Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex

被引:20
作者
Steele, NM [1 ]
Fry, SC [1 ]
机构
[1] Univ Edinburgh, Inst Cell & Mol Biol, Edinburgh Cell Wall Grp, Edinburgh EH9 3JH, Midlothian, Scotland
关键词
hemicellulose; oligosaccharide; plant cell wall; transglycosylation;
D O I
10.1042/0264-6021:3400207
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)(2)SO4, were incubated with tamarind xyloglucan (approximate to 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses > 500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximate to 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)(2)SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE.
引用
收藏
页码:207 / 211
页数:5
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