Modification of isolated subunit c of the F1F0‐ATPase from Propionigenium modestum by dicyclohexylcarbodiimide

FEBS Letters

Dimroth, Peter¹  Kluge, Claudia¹ 

[1] Mikrobiologisches Institut, Eidgenössische Technische Hochschule, ETH-Zentrum, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland

关键词: Na+ binding site;    Ion translocation;    Na+ pump;    DCCD;    N;    N'-dicyclohexylcarbodiimide;    EIPA;    N-ethyl-N-isopropylamiloride;   

DOI  :  10.1016/0014-5793(94)80147-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Subunit c of the F₁F₀-ATPase from Propionigenium modestum was extracted from the particulate cell fraction with chloroform/methanol. The protein was further purified by carboxymethyl cellulose chromatography and anion exchange HPLC in the organic solvent. SDS-PAGE of the purified protein indicated a single stained protein band migrating as expected for the c-subunit. Incubation of isolated subunit c in chlorform/methanol or aqueous buffer containing dodecyl-β-d-maltoside with [¹⁴C]dicyclohexylcarbodiimide (DCCD) resulted in the incorporation of radioactivity into the protein. The rate of this reaction depended on the external pH; it was significantly faster in the more acidic than in the alkaline pH range. In the presence of Na⁺ subunit c was partially protected from labeling with [¹⁴C]DCCD at pH 6.1 and at pH 7.5, whereas no protection was evident at pH 5.5. At pH 7.5, the rate of subunit c labeling by [¹⁴C]DCCD in the presence of 20 mM NaCl was about 50% lower than in the absence of Na⁺ ions. The isolated c-subunit therefore apparently retains in part the Na⁺ binding site which, when occupied, diminishes the reactivity of the protein towards DCCD.

【 授权许可】

Unknown