Lozier R H, Niederberger W, Bogomolni R A, Hwang S, Stoeckenius W
Biochim Biophys Acta. 1976 Sep 13;440(3):545-56. doi: 10.1016/0005-2728(76)90041-4.
We have used flash spectroscopy and pH indicator dyes to measure the kinetics and stoichiometry of light-induced proton release and uptake by purple membrane in aqueous suspension, in cell envelope vesicles and in lipid vesicles. The preferential orientation of bacteriorhodopsin in opposite directions in the envelope and lipid vesicles allows us to show that uptake of protons occurs on the cytoplasmic side of the purple membrane and release on the exterior side. In suspensions of isolated purple membrane, approximately one proton per cycling bacteriorhodopsin molecule appears transiently in the aqueous phase with a half-rise time of 0.8 ms and a half-decay time of 5.4 ms at 21degreesC. In cell envelope preparations which consist of vesicles with a preferential orientation of purple membrane, as in whole cells, and which pump protons out, the acidification of the medium has a half-rise time of less than 1.0 ms, which partially relaxes in approx. 10 ms and fully relaxes after many seconds. Phospholipid vesicles, which contain bacteriorhodopsin preferentially oriented in the opposite direction and pump protons in, show an alkalinization of the medium with a time constant of approximately 10 ms, preceded by a much smaller and faster acidification. The alkalinization relaxes over many seconds. The initial fast acidification in the lipid vesicles and the fast relaxation in the envelope vesicles are accounted for by the misoriented fractions of bacteriorhodopsin. The time constants of the main effects, acidification in the envelopes and alkalinization in the lipid vesicles correlate with the time constants for the release and uptake of protons in the isolated purple membrane, and therefore show that these must occur on the outer and inner surface respectively. The slow relaxation processes in the time range of several seconds must be attributed to the passive back diffusion of protons through the vesicle membrane.
我们利用闪光光谱法和pH指示剂染料，测量了水悬浮液、细胞膜囊泡和脂质囊泡中紫色膜光诱导质子释放和摄取的动力学及化学计量关系。细菌视紫红质在细胞膜囊泡和脂质囊泡中呈相反方向的优先取向，这使我们能够证明质子摄取发生在紫色膜的细胞质一侧，而释放发生在外侧。在分离的紫色膜悬浮液中，每个循环的细菌视紫红质分子约有一个质子瞬时出现在水相中，在21℃时，上升半衰期为0.8毫秒，衰减半衰期为5.4毫秒。在细胞膜制备物中，其由具有与完整细胞中相同的紫色膜优先取向且能将质子泵出的囊泡组成，培养基的酸化上升半衰期小于1.0毫秒，约在10毫秒时部分弛豫，数秒后完全弛豫。含有优先取向相反且能将质子泵入的细菌视紫红质的磷脂囊泡，显示培养基碱化，时间常数约为10毫秒，之前有一个小得多且更快的酸化过程。碱化在数秒内弛豫。脂质囊泡中的初始快速酸化和细胞膜囊泡中的快速弛豫是由细菌视紫红质的错误取向部分引起的。主要效应的时间常数，即细胞膜囊泡中的酸化和脂质囊泡中的碱化，与分离的紫色膜中质子释放和摄取的时间常数相关，因此表明这些过程必定分别发生在外表面和内表面。数秒时间范围内的缓慢弛豫过程必定归因于质子通过囊泡膜的被动反向扩散。
