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ヨコヤマ ケン
YOKOYAMA KEN
横山 謙 所属 京都産業大学 生命科学部 先端生命科学科 職種 教授 |
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| 言語種別 | 英語 |
| 発行・発表の年月 | 2012/07 |
| 形態種別 | 研究論文 |
| 査読 | 査読あり |
| 標題 | Reconstitution of Vacuolar-type Rotary H+-ATPase/Synthase from Thermus thermophilus |
| 執筆形態 | その他 |
| 掲載誌名 | JOURNAL OF BIOLOGICAL CHEMISTRY |
| 出版社・発行元 | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC |
| 巻・号・頁 | 287(29),pp.24597-24603 |
| 著者・共著者 | Jun-ichi Kishikawa,Ken Yokoyama |
| 概要 | Vacuolar-type rotary H+-ATPase/synthase (VoV1) from Thermus thermophilus, composed of nine subunits, A, B, D, F, C, E, G, I, and L, has been reconstituted from individually isolated V-1 (A(3)B(3)D(1)F(1)) and V-o (C(1)E(2)G(2)I(1)L(12)) subcomplexes in vitro. A(3)B(3)D and A(3)B(3) also reconstituted with V-o, resulting in a holoenzyme-like complexes. However, A(3)B(3)D-V-o and A(3)B(3)-V-o did not show ATP synthesis and dicyclohexylcarbodiimide-sensitive ATPase activity. The reconstitution process was monitored in real time by fluorescence resonance energy transfer (FRET) between an acceptor dye attached to subunit F or D in V-1 or A(3)B(3)D and a donor dye attached to subunit C in V-o. The estimated dissociation constants K-d for VoV1 and A(3)B(3)D-V-o were similar to 0.3 and similar to 1 nM at 25 degrees C, respectively. These results suggest that the A(3)B(3) domain tightly associated with the two EG peripheral stalks of V-o, even in the absence of the central shaft subunits. In addition, F subunit is essential for coupling of ATP hydrolysis and proton translocation and has a key role in the stability of whole complex. However, the contribution of the F subunit to the association of A(3)B(3) with V-o is much lower than that of the EG peripheral stalks. |
| DOI | 10.1074/jbc.M112.367813 |
| ISSN | 0021-9258 |
| PMID | 22582389 |