N-nitrosations of basic amino acid residues in polypeptide.

Changes in the electrophoretic pattern were noted in the products of polypeptides of identical basic amino acids preincubated with reactive or degraded PN, suggesting the occurrence of N-nitrosation of the epsilon-amino group of lysine, the guanido group of arginine and the imidazole group of histidine. Additionally, increase in the N-nitroso immunoreactivity of preincubated histones H2A and H2B was detected by Western blot analysis.

N-nitrosation and denitrosation on lysine residues of histones.

Increased immunoactivity of (-nitrosolysine was detected in Western blot in peroxynitrite-reacted histones H2A, H3 and H4. The N-nitrosation may be reversible as indicated by the decreased immunoactivity via either subsequent incubation with reduced glutathione or 48-hour dialysis and 48-hour storage at 4 degrees C. Nonetheless, the biological significance of N-nitrosation of protein lysine is yet to be proven.

Modifications of tyrosine and catecholamines by peroxynitrite, nitrite and nitrate.

At pH 7.0 in non-"deaired" potassium phosphate buffer, the reactions of L-tyrosine, L-dopa, dopamine, L-norepinephrine, and L-epinephrine with peroxynitrite (PN) or nitrite, generated colored products. These products displayed not only unique colors and patterns of mobility on silica thin layer chromatographic plates, but also varied increase of absorbance between 400 and 540 nm.

Further study on S-nitrosation by nitrite.

At neutral pH, S-nitrosoglutathione was formed by the reaction of reduced glutathione and sodium nitrite. The degradation of S-nitrosoglutathione, presumably by transnitrosation/denitrosation, was catalyzed by L-cysteine, or CoA-SH. Additionally, from the crude extract of rat brain, one protein with a large molecular mass was nitrosolated with nitrite, and was split into duplet peptides noted in Western blot.

Nitrosation of cysteine and reduced glutathione by nitrite at physiological pH.

Unlike the formation of nitrosothiols by nitrous acid, our study revealed that NO2- effectively reacted with L-cysteine or reduced glutathione (GSH) at pH 7.0 and 7.4, to form orange-pink products of S-nitrosocysteine (CySNO) or S-nitrosoglutathione (GSNO).

Protein denitration/modification by glutathione-S-transferase and glutathione peroxidase.

Peroxynitrite (PN)-pretreated histone III-S (NH) and reduced glutathione (GSH)-treated NH (NH(GSH)) were incubated with glutathione-S-transferase (GST) and glutathione peroxidase (GPX). Western blot analysis revealed decreased 3-nitrotyrosine immunoreactivity for NH(GSH), but not for NH. Additionally, increased nitrate was noted as an end product of these enzymatic reactions.

Regulation of catalase: inhibition by peroxynitrite and reactivation by reduced glutathione and glutathione S-transferase.

The regulation of stable catalase from Aspergillus niger was investigated. The preincubation of catalase with peroxynitrite (PN) resulted in a significant decrease in the production of O2, while the subsequent incubation with reduced glutathione (GSH, 1mM) led to restoration of the enzymatic activity. Western blot analysis revealed not only the increased immunoreactivities of 3-nitrotyrosine and S-nitrosocysteine in a PN-dose-dependent manner, but also conversely decreased immunoreactivity of 3-nitrotyrosine by the subsequent preincubation of catalase with GSH (1mM)/glutathione S-transferase (GST).

Nitration/S-nitrosation of proteins by peroxynitrite-treatment and subsequent modification by glutathione S-transferase and glutathione peroxidase.

In various peroxynitrite (PN)-treated proteins, the formations of stable 3-nitrotyrosine (nitration) and labile S-nitrosocysteine (S-nitrosation) were observed by employing rapid Western blot in 6 h. The steps of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and membrane-blotting were performed at 4 degrees C. It was noted that the intensity of immunoreactive bands specific for anti-nitrotyrosine was stronger than that specific for anti-S-nitrosocysteine.

Protein denitration/modification by Escherichia coli nitrate reductase and mammalian cytochrome P-450 reductase.

The incubation of peroxynitrite (PN)-pretreated histone III-S (NH) with Escherichia coli nitrate reductase (cytochrome, NADPH/GSH-independent) and that of NADPH-treated NH (NHNADPH) with liver cytochrome P-450 reductase (NADPH-dependent) resulted in decreased 3-nitrotyrosine immunoreactivity found in Western blot analysis. Additionally, increased nitrate was noted as an end product of these reactions. These findings imply that varied enzymatic denitration/modification of NO/PN-reacted protein, either with or without a reductant, may be important in regulating related signal transduction cascade(s) and relieving oxidative stress.