ROLE OF PHOSPHORYLATION AS A REGULATORY MECHANISM IN MUSCLE CONTRACTION

为了了解平滑肌收缩的机制以及肌动蛋白和肌动蛋白如何 肌球蛋白在非肌肉细胞中相互作用，我们一直在使用各种分析方法 肌球蛋白功能。其中一种分析涉及到在 荧光标记肌动蛋白运动的荧光显微镜 表面覆盖肌球蛋白分子的细丝。这场运动是一场 需要肌球蛋白和镁三磷酸腺苷存在的活性过程。这个 肌动蛋白细丝由平滑肌和非肌肉肌球蛋白运动 几乎完全依赖于肌球蛋白对肌球蛋白的磷酸化 轻链激酶。火鸡肌腱肌球蛋白转位肌动蛋白 细丝的移位速度大约是人类的5倍 血小板肌球蛋白。牛脑中的肌球蛋白似乎能移动肌动蛋白细丝 甚至比血小板肌球蛋白的速度还要慢。重的肌球蛋白，可溶 肌球蛋白的双头蛋白降解片段，可从血小板中制备 肌球蛋白。这种沉重的肌球蛋白还可以将肌动蛋白细丝移位到 以与完整的血小板相似的速度进行运动测定 肌球蛋白。肌球蛋白可以以细丝或细丝的形式结合到玻璃表面 (由排列紧密的肌球蛋白分子组成)或作为单体。两者都有 以相同的速率形成移位的肌动蛋白细丝，表明 平滑肌和脊椎动物的非肌肉肌球蛋白、细丝不是绝对的 活动所需的。这也增加了肌球蛋白 细丝本身可能不是细胞运动功能所必需的，并且可以 解释在以下方面遇到的一些困难 证明脊椎动物体内肌球蛋白粗丝的存在 非肌肉细胞。为了更好地了解细丝在人体内的作用 肌球蛋白与肌动蛋白的相互作用 带有EDC的细丝内的肌球蛋白分子。这些交联型肌球蛋白 细丝在通常促进的条件下不会解聚 肌球蛋白的解聚，因此代表了一种研究机制 肌动蛋白-肌球蛋白细丝的相互作用 体外不稳定。

To understand the mechanism of smooth muscle contraction and how actin and myosin interact in nonmuscle cells, we have been using various assays of myosin function. One of these assays involves the visualization in the fluorescent microscope of the movement of fluorescently-labeled actin filaments over a surface coated with myosin molecules. This movement is an active process which requires the presence of myosin and MgATP. The movement of actin filaments by smooth muscle and nonmuscle myosins is almost completely dependent upon phosphorylation of the myosin by myosin light chain kinase. Turkey gizzard smooth muscle myosin translocates actin filaments at about 5 times the rate of translocation obtained with human platelet myosin. Myosin from bovine brain appears to move actin filaments even more slowly than does platelet myosin. Heavy meromyosin, the soluble two-headed proteolytic fragment of myosin, can be prepared from platelet myosin. This heavy meromyosin can also translocate actin filaments in the motility assay at a rate similar to that obtained with the intact platelet myosin. Myosin can be bound to the glass surface as either filaments (consisting of a packed array of myosin molecules) or as monomers. Both forms translocate actin filaments at the same rate indicating that with smooth muscle and vertebrate nonmuscle myosin, filaments are not absolutely required for activity. This also raises the possibility that myosin filaments per se may not be necessary for motile functions in cells and may explain some of the difficulties that have been experienced in demonstrating the presence of myosin thick filaments in vertebrate nonmuscle cells. In order to better understand the role filaments play in the interaction of smooth muscle myosin with actin, we have cross-linked the myosin molecules within a filament with EDC. These cross-linked myosin filaments do not depolymerize under conditions that normally promote depolymerization of myosin and, thus, represent a mechanism for studying actin-myosin filament interactions under conditions where the filaments are unstable in vitro.