The mechanism of biosynthesis of plant cell walls

本研究旨在探讨高等植物细胞壁中阿拉伯半乳糖蛋白（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- focusyltransferse （α -L- fuctase）是一种催化L- focus （L- fuc）残基转移的酶，除了在亚细胞器官中的定位外，还基于其对酶活性和酶性质的特定测定进行了关注。以GDP-L-Fuc为糖供体，以2-氨基吡啶（AGG-Pa）衍生化的Araalpha1*3Galbeta1*6Gal为受体底物，以6 d老萝卜原根的微粒体（粗膜）部分为酶源构建反应混合物。25℃孵育后，用高效液相色谱法测定转移产物的酶活性。酶分析EAS也用GDP-L- [^<14>C] - fuc进行，以估计转移到高分子量化合物的作用。酶的性质：该酶在pH6.0-7.5和25℃时具有最大活性，需要Zwittergent和Mn^<21>或Mg^2。GDP-L-Fuc和AGG-PA的km值分别为0.08和3.72mM。聚焦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位置的阿拉伯糖基残基上。

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.