Harada Y, Sakurada K, Aoki T, Thomas D D, Yanagida T
Department of Biophysical Engineering, Osaka University, Japan.
J Mol Biol. 1990 Nov 5;216(1):49-68. doi: 10.1016/S0022-2836(05)80060-9.
In order to study the mechanochemical coupling in actomyosin energy transduction, the sliding distance of an actin filament induced by one ATP hydrolysis cycle was obtained by using an in vitro movement assay that permitted quantitative and simultaneous measurements of (1) the movements of single fluorescently labeled actin filaments on myosin bound to coverslip surfaces and (2) the ATPase rates. The sliding distance was determined as (the working stroke time in one ATPase cycle, tws) x (the filament velocity, v). tws was obtained from the ATPase turnover rate of myosin during the sliding (kt), the ATP hydrolysis time (delta t) and the ON-rate at which myosin heads enter into the working stroke state when they encounter actin (kON); tws approximately 1/kt-delta t-1/kON. kt was estimated from the ATPase rates of the myosin-coated surface during the sliding of actin filaments. delta t has been determined as less than 1/100 per second, kON was estimated by analyzing the movements of very short (40 nm) filaments. The resulting sliding distance during one ATP hydrolysis cycle near zero load was greater than 100 nm, which is about ten times longer than that expected for a single attachment-detachment cycle between an actin and a myosin head. This leads to the conclusion that the coupling between the ATPase and attachment-detachment cycles is not determined rigidly in a one-to-one fashion.
为了研究肌动球蛋白能量转导中的机械化学偶联，通过体外运动分析获得了由一个ATP水解循环诱导的肌动蛋白丝的滑动距离，该分析允许定量并同时测量：（1）结合在盖玻片表面的肌球蛋白上单个荧光标记的肌动蛋白丝的运动，以及（2）ATP酶活性。滑动距离确定为（一个ATP酶循环中的工作冲程时间，tws）×（丝速度，v）。tws由滑动过程中肌球蛋白的ATP酶周转速率（kt）、ATP水解时间（δt）以及肌球蛋白头部遇到肌动蛋白时进入工作冲程状态的开启速率（kON）得出；tws约为1/kt - δt - 1/kON。kt由肌动蛋白丝滑动过程中肌球蛋白包被表面的ATP酶活性估算得出。δt已确定小于每秒1/100，kON通过分析极短（40纳米）丝的运动估算得出。在接近零负荷的一个ATP水解循环期间得到的滑动距离大于100纳米，这比肌动蛋白和肌球蛋白头部之间单个附着 - 脱离循环预期的长度长约十倍。这导致得出结论：ATP酶循环与附着 - 脱离循环之间的偶联并非以一对一的方式严格确定。
