Energy transduction in muscle contraction : Study with single thick filaments

为了了解肌动球蛋白交叉桥中保持自然有序排列的力产生过程的动力学信息，我们研究了天然粗丝沿F-肌动蛋白缆索滑动时的力-速度关系。从紫贻贝的前部肌肉中分离出天然粗丝(长达30微米)，并将其附着在预先包覆了聚L赖氨酸的聚苯乙烯微珠(直径4微米)上。将珠子放置在灌流的Chara结间细胞的F-肌动蛋白缆索上，然后将其安装在离心机显微镜的转台上，测量在正(与滑动方向相反)和负(与滑动相同)离心力(高达30pN)负荷下，小球在ATP(与滑动方向相反)和负(与滑动相同)离心力作用下的稳态滑动速度。在正载荷作用下，除在0.8P0以上有一个小的“驼峰”外，大部分的力-速度关系都符合Hill方程，与蛙单肌纤维的力-速度关系非常相似。在负载力作用下，滑移速度最初随力的增大而增大，超过最大卸载速度(V0)后，滑移速度在2-3V0时趋于稳定，直至微珠从肌动蛋白缆索上脱落。结果表明，经典的力-速度关系的最小单位是几十个或数百个交叉桥，它们沿粗丝的有序排列对肌肉收缩的力学性质起着重要的决定作用。

To obtain information about the kinetics of force-generating processes in the actomyosin crossbridges which retain naturally ordered arrangement, we studied the force-velocity relations in sliding movements of native thick filaments along F-actin cables.Native thick filaments (length, up to 30 mum) were isolated from the anterior byssus retractor muscle (ABRM) of Mytilus edulis and attached to polystirene microbeads (diameter, 4mum) coated previously with poly-L-lysine. The beads were placed on F-actin cables of perfused Chara internodal cells, which was then mounted on a rotating stage of a centrifuge microscope, and the velocities of steady state, sliding movements of the beads in the presence of ATP(3mM) were measured under both positive(opposite to the direction of sliding)and negative(same as that of sliding)centrifugal loads(up to 30 pN). Under the positive load, the force-velocity relation was well described, in its most part (with the exception of a small "hump" above 0.8P0), by Hill equation and appeared markedly similar to that for frog single muscle fibers. Under the negative load, the sliding velocity initially increased with force beyond the maximal unloaded velocity (V0), then appeared to reach a steady level at 2-3V0 until the beads were eventually detached from actin cables. The results suggest that the minimal unit responsible for the classical force-velocity relations is several tens or hundreds of crossbridges, and their ordered arrangement along thick filaments plays an important role in determining the mechanical properties of muscle contraction.