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Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.

Abstract：

:The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

journal_name

Int J Mol Sci

journal_title

International journal of molecular sciences

authors

Daiber A,Daub S,Bachschmid M,Schildknecht S,Oelze M,Steven S,Schmidt P,Megner A,Wada M,Tanabe T,Münzel T,Bottari S,Ullrich V

doi

10.3390/ijms14047542

subject

Has Abstract

pub_date

2013-04-08 00:00:00

pages

7542-70

issue

issn

1422-0067

pii

ijms14047542

journal_volume

pub_type

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Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.