A simple and rapid microplate assay for glycoprotein-processing glycosidases.
A simple and convenient microplate assay for glycosidases involved in the glycoprotein-processing reactions is described. The assay is based on specific binding of high-mannose-type oligosaccharide substrates to concanavalin A-Sepharose, while monosaccharides liberated by enzymatic hydrolysis do not bind to concanavalin A-Sepharose. By the use of radiolabeled substrates [( 3H]glucose for glucosidases and [3H]mannose for mannosidases), the radioactivity in the liberated monosaccharides can be determined as a measure of the enzymatic activity. This principle was employed earlier for developing assays for glycosidases previously reported (B. Saunier et al. (1982) J. Biol. Chem. 257, 14155-14161; T. Szumilo and A. D. Elbein (1985) Anal. Biochem. 151, 32-40). These authors have reported the separation of substrate from the product by concanavalin A-Sepharose column chromatography. This procedure is handicapped by the fact that it cannot be used for a large number of samples and is time consuming. We have simplified this procedure and adapted it to the use of a microplate (96-well plate). This would help in processing a large number of samples in a short time. In this report we show that the assay is comparable to the column assay previously reported. It is linear with time and enzyme concentration and shows expected kinetics with castanospermine, a known inhibitor of alpha-glucosidase I.
Kang
M S
MS
Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Zwolshen
J H
JH
Harry
B S
BS
Sunkara
P S
PS
D000818:Animals; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000032:analysis*; D008832:Microchemistry / Q000379:methods; D012685:Sepharose / Q000031:analogs & derivatives; D013552:Swine; D014316:Tritium; D000520:alpha-Glucosidases / Q000032:analysis
D006023:Glycoproteins; C028326:concanavalin A-sepharose; D014316:Tritium; D012685:Sepharose; D006026:Glycoside Hydrolases; D000520:alpha-Glucosidases; D005947:Glucose