Druckmann S, Ottolenghi M, Korenstein R
Biophys J. 1985 Jan;47(1):115-8. doi: 10.1016/S0006-3495(85)83883-2.
The direction of the accessibility to protons of the binding site in bacteriorhodopsin is of primary importance in elucidating the proton-pump mechanism. The problem is approached via the pH-dependent equilibrium bR560 in equilibrium bR605 in vesicles with preferentially oriented purple membranes. Fast acidification (stopped-flow) experiments with inside-out, monomeric, bR vesicles were carried out with and without a buffer enclosed in the vesicle interior. The results, showing a buffer-induced delay in the formation of bR605, indicate that the binding site is accessible to protons from the inside of the vesicles. We arrive at this conclusion also by working with inside-out trimeric vesicles in the presence and in the absence of H+ (and K+) ionophores. The results suggest that in Halobacterium halobium, the binding site and thus the retinal Schiff base are exposed to the outside of the cell. This conclusion is consistent with a pumping mechanism based on a light-induced pK change.
细菌视紫红质中结合位点质子可及性的方向对于阐明质子泵机制至关重要。通过在具有优先取向的紫色膜的囊泡中，研究pH依赖的平衡态bR560与平衡态bR605之间的关系来解决这个问题。使用内外翻转的单体bR囊泡进行快速酸化（停流）实验，囊泡内部有或没有缓冲液。结果表明，缓冲液会导致bR605形成延迟，这表明结合位点可从囊泡内部接触到质子。我们在有和没有H⁺（和K⁺）离子载体的情况下，使用内外翻转的三聚体囊泡进行实验，也得出了这个结论。结果表明，在嗜盐菌中，结合位点以及视黄醛席夫碱暴露于细胞外部。这一结论与基于光诱导pK变化的泵浦机制一致。
