Dynamics of myofilament sliding studied with a newly developed assay system

为了研究离体肌丝在接近肌纤维的条件下的滑动，开发了一种新的测定系统，其中聚苯乙烯微珠（直径4 μ m;比重为1.3），具有软体动物平滑肌的单个至几个粗丝（贻贝的前肌）附着，使其沿着肌动蛋白丝阵列（肌动蛋白索）在安装在离心显微镜的转子上的细胞的结间细胞中滑动。在MgATP（5mM）存在下沿着肌动蛋白缆线移动的珠经受与珠移动相反的离心力（正载荷）或与珠移动相同方向的离心力（负载荷）。随着正载荷从零增加到最大等长力P_0（15 - 40pN，20 - 25 ℃），微珠运动速度逐渐减小到零，除载荷大于0.8P_0外，微珠运动速度与力呈双曲线关系。随着P0以上正载荷的进一步增加，珠被迫在离心力的方向上移动，最终在约1.4P_0的载荷下从肌动蛋白缆线分离。这些特征与完整单肌纤维的力-速度关系非常相似。另一方面，随着负载荷从零增加到一定值，珠粒移动的速度增加到最大无载速度（Vmax，1.5 - 3.5 μ m/s）以上，直到珠粒最终从肌动蛋白缆线分离。这些结果表明，目前的测试系统是非常有前途的未来相结合的生化和生理学研究的肌肉收缩的机制。

To investigate in vitro myofilament sliding in conditions close to those in muscle fibers, a new assay system was developed, in which a polystirene microbead (diameter, 4mum ; specific gravity, 1.3), with a single to a few thick filaments of a molluscan smooth muscle (the anteriour bysus retractor muscle of Mytilus edulis) attached, was made to slide along actin filament arrays (actin cables) in the internodal cell of an alga, mounted on the rotor of a centrifuge microscope. The bead moving along actin cables in the presence of MgATP (5mM) was subjected to centrifugal forces either opposite to the bead movement (positive loads) or in the same direction as the bead movement (negative loads). With positive loads increasing from zero to the maximum isometric force P_0 (15-40pN,20-25C), the velocity of bead movement decreased gradually to zero, exhibiting the hyperbolic force-velocity relation except for load above 0.8 P_0. With further increase of positive loads above P0, the bead was forced to move in the direction of centrifugal force, being eventually detached from actin cables at a load of around 1.4 P_0. These features are very similar to thr force-velocity relation in intact single muscle fibers. With negative loads increasing from zero to a certain value, on the other hand, the velocity of bead movement increased above the maximum unloaded velocity (Vmax, 1.5-3.5mum/s) up to around twice the Vmax, until the bead was eventually detached from actin cables. These results indicate that the present assay system is extremely promising for future combined biochemical and physiological studies on the mechanism of muscle contraction.