ROTATION OF MYOSIN CROSS-BRIDGES IN SKELETAL MUSCLES

在肌肉收缩的经典假说中，循环 肌球蛋白交叉桥的重新定位是导致 收缩。然而，这一想法最近遭到了 电子顺磁共振结果表明 只存在单一方向的肌球蛋白。因此，它是 必须通过替代技术测试是否存在 肌球蛋白的不同取向。为此目的，建议 A)制定一种方法来测量 凝胶中分离的肌动球蛋白，以及b)检测是否存在 用摄动法研究跨桥的转动问题。 所有的实验都需要一个激光光源和一个显微镜 通过测量桥梁的偏振来确定跨桥方向 肌球蛋白结合染料的荧光。在项目1中，一种新技术 凝胶定向分离蛋白取向的测量 对电泳法进行了描述，并建议将其应用于 测量分离的肌球蛋白亚片段的姿态的技术- 1(S-1)与F-肌动蛋白在不同阶段的作用 肌动蛋白激活的ATPase循环中间状态序列 S一号的活动。在项目2中，建议迅速扰乱 基于种群减少的有源跨越桥的稳态种群 一个狭窄的棱角部分的交叉桥梁由一个强大的极化 光脉冲；该扇区的重新填充将提供 关于预期过桥速度的信息 重新定向，随后将采用极化方法。在……里面 方案3，建议使用波长相关的 偏振荧光法，以确定是否 由添加镁ADP引起的角重定向是 跨桥的普适性，还是它们是 只有特定的探测器才能观察到。在项目4中，提议 利用激光光解方法快速生产毫米波 光解笼状前驱体光解浓缩三磷酸腺苷 先前扩散到肌肉中的。预计这将是 同时激活所有交叉桥，并且它们的旋转 运动，如果存在，将由极化决定 荧光法。这些项目与健康相关的是 关于肌肉收缩机制的信息将会 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.