Dynamics of myofilament sliding studied with a newly developed assay system

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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.