Steady-state actin-activated ATPase activity of M5CIQ1. The actin-activated ATPase activity of M5CIQ1 (20 nm) was measured in buffer A with 2 mm phosphoenol pyruvate, 20 units/ml pyruvate kinase, 0.2 mg/ml calmodulin, and 2 mm MgATP. The assay was done at 25 °C. The solid line is the hyperbola fit with V_max of 6.5 ± 0.4 s^-1 and K_ATPase of 62 ± 9 μm. The broken line is simulated based on our kinetic model in this study (see “Discussion”), giving a V_max value of 6.3 s^-1 and K_ATPase of 50 μm.

ATP-induced dissociation of acto-M5CIQ1. Pyrene-acto-M5CIQ1 (0.3 μm M5CIQ1 plus 0.5 μm pyrene-actin) was mixed with various concentrations of MgATP, and the changes in the pyrene fluorescence or light scattering were monitored. The second order rate constants for ATP binding to acto-M5CIQ1 () estimated from the initial slopes of the linear fits were 1.8 ± 0.1 μm^-1·s^-1 for pyrene fluorescence and 1.6 ± 0.1 μm^-1·s^-1 for light scattering. Inset, time courses of the pyrene fluorescence and light-scattering changes after mixing acto-M5CIQ1 with 10 μm MgATP. The solid lines are the best fits to single exponential kinetics, with k_obs of 14.7 s^-1 for pyrene fluorescence and 13.1 s^-1 for light scattering. The experimental conditions are as described in the legend to Fig. 3. The error bars represent the S.E. from 3–5 independent experiments.

Kinetics of ATP hydrolysis of M5CIQ1. A single turnover experiment was carried out by mixing 0.9 μm M5CIQ1 with 0.5 μm [γ-³²P]ATP, and the fraction of hydrolyzed ATP was plotted against time. The time course was fitted to double exponential kinetics. The rate constant of the burst phase (k_obs = 4.0 s^-1) with a fractional amplitude (equal to A_fast/(A_fast + A_slow)) of 0.31 ± 0.02 was obtained. The rate constant of the slow phase was k_obs of 0.064 ± 0.025 s^-1. The experimental conditions are as described in the legend to Fig. 3, except no actin was added.

Kinetics of dmant-ADP interaction with M5CIQ1. A, kinetics of dmant-ADP binding to M5CIQ1. 0.3 μm M5CIQ1 was mixed with various concentrations of dmant-ADP. The second order rate constant for dmant-ADP binding (k_-5/K₆) was 2.9 ± 0.1 μm^-1 s^-1. From the y intercept, a k₊₅ value of 3.6 ± 0.7 s^-1 was obtained. The inset shows a typical recording of the binding of dmant-ADP (5 μm) to M5CIQ1. The solid line is the best fit to single exponential kinetics, with k_obs of 9.2 s^-1. B, kinetics of dmant-ADP dissociation from M5CIQ1. 0.5 μm M5CIQ1 in the presence of 5 μm dmant-ADP was mixed with 2 mm MgADP. The solid line is the best fit to single exponential kinetics, with k_obs of 3.9 ± 0.1 s^-1. Error bars, S.E. from 3–5 independent experiments. Other experimental conditions are as described in the legend to Fig. 3.

