title: A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates.
abstract: A versatile assay for protein tyrosine phosphatases (PTP) employing 3-nitrophosphotyrosine containing peptidic substrates is described. These therapeutically important phosphatases feature in signal transduction pathways. The assay involves spectrophotometric detection of 3-nitrotyrosine production from 3-nitrophosphotyrosine containing peptidic substrates, which are accepted by many PTPs. Compared to conventional chromogenic phosphate derivatives, the more realistic peptidic substrates allow evaluating substrate specificity. The assay's applicability is demonstrated by determining kinetic parameters for several PTP-substrate combinations and inhibitor evaluation, as well as detection of PTP activity in lysates. The convenient new assay may assist further adoption of PTPs in drug development.
authors.affiliation: Medicinal Chemistry and Chemical Biology, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands; Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: van Ameijde
authors.affiliation: Hubrecht Institute, Uppsalalaan 8, P.O. Box 85164, 3508 AD Utrecht, The Netherlands.
authors.forename: John
authors.initials: J
authors.lastname: Overvoorde
authors.affiliation: Structural Genomics Consortium, Oxford University, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK.
authors.forename: Stefan
authors.initials: S
authors.lastname: Knapp
authors.affiliation: Hubrecht Institute, Uppsalalaan 8, P.O. Box 85164, 3508 AD Utrecht, The Netherlands; Institute of Biology, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: den Hertog
authors.affiliation: Pamgene International Ltd., Wolvenhoek 10, P.O. Box 1345, 5200 BJ Den Bosch, The Netherlands.
authors.forename: Rob
authors.initials: R
authors.lastname: Ruijtenbeek
authors.affiliation: Medicinal Chemistry and Chemical Biology, Faculty of Science, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands; School of Chemistry, University of Glasgow, University Avenue, Glasgow G12 8QQ, UK. Electronic address: r.m.j.liskamp@uu.nl.
authors.forename: Rob M J
authors.initials: RM
authors.lastname: Liskamp
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; C002744:3-nitrotyrosine; D014638:Vanadates; D014443:Tyrosine; D017027:Protein Tyrosine Phosphatases
keywords: Enzymatic assay; Enzyme inhibition; High-throughput screening; Phosphatases; Phosphoproteomics; Structure–activity relationships