Kulikova Natalia, Podlubnaya Zoya, Makuch Robert, Dabrowska Renata
Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur Street, Warsaw 02-093, Poland.
J Muscle Res Cell Motil. 2003;24(1):7-13. doi: 10.1023/a:1024843409429.
We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca2+ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon.
我们使用了在松弛条件下（无Ca2+且有ATP存在）具有紧密、高度有序结构的未磷酸化鸡胃肌球蛋白合成细丝，通过负染色和免疫金电子显微镜观察来可视化钙调蛋白与肌球蛋白细丝的结合模式。我们证明，将钙调蛋白添加到预先形成的肌球蛋白细丝中会导致从细丝表面弯曲伸出许多光滑的突起。添加钙调蛋白或其N端片段会部分掩盖肌球蛋白细丝的周期性。然而，它并没有改变细丝的内部结构。免疫电子显微镜观察证实，大多数钙调蛋白分子通过N端部分与肌球蛋白细丝结合，而C端部分从细丝表面突出。结合现有的关于钙调蛋白与肌动蛋白和肌球蛋白结合的生化数据，以及关于钙调蛋白以分子C端从细丝弯曲伸出的方式附着于肌动蛋白细丝的电子显微镜观察结果，钙调蛋白附着于肌球蛋白细丝的可视化完成了钙调蛋白将肌动蛋白与肌球蛋白连接起来的过程。
