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Nitric oxide (NO)-derived reactive species nitrate unsaturated fatty acids, yielding nitroalkene derivatives, including the clinically abundant nitrated oleic and linoleic acids. The olefinic nitro group renders these derivatives electrophilic at the carbon β to the nitro group, thus competent for Michael addition reactions with cysteine and histidine. By using chromatographic and mass spectrometric approaches, we characterized this reactivity by using in vitro reaction systems, and we demonstrated that nitroalkene-protein and GSH adducts are present in vivo under basal conditions in healthy human red cells. Nitro-linoleic acid (9-, 10-, 12-, and 13-nitro-9,12-octadecadienoic acids) (m/z 324.2) and nitro-oleic acid (9- and 10-nitro-9-octadecaenoic acids) (m/z 326.2) reacted with GSH (m/z 306.1), yielding adducts with m/z of 631.3 and 633.3, respectively. At physiological concentrations, nitroalkenes inhibited glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which contains a critical catalytic Cys (Cys-149). GAPDH inhibition displayed an IC50 of ∼3 μM for both nitroalkenes, an IC50 equivalent to the potent thiol oxidant peroxynitrite (ONOO-) and an IC50 30-fold less than H2O2, indicating that nitroalkenes are potent thiol-reactive species. Liquid chromatography-mass spectrometry analysis revealed covalent adducts between fatty acid nitroalkene derivatives and GAPDH, including at the catalytic Cys-149. Liquid chromatography-mass spectrometry-based proteomic analysis of human red cells confirmed that nitroalkenes readily undergo covalent, thiol-reversible post-translational modification of nucleophilic amino acids in GSH and GAPDH in vivo. The adduction of GAPDH and GSH by nitroalkenes significantly increased the hydrophobicity of these molecules, both inducing translocation to membranes and suggesting why these abundant derivatives had not been detected previously via traditional high pressure liquid chromatography analysis. The occurrence of these electrophilic nitroalkylation reactions in vivo indicates that this reversible post-translational protein modification represents a new pathway for redox regulation of enzyme function, cell signaling, and protein trafficking. [ABSTRACT FROM AUTHOR]

Human recombinant copper–zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC50) was ∼100 μM at 5 μM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 ± 1.0) × 103 M-1 s-1 per monomer at pH 7.5 and 37°C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical. [Copyright &y& Elsevier]

Trypanosoma cruzi, the causative agent of Chagas disease, has evolved particular mechanisms of gene regulation. Gene expression is regulated firstly at post-transcriptional level. This feature makes proteomic methods a promising tool for studying adaptative changes in these parasites. In this work we generated a reproducible method for protein analysis by two-dimensional electrophoresis coupled to mass spectrometry, and a protein map for T. cruzi. Western-blot analysis supported the identity of some of the proteins. This work points to proteomic approach as a powerful tool to study differential expression, stress response or drug resistance in T. cruzi. [Copyright &y& Elsevier]

Muscarinic receptors in the hippocampus are relevant to learning and memory, but the role of each subtype is poorly understood. Muscarinic toxins (MTs) from Dendroaspis snakes venom are selective for muscarinic receptor subtypes. MT2, a selective agonist for M1 receptors, given into the hippocampus immediately after training, improved memory consolidation of an inhibitory avoidance task in rats, whereas the antagonist pirenzepine was amnestic, supporting a facilitatory role of M1 receptors. Instead, MT3, a selective antagonist at M4 receptors, caused amnesia. Neither M1 nor M4 receptor appeared involved in habituation to a new environment. Thus, our results suggest that memory consolidation of an inhibitory avoidance task in the rat involves the participation of both M1 and M4 hippocampal receptors, with a positive modulatory role. [Copyright &y& Elsevier]