Hata K, Tanaka M, Tsumuraya Y, Hashimoto Y
Department of Biochemistry, Faculty of Science, Saitama University, 255 Shimo-okubo, Urawa 338, Japan.
Plant Physiol. 1992 Sep;100(1):388-96. doi: 10.1104/pp.100.1.388.
An alpha-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl alpha-l-arabinofuranoside and p-nitrophenyl beta-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding alpha-l-arabinopyranoside and beta-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg(2+), Ag(+), l-arabino-(1-->4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off alpha-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of alpha-l-arabinofuranosidase and beta-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.
已从萝卜（Raphanus sativus L.）种子中纯化出一种α-L-阿拉伯呋喃糖苷酶，纯化倍数达1043倍。变性凝胶电泳、反相或尺寸排阻高效液相色谱以及等电聚焦结果表明，纯化后的酶是一种均一的糖蛋白，由一条单链多肽组成，表观分子量为64,000，等电点为4.7。该酶的特征在于，在pH 4.5条件下，它能以恒定比例（3:1）催化对硝基苯基α-L-阿拉伯呋喃糖苷和对硝基苯基β-D-木吡喃糖苷的水解反应，而相应的α-L-阿拉伯吡喃糖苷和β-D-木呋喃糖苷则不受影响。以下证据支持具有一个催化位点的单一酶负责这种特异性：（a）酶制剂的高纯度；（b）在整个纯化步骤中，对两种底物的活性比例不变；（c）在与牛血清白蛋白激活、酶的热失活以及用重金属离子和糖类（如Hg(2+)、Ag(+)、L-阿拉伯糖-(1→4)-内酯和D-木糖）抑制时，活性具有平行性；（d）使用两种底物进行混合底物动力学分析的结果。该酶能从甜菜阿拉伯聚糖、大豆阿拉伯聚糖-4-半乳聚糖以及萝卜种子和叶片阿拉伯半乳聚糖蛋白中裂解出α-L-阿拉伯呋喃糖基残基。通过该酶对燕麦-斯佩尔特木聚糖的作用可释放出阿拉伯糖和木糖。α-L-阿拉伯呋喃糖苷酶和β-D-半乳糖苷酶对萝卜种子阿拉伯半乳聚糖蛋白的协同作用导致碳水化合物部分的广泛降解。
