A microplate assay for analysis of solution-phase glycosyltransferase reactions: Determination of kinetic constants

Brenda Jo Mengeling, Peter L. Smith, Nancy L. Stults, David F. Smith, Jacques U. Baenziger

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

We have developed a sensitive and simple method for assaying glycosyltransferase activities. This method makes use of solution-phase transferase reactions followed by capture to a microplate well coated with a substrate-specific monoclonal antibody. Sugar incorporation is quantitated by binding a saccharide-specific lectin and using bioluminescent aequorin for a reporter molecule. We demonstrate this method using the glycoprotein hormone-specific GalNAc-transferase and its acceptor substrate, agalacto-hCG. As little as 20 ng of agalacto-hCG with 32 nU of GalNAc-transferase gives a detectable signal with less than 10% of the acceptor sites substituted. In addition to this high sensitivity, by doing the transferase reactions in solution, we can assay up to 10 μg of agalacto-hCG. We show that this allows the determination of Km and Vmax kinetic constants that compare well to those obtained with radiolabeled nucleotide sugars.

Original languageEnglish
Pages (from-to)286-292
Number of pages7
JournalAnalytical Biochemistry
Volume199
Issue number2
DOIs
StatePublished - Dec 1991

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