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

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Title: Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer
Authors: Nishikata, M. Browse this author →KAKEN DB
Yoshimura, Y. Browse this author
Deyama, Y. Browse this author →KAKEN DB
Suzuki, K. Browse this author →KAKEN DB
Keywords: Protein tyrosine phosphatase
Continuous assay
Fluorogenic substrate
Fluorescence resonance energy transfer
FRET
Issue Date: Jul-2006
Publisher: Elsevier
Journal Title: Biochimie
Volume: 88
Issue: 7
Start Page: 879
End Page: 886
Publisher DOI: 10.1016/j.biochi.2006.02.002
PMID: 16540231
Abstract: 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: http://www.sciencedirect.com/science/journal/03009084
Type: article (author version)
URI: http://hdl.handle.net/2115/14548
Appears in Collections:歯学院・歯学研究院 (Graduate School of Dental Medicine / Faculty of Dental Medicine) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 西方 眞

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