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-ma nnosidase, beta-mannosidase, sucrose phosphorylase, glucosyltransferas e and fructosyltransferase were separated by isoelectric focusing and sensitively detected to be slightly diffuse and insoluble spots in thi n-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 formatio n of reduced Nitroblue Tetrazolium (NBT). Approximately 1-10 mU of enz yme was focused and the gel, after washing with a buffer, was partiall y dried and directly stained by uniformly spreading on the gel surface a staining medium containing sucrose or nitrophenyl glycosides as sub strates, intermediary enzymes such as hexokinase, mutase and/or isomer ase, NADP(+), ATP, Mg+, phenazine methosulfate (PMS) and NBT, Specific staining procedures for each of these activities, on sucrose or on th e glycosides as substrates, and staining procedures for multiple activ ities are described, with the conditions necessary for optimal develop ment.