title: A general method for assaying homo- and hetero-transglycanase activities that act on plant cell-wall polysaccharides.
abstract: Transglycanases (endotransglycosylases) cleave a polysaccharide (donor-substrate) in mid-chain, and then transfer a portion onto another poly- or oligosaccharide (acceptor-substrate). Such enzymes contribute to plant cell-wall assembly and/or re-structuring. We sought a general method for revealing novel homo- and hetero-transglycanases, applicable to diverse polysaccharides and oligosaccharides, separating transglycanase-generated (3)H-polysaccharides from unreacted (3)H-oligosaccharides--the former immobilized (on filter-paper, silica-gel or glass-fiber), the latter eluted. On filter-paper, certain polysaccharides [e.g. (1→3, 1→4)-β-D-glucans] remained satisfactorily adsorbed when water-washed; others (e.g. pectins) were partially lost. Many oligosaccharides (e.g. arabinan-, galactan-, xyloglucan-based) were successfully eluted in appropriate solvents, but others (e.g. [(3)H]xylohexaitol, [(3)H]mannohexaitol [(3)H]cellohexaitol) remained immobile. On silica-gel, all (3)H-oligosaccharides left an immobile 'ghost' spot (contaminating any (3)H-polysaccharides), which was diminished but not prevented by additives e.g. sucrose or Triton X-100. The best stratum was glass-fiber (GF), onto which the reaction-mixture was dried then washed in 75% ethanol. Washing led to minimal loss or lateral migration of (3)H-polysaccharides if conducted by slow percolation of acidified ethanol. The effectiveness of GF-blotting was well demonstrated for Chara vulgaris trans-β-mannanase. In conclusion, our novel GF-blotting technique efficiently frees transglycanase-generated (3)H-polysaccharides from unreacted (3)H-oligosaccharides, enabling high-throughput screening of multiple postulated transglycanase activities utilising chemically diverse donor- and acceptor-substrates.
authors: The Edinburgh Cell Wall Group, Institute of Molecular Plant Sciences, The University of Edinburgh, Daniel Rutherford Building, The King's Buildings, Max Born Crescent, Edinburgh EH9 3BF, UK.
authors: Lenka
authors: L
authors: Franková
authors: Stephen C
authors: SC
authors: Fry
mesh_terms: D002473:Cell Wall / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D057075:Enzyme Assays / Q000379:methods*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D010209:Paper; D059828:Plant Cells / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D013997:Time Factors; D014316:Tritium / Q000378:metabolism; D044902:beta-Mannosidase / Q000378:metabolism
chemical_list: D011134:Polysaccharides; D014316:Tritium; D006026:Glycoside Hydrolases; C020668:glycanase; D044902:beta-Mannosidase
keywords: Glass-fiber blotting; plant cell wall; polysaccharides (donor); radiolabeled oligosaccharides (acceptor); transglycanase