Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.
Paramagnetic probes attached to a light chain on the myosin head are highly disordered in active muscle fibers.
复制标题
附着在肌球蛋白头部轻链上的顺磁探针在活跃的肌纤维中高度紊乱。
DOI：
10.1016/s0006-3495(92)81717-4
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发表时间：
1992
影响因子：
3.4
通讯作者：
Cooke,R
中科院分区：
文献类型：
--
作者：
Hambly,B;Franks,K;Cooke,R
在作用力产生过程中，我们测量了靠近杆状连接处的肌球蛋白头部区域的取向。将顺磁性探针特异性地连接到纯化的肌球蛋白轻链2(LC2)的反应性半胱氨酸(Cys 125)上，并在甘油处理的兔腰大肌中交换到肌球蛋白头部。用电子顺磁共振波谱监测探针的取向。以前的工作表明，在二磷酸腺苷(ADP)存在的情况下，与LC2结合的自旋探针在僵直和肌肉中明显有序。相比之下，放松肌肉的角度分布几乎是随机的。结果表明，在等长张力的产生过程中，所有LC2束缚的自旋探针(98+/-1.6%)都呈现出与松弛肌肉相似的角度分布。这些发现与连接在交叉桥上的Cys 707上的探针的结果相反，该探针位于肌动蛋白结合位点附近，在活性作用力产生期间，部分自旋探针被排列为僵硬，而其余的探针被无序排列为松弛。为了验证这种有序成分是由于Cys 707的修饰而产生的假设，我们测量了在Cys 707修饰的纤维中连接到LC2的探针获得的光谱。Cys 707的修饰没有在这些光谱中产生有序成分。在LC2位缺少有序组分限制了活性纤维中某些态的布居。肌动蛋白/肌球蛋白/ADP状态被认为是主要的作用力状态。我们目前的结果表明，在活性纤维中，具有有序探针的态的数量为<；2%，因此，主要的作用力状态不同于在刚性纤维中添加ADP所获得的状态。
We have measured the orientation of a region of the myosin head, close to the junction with the rod, during active force generation. Paramagnetic probes were attached specifically to a reactive cysteine (Cys 125) of purified myosin light chain 2 (LC2) and exchanged into myosin heads in glycerinated rabbit psoas muscle. Electron paramagnetic resonance spectroscopy was used to monitor the orientation of the probes. Previous work has shown that the LC2 bound spin probes are significantly ordered in rigor and muscle in the presence of adenosine diphosphate (ADP). In contrast, there is a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, all of the LC2 bound spin probes (98 +/- 1.6%) show an angular distribution similar to that of relaxed muscle. These findings contrast with results obtained from probes attached to Cys 707 on the cross-bridge, located close to the actin binding site, where, during active force generation, a proportion of the spin probes were ordered as in rigor, whereas the remaining probes were disordered as in relaxation. To test the hypothesis that this ordered component is due to modification of Cys 707, we measured the spectra obtained from probes attached to LC2 in fibers modified at Cys 707. The modification of Cys 707 did not produce an ordered component in these spectra. The absence of an ordered component at the LC2 site limits the populations of some states in active fibers. An actin/myosin/ADP state is thought to be the major force-producing state. Our present results show that the populations of states with ordered probes on LC2 are < 2% in active fibers; thus, the major force-producing state is different from the one obtained by addition of ADP to rigor fibers.