The Pre-steady State of the Myosin-Adenosine Triphosphate System

The amount and rate of formation of the reactive myosin-phosphate-ADP complex, EPADP, was estimated by measuring the size and rate of the initial burst of Pi-liberation after stopping the reaction by trichloroacetic acid. Measurements on myosin-ATPase EC 3.6.1.3 were made in 0.5 M KCl and 2.5 mM MgCl2 at pH7.8 and 0°C, unless otherwise stated. The following results were obtained. 1) At ATP concentrations below 0.3μM, the values of KM and Vfm for formation of EPADP were 0.3μM and 0.70 mole Pi/min·4.8×105g myosin, respectively, while at ATP concentrations above 0.3μM, the values of KM and Vfm were much larger. 2) The values of KM and Vm of actomyosin-ATPase were measured in 50 mM KCl and 2 mM MgCl2 at pH 7.0 and 25°C. At ATP concentrations below 10μM, they were 2.9μM and 2.5×102 moles/min·4.8×105g myosin, respectively, and were equal to those for EPADP formation of myosin under the same conditions. At ATP concentrations above 10μM, the rate of the actomyosin-ATPase reaction was larger than that expected from the KM and Vm values obtained at low ATP concentrations. 3) At ATP concentrations above 0.5μM, the values of KM and Vm of myosin-ATPase in the steady state were 1μM and 0.44 mole Pi/min·4.8×105 g myosin, respectively, while at ATP concentrations below 0.3μM, the rate of the ATPase reaction was almost independent of the ATP concentration and had a Vm value of about 1/4 of that obtained at high ATP concentrations. 4) The results of Lymn and Taylor on the dependence of the burst size on the ATP concentration was reexamined in 0.5 M KCl and 10 mM MgCl2 at pH 8.0 and 20°C. Both the rate in the steady state and the size of the initial burst of Piliberation were found to be independent of the ATP concentration at concentrations of 5 to 200μM.