Patch clamp studies on H^+-pump of respiratory chain in E. coli

Research Project

Project/Area Number	13680683
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Structural biochemistry
Research Institution	University of Tokyo
Principal Investigator	YABE Isamu Institute of Molecular and Cellular Biosciences, Research Associate, 分子細胞生物学研究所, 助手 (90111575)
Co-Investigator(Kenkyū-buntansha)	KURODA Teruo Okayama University, Pharmaceutical Science, Research Associate, 薬学部, 助手 (80304327)
Project Period (FY)	2001 – 2002
Project Status	Completed (Fiscal Year 2002)
Budget Amount *help	¥3,500,000 (Direct Cost: ¥3,500,000) Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000) Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
Keywords	Giant E. coli cell / Patch clamp method / Respiratory chain / Cytochrome bo / Proton-pump activity / Provacuole / バッチクランプ法 / プロトンポンプ活性 / パッチクランプ
Research Abstract	We developed SI (Spheroplast incubation) method with which giant protoplasts of Escherichia coli could be prepared. The diameter of protoplasts was 20 μn. Also, we showed that giant vacuole-like structure found in giant protoplasts (we designated provacuole) consisted of everted cytoplasmic membranes. With protoplasts and provacuoles, we established a new method to analyze various ion transport systems electrophysiologically with patch clamp technique. It would be applicable to all ion transport systems whichever the active site of the transporter face to cytoplasm or periplasm. With the new method, we could detect H^+ pump activity of Cytochrome bo type-ubiquinol oxidase as currents induced by ubiquinol-1. And we found some characters of this activity. 1. At more negative membrane potential than reversal potential, opposite currents were not generated continuously. 2. Reversal potential was dependent on chemical H^+ gradient (50mV/ΔpH). 3. H^+ electrochemical gradient (ΔμH^+) generated vi … More a Cytochrome bo type ubiquinol oxidase was about 280 mV 4. When membrane potential was released from more negative membrane potential than reversal potential, transient currents were observed Other meaning of the result 1. It is estimated that the reaction 4H^+ + O_2 + 4e → 2H_2O is unidirectional, and opposite reaction would be stopped at Perferryl intermediates 2. H^+ is transported via Cytochrome bo type-ubiquinol oxidase 3. Generated ΔμH^+ (280 mV) is enough to produce ATP via F-type ATPase 4. After the opposite reaction is stopped at Perferryl intermediates, release of membrane potential would help us to investigate each reaction step from Perferryl intermediates With these results, we showed H^+ pump activity via Cytochrome bo type-ubiquinol oxidase was qualitatively similar to that already analyzed elsewhere biologically. Moreover, we also assessed these activities quantitatively with patch clamp technique. Those results can contribute to construct the system for computer simulation of E. coh cells Less