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

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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 mV4. When membrane potential was released from more negative membrane potential than reversal potential, transient currents were observedOther meaning of the result1. It is estimated that the reaction 4H^+ + O_2 + 4e → 2H_2O is unidirectional, and opposite reaction would be stopped at Perferryl intermediates2. H^+ is transported via Cytochrome bo type-ubiquinol oxidase3. Generated ΔμH^+ (280 mV) is enough to produce ATP via F-type ATPase4. After the opposite reaction is stopped at Perferryl intermediates, release of membrane potential would help us to investigate each reaction step from Perferryl intermediatesWith 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