Woodward S K, Eccleston J F, Geeves M A
Division of Physical Biochemistry, National Institute for Medical Research, London, U.K.
Biochemistry. 1991 Jan 15;30(2):422-30. doi: 10.1021/bi00216a017.
We have used a fluorescent analogue of ATP, mantATP [2'(3')-O-(N-methylanthraniloyl)-adenosine 5'-triphosphate; Hiratsuka T. (1983) Biochim. Biophys. Acta 742, 496-508], and made a detailed kinetic study of the interaction of mantATP and mantADP with S1 and acto-S1. We have shown that these analogues behave like ATP and ADP, respectively. In addition, we have demonstrated that this analogue can distinguish between two acto-S1 complexes, the A-M.N (attached) and A.M.N (rigor-like) states [Geeves, M. A., Good, R. S., & Gutfreund, H. (1984) J. Muscle Res. Cell Motil. 5, 351-361]. Previously, these two states were observed with a pyrene label on Cys 374 of actin. This isomerization can now be monitored at two spatially distinct sites on the ternary complex, indicative of a major conformational change in the ternary complex. Also, we have measured the rate of ADP dissociation from both A-M.N and A.M.N directly and shown these to differ by a factor of 1000. Thus the results presented here support the model of Geeves et al. and are consistent with the A-M.N to A.M.N transition being coupled to the force-generating event of the crossbridge cycle.
我们使用了ATP的荧光类似物mantATP [2'(3')-O-(N-甲基邻氨基苯甲酰基)-腺苷5'-三磷酸；平冢彻（1983年），《生物化学与生物物理学报》742卷，496 - 508页]，并对mantATP和mantADP与S1及肌动蛋白-S1的相互作用进行了详细的动力学研究。我们已经表明，这些类似物分别表现得如同ATP和ADP。此外，我们已经证明这种类似物能够区分两种肌动蛋白-S1复合物，即A-M.N（附着）态和A.M.N（类僵直）态[吉夫斯，M. A.，古德，R. S.，& 古特弗罗因德，H.（1984年），《肌肉研究与细胞运动杂志》5卷，351 - 361页]。此前，这两种状态是通过肌动蛋白第374位半胱氨酸上的芘标记观察到的。现在可以在三元复合物的两个空间上不同的位点监测这种异构化，这表明三元复合物发生了重大的构象变化。而且，我们直接测量了ADP从A-M.N和A.M.N解离的速率，结果显示二者相差1000倍。因此，这里给出的结果支持了吉夫斯等人的模型，并且与A-M.N到A.M.N的转变与横桥循环的力产生事件相偶联是一致的。
