Dantzig J A, Barsotti R J, Manz S, Sweeney H L, Goldman Y E
Department of Physiology and Pennsylvania Muscle Institute, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6083, USA.
Biophys J. 1999 Jul;77(1):386-97. doi: 10.1016/S0006-3495(99)76897-9.
When smooth muscle myosin subfragment 1 (S1) is bound to actin filaments in vitro, the light chain domain tilts upon release of MgADP, producing a approximately 3.5-nm axial motion of the head-rod junction (Whittaker et al., 1995. Nature. 378:748-751). If this motion contributes significantly to the power stroke, rigor tension of smooth muscle should decrease substantially in response to cross-bridge binding of MgADP. To test this prediction, we monitored mechanical properties of permeabilized strips of chicken gizzard muscle in rigor and in the presence of MgADP. For comparison, we also tested psoas and soleus muscle fibers. Any residual bound ADP was minimized by incubation in Mg2+-free rigor solution containing 15 mM EDTA. The addition of 2 mM MgADP, while keeping ionic strength and free Mg2+ concentration constant, resulted in a slight increase in rigor tension in both gizzard and soleus muscles, but a decrease in psoas muscle. In-phase stiffness monitored during small (<0.1%) 500-Hz sinusoidal length oscillations decreased in all three muscle types when MgADP was added. The changes in force and stiffness with the addition of MgADP were similar at ionic strengths from 50 to 200 mM and were reversible. The results with gizzard muscle were similar after thiophosphorylation of the regulatory light chain of myosin. These results suggest that the axial motion of smooth muscle S1 bound to actin, upon dissociation of MgADP, is not associated with force generation. The difference between the present mechanical data and previous structural studies of smooth S1 may be explained if geometrical constraints of the intact contractile filament array alter the motions of the myosin heads.
当平滑肌肌球蛋白亚片段1（S1）在体外与肌动蛋白丝结合时，轻链结构域在MgADP释放时发生倾斜，导致头-杆连接点产生约3.5纳米的轴向运动（惠特克等人，1995年。《自然》。378:748 - 751）。如果这种运动对动力冲程有显著贡献，那么平滑肌的强直张力应会因MgADP的横桥结合而大幅降低。为了验证这一预测，我们监测了处于强直状态以及存在MgADP时鸡砂囊肌通透化条带的力学特性。为作比较，我们还测试了腰大肌和比目鱼肌纤维。通过在含15 mM EDTA的无Mg2+强直溶液中孵育，使任何残留结合的ADP降至最低。添加2 mM MgADP时，在保持离子强度和游离Mg2+浓度恒定的情况下，砂囊肌和比目鱼肌的强直张力均略有增加，但腰大肌的强直张力却降低了。在添加MgADP时，所有三种肌肉类型在小幅度（<0.1%）500赫兹正弦长度振荡期间监测到的同相刚度均降低。在50至200 mM的离子强度下，添加MgADP时力和刚度的变化相似且是可逆的。肌球蛋白调节轻链硫代磷酸化后，砂囊肌的结果相似。这些结果表明，与肌动蛋白结合的平滑肌S1在MgADP解离时的轴向运动与力的产生无关。如果完整收缩丝阵列的几何约束改变了肌球蛋白头部的运动，那么当前力学数据与之前平滑肌S1结构研究之间的差异或许可以得到解释。
