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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.

Original publication

DOI

10.1016/j.ab.2013.11.023

Type

Journal article

Journal

Anal Biochem

Publication Date

01/03/2014

Volume

448

Pages

9 - 13

Keywords

Enzymatic assay, Enzyme inhibition, High-throughput screening, Phosphatases, Phosphoproteomics, Structure–activity relationships, Enzyme Inhibitors, HEK293 Cells, Humans, Kinetics, Protein Tyrosine Phosphatases, Spectrophotometry, Substrate Specificity, Tyrosine, Vanadates