Iwamoto Hiroyuki
Japan Synchrotron Radiation Research Institute, SPring-8, Sayo-gun, Hyogo 679-5198, Japan.
Biophys Physicobiol. 2018 Apr 27;15:111-120. doi: 10.2142/biophysico.15.0_111. eCollection 2018.
We studied the effect of myosin inhibitors, N-benzyl-p-toluenesulfonamide (BTS), blebbistatin, and butanedione monoxime (BDM) on X-ray diffraction patterns from rabbit psoas fibers under relaxing and contracting conditions. The first two inhibitors suppressed the contractile force almost completely at a 100 μM concentration, and a similar effect was obtained at 50 mM for BDM. However, still substantial changes were observed in the diffraction patterns upon calcium-activation of inhibited muscle fibers. (1) The 2nd actin layer-line reflection was enhanced normally, indicating that calcium binding to troponin and the subsequent movement of tropomyosin are not inhibited, (2) the myosin layer-line reflections became much weaker, and (3) the 1,1/1,0 intensity ratio of the equatorial reflections was increased. The observations (2) and (3) indicate that, even in the presence of the inhibitors at a saturating concentration, myosin heads leave the helix on the thick filaments and approach the thin filaments. Interestingly, the 1,0 spacing of the filament lattice remained unchanged upon activation of inhibited fibers, in contrast to the case of normal activation in which the spacing is decreased. This suggests that the normal activated myosin heads exert a pull in both axial and radial directions, but in the presence of the inhibitors, the pull is suppressed, and as a result, the heads simply bind to actin without exerting any force. The results support the idea that the inhibitors do not block the myosin binding to actin, but block the step of force-producing transition of the bound actomyosin complex.
我们研究了肌球蛋白抑制剂N-苄基对甲苯磺酰胺(BTS)、blebbistatin和丁二酮单肟(BDM)对兔腰大肌纤维在松弛和收缩条件下X射线衍射图谱的影响。前两种抑制剂在100μM浓度时几乎完全抑制了收缩力,BDM在50 mM时也得到了类似的效果。然而,在对受抑制的肌纤维进行钙激活时,衍射图谱仍观察到显著变化。(1)肌动蛋白的第二层线反射正常增强,表明钙与肌钙蛋白的结合以及随后原肌球蛋白的移动未受抑制;(2)肌球蛋白的层线反射变得弱得多;(3)赤道反射的1,1/1,0强度比增加。观察结果(2)和(3)表明,即使在抑制剂饱和浓度存在的情况下,肌球蛋白头部仍会离开粗丝上的螺旋并靠近细丝。有趣的是,与正常激活时间距减小的情况相反,受抑制纤维激活后细丝晶格的1,0间距保持不变。这表明正常激活的肌球蛋白头部在轴向和径向上都施加拉力,但在存在抑制剂的情况下,这种拉力受到抑制,结果是头部仅与肌动蛋白结合而不施加任何力。这些结果支持了这样一种观点,即抑制剂不会阻止肌球蛋白与肌动蛋白的结合,而是阻止结合的肌动球蛋白复合物产生力的转变步骤。