Lin S H, Harzelrig J B, Cheung H C
Graduate Program in Biophysical Sciences, University of Alabama at Birmingham 35294-2041.
Biophys J. 1993 Oct;65(4):1433-44. doi: 10.1016/S0006-3495(93)81209-8.
The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.
通过使用用5-（碘乙酰胺基）荧光素标记巯基SH1的肌球蛋白亚片段-1（S1）（S1-AF），研究了肌动蛋白与S1的结合动力学。在停流装置中快速混合后，荧光素部分的荧光强度增加了50%，随后是一个较慢的增加，这一增加得到了很好的分辨。荧光变化的这个慢相既不能用单指数函数也不能用双指数函数拟合，但可以用三个指数项的和拟合，得到三个观察到的一级速率常数（λi）。通过用天然S1从复合物中置换S1-AF来研究肌动蛋白-（S1-AF）的解离。解离动力学由一个单一的速率常数表征（20℃时约为0.012 s-1），并且这个常数与S1浓度无关。结合先前在确定的形成肌动蛋白-亚片段-1的条件下获得的平衡数据（Lin，S.-H.和H.C.Cheung.1991.Biochemistry.30:4317-4323），提出了一个六态双途径模型作为形成僵直肌动蛋白·S1的最小动力学方案。在这个模型中，未结合的亚片段-1存在于两个相互平衡的构象状态（S1'和S1）中，一个对应于先前确定的低温状态，另一个对应于高温状态。每个亚片段-1状态都可以与肌动蛋白相互作用形成碰撞复合物，随后进行两次异构化以形成两个肌动蛋白-亚片段-1状态（A.S1'和A.S1）。两次异构化在停流实验中都可见。考虑了该模型的两种特殊情况：1）初始碰撞复合物与肌动蛋白和S1的快速预平衡，以及2）碰撞复合物的微量积累。第一种情况要求三个观察到的速率常数的三种组合与肌动蛋白浓度无关。数据与这种近似不相符。另一种特殊情况要求λi的总和随肌动蛋白浓度线性变化，而λi的其他两种组合随肌动蛋白浓度呈二次方变化。目前的数据与第二种情况一致。在20℃、60 mM KCl、2 mM MgCl2、30 mM 2-（[（羟甲基）-1,1-双（羟甲基）乙基]氨基）乙烷磺酸和pH 7.5条件下，肌动蛋白与S1'和S1相互作用的双分子结合速率常数分别为8.58×105和1.11×106 M-1 s-1。
