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