Toyoshima Y Y, Kron S J, McNally E M, Niebling K R, Toyoshima C, Spudich J A
Nature. 1987;328(6130):536-9. doi: 10.1038/328536a0.
The rotating crossbridge model for muscle contraction proposes that force is produced by a change in angle of the crossbridge between the overlapping thick and thin filaments. Myosin, the major component of the thick filament, is comprised of two heavy chains and two pairs of light chains. Together they form two globular heads, which give rise to the crossbridge in muscle, and a coiled-coil rod, which forms the shaft of the thick filament. The isolated head fragment, subfragment-1 (S1), contains the ATPase and actin-binding activities of myosin (Fig. 1). Although S1 seems to have the requisite enzymatic activity, direct evidence that S1 is sufficient to drive actin movement has been lacking. It has long been recognized that in vitro movement assays are an important approach for identifying the elements in muscle responsible for force generation. Hynes et al. showed that beads coated with heavy meromyosin (HMM), a soluble proteolytic fragment of myosin consisting of a part of the rod and the two heads, can move on Nitella actin filaments. Using the myosin-coated surface assay of Kron and Spudich, Harada et al. showed that single-headed myosin filaments bound to glass support movement of actin at nearly the same speed as intact myosin filaments. These studies show that the terminal portion of the rod and the two-headed nature of myosin are not required for movement. To restrict the region responsible for movement further, we have modified the myosin-coated surface assay by replacing the glass surface with a nitrocellulose film. Here we report that myosin filaments, soluble myosin, HMM or S1, when bound to a nitrocellulose film, support actin sliding movement (Fig. 2). That S1 is sufficient to cause sliding movement of actin filaments in vitro gives strong support to models of contraction that place the site of active movement in muscle within the myosin head.
肌肉收缩的旋转横桥模型提出，力是由重叠的粗肌丝和细肌丝之间横桥角度的变化产生的。肌球蛋白是粗肌丝的主要成分，由两条重链和两对轻链组成。它们共同形成两个球状头部，这是肌肉中横桥的来源，以及一个卷曲螺旋杆，它形成粗肌丝的轴。分离的头部片段，即亚片段-1（S1），包含肌球蛋白的ATP酶和肌动蛋白结合活性（图1）。尽管S1似乎具有必要的酶活性，但一直缺乏S1足以驱动肌动蛋白运动的直接证据。长期以来，人们一直认识到体外运动测定是识别肌肉中负责产生力的元件的重要方法。海因斯等人表明，涂有重酶解肌球蛋白（HMM）的珠子，HMM是肌球蛋白的一种可溶性蛋白水解片段，由杆的一部分和两个头部组成，可以在丽藻属肌动蛋白丝上移动。利用克朗和斯普迪奇的肌球蛋白包被表面测定法，原田等人表明，与玻璃支持物结合的单头肌球蛋白丝能使肌动蛋白以与完整肌球蛋白丝几乎相同的速度移动。这些研究表明，杆的末端部分和肌球蛋白的双头性质对于运动不是必需的。为了进一步限制负责运动的区域，我们通过用硝酸纤维素膜代替玻璃表面对肌球蛋白包被表面测定法进行了改进。在此我们报告，当肌球蛋白丝、可溶性肌球蛋白、HMM或S1与硝酸纤维素膜结合时，它们支持肌动蛋白滑动运动（图2）。S1足以在体外引起肌动蛋白丝的滑动运动，这为将肌肉中主动运动位点置于肌球蛋白头部的收缩模型提供了有力支持。
