A simple and reliable assay for glycoprotein-processing glycosidases.
A simple and reproducible assay to measure the activity of the glycoprotein-processing glycosidases, i.e., glucosidases and mannosidases, is described. This assay takes advantage of the fact that high-mannose and glucose-containing high-mannose oligosaccharides bind to columns of concanavalin A-Sepharose, but the liberated glucose and mannose residues emerge from these columns in the wash. Thus, using [3H]mannose-labeled Man9-N-acetylglucosamine (Man9GlcNAc) or [3H]glucose-labeled Glc3Man7-9-GlcNAc as substrates, the amount of radioactivity in the wash can be quickly and efficiently determined as a measure of enzyme activity. Although the use of this assay was reported previously [B. Saunier et al., 1982, J. Biol. Chem. 257, 14155-14161], the details of its use, its reproducibility, and the problems with interfering materials have not been thoroughly described. In this report, we show that the assay is linear with time and protein concentration, and shows the expected kinetics with various processing inhibitors. The assay works well with the microsomal enzyme preparation and with a solubilized enzyme fraction. In addition, methods are described for the preparation of various radioactive oligosaccharide substrates (i.e., Man9GlcNAc and Glc3Man7-9GlcNAc) using appropriate glycoprotein-processing inhibitors.
D000818:Animals; D002846:Chromatography, Affinity; D005959:Glucosidases / Q000032:analysis*; D008099:Liver / Q000201:enzymology; D008361:Mannosidases / Q000032:analysis*; D009844:Oligosaccharides; D051381:Rats; D012685:Sepharose / Q000031:analogs & derivatives
D009844:Oligosaccharides; C028326:concanavalin A-sepharose; D012685:Sepharose; D005959:Glucosidases; D008361:Mannosidases