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

大肠杆菌呼吸链H^-泵的膜片钳研究

基本信息

  • 批准号:
    13680683
  • 负责人:
  • 金额:
    $ 2.24万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Scientific Research (C)
  • 财政年份:
    2001
  • 资助国家:
    日本
  • 起止时间:
    2001 至 2002
  • 项目状态:
    已结题

项目摘要

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
我们建立了一种制备大肠杆菌巨原生质体的SI(Spheroplast incubation)法。原生质体直径为20 μ m。此外,我们还发现,在巨大的原生质体中发现的巨大的空泡状结构(我们指定为原空泡)由外翻的细胞质膜组成。以原生质体和原核细胞为材料,建立了一种用膜片钳技术分析各种离子转运系统的新方法。该方法适用于所有的离子转运系统,无论转运体的活性位点是朝向细胞质还是周质,我们都可以用泛醇-1诱导的电流来检测细胞色素bo型泛醇氧化酶的H^+泵活性。并发现了这一活动的一些特征。当膜电位比反转电位更负时,反向电流不连续产生.抑制电位依赖于化学H^+梯度(50 mV/ΔpH)。H^+电化学梯度(ΔμH^+)产生于 ...更多信息 a细胞色素bo型泛醇氧化酶为约280 mV 4。当膜电位从比反转电位更负的膜电位释放时,瞬时电流消失。推测反应4 H ^+ + O_2 + 4 e → 2H_2O是单向的,相反的反应将在Perferryl中间体2处终止。H^+通过细胞色素bo型泛醇氧化酶3转运。产生的ΔμH^+(280 mV)足以通过F型ATP酶4产生ATP。111当相反的反应在Perferryl中间体处停止后,膜电位的释放将有助于我们研究Perferryl中间体的每个反应步骤。通过这些结果,我们表明通过细胞色素bo型泛醇氧化酶的H^+泵活性与已经在其他生物学分析中定性相似。此外,我们还用膜片钳技术定量评估了这些活动。这些结果有助于构建大肠杆菌的计算机模拟系统。coh细胞较少

项目成果

期刊论文数量(3)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
矢部 勇: "細胞工学別冊、植物細胞工学シリーズ18"秀潤社. 5 (2003)
矢部勇:《细胞工程专卷,植物细胞工程丛书18》,书俊社5(2003)。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Isamu Yabe: "Analysis on ion transporetr by Giant microorganism"Saiboukougaku. 18. 206-210 (2003)
Isamu Yabe:“巨型微生物的离子转运分析”Saiboukougaku。
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
矢部勇: "巨大化微生物細胞であらゆるイオン輸送体を解析する"秀潤社、細胞工学別冊、植物細胞工学シリーズ1,8. 206-210 (2003)
Isamu Yabe:“分析巨型微生物细胞中的所有离子转运蛋白” Shujunsha,细胞工程特刊,植物细胞工程系列 1, 8. 206-210 (2003)
  • DOI:
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  • 影响因子:
    0
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