Kinetics of pyrene-actin interaction with M5CIQ1. A, kinetics of pyrene-actin binding to M5CIQ1 in the absence of ADP. The rates of pyrene-actin binding to M5CIQ1 in the absence of ADP were measured as a function of pyrene-actin concentration. M5CIQ1 was mixed with various concentrations of pyrene-actin, and time courses of the decrease in the pyrene fluorescence were monitored. The apparent rate constant obtained by single exponential fitting increased with pyrene-actin concentration to give a second order rate constant (k_-12) of 1.11 ± 0.03 μm^-1 s^-1. Inset, a typical recording of the binding of 4 μm pyrene-actin to M5CIQ1. The solid line is the best fit to single exponential kinetics, with k_obs of 4.5 s^-1. B, kinetics of pyrene-actin binding to M5CIQ1 in the presence of ADP. The rates of pyrene-actin binding to M5CIQ1 in the presence of 50 μm MgADP were measured as a function of pyrene-actin concentration. The apparent rate constant obtained by single exponential fitting increased linearly with pyrene-actin concentration to give a second order rate constant (k_-6) of 0.88 ± 0.02 μm^-1 s^-1. The inset shows a typical recording of the binding of 4 μm pyrene-actin to M5CIQ1·ADP. The solid line is the best fit to single exponential kinetics, with k_obs of 3.5 s^-1. C, kinetics of pyrene-actin dissociation from acto-M5CIQ1. The rates of pyrene-actin dissociation from acto-M5CIQ1 or acto-M5CIQ1·ADP were measured by mixing acto-M5CIQ1 (0.5 μm M5CIQ1 and 0.7 μm pyrene-actin) in the presence or absence of 50 μm MgADP with an excess amount of nonlabeled actin (25 μm). The apparent rate constants obtained by single exponential fitting were 0.011 ± 0.001 s^-1 (k_-12) and 0.0099 ± 0.0015 s^-1 (k_-10), in the absence and presence of ADP, respectively. The error bars represent the S.E. from 3–5 independent experiments. Other experimental conditions are as described in the legend to Fig. 3.

Multimolecule and single molecule motility assay of myosin Vc. A, histogram of multimolecule actin gliding velocity of GFP-M5CHMM. GFP-M5CHMM was attached to a coverslip, and the movement of rhodamine-labeled actin filaments was observed with a fluorescent microscope. The solid line shows a fit to a single Gaussian curve. The mean ± S.D. of the actin sliding velocity was 0.16 ± 0.03 μms^-1 (n = 51). B, single molecule movement of GFP-myosin Va HMM monitored by TIRF microscope. Almost all fluorescent spots moved in a continuous line and dissociated from actin filament until the photobleaching. Typical time course of the distance from the binding position of individual GFP-myosin Va HMM. Mean velocity was 370 ± 93 nm s^-1, and mean travel distance was 710 ± 25 nm at 20 °C. C, single molecule movement of GFP-M5C HMM monitored by TIRF microscope. The fluorescent spots appeared on the actin filament but disappeared without any movements. Typical time course of the distance from the binding position of individual GFP-M5C HMM. Inset, an expanded view of the lowest trace. D, histogram of dwell time of GFP-M5C HMM on the actin filament monitored by TIRF microscope. The dwell time was defined as the period during binding on the actin filament. We did not count the spots on the actin for less than 5 frames (100 ms). The distribution was followed to the single exponential function (solid line), and the time constant was 0.51 s in 10 μm ATP.

Purification of human myosin Vc construct. A, SDS-PAGE of the purified human myosin Vc (M5CIQ1). M5CIQ1 expressed in Sf9 cells was extracted and purified through an anti-FLAG affinity column, and the purified proteins were analyzed by SDS-PAGE. HC and CaM, heavy chain of M5CIQ1 and calmodulin, respectively. A, M5CIQ1 co-expressed with calmodulin; B, M5CIQ1 co-expressed with LC17b. Lane 1, EGTA condition; lane 2, calcium condition. Molecular mass markers are shown on the left.

Kinetics of dmant-ATP binding to M5CIQ1 and acto-M5CIQ1. The experiment was done in the same conditions as described in the legend to Fig. 2 except for the absence of the ATP regeneration system. A, in the absence of actin. 0.3 μm M5CIQ1 was mixed with various concentrations of dmant-ATP. The second order rate constant for dmant-ATP binding (K₁k₊₂) was 2.4 ± 0.1 μm^-1·s^-1. Inset, a typical recording of the binding of 5 μm dmant-ATP to M5CIQ1. The solid line is the best fit to single exponential kinetics with k_obs of 12.6 s^-1. B, in the presence of actin. 0.3 μm M5CIQ1 in the presence of 0.4 μm actin was mixed with various concentrations of dmant-ATP. The second order rate constant () of 1.6 ± 0.1 μm^-1·s^-1 was obtained. Inset, a typical recording of the binding of 5 μm dmant-ATP to acto-M5CIQ1. The solid line is the best fit to single exponential kinetics with k_obs of 8.1 s^-1. The error bars represent the S.E. from 3–5 independent experiments.

