TY - JOUR
T1 - A microplate assay for analysis of solution-phase glycosyltransferase reactions
T2 - Determination of kinetic constants
AU - Mengeling, Brenda Jo
AU - Smith, Peter L.
AU - Stults, Nancy L.
AU - Smith, David F.
AU - Baenziger, Jacques U.
N1 - Funding Information:
Supported by Grant ROl-DK41738 and the Monsanto Co./Washington University Biomedical Research Grant. We thank the National Hormone and Pituitary Program for providing hCG.
PY - 1991/12
Y1 - 1991/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026409549&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(91)90103-Z
DO - 10.1016/0003-2697(91)90103-Z
M3 - Article
C2 - 1812792
AN - SCOPUS:0026409549
VL - 199
SP - 286
EP - 292
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -