The mechanism of biosynthesis of plant cell walls

植物细胞壁生物合成机制

• 批准号: 08640818
• 负责人: TSUMURAYA Yoichi
• 金额: $ 0.77万
• 依托单位: Saitama University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08640818/
• 关键词: cell walls; arabinogalactan-protein; L-fucose; alpha-L-Fucosyltransferase; GDP-L-fucose; Golgi body; アラビノガラクタン-プロテイン

This study is aimed to investigate the mechanism of the biosynthesis of arabinogalactan-proteins (AGPs) involved in cell walls of higher plants, and to evaluate their physiological roles concerning to tissue differentiation in relation to the organ specific expression of their sugar chains. To approach this purpose, alpha-L-fucosyltransferse (alpha-L-FucTase), an enzyme catalyzing the transfer of L-fucose (L-Fuc) residues, is focused based on its specific assay for enzyme activity and enzymatic properties, in addition to the localization of the enzyme in sub cellular organs.1.The reaction mixture was constructed by GDP-L-Fuc as the sugar donor and and Araalpha1*3Galbeta1*6Gal derivatized with 2-aminopyridine (AGG-Pa) as the acceptor substrate together with the microsome (crude membrane) fraction as the enzyme source obtained from 6-d-old radish primary roots. After incubation at 25C,the enzyme activity was assayd by estimation of the transferred product separated by HPLC.The enzyme assay EAS also done by using GDP-L- [^<14>C] -Fuc in order to estimate the tranfer action into high molecular weight compounds.2.The enzyme properties : The enzyme is maximally active at pH6.0-7.5 and at 25oC,and requires Zwittergent and Mn^<21> or Mg^2. The km values are estimated to be 0.08 and 3.72mM for GDP-L-Fuc and AGG-PA,respectively. Chemical and enzymatic analyzes of fucosylated AGG-PA confirmed the attachment of L-Fuc to the arabinosyl residue at 0-2 by alpha-glycosidic linkage.

本研究旨在探讨高等植物细胞壁中阿拉伯半乳聚糖蛋白（AGPs）的生物合成机制，并通过其糖链的器官特异性表达来评价其在组织分化中的生理作用。为了达到这一目的，α-L-岩藻糖基转移酶α-L-岩藻糖转移酶（alpha-L-FucTase）是一种催化L-岩藻糖（L-Fuc）残基转移的酶，基于其对酶活性和酶性质的特异性测定而受到关注，1.以GDP-L-Fuc为糖供体，用2-氨基-3-半乳糖基化的Araalpha 1 * 3Galbeta 1 * 6 Gal为底物，氨基吡啶（AGG-Pa）作为受体底物，与微粒体（粗膜）组分一起作为从6-d龄萝卜初生根获得的酶源。在25 ℃下孵育后，通过HPLC分离的转移产物的估计来测定酶活性。酶测定EAS也通过使用GDP-L- [^<14>C] -Fuc来进行，以估计向高分子量化合物的转移作用。2.酶性质：该酶在pH6.0-7.5和25 ℃下具有最大活性，并且需要Zwittergent和Mn^2<21>或Mg ^2。GDP-L-Fuc和AGG-PA的Km值估计分别为0.08和3.72mM。岩藻糖基化AGG-PA的化学和酶分析证实了L-Fuc通过α-糖苷键连接到0-2位的阿拉伯糖残基上。

项目成果

H.Misawa: "α-L-Fulosyltransferases from radish primary roots" Plant Physiology. 110. 665-673 (1996)

H. Misawa：“来自萝卜初生根的 α-L-氟糖基转移酶”《植物生理学》110. 665-673 (1996)。

H.Misawa: "alpha-L-fucosyltransferases from radish primary roots" Plant physiology. 110. 665-673 (1996)

H.Misawa：“来自萝卜初生根的α-L-岩藻糖基转移酶”植物生理学。

H.Misawa: "α-L-Fucosyltransferases from radish primary roots" Plant physiol.110. 665-673 (1996)

H.Misawa：“来自萝卜初生根的 α-L-岩藻糖基转移酶”植物生理学 110（1996）。

H-Misawa: "α-L-Fuiosyltransferases from radish Primary roots" Plant Physiology. 110. 665-673 (1996)

H-Misawa：“来自萝卜初生根的 α-L-Fuiosyltransferases”植物生理学 110. 665-673 (1996)。