AVENACOSIDASE FROM OAT - PURIFICATION, SEQUENCE-ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF A NEW MEMBER OF THE BGA FAMILY OF BETA-GLUCOSIDASES
AVENACOSIDASE FROM OAT - PURIFICATION, SEQUENCE-ANALYSIS AND BIOCHEMICAL-CHARACTERIZATION OF A NEW MEMBER OF THE BGA FAMILY OF BETA-GLUCOSIDASES
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DOI：
10.1007/bf00028858
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发表时间：
1994-11-01
影响因子：
5.1
通讯作者：
RUDIGER, W
中科院分区：
文献类型：
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作者：
GUSMAYER, S;BRUNNER, H;RUDIGER, W
A.从燕麦（Avena sativa L.）其特征为燕麦糖苷酶，一种属于燕麦抗真菌感染的预先形成的防御系统的β-葡糖苷酶。该酶是高度聚集的;它由300-350 kDa的聚集体及其多聚体组成。通过冷冻/解冻的解离导致酶活性的完全丧失。以对硝基苯基衍生物为底物，研究了该酶的专一性，底物的顺序为：β-岩藻糖苷、β-葡萄糖苷、β-半乳糖苷、β-木糖苷。相应的邻硝基苯基糖苷不太被接受。未发现α-糖苷和β-硫代葡萄糖苷的水解。制备抗As-P60抗血清并用于分离编码As-P60的cDNA克隆。通过比较cDNA序列与通过Edman降解成熟蛋白确定的N-末端序列，推断出55个氨基酸残基的前序列。前序列具有基质导向信号肽的特征;通过蛋白质印迹证实了As-PGO在燕麦幼苗质体中的定位。氨基酸序列显示与双子叶植物中防御机制的组成部分β-葡糖苷酶具有显著的同源性（> 39%序列同一性）。甚至发现与BGA家族的哺乳动物和细菌β-葡糖苷酶具有34%的序列同一性。燕麦糖苷酶将该β-葡糖苷酶家族的存在扩展到单子叶植物。
A. protein consisting of 60 kDa subunits (As-PGO) was isolated from etiolated oat seedlings (Avena sativa L.) and characterized as avenacosidase, a beta-glucosidase that belongs to a preformed defence system of oat against fungal infection. The enzyme is highly aggregated; it consists of 300-350 kDa aggregates and multimers thereof. Dissociation by freezing/thawing leads to complete loss of enzyme activity. The specificity of the enzyme was investigated with para-nitrophenyI derivatives which serve as substrates, in decreasing order beta-fucoside, beta-glucoside, beta-galactoside, beta-xyloside. The corresponding orthonitrophenyl glycosides are less well accepted. No hydrolysis was found with alpha-glycosides and beta-thioglucoside. An anti-As-P60 antiserum was prepared and used for isolation of a cDNA clone coding for As-P60. A presequence of 55 amino acid residues was deduced from comparison of the cDNA sequence with the N-terminal sequence determined by Edman degradation of the mature protein. The presequence has the characteristics of a stroma-directing signal peptide; localization of As-PGO in plastids of oat seedlings was confirmed by western blotting. The amino acid sequence revealed significant homology (> 39% sequence identity) to beta-glucosidases that are constituents of a defence mechanism in dicotyledonous plants. 34% sequence identity was even found with mammalian and bacterial beta-glucosidases of the BGA family. Avenacosidase extends the occurrence of this family of beta-glucosidases to monocotyledonous plants.