The Pre-steady State of the Myosin-Adenosine Triphosphate SystemIII. Properties of the Intermediate*

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Abstract
P-Nitrothiophenyl myosin was prepared by incubation of 1 m[image] p-nitrothiophenol (NTP) with myosin in the presence of 2m[image] adenosine triphosphate (ATP) in 1.1 [image] KC1 - 22 m[image] MgCl2 at pH 6. 5 and 0[degree]. Initial Pi liberation from myosin-ATP was 1 mole/4 x 105 g myosin. Initial extra-liberation of Pi decreased linearly to 0.1 mole/4 x 105 g myosin with binding of 1. 2 moles NTP/105 g protein. Adenosine triphosphatase (ATPase) activity at steady state increased to about 5. 5 times the control myosin with increased myosin-bound NTP (0. 7 moles NTP/105 g protein). Time-course study of NTP liberation from NTP-myosin at pH 7. 0 and 25[degree] indicated 2 kinds of NTP-binding sites per molecule, 1 stabe and 1 labile. Initial Pi-liberation from NTP-myosin-ATP remained low at 0.15 moles/4 x 105 g protein; steady state ATPase activity decreased from 5. 2 to 1. 6 times the control value on removal of NTP from the labile sites (incubation for 4 hr. at pH 7.0 and 25[degree]). Decomposition rate constant for the phosphoryl intermediate into myosin, Pi and H+ remained constant at 0.14 min. -1, at pH 8-7. 5, or about 1/10 that of Pi-liberation in steady state. Phosphoryl myosin (in actomyosin-ATP system) decomposed at 100 times the rate in the myosin-ATP system. After the addition of ATP to myosin at a molar ratio of 1:1, the reaction was stopped by removing nucleotides by the charcoal treament. Myosin, precipitated at a low ionic strength or at the isoelectric point, was separated with a Millipore filter and free Pi was washed out with buffer solution. The washed precipitate was extracted with trichloroacetic acid (TCA), and the amounts of TCA-labile phosphate in the precipitate were measured after various separation times, i. e., periods from the addition of charcoal to the end of the last washing with buffer solution. The content of TCA-labile phosphate and its decrease with increase in the separation time were consistent with the assumption that TCA-labile phosphate was derived from the intermediate of the initial reaction of myosin with ATP.