McKillop D F, Geeves M A
Department of Biochemistry, School of Medical Sciences, University of Bristol, United Kingdom.
Biophys J. 1993 Aug;65(2):693-701. doi: 10.1016/S0006-3495(93)81110-X.
Equilibrium titrations and kinetic experiments were used to define the cooperative binding of myosin subfragment 1 (S1) to actin-troponin-tropomyosin. Both types of experiment require an equilibrium between two states of the thin filament in which one state (the off state) binds S1 less readily than the other. Equilibrium titrations are compatible with > 95% of the actin7.Tn.Tm units being in the off state in the absence of calcium and 80% in the off state in the presence of calcium. Kinetic binding data suggest that the presence of calcium switches the thin filament from 70% in the off state to < 5%. The two experiments, therefore, define quite different populations of the off states. We propose a three-state model of the thin filament. A "blocked state" which is unable to bind S1, a "closed state" which can only bind S1 relatively weakly and an "open state" in which the S1 can both bind and undergo an isomerization to a more strongly bound rigor-like conformation. The equilibrium between the three states is calcium-dependent; KB = [closed]/[blocked] = 0.3 and > or = 16 and KT = [open]/[closed] = 0.09 and 0.25 in the absence and presence of calcium, respectively. This model can account for both types of experimental data.
采用平衡滴定法和动力学实验来确定肌球蛋白亚片段1（S1）与肌动蛋白-肌钙蛋白-原肌球蛋白的协同结合。这两种实验都需要细肌丝的两种状态之间达到平衡，其中一种状态（失活状态）比另一种状态更难结合S1。平衡滴定法表明，在无钙情况下，超过95%的肌动蛋白7·肌钙蛋白·原肌球蛋白单元处于失活状态；在有钙情况下，80%处于失活状态。动力学结合数据表明，钙的存在使细肌丝从70%处于失活状态转变为低于5%。因此，这两种实验确定了截然不同的失活状态群体。我们提出了细肌丝的三态模型。一种“阻断状态”，它不能结合S1；一种“闭合状态”，它只能相对较弱地结合S1；还有一种“开放状态”，其中S1既能结合，又能异构化为一种结合更强的类似僵直的构象。这三种状态之间的平衡取决于钙；在无钙和有钙情况下，平衡常数KB = [闭合状态]/[阻断状态]分别为0.3和≥16，平衡常数KT = [开放状态]/[闭合状态]分别为0.09和0.25。该模型可以解释这两种类型的实验数据。
