FIG. 4.
BopE acts as a GEF for the RhoGTPases Rac1 and Cdc42. The multiple turnover kinetics of guanine nucleotide exchange by BopE was analyzed by measuring the release of mantGDP from 10 μM Rac1-191·mantGDP (A) or Cdc42 Hs1-192·mantGDP (B) in the presence of 1 mM GDP and 25 nM or 250 nM BopE using fluorescence spectrometry (excitation wavelength, 366 nm; emission wavelength, 440 nm; step size, 1; band-pass, 4) at 20°C in a buffer containing 40 mM HEPES-NaOH (pH 7.4), 100 mM NaCl, and 5 mM MgCl2. Spontaneous dissociation of the RhoGTPase·mantGDP complex in the assay buffer or in assay buffer supplemented with 10 mM EDTA was measured as the control.