A microplate assay for analysis of solution-phase glycosyltransferase reactions: determination of kinetic constants.
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 micrograms 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.
Mengeling
B J
BJ
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Smith
P L
PL
Stults
N L
NL
Smith
D F
DF
Baenziger
J U
JU
D000331:Aequorin / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D008163:Luminescent Measurements; D017350:N-Acetylgalactosaminyltransferases; D037121:Plant Lectins; D011233:Precipitin Tests; D034781:Receptors, N-Acetylglucosamine; D012680:Sensitivity and Specificity; D019809:Streptavidin
D001426:Bacterial Proteins; D006063:Chorionic Gonadotropin; D037102:Lectins; D037121:Plant Lectins; D034781:Receptors, N-Acetylglucosamine; C040129:wisteria lectin; D000331:Aequorin; D001710:Biotin; D019809:Streptavidin; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C072136:glycoprotein hormone-specific N-acetylgalactosamine transferase; D002118:Calcium