The Pre-steady State of the Myosin-Adenosine Triphosphate System:II. Initial Rapid Absorption and Liberation of Hydrogen Ion Followed by a Stopped-flow Method

1. The rate of liberation of total H⁺ by myosin-ATPase was measured using a Titri-graph. From the dependence of this rate on the ATP concentration, K_m and k₊₂ in 0.5 M KCl-1 mM MgCl₂ at pH 8.2 were found to be 3.8μM and 0.012 sec.^-1, respectively. K_m and k₊₂ in the presence of 0.1mM ethylene-diaminetetraacetic acid (EDTA) were 68 μM and 3.9 sec.^-1, respectively.<br> 2. The time-course of the change in con-centration of free H⁺ in the myosin-ATP system was measured by a stopped-flow method, using cresol red as an indicator. In the presence of 1 mM MgGl₂, the change consisted of 3 steps. In the first step, 1 mole of H⁺ per 4×10⁵g. protein was rapidly .absorbed to myosin, and in the second step the H⁺ absorbed to the protein was again liberated and the concentration of H⁺ returned to the initial level about 2 seconds after the start of the reaction. In the third step which was the steady state, H⁺ was liberated linearly with time and simultaneously ATP was hydrolyzed. On the other hand, in the presence of 0.1 mM EDTA no rapid libera-tion-absorption of H⁺ in the initial stage was observed, and H⁺ was liberated at the same time as P_i3, after a lag period, which was about 0.21 seconds with 30 μM of ATP.<br> 3. It was concluded from these two results that in the presence of MgCl₂ k_-1»k₊₂, while in the presence of ETDA k₊₂»k_-1, if -we adopt the reaction: E+S^k₊₁_??__{k_-1} ES k₊₂→E+ADP+Pi, <br> as the mechanism of the steady state ATPase activity.<br> 4. The initial extra-liberation of P_i from the myosin-ATP system was measured under various conditions, together with the rapid absorption-liberation of H⁺ mentioned above. It was found that the amount of extra P_i-liberation is essentially equal to that of rapid H⁺-absorption under all the conditions in-vestigated. Notably both the phenomena were prevented by EDTA. Furthermore, the amount of rapid absorption of H+ in 0.5M KCl decreased in proportion to the decrease in extra-liberation of P_i, when the latter was reduced to less than 1 mole per 4 × 10⁵g. protein by changing the procedures used for isolation of the protein, and it in-creased to 1 mole 4×10⁵g. protein, as did the amount of extra-liberation of P_i in 1.0 M KCl.<br> 5. From these results, the following reaction mechanism is proposed for the initial reaction of the myosin-ATP system in the presence of MgC1₂:<br> where k_-1»k₊₂, k₊₃>k₊₄»k₂+k₊₅ and E_??_P is the intermediate whose carboxyl group may be phosphorylated.