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Renaturation and activity staining of glycosidases and glycosyltransferases in gels after sodium dodecyl sulfate‐electrophoresis
Author(s) -
Mukasa Hidehiko,
Tsumori Hideaki,
Takeda Hiroyuki
Publication year - 1994
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501501131
Subject(s) - isoelectric focusing , chemistry , sodium dodecyl sulfate , gel electrophoresis , biochemistry , glucosyltransferase , invertase , enzyme , chromatography , glycosyltransferase , silver stain , staining , polyacrylamide gel electrophoresis , electrophoresis , microbiology and biotechnology , biology , genetics
Glycosidases and glycosyltransferases were electrophoresed in the presence of sodium dodecyl sulfate (SDS) in a thin‐layer gel supported by a glass plate, treated with the nonionic detergent Triton X‐100, and specifically stained for the sugar‐releasing activity of these enzymes. Staining is based on conversion of monosugars or a sugar phosphate to glucose‐6‐phosphate by the appropriate intermediary enzymes, reduction of NADP + to NADPH, and accumulation of reduced Nitroblue Tetrazolium in the gel. Among the enzymes tested, α‐glucosidase, β‐glucosidase and β‐mannosidase could not be renatured, whereas β‐fructofuranosidase and α‐mannosidase could be renatured unless heated before electrophoresis. Sucrose phosphorylase, glucosyltransferase and fructosyltransferase, which are single‐peptide proteins with no cystine bond, could be renatured even after pretreatment with SDS and/or mercaptoethanol at 100°C for 10 min. However, exclusive heating remarkably decreased the activities of these enzymes. Two‐dimensional separation of the five renaturable enzymes was done in a single thin‐layer gel, using SDS‐electrophoresis in the first dimension and isoelectric focusing in the second dimension.