The Pre-Steady State of the Myosin-Adenosine Triphosphate System*

Abstract

1. Myosin was treated with NTP by the method described in the preceding paper (2). As mentioned in the preceding paper (2), the rate of ATP-hydrolysis of myosin [ATP: phosphohydrolase, EC 3.6.1.3] in the steady state was only slightly altered by treatment with NTP, but no initial burst of P₁-liberation could be observed with NTP-myosin. The actomyosin type of ATPase activity was inhibited by treatment of myosin with NTP. At low ionic strength and in the presence of MgCl₂, no superprecipitation or clearing response could be observed with actomyosin reconstituted from NTP-myosin and F-actin. 2. At high ionic strength and low Mg⁺⁺ concentration the amount of initial rapid P₁-liberation was more than 1 mole per 4×l0⁵g of myosin; i.e., an extra-burst of P₁-liberation was observed. As mentioned in the preceding paper (2) the initial extra-liberation of hydrogen ion was accompanied by an extra-burst of P₁-liberation. The dependencies of the amount of initial extra-burst of hydrogen ion-liberation on the ATP concentration were measured in the presence of various Mg⁺⁺ concentrations. They were in good agreement with those of the amount of decrease in light-scattering intensity of reconstituted actomyosin induced by ATP. The rate of initial rapid hydrogen ion-liberation was essentially equal to that of decrease in light-scattering. 3. The dependency of actomyosin ATPase on Mg⁺⁺ concentration was studied. The ATPase activity increased with the concentration of Mg⁺⁺ until the concentration of added MgCl₂ reached 5μM. At higher concentrations the activity decreased gradually with increasing Mg⁺⁺ concentration. The rate of superprecipitation of actomyosin in the presence of 10 μM MgCl₂ was much faster than that in the presence of 1 mM MgCl₂. 4. When F-actin was added to the myosin-ATP system at various times (10–120 sec) after mixing myosin with ATP, the initial rapid ATP-splitting, seen when ATP was added to actomyosin, was not detected, but only the steady state actomyosin ATPase was observed. 5. Based on the results given in this and the preceding paper (2), the following reaction mechanism is proposed for the function of F-actin in the myosin (E)-ATP (S) system: