Martin G E, Rutherford N G, Henderson P J, Walmsley A R
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):261-8. doi: 10.1042/bj3080261.
The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.
通过平衡和时间分辨荧光测量评估了转运抑制剂福斯高林与大肠杆菌半乳糖 - H⁺同向转运体GalP的结合。福斯高林结合后观察到蛋白质荧光淬灭8 - 12%。通过荧光滴定法测定的福斯高林的总解离常数（Kd）在1.2至2.2微摩尔之间，这与[³H]福斯高林结合的平衡透析测量报道的值（Kd = 0.9 - 1.4微摩尔）相似。通过停流荧光法测量福斯高林结合的动力学。蛋白质荧光以双相方式淬灭；这两个速率中较快的速率取决于福斯高林的浓度，并被解释为初始结合步骤，由此确定了结合（kon）和解离（koff）速率常数。结合和解离速率常数分别为5.4 - 6.2微摩尔⁻¹·秒⁻¹和5.1 - 11.5秒⁻¹，计算得出的Kd为1.5微摩尔。D - 半乳糖抑制福斯高林的结合，但L - 半乳糖不抑制，糖的置换提供了另一种计算福斯高林解离速率常数（koff = 12.4 - 13.0秒⁻¹）的方法。蛋白质荧光缓慢变化的速率（3 - 5秒⁻¹）与福斯高林浓度无关，表明转运体在不同构象之间发生异构化，可能是外向和内向形式。在一系列温度下测定了这些动力学参数，以便分析福斯高林结合和转运体重定向的热力学。结合过程是由熵驱动的（ΔS = 83.7焦·开⁻¹·摩尔⁻¹；ΔH = 8.25千焦·摩尔⁻¹），类似于细胞松弛素B的情况，细胞松弛素B也是GalP的抑制剂。通过平衡透析测量[³H]福斯高林的结合揭示了细胞松弛素B对结合的福斯高林的竞争性置换，这可能表明糖、福斯高林和细胞松弛素B的结合位点重叠；福斯高林和细胞松弛素B的Kd分别计算为0.85微摩尔和4.77微摩尔，结合位点的浓度为10.2纳摩尔·毫克⁻¹。
