Kawai M, Halvorson H R
Department of Anatomy, University of Iowa, Iowa City 52242.
Biophys J. 1991 Feb;59(2):329-42. doi: 10.1016/S0006-3495(91)82227-5.
The elementary steps of contraction in rabbit fast twitch muscle fibers were investigated with particular emphasis on the mechanism of phosphate (Pi) binding/release, the mechanism of force generation, and the relation between them. We monitor the rate constant 2 pi b of a macroscopic exponential process (B) by imposing sinusoidal length oscillations. We find that the plot of 2 pi b vs. Pi concentration is curved. From this observation we infer that Pi released is a two step phenomenon: an isomerization followed by the actual Pi release. Our results fit well to the kinetic scheme: [formula: see text] where A = actin, M = myosin, S = MgATP (substrate), D = MgADP, P = phosphate, and Det is a composite of all the detached and weakly attached states. For our data to be consistent with this scheme, it is also necessary that step 4 (isomerization) is observed in process (B). By fitting this scheme to our data, we obtained the following kinetic constants: k4 = 56 s-1, k-4 = 129 s-1, and K5 = 0.069 mM-1, assuming that K2 = 4.9. Experiments were performed at pCa 4.82, pH 7.00, MgATP 5 mM, free ATP 5 mM, ionic strength 200 mM in K propionate medium, and at 20 degrees C. Based on these kinetic constants, we calculated the probability of each cross-bridge state as a function of Pi, and correlated this with the isometric tension. Our results indicate that all attached cross-bridges support equal amount of tension. From this, we infer that the force is generated at step 4. Detailed balance indicates that 50-65% of the free energy available from ATP hydrolysis is transformed to work at this step. For our data to be consistent with the above scheme, step 6 must be the slowest step of the cross-bridge cycle (the rate limiting step). Further, AM*D is a distinctly different state from the AMD state that is formed by adding D to the bathing solution. From our earlier ATP hydrolysis data, we estimated k6 to be 9 s-1.
研究了兔快肌纤维收缩的基本步骤，特别强调了磷酸盐（Pi）结合/释放机制、力产生机制以及它们之间的关系。我们通过施加正弦长度振荡来监测宏观指数过程（B）的速率常数2πb。我们发现2πb与Pi浓度的关系图是弯曲的。基于这一观察结果，我们推断Pi的释放是一个两步过程：异构化，随后是实际的Pi释放。我们的结果与动力学方案非常吻合：[公式：见原文]，其中A = 肌动蛋白，M = 肌球蛋白，S = MgATP（底物），D = MgADP，P = 磷酸盐，Det是所有分离和弱附着状态的复合物。为了使我们的数据与该方案一致，在过程（B）中也必须观察到步骤4（异构化）。通过将该方案与我们的数据拟合，我们得到了以下动力学常数：k4 = 56 s-1，k-4 = 129 s-1，K5 = 0.069 mM-1，假设K2 = 4.9。实验在pCa 4.82、pH 7.00、MgATP 5 mM、游离ATP 5 mM、离子强度200 mM的丙酸钾介质中，以及20℃下进行。基于这些动力学常数，我们计算了每个横桥状态作为Pi函数的概率，并将其与等长张力相关联。我们的结果表明，所有附着的横桥承受相等的张力。由此，我们推断力是在步骤4产生的。详细平衡表明，ATP水解产生的自由能的50 - 65%在这一步转化为功。为了使我们的数据与上述方案一致，步骤6必须是横桥循环中最慢的步骤（限速步骤）。此外，AM*D与通过向浴液中添加D形成的AMD状态明显不同。根据我们早期的ATP水解数据，我们估计k6为9 s-1。
