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+>]-regulated step in the actomyosin ATPase反应has remained to be identified. We performedexperiments to specify the step (s) which is regulated by [Ca^<2+>].We used skinned skeletal musclefibers from rabbit，and added inorganic phosphate (Pi) to the bathing solution to increase the相关population ofthe low force A . M . ADP . p_i intermediate. the relative population of this intermediate was unchangedby 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 isnot affected by [Ca^<2+>]. Therefore the forward rate constant for the formation of the A . M . adp_i中间应该规范[Ca^<2+>].Next we studied in more detail the way in which theadp_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 presentconclusion that it is A . M . adp_i，later intermediates which are more relevant to shortening，that is the primary target of [Ca^<2+>] regulation. To answer these questions我们measured the shortening velocity of muscle fibers by using a multiple shortening protocol Atsaturating [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 headsimmediately before shortening. The results indicate that The formation of A . M . ADP . p_i is regulatednot only by [Ca^<2+>]but also by the number of attached myosin heads. the [Ca^<2+] dependence of shortening velocity isnaturally understood as a consequence of this dual regulation mechanism。

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.