Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex.
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)2SO4, were incubated with tamarind xyloglucan (approximately 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 approximately 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)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE.
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The Edinburgh Cell Wall Group, Institute of Cell and Molecular Biology, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JH, UK.
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D000645:Ammonium Sulfate / Q000378:metabolism; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D002457:Cell Extracts; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005936:Glucans; D006031:Glycosylation; D016695:Glycosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011134:Polysaccharides / Q000378:metabolism; D013379:Substrate Specificity; D014990:Xylans
D002457:Cell Extracts; D005936:Glucans; D007527:Isoenzymes; D009844:Oligosaccharides; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D016695:Glycosyltransferases; C073693:xyloglucan endotransglycosylase; D000645:Ammonium Sulfate