2006 Fiscal Year Final Research Report Summary
Biosynthesis of pectic β-(1→4)-galactan occurring in cell walls of higher plants
| Project/Area Number |
16570027
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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 |
植物生理・分子
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| Research Institution | Saitama University |
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Principal Investigator |
TSUMURAYA Yoichi Saitama University, Graduate School of Science and Engineering, Professor, 理学部, 教授 (10142233)
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| Project Period (FY) |
2004 – 2006
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| Keywords | cell wall / pectin / galactan / galactosyltransferase / UDP-galactose |
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| Research Abstract |
Pectins are plant cell wall polysaccharides consisting mainly of(1→4)-linked α-galacturonic acid (GalA) residues attached with β-(1→4)-galactan and/or α-(1→5)-arabinan side chains. This study is focused to the properties of the β-(1→4)-galactosyltransferase(GalT) involved in the synthesis of pectic β-(1→4)-galactans.
1.Preparation of GalT enzyme and its activity assay : A membrane fraction prepared from seedlings of soybean (Glycine max Merr.) contained GalT capable of transferring [^<14>C] Gal from UDP-[^<14>C]Gal into exogenous acceptors. Enzyme activity was measured by a liquid scintillation counter.
2.Properties of GalT : The enzyme was maximally active at pH 6.5 and 20-25℃ in the presence of 25 mM Mn^<2+> and 0.75% Triton X-100. A high-M_r(average M_r 60,000)β-(1→4)-galactan served as the most effective acceptor substrate.
3.Solubilization and partial purification of GalT : The GalT protein was solubilized by mixing the microsomal fraction with 0.75% Triton X-100. The solubilized enzyme was partially purified by ion-exchange chromatography and affinity chromatography.
4.Enzymatic elongation of β-(1→4)-galactosyl chains: The partially purified GalT transferred more than 25 Gal residues from UDP-Gal onto β-(1→4)-galactohaptaose acceptor, leading to the formation of long β-(1→4)-galactosyl chains comparable to the length(45 Gal units) of native β-(1→4)-galactans found in soybean pectins.
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