| Project/Area Number |
13142203
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Biological Sciences
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| Research Institution | Nagoya University |
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Principal Investigator |
MAESHIMA Masayoshi Nagoya University, Graduate School of Bioagricultural Sciences, Professor, 大学院生命農学研究科, 教授 (80181577)
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| Co-Investigator(Kenkyū-buntansha) |
NAKANISHI Yoichi Nagoya University, Graduate School of Bioagricultural Sciences, Assistant Professor, 大学院生命農学研究科, 助手 (60362290)
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| Project Period (FY) |
2001 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥76,900,000 (Direct Cost: ¥76,900,000)
Fiscal Year 2005: ¥12,500,000 (Direct Cost: ¥12,500,000)
Fiscal Year 2004: ¥13,200,000 (Direct Cost: ¥13,200,000)
Fiscal Year 2003: ¥16,200,000 (Direct Cost: ¥16,200,000)
Fiscal Year 2002: ¥17,000,000 (Direct Cost: ¥17,000,000)
Fiscal Year 2001: ¥18,000,000 (Direct Cost: ¥18,000,000)
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| Keywords | H+-pyrophosphatase / Proton pump / Higher-order structure / Vacuolar membrane / Membrane topology / Functional mechanism / エネルギー共役 / プロトンポンプ / ポロトンポンプ / 分子構築 / パッチクランプ / 機能ドメイン / 結晶構造 / H^+-ピロホスファスターゼ / 液晶構造 / 結晶形成 / NMR解析 / リコンビナントタンパク質 / 結晶解析 |
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| Research Abstract |
During the project period for five years, we obtained the following results concerning H+-pyrophosphatase and metal transporters.
(1) H^+-pyrophosphatase (H^+-PPase) :
H^+-PPase acidifies plant vacuoles or acidocalcisomes and maintains the pH gradient across the membrane. Our experiments revealed that a knockout mutant of H^+-PPase in Arabidopsis could not grow normally, suggesting that H^+-PPase is one of the essential membrane transport systems in plants.
(2) Biochemical properties and membrane topology of H^+-PPase :
In addition to plant H^+-PPase, we purified and characterized Streptomyces coelicolor H^+-PPase, since it and its mutant enzymes can be expressed in E. col. We revealed that S. coelicolor enzyme has a characteristic properties such as heat stability. We also determined the membrane topology of H^+-PPase and essential motifs in the enzyme. Our structural model has been accepted as the standard of H^+-PPases in various organisms.
(3) Crystallization of H^+-PPase :
We prepared a large quantity of Fv fragment, which specifically reacts with the active form of H+-PPase. We succeeded in preparation of a large quantity of the Fv-H+-PPase complex using the Fv fragment from the solubilized plant vacuolar membranes. Now, we are determining the best conditions for crystallization of the enzyme.
(4) Cation/H^+ exchangers :
Rice has five isoforms of cation/H^+ exchanger (CAX). These five isoforms varied with metal ion selectivity and tissue specific gene expression. CAXIa among them is expressed in all tissues examined and is highly expressed in companion and endodermis cells, which has high concentration of Ca^<2+>. We estimate that CAXla functions to reserve Ca^<2+> into vacuoles and is involved in the Ca^<2+> detoxification. The membrane topology and several functional motifs were also determined.
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