A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds
A fast and gentle method for the isolation of myrosinase complexes from Brassicaceous seeds
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DOI：
10.1016/j.jprot.2007.11.006
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发表时间：
2008-04-24
影响因子：
--
通讯作者：
Sorensen, Hilmer
中科院分区：
文献类型：
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作者：
Bellostas, Natalia;Petersen, Iben Lykke;Sorensen, Hilmer
黑芥子酶是一种β-硫葡糖苷酶葡糖水解酶，其催化芥子油苷（存在于豆科植物中的化感物质）中的硫葡糖苷键的水解。已发现这些同工酶与其他蛋白质形成复合物;然而，涉及硫酸铵沉淀和/或离子交换色谱的传统分离方法不允许分离这些复合物。本文报道了一种快速而温和的分离复合形式黑芥子酶的方法。通过Con A亲和层析和Sephadex G-200凝胶过滤的部分纯化允许从埃塞俄比亚芥（Brassica carinata，B）种子中分离黑芥子酶复合物。甘蓝变种capitata，B. napus和Sinapis alba。油菜中的黑芥子酶形成不同分子量（500-600 kDa、270-350 kDa和140-200 kDa）的复合物，而S. alba，仅可能分离和检测140-200 kDa的复合物。在所有物种的复合物形成的同工酶与等电点之间的4.8和5.6，在某些情况下高达6.8。SIDS-PAGE证实黑芥子酶同工酶由分子量在10 ~ 110 kDa之间的几个蛋白亚基组成。黑芥子酶复合物的相对量和酶活性在所研究的物种之间变化。天然黑芥子酶复合物的分离对于研究硫代葡萄糖苷在自溶条件下的水解具有重要意义。(c)2007 Elsevier B. V.保留所有权利。
Myrosinase is a beta-thioglucosidase glucohydrolase that catalyses the hydrolysis of the thioglucoside bond in glucosinolates, allelochemicals present in Brassicaceous plants. These isoenzymes have been found to form complexes with other proteins; however, traditional isolation procedures involving ammonium sulphate precipitation and/or ion exchange chromatography do not allow for the isolation of these complexes. The present paper reports a fast and gentle procedure for the isolation of myrosinases in the complex form. Partial purification by Con A affinity chromatography followed by Sephadex G-200 gel filtration allowed for the isolation of myrosinase complexes from seeds of Brassica carinata, B. oleracea var. capitata, B. napus and Sinapis alba. Myrosinases in the Brassicas formed complexes of different molecular weight (500-600 kDa, 270-350 kDa and 140-200 kDa) whereas in seeds of S. alba it was only possible to isolate and detect 140-200 kDa complexes. In all species the complexes were formed by isoenzymes with isoelectric points between 4.8 and 5.6 and in some cases up to 6.8. SIDS-PAGE confirmed that the myrosinase isoenzymes were composed by several protein subunits of molecular weights ranging between 10 and I 10 kDa. The relative amount and enzymatic activity of the myrosinase complexes varied amongst the species studied. The isolation of myrosinase complexes in their native form is of great importance for the study of the hydrolysis of glucosinolates under autolysis conditions. (c) 2007 Elsevier B.V. All rights reserved.