FIG. 6.

Single-turnover measurements of RanGAP-catalyzed hydrolysis of Ran-GTP in the presence of RanBP1. (A) Ran47-GTP-RanBP1 (1.5 μM) was mixed with different concentrations of RanGAP (only four curves are shown) in the stopped-flow apparatus. Fluorescence transients were fitted as double exponentials. (B) Rate constants (•) of the first phase plotted against RanGAP concentration. Assuming a simple saturation model (16) and given that k_off ≫ k_cat, observed rate constants can be fitted to a quadratic equation (K_d^app = 0.3 μM; k_cat = 10.3 s⁻¹). (C) Rate constants (•) and amplitudes (▵) of the second phase (k₂ ≈ 0.05 s⁻¹) plotted against RanGAP concentration. Measurements were performed and analyzed as described in Materials and Methods.