Wang F, Martin B M, Sellers J R
Laboratory of Molecular Cardiology National Heart, Lung and Blood Institute, Bethesda, Maryland 20892.
J Biol Chem. 1993 Feb 15;268(5):3776-80.
Contraction of striated muscle from Limulus polyphemus, the horseshoe crab, is regulated by both calcium binding to a troponin-tropomyosin-dependent thin filament array and a myosin light chain kinase-dependent phosphorylation of myosin. We have isolated myosin from Limulus striated muscle and examined how these two regulatory systems affect the sliding velocity of actin filaments over myosin, using an in vitro motility assay. Our results show that in the presence of ATP, Limulus myosin must be phosphorylated in order to move actin filaments. No movement was observed for actin filaments interacting with dephosphorylated Limulus myosin. Calcium was not required for actin movement. In contrast, when both troponin and tropomyosin are bound to actin filaments, calcium is required for the movement of actin filaments over phosphorylated myosin. These results demonstrate that the "off" state of either the thin filament or thick filament regulatory system is dominant and that for the movement to occur, both phosphorylated Limulus myosin and an activated troponin-tropomyosin system are required. Tropomyosin by itself increases the sliding velocity of actin filaments over phosphorylated Limulus myosin about 10-fold in a calcium-independent manner. Tropomyosins from turkey gizzard smooth muscle, bovine cardiac muscle, and Limulus muscle all have a profound effect in increasing the velocity. Troponin alone does not change the velocity. Partial sequences of the tryptic phosphopeptides of Limulus myosin regulatory light chains generated following the phosphorylation by gizzard myosin light chain kinase yield ATS(PO4)NVFAMFEQNQIA for 21 kDa and SGS(PO4)NVFSMFT for 31-kDa light chain.
鲎（Limulus polyphemus）横纹肌的收缩受两种机制调节：一是钙与肌钙蛋白 - 原肌球蛋白依赖的细肌丝阵列结合，二是肌球蛋白轻链激酶依赖的肌球蛋白磷酸化。我们从鲎横纹肌中分离出肌球蛋白，并使用体外运动分析方法研究了这两种调节系统如何影响肌动蛋白丝在肌球蛋白上的滑动速度。我们的结果表明，在ATP存在的情况下，鲎肌球蛋白必须被磷酸化才能移动肌动蛋白丝。与去磷酸化的鲎肌球蛋白相互作用的肌动蛋白丝未观察到运动。肌动蛋白移动不需要钙。相反，当肌钙蛋白和原肌球蛋白都与肌动蛋白丝结合时，钙是磷酸化肌球蛋白上肌动蛋白丝移动所必需的。这些结果表明，细肌丝或粗肌丝调节系统的“关闭”状态是主导的，并且为了发生运动，需要磷酸化的鲎肌球蛋白和激活的肌钙蛋白 - 原肌球蛋白系统。原肌球蛋白自身以不依赖钙的方式使磷酸化的鲎肌球蛋白上肌动蛋白丝的滑动速度增加约10倍。来自火鸡砂囊平滑肌、牛心肌和鲎肌肉的原肌球蛋白在增加速度方面都有深远影响。单独的肌钙蛋白不会改变速度。用砂囊肌球蛋白轻链激酶磷酸化后产生的鲎肌球蛋白调节轻链的胰蛋白酶磷酸肽的部分序列，21 kDa的为ATS(PO4)NVFAMFEQNQIA，31 kDa轻链的为SGS(PO4)NVFSMFT。
