FIGURE 8.
ADP-off is the rate-limiting step for the motor activity of Myo19. A, effects of ADP on actin-activated ATPase activity of M19-1IQ, Myo5a-1IQ, and SkM-S1. Actin-activated ATPase activity was measured in the presence of 40 μm actin, 500 μm ATP, and 0–500 μm ADP and the released phosphate was determined by the malachite green method. The inhibition of ATPase activities by ADP was fitted with a hypophobic equation. B, effects of ADP on actin-gliding activity of M19–1IQ, Myo5a-ΔT, and skeletal muscle myosin (SkM-M2). Actin-gliding activity was measured in the presence of 500 μm ATP and 0–500 μm ADP. In the absence of ADP, the actin-gliding activities of M19-2IQ-Avi, Myo5a-ΔT, and skeletal muscle myosin were 40, 280, and 3.64 μm/s, respectively. C and D, dissociation of mant-ADP from M19-1IQ (C) and acto-M19-1IQ (D). The fluorescence intensity of mant-ADP was recorded in a stopped flow after mixing 1 mm ATP with and 1 μm mant-ADP and 1 μm M19–1IQ in the absence (C) or presence (D) of 1.5 μm phalloidin-stabilized actin filaments. The smooth line is the fit to single exponential kinetics, with Kobs of 13.65 s−1 (C) and 8.37 s−1 (D).