Bransburg-Zabary S, Nachliel E, Gutman M
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Israel.
Biochim Biophys Acta. 1996 Dec 4;1285(2):146-54. doi: 10.1016/s0005-2736(96)00148-4.
The effect of cholesterol on the monensin mediated proton-cation exchange reaction was measured in the time-resolved domain. The experimental system consisted of a black lipid membrane equilibrated with monensin (Nachliel, E., Finkelstein, Y. and Gutman, M. (1996) Biochim. Biophys. Acta 1285, 131-145). The membrane separated two compartments containing electrolyte solutions and pyranine (8-hydroxypyrene 1,3,6-trisulfonate) was added on to one side of the membrane. A short laser pulse was used to cause a brief transient acidification of the pyranine-containing solution and the resulting electric signal, derived from proton-cation exchange, was measured in the microsecond time domain. Incorporation of cholesterol had a clear effect on the electric transients as measured with Na+ or K+ as transportable cations. The measured transients were subjected to rigorous analysis based on numeric integration of coupled, non-linear, differential rate equations which correspond with the perturbed multi-equilibria state between all reactants present in the system. The various kinetic parameters of the reaction and their dependence on the cholesterol content had been determined. On the basis of these observations we can draw the following conclusions: (1) Cholesterol perturbed the homogeneity of the membrane and microdomains were formed, having a composition that differed from the average value. The ionophore was found in domains which were practically depleted of phosphatidylserine. (2) The diffusivity of the protonated monensin (MoH) was not affected by the presence of cholesterol, indicating that the viscosity of the central layer of the membrane was unaltered. (3) The diffusivity of the monensin metal complexes (MoNa and MoK) was significantly increased upon addition of cholesterol. As the viscosity along the cross membranal diffusion route is unchanged, the enhanced motion of the MoNa and MoK is attributed to variations of the electrostatic potential within the domains.
在时间分辨域中测量了胆固醇对莫能菌素介导的质子 - 阳离子交换反应的影响。实验系统由与莫能菌素平衡的黑色脂质膜组成（Nachliel, E., Finkelstein, Y. 和 Gutman, M. (1996) Biochim. Biophys. Acta 1285, 131 - 145）。该膜分隔两个含有电解质溶液的隔室，并且在膜的一侧加入了芘甲磺酸（8 - 羟基芘 - 1,3,6 - 三磺酸）。使用短激光脉冲使含芘甲磺酸的溶液短暂瞬时酸化，并在微秒时间域中测量源自质子 - 阳离子交换的产生的电信号。以Na⁺或K⁺作为可运输阳离子测量时，胆固醇的掺入对电瞬变有明显影响。基于与系统中所有反应物之间的扰动多平衡状态相对应的耦合、非线性、微分速率方程的数值积分，对测量的瞬变进行了严格分析。确定了反应的各种动力学参数及其对胆固醇含量的依赖性。基于这些观察结果，我们可以得出以下结论：(1) 胆固醇扰乱了膜的均匀性并形成了微区，其组成与平均值不同。离子载体存在于实际上缺乏磷脂酰丝氨酸的区域中。(2) 质子化莫能菌素（MoH）的扩散率不受胆固醇存在的影响，表明膜中心层的粘度未改变。(3) 添加胆固醇后，莫能菌素金属络合物（MoNa和MoK）的扩散率显著增加。由于沿跨膜扩散路径的粘度不变，MoNa和MoK运动的增强归因于区域内静电势的变化。
