Structure and molecular interaction of ion-translocating subunits of Na+-coupled V-ATPase
| Project/Area Number |
15570108
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Ehime University |
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Principal Investigator |
KAKINUMA Yoshimi Ehime University, Faculty of Agriculture, Professor, 農学部, 教授 (80134394)
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| Project Period (FY) |
2003 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2004: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2003: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | V-ATPase / Sodium / Ion / Enterococcus hirae |
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| Research Abstract |
(i)We found that the H626 and E634 residues in the vicinity of the membrane surface of the seventh transmembrane helix of NtpI subunit are highly conserved among those of various V-ATPases. The V-ATPases activity totally disappeared in the H626R or E634Q mutant, suggesting the functional importance of these residue in NtpI.
(ii)The acidic residue E139 (kE139) which exists in the center of the fourth transmembrane helix of NtpK subunit is essential for ion binding. In purfied kE139D mutant enzyme, the affinity for Na+ of the ATP hydrolytic activity decreased about 1/4 of that of the wild-type enzyme. The kV138L mutation was isolated as the partial suppressor mutation for kE139D mutation. Site-directed mutagenesis at kV138 residue revealed that the kV138M/kE139D double mutant fully recovered the ATPase activity like that of the wild-type enzyme. These results suggest that spatial arrangement of the carboxyl group of kE139 is strictly required for Na+ binding in E.hirae V-ATPase.
(iii)We found that the nucleotide binding site exists in NtpB subunit with the photoaffinity label experiment of ATP.
(iv)We succeeded in purification NtpK subunit ring and found that the ion-binding rotary rotor consists of NtpK heptamer by biochemical analysis and electron microscope image analysis.
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Report
(3 results)
Research Products
(3 results)
