Kawai M, Zhao Y
Department of Anatomy, College of Medicine, University of Iowa, Iowa City 52242.
Biophys J. 1993 Aug;65(2):638-51. doi: 10.1016/S0006-3495(93)81109-3.
The rate and association constants (kinetic constants) which comprise a seven state cross-bridge scheme were deduced by sinusoidal analysis in chemically skinned rabbit psoas muscle fibers at 20 degrees C, 200 mM ionic strength, and during maximal Ca2+ activation (pCa 4.54-4.82). The kinetic constants were then used to calculate the steady state probability of cross-bridges in each state as the function of MgATP, MgADP, and phosphate (Pi) concentrations. This calculation showed that 72% of available cross-bridges were (strongly) attached during our control activation (5 mM MgATP, 8 mM Pi), which agreed approximately with the stiffness ratio (active:rigor, 69 +/- 3%); active stiffness was measured during the control activation, and rigor stiffness after an induction of the rigor state. By assuming that isometric tension is a linear combination of probabilities of cross-bridges in each state, and by measuring tension as the function of MgATP, MgADP, and Pi concentrations, we deduced the force associated with each cross-bridge state. Data from the osmotic compression of muscle fibers by dextran T500 were used to deduce the force associated with one of the cross-bridge states. Our results show that force is highest in the AMADP.Pi state (A = actin, M = myosin). Since the state which leads into the AMADP.Pi state is the weakly attached AM.ADP.Pi state, we confirm that the force development occurs on Pi isomerization (AM.ADP.Pi --> AMADP.Pi). Our results also show that a minimal force change occurs with the release of Pi or MgADP, and that force declines gradually with ADP isomerization (AMADP -->AM.ADP), ATP isomerization (AM+ATP-->AM*ATP), and with cross-bridge detachment. Force of the AM state agreed well with force measured after induction of the rigor state, indicating that the AM state is a close approximation of the rigor state. The stiffness results obtained as functions of MgATP, MgADP, and Pi concentrations were generally consistent with the cross-bridge scheme.
通过正弦分析，在20℃、200 mM离子强度以及最大Ca2+激活（pCa 4.54 - 4.82）条件下，推导了包含七态横桥模型的速率常数和缔合常数（动力学常数），实验对象为化学去膜的兔腰大肌纤维。然后利用这些动力学常数，计算了各状态下横桥的稳态概率，该概率是MgATP、MgADP和磷酸（Pi）浓度的函数。计算结果表明，在我们的对照激活条件下（5 mM MgATP，8 mM Pi），72%的可用横桥处于（强）附着状态，这与刚度比（活性：僵直，69±3%）大致相符；活性刚度在对照激活过程中测量，僵直刚度在诱导僵直状态后测量。通过假设等长张力是各状态下横桥概率的线性组合，并测量张力作为MgATP、MgADP和Pi浓度的函数，我们推导了与每个横桥状态相关的力。利用右旋糖酐T500对肌肉纤维进行渗透压缩的数据，推导了与其中一个横桥状态相关的力。我们的结果表明，在AMADP.Pi状态下力最大（A = 肌动蛋白，M = 肌球蛋白）。由于进入AMADP.Pi状态的前一个状态是弱附着的AM.ADP.Pi状态，我们证实力的产生发生在Pi异构化过程中（AM.ADP.Pi→AMADP.Pi）。我们的结果还表明，Pi或MgADP释放时力的变化最小，并且随着ADP异构化（AMADP→AM.ADP）、ATP异构化（AM + ATP→AM*ATP）以及横桥解离，力逐渐下降。AM状态下的力与诱导僵直状态后测量的力非常吻合，表明AM状态非常接近僵直状态。作为MgATP、MgADP和Pi浓度函数获得的刚度结果总体上与横桥模型一致。
