STEADY-STATE FORCE VELOCITY RELATION IN THE ATP-DEPENDENT SLIDING MOVEMENT OF MYOSIN-COATED BEADS ON ACTIN CABLES INVITRO STUDIED WITH A CENTRIFUGE MICROSCOPE
STEADY-STATE FORCE VELOCITY RELATION IN THE ATP-DEPENDENT SLIDING MOVEMENT OF MYOSIN-COATED BEADS ON ACTIN CABLES INVITRO STUDIED WITH A CENTRIFUGE MICROSCOPE
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DOI：
10.1073/pnas.87.20.7893
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发表时间：
1990-10-01
影响因子：
11.1
通讯作者：
SUGI, H
中科院分区：
文献类型：
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作者：
OIWA, K;CHAEN, S;SUGI, H
为了消除肌动球蛋白在溶液中的生物化学与肌肉收缩生理学之间的差距，我们开发了一种体外力-运动测定系统，在该系统中可以研究肌动蛋白-肌球蛋白相互作用中的稳态力-速度关系。该测定系统由包含组织良好的肌动蛋白丝阵列（肌动蛋白索）的钝拟丽藻（Nitellopsis obtusa）的节间细胞;涂有骨骼肌肌球蛋白的甲苯磺酰基活化的聚苯乙烯珠（直径，2.8 μ m;比重，1.3），以及配备有频闪光源和视频系统的离心显微镜组成。将结间细胞制备物固定在离心显微镜的转子上，以便在ATP存在的情况下，将离心力施加到肌球蛋白包被的珠粒上，所述珠粒沿着肌动蛋白索移动。在与珠运动方向相反的恒定离心力（“正”负载）下，珠继续以恒定速度移动，该速度随着离心力的增加而降低。由此获得的稳态力-速度曲线类似于单肌纤维的双双曲线力-速度曲线。珠运动的无负荷速度为1.6-3.6 μ m/s（20 - 23 ℃），而由珠上的肌球蛋白分子产生的最大“等长”力为1.9-39 pN。另一方面，如果珠粒在珠粒运动方向上受到恒定的离心力（“负”载荷），则珠粒也以恒定的速度运动。出乎意料的是，珠子运动的速度并没有随着负载荷的增加而增加，而是首先下降20-60%，然后朝着初始无载荷速度增加，直到珠子最终从肌动蛋白缆索上脱离。
To eliminate the gap between the biochemistry of actomyosin in solution and the physiology of contracting muscle, we developed an in vitro force-movement assay system in which the steady-state force-velocity relation in the actin-myosin interaction can be studied. The assay system consists of the internodal cells of an alga, Nitellopsis obtusa, containing well-organized actin filament arrays (actin cables); tosyl-activated polystyrene beads (diameter, 2.8 .mu.m; specific gravity, 1.3) coated with skeletal muscle myosin, and a centrifuge microscope equipped with a stroboscopic light source and a video system. The internodal cell preparation was mounted on the rotor of the centrifuge microscope, so that centrifugal forces were applied to the myosin-coated beads moving along the actin cables in the presence of ATP. Under constant centrifugal forces directed opposite to the bead movement ("positive" loads), the beads continued to move with constant velocities, which decreased with increasing centrifugal forces. The steady state force-velocity curve thus obtained was analogous to the double-hyperbolic force-velocity curve of single muscle fibers. The unloaded velocity of bead movement was 1.6-3.6 .mu.m/s (20-23.degree.C), while the maximum "isometric" force generated by the myosin molecules on the bead was 1.9-39 pN. If, on the other hand, the beads were subjected to constant centrifugal forces in the direction of bead movement ("negative" loads), the bead also moved with constant velocities. Unexpectedly, the velocity of bead movement did not increase with increasing negative loads but first decreased by 20-60% and then increased towards the initial unloaded velocity until the beads were eventually detached from the actin cables.