Figure 2.

Kinetic analysis of the (A and B) rates of ATP binding, (C and D) P_i release, and (E and F) ADP release for myo1b^IQ in the presence (red, ■) and absence (orange, •) of 100 μM Ca²⁺_F. (A) The rate of ATP binding was measured by rapidly mixing pyrene-actomyo1b^IQ with ATP and measuring the increase in pyrene fluorescence as the myosin dissociated from actin. The plot shows the fluorescence increase as a function of time obtained after mixing 1.0 μM actomyo1b^IQ with 150 μM ATP. (B) The rates obtained from the fast phase of the fluorescence transient were plotted against ATP concentration and fit to a hyperbolic function, as represented by the continuous lines. (C) The rate of phosphate release was measured by monitoring the fluorescence produced when free phosphate bound to phosphate-binding protein. The plot shows a transient increase in phosphate-binding protein fluorescence after 1 μM ATP was mixed with 3 μM myo1b^IQ that had been premixed with 10 μM actin. (D) The rate of phosphate release measured as a function of actin concentration. Each data point is the average of one to six transients. (E) The rate of ADP release was measured from the increase in fluorescence after 1 mM ATP was mixed with 0.15 μM pyrene-actomyo1b^IQ that was preequilibrated with varying ADP concentrations. The plot shows the fluorescence transient observed in the presence 10 μM ADP. (F) The rate of the slow phase of ATP-induced dissociation as a function of ADP concentration provides a measurement of the rate of ADP release (k_+5′) at ADP concentrations > 10 μM.