Chalovich J M, Chen Y D, Dudek R, Luo H
Department of Biochemistry, East Carolina University School of Medicine, Greenville, North Carolina 27858-4354, USA.
J Biol Chem. 1995 Apr 28;270(17):9911-6. doi: 10.1074/jbc.270.17.9911.
The time course of interaction of caldesmon with actin may be monitored by fluorescence changes that occur upon the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled caldesmon to actin or to acrylodan actin. The concentration dependence of the observed rate of caldesmon-actin binding was analyzed to a first approximation as a single-step reaction using a Monte Carlo simulation. The derived association and dissociation rates were 10(7) M-1 s-1 and 18.2 s-1, respectively. Smooth muscle tropomyosin enhances the binding of caldesmon to actin, and this was found to be due to a reduction in the rate of dissociation to 6.3 s-1. There is no evidence from this study for a different mechanism of binding in the presence of tropomyosin. The fluorescence changes that occurred with the binding of 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl))-labeled caldesmon to actin or actin-tropomyosin were reversed by the addition of myosin subfragment 1 as predicted by a competitive binding mechanism.
钙调蛋白与肌动蛋白相互作用的时间进程可通过荧光变化来监测,这种荧光变化发生在12 -(N - 甲基 - N -(7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂萘 - 4 - 基))标记的钙调蛋白与肌动蛋白或丙烯罗丹明标记的肌动蛋白结合时。使用蒙特卡罗模拟,将观察到的钙调蛋白 - 肌动蛋白结合速率的浓度依赖性初步分析为单步反应。推导得到的缔合和解离速率分别为10⁷ M⁻¹ s⁻¹和18.2 s⁻¹。平滑肌原肌球蛋白增强了钙调蛋白与肌动蛋白的结合,并且发现这是由于解离速率降低到6.3 s⁻¹。这项研究没有证据表明在存在原肌球蛋白的情况下存在不同的结合机制。如竞争结合机制所预测的那样,添加肌球蛋白亚片段1可逆转12 -(N - 甲基 - N -(7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二氮杂萘 - 4 - 基))标记的钙调蛋白与肌动蛋白或肌动蛋白 - 原肌球蛋白结合时发生的荧光变化。