Kuhlman P A, Ellis J, Critchley D R, Bagshaw C R
Department of Biochemistry, University of Leicester, UK.
FEBS Lett. 1994 Feb 21;339(3):297-301. doi: 10.1016/0014-5793(94)80434-6.
Measurement of the binding equilibrium for the interaction of alpha-actinin with F-actin is complicated by secondary reactions involving cross-linking and/or bundling of the actin filaments. To quantitate the initial binding event, we studied the interaction of the bacterially expressed actin-binding domain (ABD) of chick smooth muscle alpha-actinin with F-actin. Stopped-flow measurements revealed a quench in protein fluorescence and an enhancement in light scattering when ABD binds to F-actin yielding second order rate constants for association of 2 x 10(5), 1.8 x 10(6) and 4 x 10(6) M-1.s-1 at 5 degrees C, 15 degrees C and 25 degrees C, respectively. At the latter two temperatures the dissociation rate constants were 1.5 and 9.6s-1, giving equilibrium constants of 0.83 and 2.4 microM, respectively. Optical changes on mixing intact alpha-actinin with F-actin were dominated by secondary bundling events.
α-辅肌动蛋白与F-肌动蛋白相互作用的结合平衡测量因涉及肌动蛋白丝交联和/或成束的二级反应而变得复杂。为了定量初始结合事件，我们研究了鸡平滑肌α-辅肌动蛋白的细菌表达肌动蛋白结合结构域（ABD）与F-肌动蛋白的相互作用。停流测量显示，当ABD与F-肌动蛋白结合时，蛋白质荧光猝灭，光散射增强，在5℃、15℃和25℃时，缔合的二级速率常数分别为2×10⁵、1.8×10⁶和4×10⁶M⁻¹·s⁻¹。在后两个温度下，解离速率常数分别为1.5和9.6s⁻¹，平衡常数分别为0.83和2.4μM。完整的α-辅肌动蛋白与F-肌动蛋白混合时的光学变化主要由二级成束事件主导。
