Figure 4
GTPase activity of the FtsZ mutants in the presence and absence of ZipA. (A) Progress curves for the GTPase activity in a solution containing 50 mM Mes pH 6.5, 50 mM KCl, 10 mM MgCl2 with 12.5 μM of wild type FtsZ (O), E83Q (∇ ) or R85Q (□) at 30°C. The reaction was started by the addition of GTP at 1 mM final concentration, and GDP was determined by HPLC, as described in Material and Methods. (B) Progress curves for the GTPase activity of R85Q. The hydrolysis of GTP was measured with the following R85Q concentrations: (▼ ) 6.2; (● ) 12.4; (∇ ) 18.6; (O) 24.8; (∎) 31.0 and 49.6 μM (
). The lag time (τ) was determined as the abscissa value where the line of the steepest slope of the progress curves intersects the x-axis. The inset shows the dependence of τ on the inverse of protein concentration. The values of picomoles indicated in the ordinate axe correspond to the amount of GDP in 100 μL of the reaction mixture (as indicated in Methods). (C) GTPase activity of 12.5 μM wild type FtsZ, E83Q and R85Q in the presence of 12 μM ZipA; the control with ZipA alone showed no GTPase activity (not shown). Inorganic phosphate was quantified using malachite green as described in Methods and the values of the ordinate axe are multiplied by 20.