Yasui M, Arata T, Inoue A
J Biochem. 1984 Dec;96(6):1673-80. doi: 10.1093/oxfordjournals.jbchem.a134999.
The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k1, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k-1, were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k1 was found to be 2.55 X 10(6) M-1 X S-1 at 20 degrees C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k-1 value was calculated to be 8.5 X 10(-3) S-1 (or 0.51 min-1). The value of the dissociation constant of S-1 from acto-S-1 complex, Kd, was calculated from Kd = k-1/k1 to be 3.3 X 10(-9) M at 20 degrees C. Kd was also measured at various temperatures (0-30 degrees C), and the thermodynamic parameters, delta G degree, delta H degree, and delta S degree, were estimated from the temperature dependence of Kd to be -11.3 kcal/mol, +2.5 kcal/mol, and +47 cal/deg . mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.
在不存在核苷酸的情况下，测量了肌球蛋白亚片段-1（S-1）与F-肌动蛋白结合的速率常数k1以及F-肌动蛋白-肌球蛋白亚片段-1复合物（肌动蛋白-S-1）解离的速率常数k-1。通过将S-1与不同浓度的F-肌动蛋白混合后光散射强度变化的时间进程来测量S-1与F-肌动蛋白的结合速率，发现在20℃时k1为2.55×10⁶ M⁻¹·s⁻¹。使用芘基碘乙酰胺标记的F-肌动蛋白（Pyr-FA）测定肌动蛋白-S-1的解离速率。Pyr-FA与S-1结合后荧光减弱，将其与肌动蛋白-S-1复合物混合，通过Pyr-FA荧光强度的降低来测量Pyr-FA取代F-肌动蛋白的速率。计算得出k-1值为8.5×10⁻³ s⁻¹（或0.51 min⁻¹）。根据Kd = k-1/k1计算出S-1从肌动蛋白-S-1复合物解离的解离常数Kd在20℃时为3.3×10⁻⁹ M。还在不同温度（0 - 30℃）下测量了Kd，并根据Kd对温度的依赖性估算出热力学参数ΔG°、ΔH°和ΔS°分别为-11.3 kcal/mol、+2.5 kcal/mol和+47 cal/deg·mol。因此，肌球蛋白头部与F-肌动蛋白的结合表现为吸热且由熵驱动。
