An enzymatic method of analysis for GDP-L-fucose in biological samples, involving high-performance liquid chromatography.
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.
Noda
Katsuhisa
K
Department of Biochemistry, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan.
Miyoshi
Eiji
E
Nakahara
Susumu
S
Ihara
Hideyuki
H
Gao
Cong Xiao
CX
Honke
Koichi
K
Yanagidani
Shusaku
S
Sasaki
Yutaka
Y
Kasahara
Akinori
A
Hori
Masatsugu
M
Hayashi
Norio
N
Taniguchi
Naoyuki
N
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
D000631:Aminopyridines; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D006154:Guanosine Diphosphate Fucose; D005647:Fucosyltransferases