Miyata M, Arata T, Inoue A
Department of Biology, Faculty of Science, Osaka University.
J Biochem. 1989 Feb;105(2):271-4. doi: 10.1093/oxfordjournals.jbchem.a122652.
The species and amounts of intermediates formed by myosin in myofibrils during the ATPase reaction under relaxed conditions were examined. The amount of total nucleotides (ADP + ATP) bound to myofibrils, determined by a centrifugation method or a rapid filtration method, was 0.86 mol/mol myosin head. The amount of bound ADP, determined as the ADP remaining in the mixture after free ADP had been rapidly converted into ATP by an ATP-regenerating system, was found to be 0.67 mol/mol myosin head. We examined the time courses of free-Pi and total-Pi (TCA-Pi) formation after adding ATP to the myofibrils. The amount of Pi bound to myofibrils, calculated by subtracting the burst size of free Pi (0.23 mol/mol myosin head) from that of TCA-Pi (0.60 mol/mol myosin head), was found to be 0.37 mol/mol myosin head. The amount of tightly bound ATP determined by an ATP-quenching method was very low (0.03 mol/mol myosin head). If there is no myosin-phosphate complex, then the amounts of the myosin-phosphate-ADP complex, MADPP, and the tightly bound myosin-ATP complex, MATP, are 0.37 and 0.03 mol/mol myosin head, respectively, whereas the amounts of myosin-ADP and loosely bound myosin-ATP complexes are 0.30 and 0.16 mol/mol myosin head, respectively. Thus, half of the myosin heads forms MADPP or MATP, and the equilibrium between MADPP and M*ATP shifts to the MADPP side. These results agree with those obtained for myosin in solution (Inoue, A., Takenaka, H., Arata, T., & Tonomura, Y. (1979) Adv. Biophys. 13, 1-194). Therefore, in relaxed myofibrils the active site of myosin does not interact with actin.
研究了在松弛条件下肌原纤维中肌球蛋白在ATP酶反应过程中形成的中间体的种类和数量。通过离心法或快速过滤法测定，与肌原纤维结合的总核苷酸（ADP + ATP）的量为0.86摩尔/摩尔肌球蛋白头部。通过ATP再生系统将游离ADP快速转化为ATP后，测定混合物中剩余的ADP量，即结合的ADP量，为0.67摩尔/摩尔肌球蛋白头部。我们研究了向肌原纤维中添加ATP后游离Pi和总Pi（TCA - Pi）形成的时间进程。通过从TCA - Pi（0.60摩尔/摩尔肌球蛋白头部）的爆发量中减去游离Pi的爆发量（0.23摩尔/摩尔肌球蛋白头部）计算得出，与肌原纤维结合的Pi量为0.37摩尔/摩尔肌球蛋白头部。通过ATP猝灭法测定的紧密结合的ATP量非常低（0.03摩尔/摩尔肌球蛋白头部）。如果不存在肌球蛋白 - 磷酸复合物，那么肌球蛋白 - 磷酸 - ADP复合物（MADPP）和紧密结合的肌球蛋白 - ATP复合物（MATP）的量分别为0.37和0.03摩尔/摩尔肌球蛋白头部，而肌球蛋白 - ADP和松散结合的肌球蛋白 - ATP复合物的量分别为0.30和0.16摩尔/摩尔肌球蛋白头部。因此，一半的肌球蛋白头部形成MADPP或MATP，并且MADPP和M*ATP之间的平衡向MADPP一侧移动。这些结果与在溶液中对肌球蛋白获得的结果一致（井上，A.，竹中，H.，新田，T.，& 户村，Y.（1979年）《生物物理进展》13，1 - 194）。因此，在松弛的肌原纤维中，肌球蛋白的活性位点不与肌动蛋白相互作用。
