In vitro Ras-GTP association and hydrolysis at 0°C. (a) Ras-GDP complex from E. coli was incubated with a molar excess of [α-³²P]GTP at 30°C or at 0°C for up to 4 hours followed by precipitation and quantification of Ras-associated label. Values are expressed as percent of maximal association, as determined by equilibration in magnesium-free conditions. Values shown are averages of duplicate data points. (b) Ras from Sf9 cells was complexed with [α-³²P]GTP and then incubated either with (+) or without (–) recombinant p120GAP at 0°C for 4 hours or at 30°C for 15 minutes. Ras-associated nucleotide was then precipitated and quantified. Lane S represents the starting material, substrate Ras-GTP complex that was precipitated immediately. The percent of total radiolabel that was present as GTP is shown above each lane. Chromatographic positions of GDP and GTP are shown on the right. (c) Ras-GDP from Sf9 cells was incubated at 0°C with a molar excess of [α-³²P]GTP in the presence (p30) or absence (buffer) of the catalytic domain of Ras-GRF1 for 4 hours. Newly associated radiolabeled nucleotide was precipitated and quantified. Values are expressed as percent of maximal association and are the averages of triplicate data points with the standard deviation indicated. Note that when parallel reactions were performed at 30°C for 15 minutes, spontaneous and catalyzed association values of 4% and 43%, respectively, were observed.