title: N-acetylglucosaminyltransferase (GnT) assays using fluorescent oligosaccharide acceptor substrates: GnT-III, IV, V, and IX (GnT-Vb).
abstract: Determining glycosyltransferase activities gives a clue for better understanding an underlying mechanism for glycomic alterations of carrier molecules. N-glycan branch formation is concertedly regulated by cooperative and competitive activities of N-acetylglucosaminyltransferases (GnTs). Here, we describe methods for large scale preparation of the oligosaccharide acceptor substrate, fluorescence-labeling of oligosaccharides by pyridylamination, quality control, and reversed phase HPLC-based measurement of GnT activities including GnT-III, IV, V, and IX.
authors.affiliation: Division of Systems Glycobiology Research Group, RIKEN Global Research Cluster, Wako, Saitama, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takamatsu
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Korekane
authors.forename: Kazuaki
authors.initials: K
authors.lastname: Ohtsubo
authors.forename: Suguru
authors.initials: S
authors.lastname: Oguri
authors.forename: Jong Yi
authors.initials: JY
authors.lastname: Park
authors.forename: Akio
authors.initials: A
authors.lastname: Matsumoto
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000586:Amination; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D009844:Oligosaccharides; D017351:N-Acetylglucosaminyltransferases