Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain
Physical integrity of smooth muscle myosin filaments is enhanced by phosphorylation of the regulatory myosin light chain
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DOI：
10.1159/000107548
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发表时间：
2007-01-01
影响因子：
--
通讯作者：
Seow, Chun Y.
中科院分区：
文献类型：
--
作者：
Ip, Kelvin;Sobieszek, Apolinary;Seow, Chun Y.
背景和目的：平滑肌肌球蛋白单体在溶液中自组装形成丝。 20-kD 调节性肌球蛋白轻链 (MLC20) 的磷酸化可增强肌丝形成。目前尚不清楚磷酸化和非磷酸化丝是否具有相同的结构完整性。方法：我们从牛气管中纯化肌球蛋白，在含 ATP 的零钙溶液中进行缓慢透析，逐渐降低离子强度，从而形成细丝。肌球蛋白纯化后保留了足够的肌球蛋白轻链激酶和磷酸酶以及钙调蛋白，这使得MLC20能够在向细丝悬浮液中添加钙后20-40秒内磷酸化。然后通过超声波部分分解磷酸化和非磷酸化的丝。通过原子力显微镜观察和量化细丝崩解的程度。结果：MLC20 磷酸化降低了细丝的直径，并使细丝对超声波搅拌具有更强的抵抗力。电子显微镜显示，当细胞被激活时，完整平滑肌的细丝直径也出现类似的减小。结论：MLC20 磷酸化对肌球蛋白丝结构和物理特性的修饰可能是平滑肌的关键调节步骤，平滑肌中肌球蛋白丝的形成和溶解是细胞适应不同细胞长度所必需的。
Background and Aims: Smooth muscle myosin monomers self- assemble in solution to form filaments. Phosphorylation of the 20- kD regulatory myosin light chain ( MLC20) enhances filament formation. It is not known whether the phosphorylated and non-phosphorylated filaments possess the same structural integrity. Methods: We purified myosin from bovine trachealis to form filaments, in ATP- containing zerocalcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of MLC20 within 20- 40s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: MLC20 phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Modification of the structural and physical properties of myosin filaments by MLC20 phosphorylation may be a key regulation step in smooth muscle where formation and dissolution of the filaments are required in the cells' adaptation to different cell length.