Kinetics of adenosine triphosphate hydrolysis by shortening myofibrils from rabbit psoas muscle.
Kinetics of adenosine triphosphate hydrolysis by shortening myofibrils from rabbit psoas muscle.
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DOI：
10.1113/jphysiol.1991.sp018773
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发表时间：
1991-09
期刊：
影响因子：
--
通讯作者：
T. Ohno;T. Kodama
中科院分区：
文献类型：
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作者：
T. Ohno;T. Kodama
1. 使用螺线管操作的混合装置，对兔腰肌制备的肌原纤维在 20 摄氏度下的缩短和伴随的 ATP 水解进行了时间分辨测量。 2. ATP 水解程度通过改进的孔雀石绿法测定，用于在大量过量 ATP 存在下测定无机磷酸盐 (Pi)。为了通过相差显微镜测量肌节长度的变化，用 0.2 M 乙酸盐 (pH 4.6) 和 1.25% (v/v) 戊二醛的混合物毫不延迟和伪影地终止缩短。 3. 对于肌节长度超过 1.4 微米的情况，每个半肌节的缩短速度在 25 mM-KCl 中为 10 微米 s-1，对于肌节长度超过 1.7 微米的情况，在 150 mM-KCl 中每个半肌节的缩短速度至少为 12 微米 s-1。在这种快速缩短过程中，跨桥没有显着的 ATP 周转（95% 置信上限：25 mM-KCl 中为 0.14 mol（肌球蛋白头的 mol）-1；大于或等于 100 mM KCl 溶液中为 0.12 mol mol-1）。 4. 当肌节在 KCl 浓度大于 100 mM 中缩短至 1.7 微米以下或在 25 mM-KCl 中缩短至 1.4 微米以下时，ATP 水解会出现短暂加速（ATP 水解延迟），然后是稳定的缓慢水解。 5. 在 25 mM、100 mM 和 150 mM-KCl 中，初始肌节长度 2.4 微米的肌原纤维延迟 ATP 水解的幅度（+/-估计标准差）为 0.42 +/- 0.19、0.31 +/- 0.10 和 0.17 +/- 0.09 mol（mol 肌球蛋白头）-1，分别。然而，对于肌节长度为 2.0 微米的肌原纤维，它在 25 mM-KCl 中降至 0.24 +/- 0.10 mol mol-1，或在 150 mM-KCl 中降至微不足道的水平。 6. 这些结果表明，在快速缩短过程中，大多数 ATP 水解产物仍然与横桥结合，并且当对抗缩短的力增加时，一部分横桥迅速解离产物并进入下一个 ATP 循环，随着缩短距离的减小以及离子强度的增加，横桥的数量减少。跨桥的这种行为可能是其在零负载和从零负载到非零负载转变时与肌动蛋白丝相互作用的结合 ADP 和 Pi 状态下的能量和动力学特性的表现。
1. Using a solenoid‐operated mixing device, time‐resolved measurements were made of shortening and accompanying ATP hydrolysis at 20 degrees C by myofibrils prepared from rabbit psoas muscle. 2. The extent of ATP hydrolysis was determined by an improved Malachite Green method for determination of inorganic phosphate (Pi) in the presence of a large excess of ATP. For the measurement of the change in sarcomere length by phase contrast microscopy, shortening was terminated without delay and artifact by a mixture of 0.2 M‐acetate (pH 4.6) and 1.25% (v/v) glutaraldehyde. 3. The shortening velocity per half‐sarcomere was 10 microns s‐1 in 25 mM‐KCl for sarcomere lengths above 1.4 microns, and at least 12 microns s‐1 in 150 mM‐KCl for sarcomere lengths above 1.7 microns. During this rapid shortening, there was no significant ATP turnover by cross‐bridges (upper 95% confidence limit: 0.14 mol (mol of myosin head)‐1 in 25 mM‐KCl; 0.12 mol mol‐1 in KCl solutions greater than or equal to 100 mM). 4. When the sarcomeres shortened below 1.7 microns in KCl concentrations greater than 100 mM or below 1.4 microns in 25 mM‐KCl, there was a transient acceleration of ATP hydrolysis (delayed ATP hydrolysis), which was then followed by a steady slow hydrolysis. 5. The magnitudes (+/‐ estimated standard deviation) of delayed ATP hydrolysis by myofibrils of initial sarcomere length 2.4 microns were 0.42 +/‐ 0.19, 0.31 +/‐ 0.10 and 0.17 +/‐ 0.09 mol (mol myosin head)‐1 in 25 mM, 100 mM and 150 mM‐KCl, respectively. For myofibrils of sarcomere length 2.0 microns, however, it decreased to 0.24 +/‐ 0.10 mol mol‐1 in 25 mM‐KCl or to an insignificant level in 150 mM‐KCl. 6. These results indicate that most of the ATP hydrolysis products remain bound to cross‐bridges during rapid shortening, and that when the force opposing shortening increases, a proportion of cross‐bridges rapidly dissociate the products and enter the next ATP cycle, which diminishes with the decrease in shortening distance as well as the increase in ionic strength. Such behaviour of the cross‐bridge is probably a manifestation of its energetic and kinetic properties in the state with bound ADP and Pi interacting with actin filaments at zero load and at a transition from zero to non‐zero loads.