Novel ‘dot-blot’ assays for glycosyltransferases and glycosylhydrolases: optimization for xyloglucan endotransglycosylase (XET) activity

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Abstract

A novel dot-blot method is described for the rapid, semi-quantitative assay of xyloglucan endotransglycosylase (XET) activity. Test paper was prepared by impregnating Whatman No. 1 filter paper with xyloglucan (2.5 g m−2 paper) plus a sulphorhodamine conjugate of the xyloglucan oligosaccharide, XLLG (1 µmol m−2). Spots of putative enzyme solution were applied to the test paper, which was incubated under humid conditions. XET-catalysed transglycosylation produced a xyloglucan-sulphorhodamine conjugate. The test paper was washed to remove unreacted oligosaccharide, leaving fluorescent spots of the xyloglucan-sulphorhodamine reaction product hydrogen-bonded to the paper. The assay is suitable for testing crude plant extracts (including Arabidopsis stems squashed directly on to the test paper). The test paper is also suitable for tissue-printing of plant specimens and for preparation of zymograms after non-denaturing gel electrophoresis. Tissue prints indicated that extractable XET activity tends to be concentrated in the outer tissues and sometimes in or near the vascular bundles. The method reported can be quantified by fluorimetry and can potentially be extended to the assay of other transglycosylases (including polysaccharide synthases, fructosyl-transferase and dextransucrase) and hydrolases.

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