ATP-induced intrinsic tryptophan fluorescence change of M5CIQ1. 0.8 μm M5CIQ1 was mixed with various concentrations of MgATP, and the tryptophan fluorescence change was monitored. The observed rates (k_obs) were saturated at 59 ± 3 s^-1 (k₊₃ + k_-3). The second order rate constant for ATP binding can be estimated from the initial slope of the linear fit, and the obtained value (K₁k₊₂ = 2.5 ± 0.1 μm^-1 s^-1) was consistent with that obtained in Fig. 3. The inset shows a typical recording of the intrinsic tryptophan fluorescence change after mixing M5CIQ1 with 10 μm MgATP. The solid line is the best fit to single exponential kinetics, with k_obs of 23.7 s^-1. The experimental conditions are as described in the legend to Fig. 3. The error bars represent the S.E. from 3–5 independent experiments.

Kinetics of phosphate release from M5CIQ1. The rate of phosphate release from M5CIQ1 was measured by using MDCC-PBP. 1.2 μm M5CIQ1 was mixed with 0.9 μm MgATP, aged for 5 s, and then mixed with various concentrations of actin. Other experimental conditions are as described in the legend to Fig. 3. A, a typical recording of the MDCC-PBP fluorescence change at 50 μm actin. The time courses were fitted to double exponential kinetics. The fast and slow rates were 23.8 and 1.7 s^-1, respectively. The inset shows a typical recording in the absence of actin. The fluorescence change in the absence of actin was best fitted to single exponential kinetics, with k_obs of 0.11 s^-1. B, actin concentration dependence of the rate of phosphate release. The apparent rate of the fast phase was linearly increased with actin concentration. The apparent second order rate constant was 0.46 ± 0.05 μm^-1 s^-1. The rates of the slow phase (0.2–1.7 s^-1) were actin-independent. The error bars represent the S.E. from 4–6 independent experiments.

Kinetics of dmant-ADP interaction with acto-M5CIQ1. A, kinetics of dmant-ADP binding to acto-M5CIQ1. 0.3 μm M5CIQ1 in the presence of 0.4 μm actin was mixed with various concentrations of dmant-ADP. The second order rate constant () of 6.0 ± 0.4 μm^-1·s^-1 and of 17.1 ± 1.3 s^-1 from the y intercept were obtained. Inset, a typical recording of the binding of dmant-ADP (5 μm) to acto-M5CIQ1. The solid line is the best fit to single exponential kinetics with k_obs of 46.8 s^-1. B, kinetics of dmant-ADP dissociation from acto-M5CIQ1. 0.5 μm M5CIQ1 in the presence of 5 μm dmant-ADP and 0.6 μm actin was mixed with 2 mm MgADP. The solid line is the best fit to single exponential kinetics, with k_obs of 17.7 ± 0.6 s^-1. C, the rate of ADP dissociation from acto-M5CIQ1 was determined by measuring the time course of change in the light-scattering intensity of acto-M5CIQ1. Acto-M5CIQ1 (0.3 μm M5CIQ1 and 0.4 μm actin) in the presence of 50 μm MgADP was mixed with various concentrations of MgATP. The apparent dissociation rates were saturated at 12.7 ± 0.9 s^-1 (), which reflected the ADP dissociation rate from acto-M5CIQ1. The error bars represent the S.E. from 3–5 independent experiments. Other experimental conditions are as described in the legend to Fig. 3.

Apparent binding affinities of M5CIQ1 for actin in the presence of ATP analogues. Actin cosedimentation assays were performed in the presence of 2.5 mm Mg-ATPγS or 5 mm Mg-AMPPNP at 25 °C. The experimental conditions are as described in the legend to Fig. 3. Apparent binding affinities of M5CIQ1 for actin were >70 μm in the presence of ATPγS(K_d(ATPγS)) and 6.5 ± 0.6 μm in the presence of AMPPNP (K_d(AMPPNP)).

A, reaction scheme of myosin Vc ATPase cycle. A, actin; M, myosin; T, ATP; D, ADP; P, phosphate. The major kinetic pathway is shown in gray shading. B, actin concentration dependence of the duty ratio of M5CIQ1. The duty ratio was calculated based on our kinetic model using experimentally obtained parameters as described under “Discussion.” The duty ratio was 0.33 at saturating actin concentration.