Extra Burst of Pi Liberation and Formation of the Myosin-Phosphate-ADP Complex at Various Concentrations of Mg2+ Ions1

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Abstract
We [Kanazawa, T. & Tonbmura, Y. (1964) J. Biochem. 57, 604–615; Furukawa, K.-L, Ikebe, M., Inoue, A., & Tonomura, Y. (1980) J. Biochem. 88, 1629–1641] previously reported that the initial P1burst size of the myosin-ATPase reaction was about 11 mol/mol Myo head at 1 μm of free Mg2+ ions which decreased with an increase in the free Mg2+ concentration, but was still larger than the stoichiometric value (0.5 mol/mol Myo head) at 10 mM of Mg2+ ions. We attributed the P1-burst size larger than the stoichiometric value to a so-called extra P1 burst. The results of our present study support our hypothesis that the larger burst size is due to contamination of the true P1 burst by an extra P1 burst. The rate of fluorescence enhancement of myosin induced by ATP increased with an increase in the free Mg2+ concentration, while the final extent of fluorescence enhancement in the steady state was independent of the free Mg2+ concentration (1 μM to 10 mM). When excess EDTA was added to the reaction mixture in the steady state, the fluorescence decreased to the original level. The rate of decay was independent of the free Mg2+ concentration which had been used for the fluorescence enhancement. Furthermore, the time course of fluorescence decay was equal to that of the transition of ATPase from the Mg2+ to the EDTA(K+) type. The amount of free P1 liberated (free P1) was measured by a double-membrane filtration method at various free Mg2+ concentrations, and compared with the amount of P1 liberated when the reaction was stopped by addition of TCA (TCA-P1). The steady-state rates of free P1 liberation were equal to those of TCA-P1 liberation at various free Mg2+ concentrations. The difference between the amounts of TCA-P1 and free P1, i.e., the amount of P bound to myosin, was 0.49–0.51 mol/mol Myo head regardless of the free Mg2+ concentration. Furthermore, at the completion of the initial P1 burst, the amount of ADP bound to myosin was 0.48–0.50 mol/mol Myo head at various free Mg2+ concentrations.