ROTATION OF MYOSIN CROSS-BRIDGES IN SKELETAL MUSCLES

在肌肉收缩的经典假设中， 肌球蛋白跨桥的重新定向是导致 收缩。 然而，这一想法最近受到了 电子顺磁共振结果表明， 肌球蛋白只有一个方向。 因此 必须通过替代技术测试是否存在 肌球蛋白的不同取向。 为此，建议 （a）制定一种测量空间姿态的方法， 凝胶中的分离的肌动球蛋白，和B）测试所述肌动球蛋白的存在， 旋转运动的交叉桥梁的摄动技术。 所有实验都需要激光源和显微镜， 通过测量 肌球蛋白结合染料的荧光。 在项目1中，一种新技术 用凝胶法测量分离蛋白质的方向 电泳进行了描述，并建议将其应用于 技术来测量分离的肌球蛋白亚片段的姿态， 1（S-1）在不同步骤中的F-肌动蛋白 ATP酶肌动蛋白激活循环的中间状态序列 S-1的活性。 在项目2中，建议快速扰动 一个稳定状态的人口活跃的跨桥梁， 一个狭窄的角部门的交叉桥梁由一个强大的极化 光脉冲;这一部门的重新人口将提供 关于预期过桥率的信息 重定向，并将遵循极化方法。 在 项目3，建议使用波长相关的 荧光偏振法，以确定是否 MgADP的加入引起的角取向是一个 跨桥的普遍属性，或者它们是否是 仅对特定探针可观察到。 在项目4中，建议 利用激光光解法快速制备毫微摩尔 通过光解“笼状”前体的光解浓缩ATP 三磷酸腺苷之前扩散到肌肉。 这预计将 同时激活所有跨桥， 运动，如果存在的话，将由极化决定， 荧光法 这些项目的健康相关性是 关于肌肉收缩机制的信息 最终导致对肌肉疾病的积极干预。

In a classical hypothesis of muscle contraction, the cyclic reorientations of myosin cross-bridges are the cause of contraction. However, this idea has recently been contested by the Electron Paramagnetic Resonance results which suggest that there exists only a single orientation of myosin. It is therefore essential to test, by alternative techniques, for the presence of different orientations of myosin. To this end, it is proposed to a) develop a method of measuring the spatial attitude of the isolated actomyosin in a gel, and b) test for the presence of the rotational motions of the cross-bridges by perturbation techniques. All experiments require a laser source and a microscope to determine cross-bridge orientation by measuring polarization of fluorescence of myosin-bound dye. In project 1, a new technique of measuring the orientation of isolated proteins oriented by gel electrophoresis is described, and it is proposed to apply this technique to measure the attitude of isolated myosin subfragment- 1 (S-1) with respect of F-actin during different steps in a sequence of intermediate states of actin-activated cycle of ATPase activity of S-1. In project 2, it is proposed to rapidly perturb a steady state population of active cross-bridges by depopulating a narrow angular sector of cross-bridges by a powerful polarized light pulse; the repopulation of this sector will provide the information about the rate of anticipated cross-bridge reorientations and will be followed by polarization methods. In project 3, it is proposed to use the wavelength-dependent polarization of fluorescence method in order to establish whether the angular reorientations induced by the addition of MgADP are a universal property of the cross-bridges, or whether they are observable only for specific probes. In project 4, it is proposed to use laser photolysis method to rapidly produce millimolar concentration of ATP by photolysis of photolysis "caged" precursor of ATP previously diffused into muscle. This is expected to activate all cross-bridges simultaneously, and their rotational motion, if present, will be determined by polarization of fluorescence method. The health relatedness of these projects are that the information about the mechanism muscle contraction will ultimately lead to the positive intervention in diseases of muscle.