A Fluorescent Assay Method for GDP-L-Fuc:N-Acetyl-.BETA.- D-Glucosaminide .ALPHA.1-6Fucosyltransferase Activity, Involving High Performance Liquid Chromatography.
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概要
- 論文の詳細を見る
An assay method for GDP-L-Fuc: N-acetyl-β-D-glucosaminide α1-6fucosyltransferase (α1-6FucT; EC 2. 4. 1. 68) activity has been developed, involving a fluorescent pyridylaminated substrate. A glycopeptide derived from bovine γ-globulin was coupled with 4-(2-pyridylamino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. _??_ 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 α1-6fucose at the reducing end GlcNAc. This method is highly specific for α1-6FucT and is applicable for various experiments, including purification and cell culture ones.
- 社団法人 日本生化学会の論文
著者
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Fujii Shigeru
Laboratory Of Chemistry Kansai Medical University
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Ihara Yoshito
Department of Biochemistry and Molecular Biology in Disease
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Taniguchi Naoyuki
Department Of Biochemistry Osaka University Graduate School Of Medicine
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Islam Kazi
Department Of Biochemistry And Second Department Of Internal Medicine Osaka University Medical Schoo
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NODA Katsuhisa
Department of Biochemistry, Osaka University Graduate School of Medicine
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Uozumi Naofumi
Department Of Biochemistry Osaka University Medical School
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Gao Yu-e
Department Of Biochemistry Osaka University Medical School
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Shiba Tetsuo
Peptide Insitute Inc.
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Miyoshi Eiji
Department Of Biochemistry School Of Allied Health Science Osaka University Faculty Of Medicine
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Gao Cong-xiao
Department Of Disease Glycomics Institute For Microbial Diseases Osaka University
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Teshima Tadashi
Peptide Institute Protein Research Foundation
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Nishikawa Atsushi
Department Of Applied Biological Science Tokyo University Of Agriculture And Technology
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Yamamoto Toshihiro
Peptide Institute Protein Research Foundation
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Ihara Yoshito
Department of Biochemistry, Osaka University Medical School
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