Walmsley A R, Lowe A G, Henderson P J
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Eur J Biochem. 1994 Apr 1;221(1):513-22. doi: 10.1111/j.1432-1033.1994.tb18763.x.
The kinetics of the binding of cytochalasin B to the proton-linked L-arabinose (AraE) and D-galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluorescence that occur upon binding the ligand. Steady-state measurements yielded Kd values of 1.1, 1.9 and 0.14 microM for the AraE, GalP and GLUT1 proteins, respectively. The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped-flow spectroscopy. In each case, the apparent Kd was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 microM for AraE, GalP and GLUT1, respectively. The differences between these apparent Kd values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B: [formula: see text] The transporter is proposed to alternate between two different conformational forms (T1 and T2), with cytochalasin B binding only to the T2 conformation, to induce a further conformational transition of the transporter to the T3 form. The values for the overall dissociation constants show that the T1 conformation is favoured by AraE and GalP in the absence of ligands, but the T2 conformation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP. A thermodynamic analysis of these conformational transitions has been performed. The T1 and T2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively.
通过利用配体结合时蛋白质荧光的变化，研究了细胞松弛素B与大肠杆菌质子偶联的L-阿拉伯糖（AraE）和D-半乳糖（GalP）同向转运体以及人红细胞葡萄糖转运体（GLUT1）的结合动力学。稳态测量得出AraE、GalP和GLUT1蛋白的解离常数（Kd）值分别为1.1、1.9和0.14微摩尔。细胞松弛素B结合的缔合和解离速率常数已通过停流光谱法测定。在每种情况下，表观Kd由相应的速率常数计算得出，AraE、GalP和GLUT1的表观Kd值分别为1.5、0.4和1.6微摩尔。这些表观Kd值与荧光滴定法测得的值之间的差异，根据以下三步机制进行解释，其中CB代表细胞松弛素B：[公式：见原文] 提出转运体在两种不同的构象形式（T1和T2）之间交替，细胞松弛素B仅与T2构象结合，诱导转运体进一步构象转变为T3形式。整体解离常数的值表明，在没有配体的情况下，AraE和GalP有利于T1构象，但GLUT1有利于T2构象。因此，细胞松弛素B与GLUT1的结合使平衡向T3（CB）构象状态移动，产生观察到的紧密结合，这与细胞松弛素B与AraE和GalP结合时观察到的平衡变化相反。已对这些构象转变进行了热力学分析。T1和T2构象可能分别代表转运体的结合位点面向外侧和内侧的状态。
