Van Walraven H S, Marvin H J, Koppenaal E, Kraayenhof R
Eur J Biochem. 1984 Nov 2;144(3):555-61. doi: 10.1111/j.1432-1033.1984.tb08501.x.
ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.
利用各种光学探针，在ATP酶蛋白脂质体中研究了ATP水解诱导的质子转运和电势产生。蛋白脂质体由重组的ATP酶复合物和从嗜热蓝藻集胞藻6716中分离出的天然脂质混合物组成[Van Walraven等人（1983年），《欧洲生物化学杂志》137卷，第101 - 106页]。天然类胡萝卜素和添加的氧杂萘酚VI用作净电荷分离产生的膜电势的探针（外侧为负，内侧为正）。正如预期的那样，它们的响应与9 - 十四烷基氨基 - 6 - 氯 - 2 - 甲氧基吖啶具有相似的半衰期，对缬氨霉素敏感并受尼日利亚菌素刺激。通过中性红和甲酚红监测脂质体外部和内部水相空间的质子浓度；对于内部测量，这些pH指示剂在去污剂透析过程中被困在囊泡内。由ATP水解引起的内部酸化和外部碱化受到尼日利亚菌素的抑制，并因缬氨霉素而增强；在常用的较高缬氨霉素浓度下，中性红响应变得短暂，而慢得多的甲酚红响应从一开始就减弱。在较小的预设pH梯度下，ATP水解活性和中性红响应都会降低。随着MgCl2浓度增加，中性红响应减慢，甲酚红响应略有增强；在内部和外部染料响应中均观察到这种情况。在我们的实验条件下，中性红透过膜的渗透率微不足道，但在低于脂质相变温度时会增强。对于外部添加的中性红，非渗透性缓冲液Hepes仅在高MgCl2浓度下有效，而一些外部甲酚红响应仅在存在Hepes的高MgCl2浓度下可见。pH指示剂和电势探针响应的动力学清楚地区分了快速的界面和膜内质子位移与缓慢的整体质子平衡。数据总结在一个模型中，该模型支持局部质子位移对主要能量转换事件的重要性。
