Stehle R, Brenner B
Molekular- und Zellphysiologie, Medizinische Hochschule Hannover, D-30625 Hannover, Germany.
Biophys J. 2000 Mar;78(3):1458-73. doi: 10.1016/S0006-3495(00)76699-9.
To characterize the kinetics of cross-bridge attachment to actin during unloaded contraction (maximum velocity of filament sliding), ramp-shaped stretches with different stretch-velocities (2-40,000 nm per half-sarcomere per s) were applied to actively contracting skinned fibers of the rabbit psoas muscle. Apparent fiber stiffness observed during such stretches was plotted versus the speed of the imposed stretch (stiffness-speed relation) to derive the rate constants for cross-bridge dissociation from actin. The stiffness-speed relation obtained for unloaded shortening conditions was shifted by about two orders of magnitude to faster stretch velocities compared to isometric conditions and was almost identical to the stiffness-speed relation observed in the presence of MgATPgammaS at high Ca(2+) concentrations, i.e., under conditions where cross-bridges are weakly attached to the fully Ca(2+) activated thin filaments. These data together with several control experiments suggest that, in contrast to previous assumptions, most of the fiber stiffness observed during high-speed shortening results from weak cross-bridge attachment to actin. The fraction of strongly attached cross-bridges during unloaded shortening appears to be as low as some 1-5% of the fraction present during isometric contraction. This is about an order of magnitude less than previous estimates in which contribution of weak cross-bridge attachment to observed fiber stiffness was not considered. Our findings imply that 1) the interaction distance of strongly attached cross-bridges during high-speed shortening is well within the range consistent with conventional cross-bridge models, i.e., that no repetitive power strokes need to be assumed, and 2) that a significant part of the negative forces that limit the maximum speed of filament sliding might originate from weak cross-bridge interactions with actin.
为了描述在无负荷收缩（细丝滑动的最大速度）过程中横桥与肌动蛋白结合的动力学，对兔腰大肌的活性化去皮纤维施加了不同拉伸速度（每半个肌节每秒2 - 40000纳米）的斜坡形拉伸。将在这种拉伸过程中观察到的表观纤维刚度与施加的拉伸速度作图（刚度 - 速度关系），以推导横桥从肌动蛋白解离的速率常数。与等长收缩条件相比，无负荷缩短条件下获得的刚度 - 速度关系向更快的拉伸速度方向移动了约两个数量级，并且几乎与在高钙（2 +）浓度下存在MgATPγS时观察到的刚度 - 速度关系相同，即在横桥与完全钙（2 +）激活的细肌丝弱结合的条件下。这些数据以及几个对照实验表明，与先前的假设相反，在高速缩短过程中观察到的大部分纤维刚度是由于横桥与肌动蛋白的弱结合。在无负荷缩短过程中强结合横桥的比例似乎低至等长收缩过程中存在比例的约1 - 5％。这比先前的估计低约一个数量级，先前的估计未考虑弱横桥结合对观察到的纤维刚度的贡献。我们的研究结果意味着：1）高速缩短过程中强结合横桥的相互作用距离完全在与传统横桥模型一致的范围内，即无需假设重复的动力冲程；2）限制细丝滑动最大速度的负力的很大一部分可能源于横桥与肌动蛋白的弱相互作用。
