Studies on function of myosin subfragment-2 in skeletal muscle contraction

Huxley，H.C.和Huxley，A.F.独立地提出，肌球蛋白亚片段-1(S-1)区域的结构变化(头部旋转)激活了肌纤维，同时它附着在肌动蛋白上，有助于力的产生。然而，到目前为止，没有直接证据支持这一机制。另一方面，Harrington，W.F.提出，位于短亚片段-2和轻质肌球蛋白片段之间的铰链区的融化和缩短有助于激活的肌肉纤维中的力产生。我们发现，抗肌球蛋白亚片段-2(S-2)的抗体可完全降低甘油化兔腰肌纤维的钙激活力，而不影响钙激活的ATPase活性。为了进一步了解S-2在肌球蛋白铰链区的肌肉收缩中的作用，我们提纯了针对骨骼肌肌球蛋白铰链区20个氨基酸的多肽片段的抗体。电子显微镜显示…在松弛的单个甘油纤维中，经抗S-2铰链抗体(Ig G，3 mg/ml，60min)处理后，钙激活的等长力和肌纤维硬度平行降低，但没有完全抑制，相反，钙激活的ATPase活性保持不变。在僵直的单一纤维中，等长僵直的力量和肌肉纤维的僵硬都没有改变。这些结果表明，铰链区对肌肉收缩功能的影响不大，因此，没有铰链区的肌球蛋白亚片段-2的另一个区域可能与肌肉收缩有关。然而，抗S-2抗体不太可能靠近S-2-S-2结合处，更不可能降低钙离子激活力。肌球蛋白亚片段-2的整个区域可能在肌肉收缩机制中起重要作用，或者在非常有限的小区域针对抗原的抗体可能无效。较少

Huxley, H.C.and Huxley, A.F.have independently proposed that structural changes (head rotation) in myosin subfragment-1 (S-1) region activated muscle fibers, whilee it is attached to actin, contribute to force generation. However there is no direct evidence supporting this mechanism until now. On the other hand, Harrington, W.F.has proposed that melting and shortening in the hinge region located between the short subfragment-2 and light meromyosin segments contribute to force generation in activated muscle fibers. We have shown that the antibody to whole myosin subfragment-2 (S-2) completely reduces the Ca^<2+>-activated force in glycerinated rabbit psoas muscle fibers without affecting the Ca^<2+>-activated ATPase activity. To obtain further information about the function of S-2 in muscle contraction coupled to the myosin hinge region, we purified an antibody directed against a 20-amino acid peptide segment within the hinge region of skeletal muscle myosin. Electron microscopy showed … More that a globular structure corresponding to an anti-hinge antibody bound at the hinge region of the myosin molecule.In relaxd single glycerinated fibers treated with anti S-2 hinge antibody (IgG,3mg/ml, 60min) , Ca^<2+>-activated isometric force and muscle fiber stiffness decreased in parallel without complete suppresion, in contrast Ca^<2+>-activated ATPase activity remained unchanged. In rigor single fibers done with, both isometric rigor force and muscle fiber stiffness did not changed. These results suggest that the hinge region does not strongly affect the function of muscle contraction, therefore another region of myosin subfragment-2 without the hinge region may contribute to muscle contraction. However, this possibility is unlikely that anti S-2 antibody against to antigen in 20-amino acid peptide of S-2 region closed to S-2-S-2 junction, much less reduces in Ca^<2+>-activated force. Whole region of myosin subfragment-2 may be important to muscle contraction mechanism, or antibody against to antigen in very limited small region may be no effective. Less