An ELISA-Based Assay for Detergent-solubilized Cellular β1,4-Galactosyltransferase Activity. Use of a Polyacrylamide Derivative with GlcNAc-β Side Chains as a Solid Phase Acceptor Substrate

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概要

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• In our previous paper [Oubihi et al. (1998) Anal. Biochem., 257,169-175], we have shown that a polyacrylamide-derived synthetic glycopolymer with GlcNAcβ side chains, termed PAP(GlcNAcβ), is useful as a solid phase acceptor substrate for the ELISA-based analyses of soluble β1,4(-)galactosyltransferase (GalT) activity in milk. This method is now used to assay detergent-solubilized cellular GalT. The giycopolymer coated on polystyrene plates was shown to be highly stable against the non-ionic and ionic detergents tested (0〜5% solutions of Triton X-100 and SDS). Such stability made it possible to incubate the ELISA plate with detergent-solubilized GalT and to wash the ELISA plate with SDS solution after the GalT reaction, leading to high accuracy and sensitivity of this assay. The GalT activity was assayed using this method for 1% Triton X-100 extracts of various tissue samples of mice and several cultured cell lines. The results showed that the specific GalT activity of tissue extracts was low in brain and intestine, and high in ovary, muscle, and kidney. As for the cultured cell lines, COS7,COMMA-1D and C2C12 cells showed high specific activity, while CHO and MDCK cells showed low activity. The myoblast C2C12 had a slight increase in GalT activity during starvation-induced cell differentiation. On the other hand, GalT-I transcript estimated by RT-PCR rather decreased during C2C12 cell differentiation, suggesting a differentiation-dependent switch in GalT isozymes. Taken all together, the ELISA-based assay using PAP(GlcNAcβ) as a solid phase acceptor substrate was demonstrated to be a useful method for the assay of membrane-bound galactosyltransferases.

• 社団法人日本農芸化学会の論文
• 2000-04-23

著者

• Aoki Naohito

  Department of Life Sciences, Faculty of Bioresources, Mie University

• MATSUDA Tsukasa

  Department of Food Science ande Tecknology, School of Agriculture, Nagoya University

• Aoki N

  Yamaguchi Univ. Yamaguchi Jpn

• Aoki Naohito

  Department Of Applied Biological Sciences School Of Agricultural Sciences Nagoya University

• Kitajima Ken

  Department of Cardiology, Fukuoka University School of Medicine

• KOBAYASHI Kazukiyo

  Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, C

• Matsuda T

  Department Of Applied Molecular Biosciences School Of Bioagricultural Sciences Nagoya University

• Matsuda T

  Department Of Applied Molecular Biosciences Graduate School Of Bioagricultural Sciences Nagoya Unive

• Matsuda Tsukasa

  School Of Agriculture Nagoya University

• Kitajima Ken

  Bioscience And Biotechnology Center And Graduate School Of Bioagricultural Sciences Nagoya Universit

• OSHIMA Kenji

  Department of Ophthalmology, Fukuoka University School of Medicine

• Matsuda Tsukasa

  Department Of Applied Biological Sciences School Of Agricultural Sciences Nagoya University

• Kitajima K

  Laboratory Of Animal Cell Function Bioscience And Biotechnology Center Nagoya University

• Kitajima Ken

  Department Of Applied Molecular Biosciences Graduate School Of Bioagricultural Sciences Nagoya Unive

• Oshima Kenji

  Department Of Bioengineering Nagaoka University Of Technology

• Kobayashi Kazukiyo

  Department Of Engineering Nagoya University

• Kobayashi Kazukiyo

  Department Of Molecular Design And Biotechnology Graduate School Of Engineering Nagoya University

• OUBIHI Mohamed

  Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya Uni

• Oubihi Mohamed

  Department Of Applied Molecular Biosciences Graduate School Of Bioagricultural Sciences Nagoya Unive

• Oshima K

  Department Of Applied Molecular Biosciences Graduate School Of Bioagricultural Sciences Nagoya Unive

• Matsuda Tsukasa

  Department Of Applied Biological Science School Of Agricultural Science Nagoya University

• Oshima Kenji

  Department Of Applied Chemistry Kyushu Institute Of Technology

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