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Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

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タイトル: Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer
著者: Nishikata, M 著作を一覧する
Yoshimura, Y 著作を一覧する
Deyama, Y 著作を一覧する
Suzuki, K 著作を一覧する
キーワード: Protein tyrosine phosphatase
Continuous assay
Fluorogenic substrate
Fluorescence resonance energy transfer
発行日: 2006年 7月
出版者: Elsevier
誌名: Biochimie
巻: 88
号: 7
開始ページ: 879
終了ページ: 886
出版社 DOI: 10.1016/j.biochi.2006.02.002
抄録: An assay method that continuously measures the protein tyrosine phosphatase (PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energy transfer (FRET) was developed as an improvement of our previously reported discontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A. Matsumoto, Biochem. J. 343 (1999) 385-391]. The assay uses oligopeptide substrates that contain Mca [(7-methoxycoumarin-4-yl)acetyl] group as a fluorescence donor and DNP (2,4-dinitrophenyl) group as a fluorescence acceptor, in addition to a phosphotyrosine residue located between these two groups. In the assay, a PTP solution is added to a buffer solution containing a FRET substrate and chymotrypsin. The PTP-catalyzed dephosphorylation of the substrate and subsequent chymotryptic cleavage of the dephosphorylated substrate results in a disruption of FRET, thereby increasing Mca fluorescence. In this study, we used FRET substrates that are much more susceptible to chymotryptic cleavage after dephosphorylation than the substrate used in our discontinuous assay, thus enabling the continuous assay without significant PTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflected the rate of dephosphorylation at appropriate chymotrypsin concentrations. Since the continuous assay allows the measurement of initial rate of dephosphorylation reaction, kinetic parameters for the dephosphorylation reactions of a FRET substrate by Yersinia, T-cell and LAR PTPs were determined. The continuous assay was compatible with the measurement of very low PTP activity in a crude enzyme preparation and was comparable in sensitivity to assays that use radiolabeled substrates.
Relation (URI):
資料タイプ: article (author version)
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 西方 眞


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