29] Redox regulation of protein tyrosine phosphatases by hydrogen peroxide: Detecting sulfenic acid intermediates and examining reversible inactivation

Abstract

Publisher Summary This chapter describes a mechanism of redox reduction for protein tyrosine phosphatases (PTPs) in which the transient intracellular production of hydrogen peroxide, as a result of growth factor/mitogen stimulation of cell surface receptors, leads to the rapid inactivation of PTPs by the modification of their invariant catalytic cysteine thiolate (R–S) to an inactive sulfenic acid (R–SOH). The chapter describes methods to test if specific PTPs can be regulated by reversible inactivation with hydrogen peroxide. These techniques include a continuous and end point spectrophotometric assay for the inactivation of PTPs by hydrogen peroxide, a method to examine the reversibility of the hydrogen peroxide inactivation of PTPs by monitoring the reactivation kinetics with thiol compounds, three separate techniques to verify that catalytic cysteine thiolate is the target for hydrogen peroxide inactivation, a chemical modification technique to trap the cysteine sulfenic acid intermediate, and a method to examine if growth factor treatment of cells results in a reversible inactivation of the PTP of interest.