Identifying the calcium-regulated step in the actomyosin ATPase reaction in muscle

Contraction of skeletal muscle is regulated by intracellular calcium levels [Ca^<2+>]。[Ca^<2+>]-在actomyosin ATPase反应中的调节步骤已被识别。我们进行了实验，确定了步骤（s）是由[Ca^<2+>]调节的，我们使用了来自兔子的皮肤肌肉纤维，并添加了非有机磷酸盐（Pi）以增加相对较低力的A. M. ADP和P_i中间人群的沐浴解决方案。这个中间人的相对人口是由lowering [Ca^<2+>]改变的，这是由[Ca^<2+>]调节的低强度中间人的步骤。我们进一步展示了这种低力actomyosin复合体的比率不受影响的[Ca^<2+>]。因此，A·M·ADP·P_i中间体的形成应按[Ca^<2+>]进行调整。下一步，我们研究了更多细节，以了解A·M·ADP·P_i中间体的形成的方法。所提出的问题是： （1）是[Ca^<2+>]是调节A. M. ADP和P_i中间体形成的唯一因素吗？（2） How can the [Ca^<2+>] dependence of shortening velocity by reconciled with the present conclusion that it is A·M·ADP·P_i， not the later intermediates which are more relevant to shortening， that is the primary target of [Ca^<2+>] regulation。为了回答这些问题，我们通过使用多个缩短协议来测量肌肉纤维的短速度。At saturating [Ca^<2+>]一个快速的shortening pattern被重复了很多次。在亚最大值[Ca^<2+>]中，短速度已被消除，且速度取决于张力水平，如：附着的肌红蛋白头的数量，几乎在短时间前。结果表明，A. M. ADP和P_i的形成并不只是由[Ca^<2+>]调节，但也是由附着的肌红蛋白头的数目决定的。The [Ca^<2+\] dependence of shortening Velocity is naturally understood as a consequence of this dual regulation mManagement ism。

Contraction of skeletal muscle is regulated by intracellular calcium levels [Ca^<2+>]. The [Ca^<2+>] -regulated step in the actomyosin ATPase reaction has remained to be identified. We performed experiments to specify the step (s) which is regulated by [Ca^<2+>].We used skinned skeletal muscle fibers from rabbit, and added inorganic phosphate (Pi) to the bathing solution to increase the relative population of the low force A・M・ADP・P_i intermediate. The relative population of this intermediate was unchanged by lowering [Ca^<2+>], indicating that it is the step of formation of the low force intermediate that is regulated by [Ca^<2+>]. We further showed that the rate of dissociation of this low force actomyosin complex is not affected by [Ca^<2+>]. Therefore the forward rate constant for the formation of the A・M・ADP・P_i intermediate should be regulated by [Ca^<2+>].Next we studied in more detail the way in which the formation of the A・M・ADP・P_i intermediate is regulated. The questions addressed were : (1) Is [Ca^<2+>] the only factor that regulates the formation of the A・M・ADP・P_i intermediate? (2) How can the [Ca^<2+>] dependence of shortening velocity by reconciled with the present conclusion that it is A・M・ADP・P_i, not the later intermediates which are more relevant to shortening, that is the primary target of [Ca^<2+>] regulation. To answer these questions, we measured the shortening velocity of muscle fibers by using a multiple shortening protocol. At saturating [Ca^<2+>] a fast shortening pattern was repeated many times. At submaximal [Ca^<2+>], the shortening velocity was decreased, and the velocity depended on the tension levle, i.e., the number of attached myosin heads, immediately before shortening. The results indicate that the formation of A・M・ADP・P_i is regulated not only by [Ca^<2+>], but also by the number of attached myosin heads. The [Ca^<2+>] dependence of shortening velocity is naturally understood as a consequence of this dual regulation mechanism.