Ramos S, Kaback H R
Biochemistry. 1977 Mar 8;16(5):848-54. doi: 10.1021/bi00624a006.
Membrane vesicles isolated from Escherichia coli grown under various conditions generate a transmembrane pH gradient (delta pH) of about 2 pH units (interior alkaline) under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure delta pH and the distribution of the lipophilic cation triphenylmethylphosphonium to measure the electrical potential (delta psi) across the membrane, the vesicles are demonstrated to develop an electrochemical proton gradient (delta-muH+) of almost - 200 mV (interior negative and alkaline) at pH 5.5 in the presence of reduced phenazine methosulfate or D-lactate, the major component of which is a deltapH of about - 120 mV. As external pH is increased, deltapH decreases, reaching 0 at about pH 7.5 and above, while delta psi remains at about - 75 mV and internal pH remains at pH 7.5-7.8. The variations in deltapH correlate with changes in the oxidation of reduced phenazine methosulfate or D-lactate, both of which vary with external pH in a manner similar to that described for deltapH. Finally, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin with little change in delta-muH+ and no change in respiratory activity. These data and those presented in the following paper (Ramos and Kaback 1976) provide strong support for the role of chemiosmotic phenomena in active transport and extend certain aspects of the chemiosmotic hypothesis.
从在各种条件下生长的大肠杆菌中分离出的膜囊泡,在适当条件下通过流动透析测定时,会产生约2个pH单位的跨膜pH梯度(ΔpH)(内部呈碱性)。利用弱酸的分布来测量ΔpH,以及利用亲脂性阳离子三苯基甲基鏻的分布来测量跨膜电势(Δψ),结果表明,在存在还原型吩嗪硫酸甲酯或D-乳酸的情况下,膜囊泡在pH 5.5时会形成近-200 mV的电化学质子梯度(ΔμH⁺)(内部呈负且碱性),其主要成分是约-120 mV的ΔpH。随着外部pH升高,ΔpH降低,在约pH 7.5及以上时达到0,而Δψ保持在约-75 mV,内部pH保持在pH 7.5 - 7.8。ΔpH的变化与还原型吩嗪硫酸甲酯或D-乳酸氧化的变化相关,这两者都随外部pH的变化方式与ΔpH的描述相似。最后,在缬氨霉素和尼日利亚菌素存在的情况下,ΔpH和Δψ可以相互变化,而ΔμH⁺变化很小,呼吸活性没有变化。这些数据以及下一篇论文(拉莫斯和卡巴克,1976年)中呈现的数据,为化学渗透现象在主动运输中的作用提供了有力支持,并扩展了化学渗透假说的某些方面。