pull down' experiment was performed using Forskolin-agarose (see 'materials and methods'). Numbers represent the timing of incubation prior to solubilization. The arrow corresponds to MW = 45 kDa. B: Quantification of ADP ribosylated Gás in Forskolin agarose 'pull down' Same experiment as in A except that the 45 kDa band was excised and its radioactivity counted. Values are means +/- SE of 3 determinations. * corresponds to values iso+bac (isoproterenol + baclofen) statistically different from iso (isoproterenol), at P < 0.01. C: electrophoresis analysis of the 'pull down' material Electrophoretic analysis of the 'pull down' material using Forskolin–agarose was carried out with solubilized membranes previously incubated with isoproterenol (lane1), baclofen (lane 2) or baclofen + isoproterenol (lane 3) at 100 nM for each. Control experiments were carried out with membranes incubated with an excess of GDP (lane 4), and also with membranes treated with pertussis toxin (see 'materials and methods') then incubated with baclofen + isoproterenol at 100 nM (lane 5). The upper band corresponds to MW = 45 kDa, the lower band to MW = 35 kDa. Controls of chromatography procedure was carried out as follows: membranes were incubated with GDP (1 mM) (lane 6) and GTPγS (50 μM) plus bac+iso (100 nM each) (lane 7) then solubilized and proteins isolated with Forskolin-agarose were directly analyzed (without washing) in acrylamide gel electrophoresis.