Reconstitution of the metal‐tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1‐ATPase

FEBS Letters

Someya, Yuichi²  Futai, Masamitsu¹  Yamaguchi, Akihito²  Moriyama, Yoshinori¹  Sawai, Tetsuo² 

[1] Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567, Japan;Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Chiba 263, Japan

关键词: Tetracycline;    Antiporter;    Tetracycline/H+ antiporter;    H+-ATPase;    Reconstitution;    Proteoliposome;    TetA;    tetracycline resistance protein;    MOPS;    3-(N-morpholino)propanesulfonic acid;    MES;    2-(N-morpholino)ethanesulfonic acid;    tricine;    N-tris(hydroxymethyl)methylglycine;    octylglucoside;    NEM;    N-ethylmaleimide;    TC;    tetracycline;   

DOI  :  10.1016/0014-5793(95)01079-T

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The tetracycline resistance gene (tetA) was cloned downstream of the lac promoter. When expression of the tetA gene in E. coli cells carrying the lac I^q gene was induced with isopropyl β-d-thiogalactopyranoside, the tetracycline resistance protein (TetA) was overproduced, amounting to about 30% of the integral cytoplasmic membrane protein. Essentially pure TetA protein could be obtained by solubilization with 1.25% and one-step purification by DEAE Sepharose CL-6B column chromatography. The TetA protein was incorporated into proteoliposomes with F₀F₁-ATPase. The proteoliposomes exhibited [³H]tetracycline transport dependent on ATP hydrolysis. The specific activity was about 2 nmol/mg protein/min. The proteoliposomes also showed H⁺ efflux coupled with tetracycline influx. Tetracycline/H⁺ antiport by proteoliposomes reconstituted with the Ser-65 → Cys mutant TetA protein was inhibited by N-ethylmaleimide. These results proved for the first time that the tetracycline/H⁺ antiport is only mediated by the TetA protein.

【 授权许可】

Unknown