Reedy M C, Reedy M K, Tregear R T
Department of Anatomy, Duke University Medical Center, Durham, NC 27710.
J Mol Biol. 1988 Nov 20;204(2):357-83. doi: 10.1016/0022-2836(88)90582-7.
We have performed thin-section electron microscopy on muscle fibers fixed in different mechanically monitored states, in order to identify structural changes in myosin crossbridges associated with force production and maintenance. Tension and stiffness of fibers from glycerinated Lethocerus flight muscle were monitored during a sequence of conditions using AMPPNP and then AMPPNP plus increasing concentrations of ethylene glycol, which brought fibers through a graded sequence from rigor relaxation. Two intermediate crossbridge forms distinct from the rigor or relaxed forms were observed. The first was produced by AMPPNP at 20 degrees C, which reduced isometric tension 60 to 70% below rigor level without reducing rigor stiffness. Electron microscopy of these fibers showed that, in spite of the drop in tension, no obvious change from the 45 degrees crossbridge angle characteristic of rigor occurred. However, the thick filament ends of the crossbridges were altered from their rigor positions, so that they now marked a 14.5 nm repeat, and formed four separate origins at each crossbridge level. The bridges were also less slewed and bent than rigor bridges, as seen in transverse sections. The second crossbridge form was seen in glycol-AMPPNP at 4 degrees C, just below the glycol concentration that produced mechanical relaxation. These fibers retained 90% of rigor stiffness at 40 Hz oscillation, but would not bear sustained tension. Stiffness was also high in the presence of calcium at room temperature under similar conditions. Electron microscopy showed crossbridges projecting from the thick filaments at an angle that centered around 90 degrees, rather than the 45 degree angle familiar from rigor. This coupling of relaxed appearance with persistent stiffness suggests that the 90 degree form may represent a weakly attached crossbridge state like that proposed to precede force development in current models of the crossbridge power stroke.
我们对处于不同机械监测状态下固定的肌纤维进行了薄切片电子显微镜检查，以确定与力产生和维持相关的肌球蛋白横桥的结构变化。在一系列条件下，使用AMPPNP，然后是AMPPNP加浓度不断增加的乙二醇，监测甘油化大负蝗飞行肌纤维的张力和刚度，这使纤维经历了从强直松弛的分级序列。观察到两种不同于强直或松弛形式的中间横桥形式。第一种是由20℃的AMPPNP产生的，它使等长张力比强直水平降低了60%至70%，而不降低强直刚度。这些纤维的电子显微镜检查表明，尽管张力下降，但与强直特征性的45度横桥角度没有明显变化。然而，横桥的粗丝末端从其强直位置发生了改变，因此它们现在标记为14.5nm的重复，并在每个横桥水平形成四个独立的起点。如在横切面中所见，这些桥也比强直桥更少扭转和弯曲。第二种横桥形式出现在4℃的乙二醇-AMPPNP中，略低于产生机械松弛的乙二醇浓度。这些纤维在40Hz振荡时保留了90%的强直刚度，但不能承受持续张力。在类似条件下，室温下存在钙时刚度也很高。电子显微镜显示横桥从粗丝以大约90度的角度伸出，而不是强直时常见的45度角。这种松弛外观与持续刚度的耦合表明，90度形式可能代表一种弱附着的横桥状态，类似于当前横桥动力冲程模型中提出的在力发展之前的状态。
