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A simple and rapid microplate assay for glycoprotein-processing glycosidases
M S Kang1, J H Zwolshen, B S Harry
1Merrell Dow Research Institute, Cincinnati, Ohio 45215.
Analytical Biochemistry
|August 15, 1989
Summary
A new microplate assay simplifies measuring glycosidases crucial for glycoprotein processing. This method uses concanavalin A-Sepharose to separate released sugars, enabling high-throughput analysis of enzyme activity.
Area of Science:
- Biochemistry
- Enzymology
- Glycobiology
Background:
- Glycosidases play a vital role in glycoprotein processing.
- Previous assays using concanavalin A-Sepharose column chromatography are time-consuming and not suitable for high-throughput analysis.
Purpose of the Study:
- To develop a simple, convenient, and high-throughput microplate assay for glycosidases involved in glycoprotein processing.
- To adapt the concanavalin A-Sepharose binding principle to a 96-well microplate format.
Main Methods:
- Utilized radiolabeled substrates ([3H]glucose for glucosidases, [3H]mannose for mannosidases).
- Employed specific binding of high-mannose oligosaccharide substrates to concanavalin A-Sepharose.
- Separated liberated monosaccharides (which do not bind) from the substrate using a microplate format.
Main Results:
- The microplate assay demonstrated comparable results to the established column chromatography method.
- Assay linearity was confirmed with respect to time and enzyme concentration.
- Expected enzyme kinetics were observed when using castanospermine, an inhibitor of alpha-glucosidase I.
Conclusions:
- The developed microplate assay is a simplified and efficient method for quantifying glycosidase activity.
- This assay facilitates the processing of a large number of samples rapidly.
- The assay is suitable for studying enzymes involved in glycoprotein processing and can be used for kinetic studies and inhibitor screening.