Geeves M A, Jeffries T E
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Biochem J. 1988 Nov 15;256(1):41-6. doi: 10.1042/bj2560041.
The binding of actin to myosin subfragment 1 (S1) has been shown to occur as a two-step reaction [Coates, Criddle & Geeves (1985) Biochem. J. 232, 351-356]. In the first step actin is weakly bound and the second step involves the complex isomerizing to a more tightly bound state. This isomerization can be followed specifically by monitoring the fluorescence of actin that has been covalently labelled with N-(pyren-1-yl)-iodoacetamide at Cys-374 [Geeves, Jeffries & Millar (1986) Biochemistry 25, 8454-8458]. We report here that the presence of nucleotides and nucleotide analogues affects the equilibrium between the strongly bound and weakly bound states (referred to as K2). In the presence of ATP, [gamma-thio]ATP or ADP and vanadate a value of approx. less than 10(-2) was estimated for K2. In the presence of PPi or ADP a value of approx. 2.3 or 10 respectively was obtained. An increase in KCl concentration or the presence of 40% ethylene glycol was found to decrease K2 in the presence of ADP. The data presented here are consistent with the two-step binding model proposed by Geeves, Goody & Gutfreund [(1984) J. Muscle Res. Cell Motil. 5, 351-361], where it was suggested that the transition between weakly bound and strongly bound states is closely associated with the force-generating event in whole muscle.
肌动蛋白与肌球蛋白亚片段1（S1）的结合已被证明是一个两步反应[科茨、克里德尔和吉夫斯（1985年）《生物化学杂志》232卷，351 - 356页]。第一步，肌动蛋白被弱结合，第二步涉及复合物异构化为更紧密结合的状态。这种异构化可以通过监测在半胱氨酸374处用N -（芘 - 1 - 基） - 碘乙酰胺共价标记的肌动蛋白的荧光来特异性跟踪[吉夫斯、杰弗里斯和米勒（1986年）《生物化学》25卷，8454 - 8458页]。我们在此报告，核苷酸和核苷酸类似物的存在会影响强结合态和弱结合态之间的平衡（称为K2）。在ATP、[γ - 硫代]ATP或ADP与钒酸盐存在的情况下，估计K2的值约小于10^(-2)。在焦磷酸（PPi）或ADP存在的情况下，分别得到约2.3或10的值。发现在ADP存在时，氯化钾浓度的增加或40%乙二醇的存在会降低K2。此处呈现的数据与吉夫斯、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]提出的两步结合模型一致，该模型认为弱结合态和强结合态之间的转变与完整肌肉中产生力的事件密切相关。
