CHARGERIN---ITS TERTIARY STRUCTURE AND THE MECHANISM OF ENERGY TRANSDUCTION

最近，我们从大鼠肝脏线粒体中纯化了一种名为chargerin 11的疏水蛋白[Higuti， T., et al.]。生物。化学，263,6772-6776(1988)]，由线粒体DNA的未知阅读框URFA6L编码。虽然chargerin IT是疏水的，可溶于氯仿和甲醇的混合物（2:1），但它在其序列中具有不平衡的正电荷。电荷素11是H^+-ATP合酶的Fo亚基之一。此外。一种抗电荷素II的抗体以能量依赖的方式抑制有丝分裂体中H^+-ATP合酶的能量转导。这表明H^+-ATP合酶的通电引起了充电素II的构象变化[Uchida]。J.等人。物化学。Biophys。《共同法典》，165,449-456(1987)]。电荷素II的这些独特特征表明，它在线粒体ATP合酶的能量转导中起着至关重要的作用，这与电荷转移偶联假说很好地符合[Higuti， T., Mol. Cell.][j].生物化学学报，2004,27(5):387 - 391。更多的是目前的研究项目。我们得到了以下重要的发现。利用抗体检测了大鼠肝脏线粒体ATP合酶Fo区电荷素II的取向。结果表明，它的n端区（约8个氨基酸残基）暴露在Fo的c侧表面，但其c端和电荷簇区域隐藏在Fo.2中。利用能量传导的各向异性抑制剂。我们发现H^+转运氧化还原复合物和ATP合酶在通电状态下形成了“内部电场”。很好地符合电荷转移耦合假设。用放射免疫法测定了大鼠肝脏线粒体纯化的H^+-ATP合酶和亚线粒体颗粒中电荷素If的含量。结果表明，H^+-ATP合酶中电荷素II的摩尔比为1比1。这是关于线粒体H^+-ATP合成酶a6l产物化学计量学的首次报道。用反相色谱柱纯化大鼠肝脏线粒体H^+-ATP合成酶亚基。这些亚基的序列由蛋白质序列决定。亚基b和因子6首次在大鼠体内发现。然后我们合成了b亚基的探针DNA，并成功地明确了b亚基的导入前体cDNA的序列。该序列包含输入信号重复的42个氨基酸编码区和成熟蛋白的214个氨基酸编码区。少

Recently we purified a hydrophobic protein named chargerin 11 from rat liver mitochondria [Higuti, T., et al. J. Biol. Chem. 263, 6772-6776 (1988)] , which was encoded by the unidentified reading frame URFA6L of mitochondrial DNA. Although chargerin IT is hydrophobic and soluble in a mixture of chloroform and methanol (2:1), it has unbalanced positive charges in its sequence. Chargerin 11 is one of the subunits of Fo of H^+-ATP synthase. Furthermore. an antibody against chargerin II inhibited energy-transduction by H^+-ATP synthase in mitoplasts in an energy-dependent fashion. suggesting that energization of H^+-ATP synthase causes a conformational change in chargerin II [Uchida. J. et al. Biochem. Biophys. Res. Commun. 165, 449-456 (1987)].These unique features of chargerin II suggest that it has an essential role in the energy transduction by mitochondrial ATP synthase, in good accord with the charge-transfer coupling hypothesis [Higuti, T. , Mol. Cell. Biochem. 166, 37-61 (1984)].In … More the present research project. we have obtained the following important findings.1. The orientation of chargerin II in Fo of the ATP synthase of rat liver mitochondria was examined using antibodies against peptides of chargerin II. Results showed that its N-terminal region (about 8 amino acid residues) was exposed on the surface of the C-side of Fo, but its C-terminal and charge-cluster regions were buried in Fo.2. By using the anisotropic inhibitors of energy transduction. which were found by us previously, we found that "an internal electric field" in H^+-transport redox complexes and ATP synthase is formed in their energized state. in good accord with the charge-transfer coupling hypothesis.3. The contents of chargerin If in the H^+-ATP synthase purified from rat liver mitochondria and in submitochondrial particles were determined by radioimmunoassay. Results showed that the H^+-ATP synthase contained chargerin II in a molar ratio of one to one. This is the first report on the stoichiometry of the A6L-product in mitochondrial H^+-ATP synthase.4. The subunits of H^+-ATP synthase from rat liver mitochondria were purified on a reverse-phase column. The sequences of these subunits were determined by the protein sequences. The subunit b and factor 6 were firstly found in rat. Then we synthesized the probe DNA for the subunit b and succeeded to clarify the sequence of cDNA for the import precursor of subunit b. The cDNA of subunit b contained 1,124 base pairs. The sequence contained the coding region of 42 amino acids of the import signal reptide and 214 amino acids of the mature protein. Less