ATP-dependent movement of myosin in vitro: Characterization of a quantitative assay

Sheetz and Spudich (1983, Nature (Lond.), 303:31-35) showed that ATP-dependent movement on myosin along actin filaments can be measured in vitro using myosin-coated beads and oriented actin cables from Nitella. To establish this in vitro movement as a quantitative assay and to understand better the basis for the movement, we have defined the factors that affect the myosin-bead velocity. Beads coated with skeletal muscle myosin move at a rate of 2-6 μm/s, depending on the myosin preparation. This velocity is independent of myosin concentration on the bead surface for concentrations above a critical value (~ 20 μg myosin/2.5 x 10⁹ beads of 1 μm in diameter). Movement is optimal between pH 6.8 and 7.5, at KCl concentrations <70 mM, ATP concentrations >0.1 mM, and at Mg²⁺ concentrations between 2 and 6 mM. From the temperature dependence of bead velocity, we calculate activation energies of 91 kJ/mol below 22°C and 40 kJ/mol above 22° C. Different myosin species move at their own characteristic velocities, and these velocities are proportional to their actin-activated ATPase activities. Further, the velocities of beads coated with smooth or skeletal muscle myosin correlate well with the known in vivo rates of myosin movement along actin filaments in these muscles. This in vitro assay, therefore, provides a rapid, reproducible method for quantitating the ATP-dependent movement of myosin molecules on actin.