Regulation of binding of subfragment 1 in isolated rigor myofibrils.
Regulation of binding of subfragment 1 in isolated rigor myofibrils.
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分离的僵硬肌原纤维中亚片段 1 结合的调节。
DOI：
10.1083/jcb.111.6.2989
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发表时间：
1990
期刊：
影响因子：
--
通讯作者：
Marsh,BB
中科院分区：
文献类型：
--
作者：
Swartz,DR;Greaser,ML;Marsh,BB
空间位阻模型已被用来解释原肌球蛋白-肌钙蛋白复合物对肌肉收缩的调节。通过显微镜观察不同肌动蛋白与 S1 比例以及存在和不存在钙的情况下荧光亚片段 1 (S1) 与严格肌原纤维的混合物来研究结合的调节。在与硫醇特异性荧光染料缀合之前，对程序进行了调整以保护 S1 的关键硫醇，这使得荧光 S1 具有未改变的酶活性。无论[S1]如何，在钙存在的情况下，S1 结合在 I 带（Z 线除外）中最强。缺乏钙时的模式取决于肌动蛋白与 S1 的比率：低 [S1]，结合在肌球蛋白 - 肌动蛋白重叠区域；低 [S1]，结合在肌球蛋白 - 肌动蛋白重叠区域；中间体 [S1]，AI 连接处结合力最高；高 [S1]，I 带中最大结合力。当肌原纤维依次与不含钙的荧光 S1 和含钙的不同荧光 S1 孵育时，双通道荧光显微镜证实了在低 [S1] 下观察到的两种不同的结合模式。这些观察结果支持肌动蛋白位点严格激活的概念。在没有钙的情况下增加IS1]后模式的变化证明了沿着细丝的协同相互作用。然而，这些相互作用（在没有钙的条件下）似乎不会延伸到 > 2-3 个原肌钙蛋白-7 肌动蛋白功能单元。M USC LE 通过含肌动蛋白的细丝在含肌球蛋白的粗丝上滑动而收缩（Huxley 和 Neidergerke，1954；Huxley 和 Hanson，1954），这是一个使用 ATP 驱动的肌动蛋白和肌球蛋白头之间的循环相互作用的过程产生运动和力。脊椎动物骨骼肌收缩的调节是一个复杂的过程，至少使用六种不同的蛋白质种类和钙（Ebashi et al., 1969; Weber and Murray, 1973）。原肌球蛋白和肌钙蛋白都是证明肌动球蛋白制剂中 ATP 酶活性的钙敏感性所必需的。原肌球蛋白-肌钙蛋白复合物跨越七个肌动蛋白单体，该基团被定义为细丝的功能单元（Bremel 和 Weber，1972）。肌钙蛋白复合物由三个亚基组成：肌钙蛋白-T、肌钙蛋白-I 和肌钙蛋白-C。后者赋予钙对受调节的肌动球蛋白 ATP 酶活性的敏感性，而肌钙蛋白-T 和肌钙蛋白-I 的组合（不含肌钙蛋白-C）会抑制 ATP 酶活性（Greaser 和 Gergely，1973）。原肌球蛋白和原蛋白调节收缩的结构机制已通过完整肌纤维的 X 射线衍射进行了研究。早期研究表明，放松和收缩的肌肉之间肌动蛋白层线的强度模式存在差异。这是通过原肌球蛋白从细丝上的外围位置到更靠近肌动蛋白丝凹槽的位置的小幅移动来解释的（Haselgrove，1972；Huxley，1972；Parry 和 Squire，1973）。这些结构性观察结果和拟议的监管机制
A steric-hindrance model has been used to explain the regulation of muscle contraction by tropomyosin-troponin complex. The regulation of binding was studied by microscopic observation of mixtures of fluorescent subfragment 1 (S1) with rigor myofibrils at different actin-to-S1 ratios and in the presence and absence of calcium. Procedures were adapted to protect the critical thiols of S1 before conjugation to thiol-specific fluorochromes, this giving fluorescent S1 with unaltered enzyme activity. S1 binding was greatest in the I band (except at the Z-lines) in the presence of calcium regardless of the [S1]. The patterns in the absence of calcium depended on the actin-to-S1 ratios: low [S1], binding in the myosin-actin overlap region; intermediate [S1], highest binding at the AI junction; high [S1], greatest binding in the I-band. The two distinct binding patterns observed at low [S1] were demonstrated by dual-channel fluorescence microscopy when myofibrils were sequentially incubated with fluorescent S1 without calcium followed by a different fluorescent S1 with calcium. These observations support the concept of rigor activation of actin sites. The change in the pattern upon increasing IS1] without calcium demonstrate cooperative interactions along the thin filament. However, these interactions (under the conditions used without calcium) do not appear to extend over> 2-3 tropomyosintroponin-7 actin functional units.M USC LE contracts by a sliding of the actin-containing thin filaments over the myosin-containing thick filaments (Huxley and Neidergerke, 1954; Huxley and Hanson, 1954), a process using the ATP driven, cyclical interaction between actin and the myosin head to produce both movement and force. The regulation of contraction in vertebrate skeletal muscle is a complex process employing a minimum of six different protein species and calcium (Ebashi et al., 1969; Weber and Murray, 1973). Both tropomyosin and troponin are required for demonstrating calcium sensitivity of ATPase activity in acto-myosin preparations. The tropomyosin-troponin complex spans seven actin monomers, and this group has been defined as the functional unit of the thin filament (Bremel and Weber, 1972). The troponin complex is composed of three subunits, troponin-T, troponin-I, and troponin-C. The latter confers calcium sensitivity to regulated acto-myosin ATPase activity, while the combination of troponin-T and troponin-I without troponin-C inhibits ATPase activity (Greaser and Gergely, 1973). The structural mechanism by which tropomyosin and tropouin regulate contraction has been studied by x-ray diffraction of intact muscle fibers. Early studies demonstrated that there was a difference in the intensity pattern of the actin layer lines between relaxed and contracted muscles; it was explained by a small movement of tropomyosin from a peripheral position on the thin filament to a position nearer to the groove of the actin filament (Haselgrove, 1972; Huxley, 1972; Parry and Squire, 1973). These structural observations and the proposed mechanism for the regulation of