Coates J H, Criddle A H, Geeves M A
Biochem J. 1985 Dec 1;232(2):351-6. doi: 10.1042/bj2320351.
We have used actin labelled at Cys-374 with N-(1-pyrenyl)iodoacetamide [Kouyama & Mihashi (1981) Eur. J. Biochem. 114, 33-38] to monitor pressure-induced relaxations of acto-myosin subfragment 1. This label greatly increases the sensitivity of measurement of dissociated actin and reveals the presence of two relaxations. The experimental data can be fitted by a model in which actin binds subfragment 1 relatively weakly (K = 5.9 X 10(4) M-1) and then isomerizes to a more tightly bound complex (K = 1.7 X 10(7) M-1). This directly observed isomerization supports the model of Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell. Motil. 5, 351-361]. The rate of the isomerization is too high to be observed in the pressure-jump apparatus (less than 200 microseconds), but analysis of the amplitudes allows estimation of the equilibrium constant of the isomerization as 280 (20 degrees C, 0.1 M-KCl, pH 7). The equilibrium is sensitive to temperature, pressure, ionic strength and the presence of ethylene glycol. The pressure-sensitivity of the isomerization suggests a significant conformational change of the acto-myosin subfragment 1 complex.
我们使用用N-(1-芘基)碘乙酰胺标记在Cys-374位点的肌动蛋白[小山和三桥(1981年)《欧洲生物化学杂志》114卷，33 - 38页]来监测压力诱导的肌动蛋白-肌球蛋白亚片段1的松弛。这种标记大大提高了对解离肌动蛋白测量的灵敏度，并揭示了两种松弛的存在。实验数据可以用一个模型来拟合，在该模型中，肌动蛋白与亚片段1的结合相对较弱(K = 5.9×10⁴ M⁻¹)，然后异构化为一种结合更紧密的复合物(K = 1.7×10⁷ M⁻¹)。这种直接观察到的异构化支持了吉夫斯、古迪和古特弗伦德[(1984年)《肌肉研究与细胞运动杂志》5卷，351 - 361页]的模型。异构化速率太高以至于在压力跳跃装置中无法观察到(小于200微秒)，但对振幅的分析允许估计异构化的平衡常数为280(20℃，0.1 M - KCl，pH 7)。该平衡对温度、压力、离子强度和乙二醇的存在敏感。异构化的压力敏感性表明肌动蛋白-肌球蛋白亚片段1复合物发生了显著的构象变化。
