1999 Fiscal Year Final Research Report Summary
Structure and function of NaィイD1+ィエD1-translocating V-type ATPase
| Project/Area Number |
09480166
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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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 | Science University of Tokyo |
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Principal Investigator |
YAMATO Ichiro Science University of Tokyo, Department of Biological Science and Technology, Professor, 基礎工学部, 教授 (70111458)
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| Co-Investigator(Kenkyū-buntansha) |
KAKINUMA Yoshimi Chiba University, Faculty of Pharmaceutical Science, Associate Professor, 薬学部, 助教授 (80134394)
MEGURO Toshiyuki Science University of Tokyo, Department of Biological Science and Technology, Assistant Professor, 基礎工学部, 助手
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| Project Period (FY) |
1997 – 1999
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| Keywords | V-type ATPase / NaィイD1+ィエD1-translocating ATPase / Enterococcus hirae / purification & reconstitution / NaィイD1+ィエD1 binding / energy coupling mechanism / osmotolerance |
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| Research Abstract |
We have almost accomplished our proposed projects.
1.By characterizing gene-disrupted mutants of the NaィイD1+ィエD1-ATPase subunits, we have demonstrated that all of the nine subunits are necessary for the enzyme activity. Site-directed mutagenesis of A and K subunits revealed that Cys locating near the ATP binding site was related with the sensitivity for SH reagents of the V-type ATPase and that the primary structure around the conserved Glu residue (DCCD binding site) was essential for the ion-translocating function.
2.We characterized the purified ATPase. Subunits A to G and K plus I were the constituents. Subunits I and K were membrane-bound and others were releasable from the complex (VィイD21ィエD2VィイD20ィエD2) by EDTA treatment which belongs to the catalytic head piece (VィイD21ィエD2). The reconstituted ATPase was strictly NaィイD1+ィエD1 dependent.
3.The reconstituted ATPase pumped out NaィイD1+ィエD1electrogenically. While the membrane-bound portion (VィイD20ィエD2) functioned as a facilitated diffusion carrier transporting NaィイD1+ィエD1driven by membrane potential.
4.By fusing CAT gene under the ntp operon, we characterized the gene expression regulation. The gene expression was dependent on the NaィイD1+ィエD1concentration and also on alkaline pH.
5.Bindings of NaィイD1+ィエD1, ATP, ADP etc were characterized Membrane-bound portion had always a high binding affinity for NaィイD1+ィエD1. Addition of ATP caused the lowering of the affinity, which indicated that the energy of ATP hydrolysis works at the step to release NaィイD1+ィエD1 from the binding site.
6.We purified the head piece (VィイD21ィエD2) of this ATPase. For X-ray crystallography, we crystallized the VィイD21ィエD2 part. Now we are determining the three dimensional structure of it.
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