An integrated in vitro and in situ study of kinetics of myosin II from frog skeletal muscle

Abstract

A new efficient protocol for extraction and conservation of myosin II from frog skeletal muscle made it possible to preserve the myosin functionality for a week and apply single molecule techniques to the molecular motor that has been best characterized for its mechanical, structural and energetic parameters in situ.With the in vitro motility assay, we estimated the sliding velocity of actin on frog myosin II (VF) and its modulation by pH, myosin density, temperature (range 4-30◦C) and substrate concentration. VF was 8.88 ± 0.26 μms⁻¹ at 30.6◦C and decreased to 1.60 ± 0.09 μms⁻¹ at 4.5◦C. The in vitro mechanical and kinetic parameters were integrated with the in situ parameters of frog muscle myosin working in arrays in each half-sarcomere. By comparing VF with the shortening velocities determined in intact frog muscle fibres under different loads and their dependence on temperature, we found that VF is 40-50% less than the fibre unloaded shortening velocity (V0) at the same temperature and we determined the load that explains the reduced value of VF. With this integrated approach we could define fundamental kinetic steps of the acto-myosin ATPase cycle in situ and their relation with mechanical steps. In particular we found that at 5◦C the rate of ADP release calculated using the step size estimated from in situ experiments accounts for the rate of detachment of motors during steady shortening under low loads.

Figure 1. Polyacrylamide-gel electrophoresis (15% w/v) of frog myosin in the presence of sodium dodecyl sulphate (0.1%)

Lane 1: protein marker. Lane 2: proteins extracted from frog muscle. Lanes 3 and 4: final myosin preparation, with 1× and 2× concentration loadings. Myosin heavy chain: 200 kDa; myosin light chains: 25, 20 and 16.5 kDa.

Figure 2. Histogram of observed sliding velocities (V_F, μm s⁻¹) of actin over myosin molecules from frog skeletal muscle under control conditions

V_F was grouped in classes of 0.5 μm s⁻¹. The continuous line is the least-square fit, using the Gaussian equation (eqn (S2) in Supplemental Material), of the number of the observations (n) as a function of V_F. Estimated Gaussian parameters are: mean 5.25 μm s⁻¹ and σ 0.79 μm s⁻¹. Statistical mean ± SD is 5.28 ± 0.35 μm s⁻¹.

Figure 3. Effect of the concentration of myosin on V_F and on the length of the actin filaments moving in the IVMA

A, relation between the sliding velocity and the myosin concentration. All the data points (mean ± SEM from at least 20 filaments) belong to the same preparation. Continuous line is the linear fit to data, slope 1.05 ± 0.20 μm s⁻¹ ml mg⁻¹. B, relation between the length of moving actin filaments and the myosin concentration. Data points (mean ± SEM) are from the same slides as in A. The dotted lines are drawn to facilitate the visualization of the corner at 0.6 mg ml⁻¹. The average filament length for the three points above the myosin concentration 0.6 mg ml⁻¹ is 3.04 ± 0.18 μm.

Figure 6. Dependence of V_F on MgATP concentration at two temperatures

A, relation of V_Fversus[MgATP] at 23°C (circles) and at 5°C (triangles). Each data point (mean ± SD) is from at least 3 slides. The lines are data fits with the Michaelis–Menten equation (eqn (2)): the best fit parameters are at 23°C (continuous line): V_max = 5.92 ± 0.08 μm s⁻¹ and K_m = 0.174 ± 0.004 mm; at 5°C (dashed line) the values are: V_max = 1.41 ± 0.14 μm s⁻¹ and K_m = 0.051 ± 0.029 mm. B, plots of the reciprocal of V_Fversus the reciprocal of [MgATP]. Symbols as in A. The Michaelis–Menten parameters estimated from the linear fit (eqn (3)) are shown in the following table. For the relation at 5°C separate fits are done for [MgATP] < 0.4 mm (dashed line) and for [MgATP] > 0.4 mm (dotted line).

Figure 4. Effect of pH on sliding velocity

In brackets the number of slides contributing to the data points (mean and SD) are indicated.

Figure 5. Effect of temperature on filament sliding velocity (V)

A, dependence on temperature of V_F (filled triangles, mean and SD) and of unloaded shortening velocity V₀ in fibres (filled circles, calculated as reported in the text from 11 fibres grouped in classes of temperature (°C) 2.5–5.5, 10–13, 15–18, 19–22). B, Arrhenius plot of data in A. The continuous line is the linear regression to V₀ data; the dashed line is the linear regression to V_F data for temperatures ≤ 20°C (1/K≥ 3.4 × 10⁻³); the dotted line is the linear regression to V_F data for temperatures ≥ 20°C (1/K≤ 3.4 × 10⁻³).

Figure 7. Dependence of the force–velocity relation on temperature

A, force–velocity (T–V) relation for a single muscle fibre at three different temperatures: circles, 7°C; triangles, 14.5°C; squares, 21°C. Data from a fibre with CSA 7600 μm², sarcomere length 2.11 μm. Continuous lines are Hill hyperbolic fits to data. Vertical dotted lines are drawn to intersect velocity points at constant load at the three temperatures. B, Arrhenius plots of V_F (triangles and dashed line from Fig. 5B), V₀ (black circles and continuous line from Fig. 5B) and V for loads of 20 kPa (green circles), 40 kPa (blue circles) and 60 kPa (red circles) estimated as shown in A in a total of 11 fibres. Coloured lines are the linear regressions to data points with corresponding colours.

Figure 8. Time course of a single interaction between the myosin motor and the actin, showing the length step d and the underlying biochemical transitions

During the ATPase cycle time (τ_c), the motor may be detached (or weakly attached, M-ADP-P_i state) or attached (A-M states). The duration of the attachment (τ_on) is divided in the time for the ADP release (τ_-ADP) and the time for the ATP binding and actin dissociation (τ_+ATP) (modified from Tyska & Warshaw (2002)).

Figure 9. Plot of d/V_F (τ_on) versus the reciprocal of [MgATP] in the range of [MgATP] > 0.4 mm

Data from triangles in Fig. 6B. The line is drawn according to the parameters from the fit in Fig. 6B multiplied by d (see text).