A fluorescent assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase activity, involving high performance liquid chromatography.
An assay method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1-6fucosyltransferase (alpha 1-6FucT; EC 2.4.1.68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine gamma-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. [equation: see text] The substrate and guanosine diphospho-fucopyranoside (GDP-Fuc) were incubated with a crude enzyme extract for 2 h, and then the enzymatic product was separated by reversed phase HPLC. Quantitation of the product involved measurement of the fluorescence intensity of the fucosylated pyridylaminated sugar. The structures of both synthesized GnGn-bi-Asn-PABA (substrate), and synthesized GnGnF-bi-Asn-PABA (product) were analyzed by 1H NMR. The enzymatic product was also analyzed by 1H NMR and was found to have alpha 1-6fucose at the reducing end GlcNAc. This method is highly specific for alpha 1-6FucT and is applicable for various experiments, including purification and cell culture ones.
Department of Biochemistry, Osaka University Medical School.
N
N
Uozumi
T
T
Teshima
T
T
Yamamoto
A
A
Nishikawa
Y E
YE
Gao
E
E
Miyoshi
C X
CX
Gao
K
K
Noda
K N
KN
Islam
Y
Y
Ihara
S
S
Fujii
T
T
Shiba
N
N
Taniguchi
D000818:Animals; D001921:Brain / Q000201:enzymology; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry; D005647:Fucosyltransferases / Q000032:analysis* / Q000378:metabolism; D006020:Glycopeptides / Q000737:chemistry; D008099:Liver / Q000201:enzymology; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D051381:Rats; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013552:Swine
D005456:Fluorescent Dyes; D006020:Glycopeptides; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase