A rapid method for assay of glycosidases involved in glycoprotein biosynthesis.
A rapid procedure to measure processing glycosidases with labeled oligosaccharide as substrate is described, using assay of the specific processing alpha-mannosidase from Saccharomyces cerevisiae as an example. After incubation of [3H]mannose-labeled Man9GlcNAc with the mannosidase, a solution of concanavalin A is added, followed by polyethylene glycol to precipitate the oligosaccharide-lectin complex. The radioactivity present in the supernatant after centrifugation is then measured to determine the amount of labeled mannose released. It is shown that the results of this procedure are similar to those obtained previously using small columns of concanavalin A-Sepharose (B. Saunier, R. D. Kilker, Jr., J. S. Tkacz, A. Quaroni, and A. Herscovics (1982) J. Biol. Chem. 257, 14155-14161). The precipitation procedure, which can be applied to the assays of other processing enzymes, is much more convenient when a large number of samples must be analyzed.
Herscovics
A
A
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Jelinek-Kelly
S
S
D003208:Concanavalin A; D005959:Glucosidases / Q000032:analysis*; D006023:Glycoproteins / Q000096:biosynthesis*; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000032:analysis; D011092:Polyethylene Glycols; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D012685:Sepharose; D013997:Time Factors; D043323:alpha-Mannosidase
D006023:Glycoproteins; D003208:Concanavalin A; D011092:Polyethylene Glycols; D012685:Sepharose; D005959:Glucosidases; D008361:Mannosidases; D043323:alpha-Mannosidase; D008358:Mannose