Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer
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概要
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An assay method that continuously measures the protein tyrosine phosphatase(PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energytransfer (FRET) was developed as an improvement of our previously reporteddiscontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A.Matsumoto, Biochem. J. 343 (1999) 385-391]. The assay uses oligopeptide substratesthat contain Mca [(7-methoxycoumarin-4-yl)acetyl] group as a fluorescence donor andDNP (2,4-dinitrophenyl) group as a fluorescence acceptor, in addition to aphosphotyrosine residue located between these two groups. In the assay, a PTP solutionis added to a buffer solution containing a FRET substrate and chymotrypsin. ThePTP-catalyzed dephosphorylation of the substrate and subsequent chymotrypticcleavage of the dephosphorylated substrate results in a disruption of FRET, therebyincreasing Mca fluorescence. In this study, we used FRET substrates that are muchmore susceptible to chymotryptic cleavage after dephosphorylation than the substrateused in our discontinuous assay, thus enabling the continuous assay without significantPTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflectedthe rate of dephosphorylation at appropriate chymotrypsin concentrations. Since thecontinuous 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 themeasurement of very low PTP activity in a crude enzyme preparation and wascomparable in sensitivity to assays that use radiolabeled substrates.
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