期刊
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
卷 1777, 期 10, 页码 1301-1310出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbabio.2008.06.008
关键词
ATP synthase; F-0 part; H+-transport; Pyranine; Driving force
资金
- Swiss National Science Foundation
The membrane-embedded F-0 part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F-0 complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F-0 part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Delta psi or Delta pH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F1F0 holoenzyme, no significant difference was observed in the efficiency of Delta pH or Delta psi as driving forces for H+-transport through F-0. Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H+/(s x F-0) at Delta psi of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H+-transport at initial rates of 6300 H+/(s x F-0) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms. (C) 2008 Elsevier B.V. All rights reserved.
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