title: Universal phosphatase-coupled glycosyltransferase assay.
abstract: A nonradioactive glycosyltransferase assay is described here. This method takes advantage of specific phosphatases that can be added into glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving groups of glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multiwell plates and quantitated by a plate reader, thus making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. As examples, we first assayed Clostridium difficile toxin B, a protein O-glucosyltransferase that specifically monoglucosylates and inactivates Rho family small GTPases; we then showed that human KTELC1, a homolog of Rumi from Drosophila, was able to hydrolyze UDP-Glc; and finally, we measured the kinetic parameters of human sialyltransferase ST6GAL1.
authors.affiliation: R&D Systems Inc., Minneapolis, MN 55413, USA. leon.wu@rndsystems.com
authors.forename: Zhengliang L
authors.initials: ZL
authors.lastname: Wu
authors.forename: Cheryl M
authors.initials: CM
authors.lastname: Ethen
authors.forename: Brittany
authors.initials: B
authors.lastname: Prather
authors.forename: Miranda
authors.initials: M
authors.lastname: Machacek
authors.forename: Weiping
authors.initials: W
authors.lastname: Jiang
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D001427:Bacterial Toxins / Q000032:analysis / Q000378:metabolism; D003124:Colorimetry; D057075:Enzyme Assays / Q000379:methods*; D005964:Glucosyltransferases; D016695:Glycosyltransferases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010710:Phosphates / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D012394:Rosaniline Dyes / Q000737:chemistry; D012799:Sialyltransferases / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D010710:Phosphates; D011506:Proteins; D012394:Rosaniline Dyes; C031815:toxB protein, Clostridium difficile; C005095:malachite green; D016695:Glycosyltransferases; D005964:Glucosyltransferases; C510651:POGLUT1 protein, human; D012799:Sialyltransferases; D010744:Phosphoric Monoester Hydrolases