Uyeda T Q, Kron S J, Spudich J A
Department of Cell Biology, Stanford University School of Medicine, CA 94305.
J Mol Biol. 1990 Aug 5;214(3):699-710. doi: 10.1016/0022-2836(90)90287-V.
We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.
我们通过在硝酸纤维素表面测量低密度重酶解肌球蛋白上缓慢移动的肌动蛋白丝的速度，在体外运动分析系统中估算了肌球蛋白横桥的步长（d，每一次ATP水解时肌动蛋白丝的位移）。在先前的研究中，仅观察到大于最小长度的丝进行连续滑动运动。这些丝以最大速度（Vo）移动，而较短的丝则从表面解离。我们现在通过在ATP溶液中加入0.8%的甲基纤维素来改进分析系统。在这些条件下，短于先前最小长度的丝会移动，但速度明显慢于Vo，因为它们由有限数量的肌球蛋白头部推动。这些数据与一个模型一致，该模型预测缓慢移动的丝的滑动速度（v）由Vo与至少一个肌球蛋白头部推动丝的时间分数的乘积决定，即v = vo [1-(1-ts/tc)N]，其中ts是头部与肌动蛋白紧密结合的时间，tc是ATP水解的循环时间，N是可与丝相互作用的肌球蛋白头部的平均数。使用这个方程计算得出，拟合测量的v与N之间关系的ts/tc的最佳值为0.050。假设d = vots，然后计算出每水解一个ATP的步长在10nm至28nm之间，后者代表上限。这个范围在肌球蛋白头部大小对构象变化的几何约束范围内，因此与紧密耦合ATP水解的摆动横桥模型并不矛盾。
