Glucose, fructose, mannose and/or glucose-1-phosphate-releasing activity stains for glycosidases and glycosyltransferases in gels after isoelectric focusing.
beta-Fructofuranosidase, alpha-glucosidase, beta-glucosidase, alpha-mannosidase, beta-mannosidase, sucrose phosphorylase, glucosyltransferase and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thin-layer gels, supported by a glass plate, by release of monosugars or a sugar phosphate, followed by conversion to glucose-6-phosphate (G6P) and then by reduction of NADP+ to NADPH, terminated by the formation of reduced Nitroblue Tetrazolium (NBT). Approximately 1-10 mU of enzyme was focused and the gel, after washing with a buffer, was partially dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as substrates, intermediary enzymes such as hexokinase, mutase and/or isomerase, NADP+, ATP, Mg+, phenazine methosulfate (PMS) and NBT. Specific staining procedures for each of these activities, on sucrose or on the glycosides as substrates, and staining procedures for multiple activities are described, with the conditions necessary for optimal development.
Mukasa
H
H
Department of Chemistry, National Defense Medical College, Saitama, Japan.
Tsumori
H
H
Uezono
Y
Y
D005632:Fructose / Q000737:chemistry; D005782:Gels; D005947:Glucose / Q000737:chemistry; D005958:Glucosephosphates / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000032:analysis*; D006027:Glycosides / Q000032:analysis; D016695:Glycosyltransferases / Q000032:analysis*; D006601:Hexoses / Q000737:chemistry*; D007525:Isoelectric Focusing / Q000379:methods*; D008358:Mannose / Q000737:chemistry; D013194:Staining and Labeling; D013395:Sucrose / Q000032:analysis
D005782:Gels; D005958:Glucosephosphates; D006027:Glycosides; D006601:Hexoses; D005632:Fructose; D013395:Sucrose; C031590:glucose-1-phosphate; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D005947:Glucose; D008358:Mannose