title: An enzymatic method of analysis for GDP-L-fucose in biological samples, involving high-performance liquid chromatography.
abstract: To investigate the biological significance of GDP-L-fucose, we established a unique method for the determination of GDP-L-fucose levels in microsomal fractions, using an HPLC assay of alpha 1-6-fucosyltransferase (alpha1-6-FucT), an enzyme that catalyzes the synthesis of core fucosylation in N-glycans. A microsomal protein and a large excess of fluorescence-labeled synthetic oligosaccharide (a substrate) were incubated with a large excess of alpha1-6-FucT. The fluorescent intensity of the fucosylated reaction product, which was analyzed by isocratic reverse phase HPLC, was proportional to the level of GDP-L-fucose in the microsomal fractions over the range 0.20-10 pmol. This assay is applicable to the determination of the GDP-L-fucose content in various cancer cell lines as well as rat liver and would be useful in developing a better understanding of the fucosylation potential of such cells and tissues.
authors.affiliation: Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Noda
authors.forename: Eiji
authors.initials: E
authors.lastname: Miyoshi
authors.forename: Susumu
authors.initials: S
authors.lastname: Nakahara
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Ihara
authors.forename: Cong Xiao
authors.initials: CX
authors.lastname: Gao
authors.forename: Koichi
authors.initials: K
authors.lastname: Honke
authors.forename: Shusaku
authors.initials: S
authors.lastname: Yanagidani
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Akinori
authors.initials: A
authors.lastname: Kasahara
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Hori
authors.forename: Norio
authors.initials: N
authors.lastname: Hayashi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Taniguchi
mesh_terms: D000631:Aminopyridines / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003110:Colonic Neoplasms / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005647:Fucosyltransferases / Q000378:metabolism; D006154:Guanosine Diphosphate Fucose / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014407:Tumor Cells, Cultured
chemical_list: D000631:Aminopyridines; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases