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Dynamics of myofilament sliding studied with a newly developed assay system

使用新开发的分析系统研究肌丝滑动动力学

基本信息

批准号：
06640876
负责人：
ISHII Naokata
金额：
$ 1.22万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

项目摘要

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.

为了研究在接近肌肉纤维的条件下肌丝的体外滑动，建立了一种新的测试系统，在该系统中，将直径为4微米的聚苯乙烯微球与一条或几条粗大的软体肌肉(紫贻贝的牵引肌的前肌丝)连接在一起，使其沿安装在离心机显微镜转子上的藻节间细胞中的肌动蛋白微丝阵列(肌动蛋白缆线)滑动。在镁ATP(5 Mm)存在的情况下，沿着肌动蛋白缆索移动的珠子受到与珠子运动相反的离心力(正载荷)或与珠子运动方向相同的离心力(负载荷)。当正载荷从零增加到最大等轴向力P0(15-40pN，20-25C)时，钢珠的运动速度逐渐减小到零，除0.8P0以上的载荷外，其余均呈双曲线力-速度关系。随着P0以上正载荷的进一步增加，珠子被迫沿离心力方向移动，最终在约1.4P0的载荷下从肌动蛋白缆索上脱落。这些特征与完整的单肌纤维的力-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，微珠的移动速度增加到最大卸载速度(Vmax，1.5-3.5微米/S)以上，直到微珠最终从肌动蛋白缆索上分离出来。这些结果表明，目前的检测系统对于未来肌肉收缩机制的生物化学和生理学联合研究是非常有前途的。