Tu S I, Hutchinson H
Arch Biochem Biophys. 1984 Feb 1;228(2):609-16. doi: 10.1016/0003-9861(84)90029-8.
Bacteriorhodopsin (BR) was incorporated into phosphatidylcholine (PC) vesicles containing different amounts of other lipids. Under the conditions of nullified membrane potential, light-induced proton movement seemed to follow a kinetic scheme which assumed the existence of a proton-pumping inhibition process characterized by a rate constant, kI. The temperature dependence of both kI and the membrane proton leak rate constant (kD) obeyed a simple Arrhenius equation. The presence of cholesterol in the membrane significantly increased the activation energy (Ea) of both the inhibition and leak process. However, further addition of phosphatidic acid (PA) suppressed the increase of Ea associated with kI. The initial proton pumping rate (R0) of vesicles reconstituted with PC showed a bell-shaped temperature dependence with a maximum at approximately 20 degrees C. The addition of cholesterol abolished this dependence. These results suggest that the molecular origin of the inhibition process characterized by kI is different from that of R0 or kD. The temperature dependence of the steady-state fluorescence polarization of dansylated bacteriorhodopsin in vesicles was also investigated. The polarization of the labels in the vesicles without cholesterol showed a bell-shaped temperature dependence with a maximum at approximately 20 degrees C. However, in the presence of cholesterol, the polarization increased linearly as temperature decreased. A comparison of these results with the observed proton movement in similarly reconstituted systems with unmodified protein indicates that membranes with a low fluidity and negatively charged surfaces enhance proton pumping efficiency of bacteriorhodopsin.
细菌视紫红质（BR）被整合到含有不同量其他脂质的磷脂酰胆碱（PC）囊泡中。在膜电位消除的条件下，光诱导的质子移动似乎遵循一种动力学模式，该模式假定存在一个以速率常数kI为特征的质子泵浦抑制过程。kI和膜质子泄漏速率常数（kD）的温度依赖性均遵循简单的阿伦尼乌斯方程。膜中胆固醇的存在显著增加了抑制和泄漏过程的活化能（Ea）。然而，进一步添加磷脂酸（PA）抑制了与kI相关的Ea的增加。用PC重构的囊泡的初始质子泵浦速率（R0）呈现出钟形的温度依赖性，在约20℃时达到最大值。胆固醇的添加消除了这种依赖性。这些结果表明，以kI为特征的抑制过程的分子起源与R0或kD的不同。还研究了囊泡中丹磺酰化细菌视紫红质稳态荧光偏振的温度依赖性。不含胆固醇的囊泡中标记物的偏振呈现出钟形的温度依赖性，在约20℃时达到最大值。然而，在胆固醇存在的情况下，偏振随温度降低呈线性增加。将这些结果与在具有未修饰蛋白质的类似重构系统中观察到的质子移动进行比较表明，具有低流动性和带负电荷表面的膜可提高细菌视紫红质的质子泵浦效率。
