2005 Fiscal Year Final Research Report Summary
Functional analysis of plant magnesium transport proteins
| Project/Area Number |
15580096
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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 |
Bioproduction chemistry/Bioorganic chemistry
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| Research Institution | Kyoto Prefectural University |
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Principal Investigator |
ISHIJIMA Sumio Kyoto University, Research Division of Agriculture, Associate Professor, 農学研究科, 助教授 (70184520)
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| Project Period (FY) |
2003 – 2005
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| Keywords | Magnesium / Transport proteins / Chloroplast / Arabidopsis thaliana / Liposome |
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| Research Abstract |
1.Analysis of changing mechanisms of free Mg^<2+> concentration ([Mg^<2+>]) in plant chloroplasts.
We measured internal chloroplast [Mg^<2+>] and characterized Mg^<2+> transport systems in chloroplast membranes. We indicated that stromal alkalinization can induce an increase in stromal [Mg^<2+>] without illumination. Some inhibitors of envelope proton-translocating ATPase activity involved in H^+ efflux inhibited the alkalinization -induced increase in [Mg^<2+>].
2.Measurement of Mg^<2+> transport activity across membrane.
We have measured Mg^<2+> transport activity following the reconstitution of the Mg^<2+> transport protein into proteoliposomes. Since radioactive ^<28>Mg is difficult to use, Mg^<2+> transport activity across phospholipid membrane is measured using a fluorescent indicator, mag-fura-2. Liposomes were constituted in the presence of mag-fura-2. More than 30% of mag-fura-2 was retained into liposome. This makes measurement of Mg^<2+> transport activity possible using mag-fura-2.
3.Recombinant plant Mg^<2+> transport proteins
We have expressed an Arabidopsis thaliana protein, AtMRS2-10. The Arabidopsis thaliana MRS2 gene family belongs to a subset of the CorA super-family of Mg^<2+> transport proteins. We have expressed AtMRS2-10 with an additional 6 x His tag in Escherichia coli cells. The recombinant protein was solubilized from the E.coli membrane fraction by 0.3% sarkosyl and then purified. This makes molecular analysis of Mg^<2+> transport proteins possible by reconstitution of the purified proteins into liposomes and characterization.
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