Activation of R-Ras GTPase by GTPase activating proteins for Ras, Gap1(m), and p120GAP

The enzymatic properties of Gap1(m) were characterized using three Ras and R-Ras proteins as substrates and were compared with those of p120GAP. Gap1(m) stimulated the GTPase of Ras better than that of R-Ras, in contrast to p120GAP which promoted the GTPase of R-Ras better than that of Ras, The EC50 values of Gap1(m) for Ha-Ras and R-Ras were 0.48 +/- 0.02 and 1.13 +/- 0.12 nM, respectively, whereas the EC50 values of p120GAP for Ha-Ras and R-Ras were 23.1 +/- 1.9 and 3.86 +/- 0.38 nM, respectively, The affinities of Gap1(m) and p120GAP to the substrates determined by competitive inhibition by using Ha-Ras.GTP gamma S (guanosine 5'-O-(3-thiotriphosphate)) or R-Ras.GTP gamma S as a competitor agreed well with the substrate specificities of these GTPase-activating proteins, The K-m values of Gap1(m) for Ha-Ras and R-Ras were 1.53 +/- 0.27 and 3.38 +/- 0.53 mu M, respectively, which were lower than that of p120GAP for Ha-Ras (145 +/- 11 mu m) by almost 2 orders of magnitude, The high affinity of Gap1(m) to the substrates and its membrane localization suggest that Gap1(m) may act as a regulator of the basal activity of Ha-Ras and R-Bas.