Misawa H, Tsumuraya Y, Kaneko Y, Hashimoto Y
Department of Biochemistry, Saitama University, Urawa, Japan.
Plant Physiol. 1996 Feb;110(2):665-73. doi: 10.1104/pp.110.2.665.
A novel alpha-L-fucosyltransferase capable of transferring L-fucose (L-Fuc) from GDP-L-Fuc to the O-2 of alpha-L-arabinofuranosyl residue (GDP-L-Fuc:alpha-L-arabinofuranoside 2-alpha-L-fucosyltransferase) has been found in the microsomal fraction of primary roots from 6-d-old radish (Raphanus sativus L.) seedlings. Enzyme activity was measured fluorometrically at 25 degrees C using a pyridylaminated trisaccharide, L-arabinofuranosylf alpha(1-->3)D-galactopyranosyl beta(1-->6)D-galactose (AraGalGal-PA) as the acceptor. This enzyme found in the microsomal fraction is maximally active at pH 6.8 and requires 0.1% (w/v) Zwittergent 3-16 and 5 mM Mn2+. Chemical and enzymatic analyses of fucosylated AraGalGal-PA confirmed the attachment of L-Fuc to the L-arabinofuranosyl (L-Araf) residue at O-2 by alpha-glycosidic linkage. Radiolabeling was used to assay L-Fuc transfer to L-Araf-containing galacto-oligomers and tamarind xyloglucan. The enzyme specific for the L-Araf residue undergoes development- and organ-specific expression in root tissue, whereas the L-Fuc transfer to tamarind xyloglucan can be detected in microsomal fractions from various organs in developing radish plants. Enzyme assays of membranes fractionated from microsomal fractions revealed that two distinct alpha-L-fucosyltransferases with different acceptor specificity are associated with Golgi membranes from primary roots, whereas hypocotyl Golgi membranes completely lack the enzyme specific for the L-Araf residue.
在6日龄萝卜（Raphanus sativus L.）幼苗初生根的微粒体部分发现了一种新型α-L-岩藻糖基转移酶，它能够将L-岩藻糖（L-Fuc）从GDP-L-Fuc转移至α-L-阿拉伯呋喃糖基残基的O-2位（GDP-L-Fuc：α-L-阿拉伯呋喃糖苷2-α-L-岩藻糖基转移酶）。使用吡啶胺化三糖L-阿拉伯呋喃糖基α(1→3)D-吡喃半乳糖基β(1→6)D-半乳糖（AraGalGal-PA）作为受体，于25℃通过荧光法测定酶活性。在微粒体部分发现的这种酶在pH 6.8时活性最高，需要0.1%（w/v）两性离子去污剂3-16和5 mM Mn2+。对岩藻糖基化的AraGalGal-PA进行化学和酶学分析，证实L-Fuc通过α-糖苷键连接到O-2位的L-阿拉伯呋喃糖基（L-Araf）残基上。使用放射性标记来测定L-Fuc向含L-Araf的半乳糖寡聚物和罗望子木葡聚糖的转移。对L-Araf残基具有特异性的这种酶在根组织中呈现发育和器官特异性表达，而在发育中的萝卜植株的各个器官的微粒体部分都能检测到L-Fuc向罗望子木葡聚糖的转移。对从微粒体部分分级分离的膜进行酶活性测定表明，两种具有不同受体特异性的不同α-L-岩藻糖基转移酶与初生根的高尔基体膜相关，而下胚轴高尔基体膜完全缺乏对L-Araf残基具有特异性的酶。
