The Pre-steady State of the Myosin-Adenosine Triphosphate System:I. Initial Rapid Liberation of Inorganic Phosphate

The initial burst of Pi-liberation from the myosin-ATP system was measured under various experimental conditions after stopping the ATPase reaction by addition of trichloro-acetic acid and the following results were obtained.<br> 1. The amount of the initial burst re-mained almost constant during purification procedures and repetition of the dilution-pre-cipitation procedure of myosin, and was unaf-fected by removal of the water-soluble fraction from myosin by DEAE-cellulose treatment.<br> 2. The amount of the initial burst was usually 1 mole of Pi/mole (4×10⁵g.) of myosin over wide ranges of KCl concentration (0.04-1.1 M), pH (6.5-8.5) and temperature (0-30°C), if the Mg⁺⁺ concentration was higher than 1 mM. Although the amount of the initial burst was often less than the above value at low KCl concentration or at low temperature, it could always be restored to this value by raising the KCl concentration or temperature.<br> 3. When the concentration of Mg⁺⁺ was higher than 1 mM, the amount of extra-libera-tion of Pi from the myosin-ATP system in-creased linearly with the ATP concentration until the amount of added ATP reached 1 mole/mole of myosin. At higher ATP con-centrations than this, the amount of the initial burst remained constant at the level of 1 mole/ mole of myosin. The time-course of Pi-libera-tion was unaffected by the addition of a large amount of ADP immediately after the com-pletion of the initial burst of Pi-liberation. The initial reaction of myosin ATPase was thus shown to be a stoichiometric and irrevers-ible interaction of ATP with myosin.<br> 4. At low Mg⁺⁺ concentration, the timecourse of Pi-liberation consisted of 3 steps. In the i st step extra Pi was liberated instantane-ously after ATP addition. The velocity of Pi-liberation decreased gradually in the 2 nd step, and finally reached a constant steady value in the 3 rd step.<br> 5. The amount of extra Pi-liberation in the 1 st step was 1 mole/mole of myosin when the Mg⁺⁺ concentration was higher than 0.05mM and it diminished when the Mg⁺⁺ con-centration was decreased to below 0.05 mM, and became zero in the absence of Mg⁺⁺. The amount of extra Pi-liberation in the 2 nd step was zero when the Mg⁺⁺ concentration was higher than 1 mM. When the Mg⁺⁺ con-centration was decreased below 1mM, it increased at first, showing a maximum at about 0.01mM Mg⁺⁺, and then decreased with decreasing Mg⁺⁺ concentration.<br> 6. The amount of extra-liberation from synthetic actomyosin ATPase was large at low ionic strength. It decreased toward a constant value of 1 mole/mole of myosin when the ionic strength was increased. On the contrary, the amount of extra Pi -liberation of myosin ATPase used in the control experiment was small at low ionic strength. It approached the value of 1mole/mole of myosin as the ionic strength was increased.<br> 7. It was confirmed that the initial burst was inhibited by p-chloromercuribenzoate and ethylenediaminetetraacetic acid under the present experimental conditions, as reported in our previous papers.