Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.
Direct visualization by electron microscopy of the weakly bound intermediates in the actomyosin adenosine triphosphatase cycle.
复制标题
通过电子显微镜直接观察肌动球蛋白三磷酸酶循环中弱结合的中间体。
DOI：
10.1016/s0006-3495(93)81387-0
复制
发表时间：
1993
影响因子：
3.4
通讯作者：
Zot,HG
中科院分区：
文献类型：
--
作者：
Pollard,TD;Bhandari,D;Maupin,P;Wachsstock,D;Weeds,AG;Zot,HG
我们使用了一种新型的停流/快速冷冻机，以制备肌动蛋白-肌球蛋白腺苷三磷酸酶（ATP酶）循环中的瞬时中间产物，并通过电子显微镜进行直接观察。我们专注于肌球蛋白-三磷酸腺苷（ATP）和肌球蛋白-二磷酸腺苷（ADP）-Pi与肌动蛋白丝的低亲和力复合物，因为从这些状态到高亲和力肌动蛋白-肌球蛋白-ADP和肌动蛋白-肌球蛋白状态的转变被假定为产生驱动肌肉收缩和其他类型细胞运动的分子运动。肌球蛋白亚片段-1、肌动蛋白丝和ATP的混合物在快速冷冻和金属复制后，弱结合中间体的结构与无核苷酸的刚性复合物没有区别。特别是，在这两种情况下，肌球蛋白头与肌动蛋白丝的平均附着角约为40度。在ATP酶循环的所有阶段，大多数与肌动蛋白丝结合的肌球蛋白头的构型是相似的，并且在从低亲和力到高亲和力状态的过渡期间，保存在冷冻断裂复制品中的肌球蛋白头的部分不会倾斜超过几度。相比之下，当通过负染色观察时，与肌动蛋白丝化学交联的肌球蛋白头在它们的附着角度上不同，从没有ATP时的40度有序到存在ATP时的几乎随机（克雷格，R.，L.E.格林和E.艾森伯格1985. Proc. Natl. Acad. Sci. USA. 82：3247-3251，并在此确认），在玻璃冰中冷冻（Applegate，D.，和P·弗里克1987. J.Biol.Chem.262：6856-6863）和快速冷冻样品的复制品中。这表明，在这些制剂中的许多交联的头部是解离的，但在ATP的存在下拴在肌动蛋白丝。这些观察结果表明，由肌球蛋白和肌动蛋白产生的分子运动发生在与肌动蛋白结合位点有一定距离的点处，或者不涉及肌球蛋白头部形状的大的变化。
We used a novel stopped-flow/rapid-freezing machine to prepare the transient intermediates in the actin-myosin adenosine triphosphatase (ATPase) cycle for direct observation by electron microscopy. We focused on the low affinity complexes of myosin-adenosine triphosphate (ATP) and myosin-adenosine diphosphate (ADP)-Pi with actin filaments since the transition from these states to the high affinity actin-myosin-ADP and actin-myosin states is postulated to generate the molecular motion that drives muscle contraction and other types of cellular movements. After rapid freezing and metal replication of mixtures of myosin subfragment-1, actin filaments, and ATP, the structure of the weakly bound intermediates is indistinguishable from nucleotide-free rigor complexes. In particular, the average angle of attachment of the myosin head to the actin filament is approximately 40 degrees in both cases. At all stages in the ATPase cycle, the configuration of most of the myosin heads bound to actin filaments is similar, and the part of the myosin head preserved in freeze-fracture replicas does not tilt by more than a few degrees during the transition from the low affinity to high affinity states. In contrast, myosin heads chemically cross-linked to actin filaments differ in their attachment angles from ordered at 40 degrees without ATP to nearly random in the presence of ATP when viewed by negative staining (Craig, R., L.E. Greene, and E. Eisenberg. 1985. Proc. Natl. Acad. Sci. USA. 82:3247–3251, and confirmed here), freezing in vitreous ice (Applegate, D., and P. Flicker. 1987. J. Biol. Chem. 262:6856–6863), and in replicas of rapidly frozen samples. This suggests that many of the cross-linked heads in these preparations are dissociated from but tethered to the actin filaments in the presence of ATP. These observations suggest that the molecular motion produced by myosin and actin takes place with the myosin head at a point some distance from the actin binding site or does not involve a large change in the shape of the myosin head.