Pate E, Cooke R
Department of Mathematics, Washington State University, Pullman 99164.
Biophys J. 1988 Apr;53(4):561-73. doi: 10.1016/S0006-3495(88)83136-9.
The interaction between actin and myosin in the filament array of glycerinated muscle fibers has been monitored using paramagnetic probes and mechanical measurements. Both fiber stiffness and the spectra of probes bound to a reactive sulfydral on the myosin head were measured as the actomyosin bond was weakened by addition of magnesium pyrophosphate (MgPPi) and glycerol. In the absence of MgPPi, all myosin heads are attached to actin with oriented probes. When fibers were incubated in buffers containing MgPPi, a fraction of the probes became disordered, and this effect was greater in the presence of glycerol. To determine whether the heads with disordered probes were detached from actin, spin-labeled myosin subfragment-1 (MSL-S1) was diffused into unlabeled fibers, and the fractions bound to actin and free in the medium were correlated with the oriented and disordered spectral components. These experiments showed that the label was oriented when MSL-S1 was attached to actin in a ternary complex with the ligand and that all heads with disordered probes were detached from actin. Thus the fraction of oriented labels could be used to determine the fraction of heads attached to actin in a fiber in the presence of ligand. The fraction of myosin heads attached to actin decreased with increasing [MgPPi], and in the absence of glycerol approximately 50% of the myosin heads were dissociated at 3.3 mM ligand with little change in fiber stiffness. In the presence of 37% glycerol plus ligand, up to 80% of the heads could be detached with a 50% decrease in fiber stiffness. The data indicate that there are two populations of myosin heads in the fiber. All the data could be fit with a model in which one population of myosin heads (comprising approximately 50% of the total) sees an apparent actin concentration of 0.1 mM and can be released from actin with little change in fiber stiffness. A second population of myosin heads (approximately 50%) sees a higher actin concentration (5 mM) and is only released in the presence of both glycerol and ligand.
利用顺磁探针和力学测量手段，监测了甘油处理的肌纤维细丝阵列中肌动蛋白和肌球蛋白之间的相互作用。随着焦磷酸镁（MgPPi）和甘油的加入使肌动球蛋白键减弱，同时测量了纤维刚度以及与肌球蛋白头部上一个反应性巯基结合的探针的光谱。在没有MgPPi的情况下，所有肌球蛋白头部都通过定向探针附着于肌动蛋白。当纤维在含有MgPPi的缓冲液中孵育时，一部分探针变得无序，并且在甘油存在的情况下这种效应更明显。为了确定探针无序的头部是否从肌动蛋白上脱离，将自旋标记的肌球蛋白亚片段-1（MSL-S1）扩散到未标记的纤维中，并且与肌动蛋白结合的部分和在介质中游离的部分与定向和无序的光谱成分相关联。这些实验表明，当MSL-S1在与配体形成的三元复合物中附着于肌动蛋白时，标记是定向的，并且所有探针无序的头部都从肌动蛋白上脱离。因此，在存在配体的情况下，定向标记的部分可用于确定纤维中附着于肌动蛋白的头部的部分。附着于肌动蛋白的肌球蛋白头部的部分随着[MgPPi]的增加而减少，并且在没有甘油的情况下，在3.3 mM配体时约50%的肌球蛋白头部解离，而纤维刚度几乎没有变化。在37%甘油加配体存在的情况下，高达80%的头部可以脱离，纤维刚度降低50%。数据表明纤维中存在两种肌球蛋白头部群体。所有数据都可以用一个模型拟合，其中一种肌球蛋白头部群体（约占总数的50%）看到的肌动蛋白表观浓度为0.1 mM，并且可以从肌动蛋白上释放，而纤维刚度几乎没有变化。第二种肌球蛋白头部群体（约50%）看到更高的肌动蛋白浓度（5 mM），并且仅在甘油和配体都存在时才被释放。
