The Pre-steady State of the Myosin-Adenosine Triphosphate SystemI. Initial Rapid Liberation of Inorganic Phosphate*

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Abstract
The initial burst of Pi-liberation from the myosin-ATP [adenosine triphosphate] system was measured under various experimental conditions after stopping the ATPase reaction by addition of trichloroacetic acid. The amount of initial burst remained almost constant during purification and dilution-precipitation procedures of myosin, and was unaffected by removal of the water-soluble fraction from myosin by diethylaminoethyl-cellulose treatment. The initial burst was usually 1 mole of Pi/mole (4 x 105 g) of myosin over wide ranges of KC1 concentration (0. 04-1.1 [image]), pH (6. 5-8. 5) and temperature (0-30[degree]C), with Mg++ concentration more than 1m[image]. Although the initial burst was often less than the above value at low KC1 concentration or at low temperature, it was restored to this value by raising KC1 concentration or temperature. With Mg++ concentration more than 1 m[image], the amount of extra-liberation of Pi from the myosin-ATP system increased linearly with ATP concentration up to a maximum at 1 mole ATP/mole of myosin. The time-course of Pi-liberation was unaffected by the addition of a large amount of adenosine diphosphate immediately after the completion of the initial burst of Pi-liberation. The initial reaction of myosin ATPase was thus shown to be a stoichiometric and irreversible interaction of ATP with myosin. At low Mg++ concentration, the time-course of Pi-liberation consisted of 3 steps. In the 1st step extra Pi was liberated instantaneously after ATP addition. The velocity of Pi -liberation decreased gradually in the 2nd step, and finally reached a constant steady value in the 3rd step. The amount of extra Pi-liberation in the 1st step was 1 mole/mole of myosin with Mg++ concentration more than 0. 05 m[image]; it diminished when Mg++ concentration was below 0. 05 m[image] and became 0 in the absence of Mg++. The amount of extra Pi-liberation in the 2nd step was 0 at Mg++ concentration more than lm[image]. At Mg++ concentration below lmM, it increased at 1st, showing a maximum at about 0. 01 m[image] Mg++, and then decreased with decreasing Mg++ concentration. 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 at increased ionic strength. The amount of extra Pi-liberation of myosin ATPase in the control experiment was small at low ionic strength. It approached the value of 1 mole/mole of myosin with increased ionic strength. The initial burst was inhibited by p-chloromercuribenzoate and ethylenedia-minetetraacetic acid under the present experimental conditions, as reported in our previous papers.

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