Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.
Simultaneous measurement of rotations of myosin, actin and ADP in a contracting skeletal muscle fiber.
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同时测量收缩骨骼肌纤维中肌球蛋白、肌动蛋白和 ADP 的旋转。
DOI：
10.1007/s10974-004-5073-6
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发表时间：
2004
影响因子：
2.7
通讯作者：
Borejdo,J
中科院分区：
文献类型：
--
作者：
Shepard,AA;Dumka,D;Akopova,I;Talent,J;Borejdo,J
同时测量肌球蛋白头和肌动蛋白的旋转与肌球蛋白的酶活性的指示剂。为了最小化由于来自许多分子的信号的平均化而引起的并发症，在驻留在肌肉纤维的毫微微升体积中的小群体中测量信号。旋转的开始是同步的突然释放笼ATP。通过与天然调节轻链交换的重组荧光调节轻链的各向异性来测量交叉桥的取向。肌动蛋白的取向是通过加入到肌动蛋白丝中的鬼笔环肽的各向异性来测量的。肌球蛋白的酶活性通过荧光ADP从活性位点的解离来测量。所有3起事件均同时发生。这表明，在收缩肌肉，肌动蛋白并不独立于肌球蛋白和ATP水解强烈耦合到旋转的横桥。
The rotation of myosin heads and actin were measured simultaneously with an indicator of the enzymatic activity of myosin. To minimize complications due to averaging of signals from many molecules, the signal was measured in a small population residing in a femtoliter volume of a muscle fiber. The onset of rotation was synchronized by a sudden release of caged ATP. The orientation of cross-bridges was measured by anisotropy of recombinant fluorescent regulatory light chains exchanged with native regulatory light chains. The orientation of actin was measured by anisotropy of phalloidin added to actin filaments. The enzymatic activity of myosin was measured by dissociation of fluorescent ADP from the active site. The onset of all three events occurred at the same time. This suggests that in contracting muscle, actin does not move independently of myosin and that ATP hydrolysis is strongly coupled to the rotation of cross-bridges.