Renaturation and activity staining of glycosidases and glycosyltransferases in gels after sodium dodecyl sulfate-electrophoresis.
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, alpha-glucosidase, beta-glucosidase and beta-mannosidase could not be renatured, whereas beta-fructofuranosidase and alpha-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 degrees 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.
Mukasa
H
H
Department of Chemistry, National Defense Medical College, Tokorozawa, Japan.
Tsumori
H
H
Takeda
H
H
D004591:Electrophoresis, Polyacrylamide Gel; D006026:Glycoside Hydrolases / Q000032:analysis / Q000737:chemistry*; D016695:Glycosyltransferases / Q000032:analysis / Q000737:chemistry*; D006358:Hot Temperature; D007525:Isoelectric Focusing; D008623:Mercaptoethanol; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling
D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases