caa a22 4500 397556810 CHVBK 20180308164804.0 cr unu---uuuuu 161202e199608 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021424 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021424 A Fluorescent Assay Method for GDP-L-Fuc: N-Acetyl-βD- Glucosaminide α1-6Fucosyltransferase Activity, Involving High Performance Liquid Chromatography [Elektronische Daten] [Naofumi Uozumi, Tadashi Teshima, Toshihiro Yamamoto, Atsushi Nishikawa, Yu-E Gao, Eiji Miyoshi, Cong-Xiao Gao, Katsuhisa Noda, Kazi Nazrul Islam, Yoshito Ihara, Shigeru Fujii, Tetsuo Shiba, Naoyuki Taniguchi] 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-pyridy1amino)butylamine (PABA) through the peptide bond, and the following substrate was obtained. GlcNAc′15′-2Manα1⋈64′GlcNAcβ15-2Manα1⋈34Manβ13-4GlcNAcβ12-4GlcNAc-Asn-PABA1 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. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence carbohydrate structure nationallicence fluorescent labeling nationallicence α1-6fucosyltramferase nationallicence glycosyltransferase activity nationallicence HPLC nationallicence Uozumi Naofumi Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Teshima Tadashi Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut Yamamoto Toshihiro Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut Nishikawa Atsushi Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Gao Yu-E Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Miyoshi Eiji Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Gao Cong-Xiao Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Noda Katsuhisa Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Islam Kazi Nazrul Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Ihara Yoshito Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut Fujii Shigeru Laboratory of Chemistry, Kansai Medical University Hirakata, Osaka 573 aut Shiba Tetsuo Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut Taniguchi Naoyuki Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut The Journal of Biochemistry Oxford University Press 120/2(1996-08), 385-392 0021-924X 120:2<385 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021424 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021424 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Uozumi Naofumi Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Teshima Tadashi Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut NATIONALLICENCE 700 1- Yamamoto Toshihiro Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut NATIONALLICENCE 700 1- Nishikawa Atsushi Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Gao Yu-E Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Miyoshi Eiji Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Gao Cong-Xiao Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Noda Katsuhisa Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Islam Kazi Nazrul Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Ihara Yoshito Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 700 1- Fujii Shigeru Laboratory of Chemistry, Kansai Medical University Hirakata, Osaka 573 aut NATIONALLICENCE 700 1- Shiba Tetsuo Peptide Institute, Protein Research Foundation 4-1-2 Ina, Minoh, Osaka 562 aut NATIONALLICENCE 700 1- Taniguchi Naoyuki Department of Biochemistry, Osaka University Medical School 2-2 Yamadaoka, Suita, Osaka 565 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/2(1996-08), 385-392 0021-924X 120:2<385 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford