Linari Marco, Caremani Marco, Piperio Claudia, Brandt Philip, Lombardi Vincenzo
Laboratorio di Fisiologia, Dipartimento di Biologia Animale e Genetica, Università degli Studi di Firenze, Firenze, Italy.
Biophys J. 2007 Apr 1;92(7):2476-90. doi: 10.1529/biophysj.106.099549. Epub 2007 Jan 19.
The stiffness of the single myosin motor (epsilon) is determined in skinned fibers from rabbit psoas muscle by both mechanical and thermodynamic approaches. Changes in the elastic strain of the half-sarcomere (hs) are measured by fast mechanics both in rigor, when all myosin heads are attached, and during active contraction, with the isometric force (T0) modulated by changing either [Ca2+] or temperature. The hs compliance is 43.0+/-0.8 nm MPa-1 in isometric contraction at saturating [Ca2+], whereas in rigor it is 28.2+/-1.1 nm MPa-1. The equivalent compliance of myofilaments is 21.0+/-3.3 nm MPa-1. Accordingly, the stiffness of the ensemble of myosin heads attached in the hs is 45.5+/-1.7 kPa nm-1 in isometric contraction at saturating [Ca2+] (e0), and in rigor (er) it rises to 138.9+/-21.2 kPa nm-1. Epsilon, calculated from er and the lattice molecular dimensions, is 1.21+/-0.18 pN nm-1. epsilon estimated, using a thermodynamic approach, from the relation of T0 at saturating [Ca2+] versus the reciprocal of absolute temperature is 1.25+/-0.14 pN nm-1, similar to that estimated for fibers in rigor. Consequently, the ratio e0/er (0.33+/-0.05) can be used to estimate the fraction of attached heads during isometric contraction at saturating [Ca2+]. If the osmotic agent dextran T-500 (4 g/100 ml) is used to reduce the lateral filament spacing of the relaxed fiber to the value before skinning, both e0 and er increase by approximately 40%. Epsilon becomes approximately 1.7 pN nm-1 and the fraction and the force of myosin heads attached in the isometric contraction remain the same as before dextran application. The finding that the fraction of myosin heads attached to actin in an isometric contraction is 0.33 rules out the hypothesis of multiple mechanical cycles per ATP hydrolyzed.
通过机械和热力学方法，在兔腰大肌的去膜纤维中测定了单个肌球蛋白马达的刚度（ε）。在强直收缩时（此时所有肌球蛋白头部均已附着）以及主动收缩过程中，通过快速力学测量半肌小节（hs）的弹性应变变化，主动收缩时通过改变[Ca2+]或温度来调节等长力（T0）。在饱和[Ca2+]的等长收缩中，hs的顺应性为43.0±0.8 nm MPa-1，而在强直收缩时为28.2±1.1 nm MPa-1。肌丝的等效顺应性为21.0±3.3 nm MPa-1。因此, 在饱和[Ca2+]的等长收缩中，附着在hs中的肌球蛋白头部集合的刚度为45.5±1.7 kPa nm-1（e0），在强直收缩时（er）则升至138.9±21.2 kPa nm-1。根据er和晶格分子尺寸计算得出的ε为1.21±0.18 pN nm-1。使用热力学方法，根据饱和[Ca2+]时T0与绝对温度倒数的关系估算出的ε为1.25±0.14 pN nm-1，与强直收缩时纤维的估算值相似。因此，e0/er的比值（0.33±0.05）可用于估算饱和[Ca2+]等长收缩过程中附着头部的比例。如果使用渗透剂葡聚糖T-500（4 g/100 ml）将松弛纤维的横向细丝间距减小到去膜前的值，则e0和er均增加约40%。ε约为1.7 pN nm-1，等长收缩中附着的肌球蛋白头部的比例和力与施加葡聚糖之前相同。在等长收缩中，附着在肌动蛋白上的肌球蛋白头部比例为0.33这一发现排除了每水解一个ATP存在多个机械循环的假设。
