Zhao Y, Swamy P M, Humphries K A, Kawai M
Department of Anatomy, College of Medicine, University of lowa, lowa City 52242, USA.
Biophys J. 1996 Nov;71(5):2759-73. doi: 10.1016/S0006-3495(96)79469-9.
The elementary steps of the cross-bridge cycle in which troponin C (TnC) was partially extracted were investigated by sinusoidal analysis in rabbit psoas muscle fibers. The effects of MgATP and phosphate on the rate constants of exponential processes were studied at 200 mM ionic strength, pCa 4.20, pH 7.00, and at 20 degrees C. The results were analyzed with the following cross-bridge scheme: [formula: see text] where A is actin, M is myosin, S is MgATP, D is MgADP, and P is phosphate (Pi). When TnC was extracted so that the average remaining tension was 11% (range 8-15%), K1 (MgATP association constant) increased to 7x, k2 (rate constant of cross-bridge detachment) increased to 1.55x, k-2 (reversal of detachment) decreased to 0.27x, and K2 (= k2/k-2: equilibrium constant of cross-bridge detachment) increased to 6.6x, k4 (rate constant of force generation) decreased to 0.4x, k-4 (reversal of force generation) increased to 2x, K4 (= k4/k-4) decreased to 0.17x, and K5 (Pi association constant) did not change. The activation factor alpha, which represents the fraction of cross-bridges participating in the cycling, decreased from 1 to 0.14 with TnC extraction. The fact that K1 increased with TnC extraction implies that the condition of the thin filament modifies the contour of the substrate binding site on the myosin head and is consistent with the Fenn effect. The fact that alpha decreased to 0.14 is consistent with the steric blocking mechanism (recruitment hypothesis) and indicates that some of the cross-bridges disappear from the active cycling pool. The fact that the equilibrium constants changed is consistent with the cooperative activation mechanism (graded activation hypothesis) among thin-filament regulatory units that consist of troponin (TnC, Tnl, TnT), tropomyosin, and seven actin molecules, and possibly include cross-bridges.
通过正弦分析研究了兔腰大肌纤维中肌钙蛋白C（TnC）部分提取时横桥循环的基本步骤。在200 mM离子强度、pCa 4.20、pH 7.00和20℃条件下，研究了MgATP和磷酸盐对指数过程速率常数的影响。结果采用以下横桥模式进行分析：[公式：见正文]，其中A为肌动蛋白，M为肌球蛋白，S为MgATP，D为MgADP，P为磷酸盐（Pi）。当TnC被提取后，平均剩余张力为11%（范围8 - 15%）时，K1（MgATP结合常数）增加到7倍，k2（横桥解离速率常数）增加到1.55倍，k - 2（解离逆转）降低到0.27倍，K2（= k2/k - 2：横桥解离平衡常数）增加到6.6倍，k4（力产生速率常数）降低到0.4倍，k - 4（力产生逆转）增加到2倍，K4（= k4/k - 4）降低到0.17倍，K5（Pi结合常数）不变。代表参与循环的横桥比例的激活因子α随着TnC的提取从1降低到0.14。K1随TnC提取而增加这一事实意味着细肌丝的状态改变了肌球蛋白头部底物结合位点的轮廓，这与芬恩效应一致。α降低到0.14这一事实与空间位阻机制（募集假说）一致，表明一些横桥从活跃循环池中消失。平衡常数发生变化这一事实与由肌钙蛋白（TnC、Tnl、TnT）、原肌球蛋白和七个肌动蛋白分子组成的细肌丝调节单位之间的协同激活机制（分级激活假说）一致，并且可能包括横桥。
