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

ABSTRACT: 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.

SUBMITTER: Steele NM

PROVIDER: S-EPMC1220239 | biostudies-other | 1999 May

REPOSITORIES: biostudies-other

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