Figure 5.

Mictrotubule dependence of mant-ADP release from HK560 kinesin. To determine the ADP release rate, mant-ADP-loaded HK560 was mixed with MTs in a stopped-flow apparatus at post-mixing concentrations of 1 μM HK560, 2 μM mant-ADP, 0.25–10 μM wild-type or α-E415A MTs, and 1 mM ATP. The apparent rate of ADP dissociation was derived by fitting the fluorescence decay by double exponentials, giving fast- and slow-phase rates for ADP release (see Materials and methods section for details). Fast-phase rates (k_obs) are plotted as a function of MT concentration. Slow-phase rates were low and independent of MT concentrations, and are omitted from the graphic and analysis. MT dependence of k_obs was fit to a hyperbolic model, from which values for parameters k_max and K_0.5MT were extracted (Table II).