A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

Jeroen van Ameijde; John Overvoorde; Stefan Knapp; Jeroen den Hertog; Rob Ruijtenbeek; Rob M J Liskamp

doi:10.1016/j.ab.2013.11.023

A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

Jeroen van Ameijde, John Overvoorde, Stefan Knapp, Jeroen den Hertog, Rob Ruijtenbeek, Rob M J Liskamp

Hubrecht Institute

Research output: Contribution to journal/periodical › Article › Scientific › peer-review

9 Citations (Scopus)

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.

Original language	English
Pages (from-to)	9-13
Number of pages	5
Journal	Analytical Biochemistry
Volume	448
Early online date	03 Dec 2013
DOIs	https://doi.org/10.1016/j.ab.2013.11.023
Publication status	Published - 01 Mar 2014

Keywords

Enzyme Inhibitors
HEK293 Cells
Humans
Kinetics
Protein Tyrosine Phosphatases
Spectrophotometry
Substrate Specificity
Tyrosine
Vanadates

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10.1016/j.ab.2013.11.023

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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.",

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doi = "10.1016/j.ab.2013.11.023",

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T1 - A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

AU - van Ameijde, Jeroen

AU - Overvoorde, John

AU - Knapp, Stefan

AU - den Hertog, Jeroen

AU - Ruijtenbeek, Rob

AU - Liskamp, Rob M J

N1 - Reporting year: 2013

PY - 2014/3/1

Y1 - 2014/3/1

N2 - 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.

AB - 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.

KW - Enzyme Inhibitors

KW - HEK293 Cells

KW - Humans

KW - Kinetics

KW - Protein Tyrosine Phosphatases

KW - Spectrophotometry

KW - Substrate Specificity

KW - Tyrosine

KW - Vanadates

U2 - 10.1016/j.ab.2013.11.023

DO - 10.1016/j.ab.2013.11.023

M3 - Article

C2 - 24309020

SN - 0003-2697

VL - 448

SP - 9

EP - 13

JO - Analytical Biochemistry

JF - Analytical Biochemistry

ER -

A versatile spectrophotometric protein tyrosine phosphatase assay based on 3-nitrophosphotyrosine containing substrates

Abstract

Keywords

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