Structure, properties, and tissue localization of apoplastic α-glucosidase in crucifers
Structure, properties, and tissue localization of apoplastic α-glucosidase in crucifers
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DOI：
10.1104/pp.119.2.385
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发表时间：
1999-02-01
期刊：
影响因子：
7.4
通讯作者：
Wright, PW
中科院分区：
文献类型：
--
作者：
Monroe, JD;Gough, CM;Wright, PW
质外体α-葡萄糖苷酶广泛存在于植物中，但它们的功能尚不清楚，因为质外体中合适的底物尚未被鉴定。拟南芥含有至少三个α-葡糖苷酶基因; Aglu-1和Aglu-3被测序，并且从六个表达的序列标签已知Aglu-2。针对在大肠杆菌中表达的Aglu-1的一部分产生的抗体分别识别拟南芥、花椰菜（Brassica oleracea L.）和芥菜（Brassica napus L.）。用刀豆球蛋白A和离子交换层析法从青花菜花蕾中纯化酸性α-葡萄糖苷酶活性。两个活性组分被解决，都含有96 kD的免疫反应性多肽。来自96-kD青花菜α-葡糖苷酶的N-末端序列表明其对应于拟南芥Aglu-2基因，并且预测的N末端的约15 kD被切割。81-kD蛋白比96-kD蛋白更丰富，但它对作为底物的4-甲基伞形酮基-α-D-吡喃葡萄糖苷没有活性，并且它不与伴刀豆球蛋白A结合。使用5-溴-4-氯-3-吲哚基-α-D-吡喃葡萄糖苷的原位活性染色显示，酸性α-葡萄糖苷酶活性主要位于西兰花茎的外皮层和维管组织中，特别是在叶迹中。
Apoplastic alpha-glucosidases occur widely in plants but their function is unknown because appropriate substrates in the apoplast have not been identified. Arabidopsis contains at least three alpha-glucosidase genes; Aglu-1 and Aglu-3 are sequenced and Aglu-2 is known from six expressed sequence tags. Antibodies raised to a portion of Aglu-1 expressed in Escherichia coli recognize two proteins of 96 and 81 kD, respectively, in vegetative tissues of Arabidopsis, broccoli (Brassica oleracea L.), and mustard (Brassica napus L.). The acidic alpha-glucosidase activity from broccoli flower buds was purified using concanavalin A and ion-exchange chromatography. Two active fractions were resolved and both contained a 96-kD immunoreactive polypeptide. The N-terminal sequence from the 96-kD broccoli alpha-glucosidase indicated that it corresponds to the Arabidopsis Aglu-2 gene and that approximately 15 kD of the predicted N terminus was cleaved. The 81-kD protein was more abundant than the 96-kD protein, but it was not active with 4-methylumbelliferyl-alpha-D-glucopyranoside as the substrate and it did not bind to concanavalin A. In situ activity staining using 5-bromo-4-chloro-3-indolyl-alpha-D-glucopyranoside revealed that the acidic alpha-glucosidase activity is predominantly located in the outer cortex of broccoli stems and in vascular tissue, especially in leaf traces.