Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.
Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.
复制标题
肌钙蛋白 C 与肌原纤维细丝结合的特征：肌钙蛋白 C 的提取沿着细丝的长度不是随机的。
DOI：
10.1016/s0006-3495(97)78070-6
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Greaser,ML
中科院分区：
文献类型：
--
作者：
Swartz,DR;Moss,RL;Greaser,ML
肌钙蛋白 C (TnC) 是肌钙蛋白的 Ca(2+) 感应亚基，负责启动导致横纹肌收缩的级联事件。使用低离子强度的含 EDTA 缓冲液可以轻松从肌原纤维中提取该蛋白质。 TnC 提取的特性尚未在结构水平上得到表征，也没有研究 TnC 与天然肌原纤维细丝的相互作用。为了解决这些问题，荧光素标记的 TnC 与高分辨率数字荧光显微镜相结合，用于表征 TnC 与肌原纤维的结合，并确定 TnC 提取的随机性。荧光素-5-马来酰亚胺 TnC (F5M TnC) 保留了生物活性，这通过提取的肌原纤维中 Ca(2+) 依赖性 ATP 酶活性的重建以及以 Ca(2+) 敏感方式与 TnI 结合来证明。在严格条件下，F5M TnC 与低 Ca2+ 下高度提取的肌原纤维的结合仅限于重叠区域，并且结合位置不受 F5M TnC 浓度的影响。添加肌球蛋白亚片段 1 以占据所有肌动蛋白位点导致 F5M TnC 结合在重叠和非重叠区域中。然而，在松弛条件下，很少有 F5M TnC 与肌原纤维结合。这些结果表明肌球蛋白头的强结合增强了 TnC 结合。在高 Ca2+ 下，F5M TnC 结合模式是浓度依赖性的：在低 F5M TnC 浓度下，结合仅限于重叠区域，而在较高水平下，结合传播到非重叠区域。荧光强度分析显示高 Ca2+ 下 F5M TnC 与 S1 的结合最强，这些条件用于表征部分 TnC 提取的肌原纤维。部分提取的肌原纤维的比较表明，相对于较高水平的提取，低水平的提取与非重叠区域中结合的F5M TnC比重叠区域中更多的F5M TnC相关。这些结果表明，TnC 提取并不是沿着细丝的长度随机进行的，而是更容易发生在非重叠区域。这一观察结果与刚性头部对 F5M TnC 结合模式的影响相结合，表明肌球蛋白与肌动蛋白的强结合可在低 Ca2+ 条件下稳定 TnC 结合。
Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.