muramidase and 3-nitrotyrosine

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Chickens; Cytochromes c; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Free Radicals; Horses; Hydrogen Peroxide; Immunoblotting; Magnetics; Models, Chemical; Muramidase; Nitric Oxide; Nitrites; Oxygen; Peroxynitrous Acid; Rats; Superoxide Dismutase; Time Factors; Tyrosine

Tyrosine nitration is a covalent posttranslational protein modification derived from the reaction of proteins with nitrating agents. Protein nitration appears to be a selective process since not all tyrosine residues in proteins or all proteins are nitrated in vivo. To investigate factors that may determine the biological selectivity of protein tyrosine nitration, we developed an in vitro model consisting of three proteins with similar size but different three-dimensional structure and tyrosine content. Exposure of ribonuclease A to putative in vivo nitrating agents revealed preferential nitration of tyrosine residue Y(115). Tyrosine residue Y(23) and to a lesser extent residue Y(20) were preferentially nitrated in lysozyme, whereas tyrosine Y(102) was the only residue modified by nitration in phospholipase A(2). Tyrosine Y(115) was the residue modified by nitration after exposure of ribonuclease A to different nitrating agents: chemically synthesized peroxynitrite, nitric oxide, and superoxide generated by SIN-1 or myeloperoxidase (MPO)/H(2)O(2) plus nitrite (NO(-2)) in the presence of bicarbonate/CO(2). The nature of the nitrating agent determined in part the protein that would be predominantly modified by nitration in a mixture of all three proteins. Ribonuclease A was preferentially nitrated upon exposure to MPO/H(2)O(2)/NO(-2), whereas phospholipase A(2) was the primary target for nitration upon exposure to peroxynitrite. The data also suggest that the exposure of the aromatic ring to the surface of the protein, the location of the tyrosine on a loop structure, and its association with a neighboring negative charge are some of the factors determining the selectivity of tyrosine nitration in proteins.

Topics: Amino Acid Sequence; Models, Molecular; Molecular Sequence Data; Muramidase; Nitrates; Nitric Oxide; Phospholipases A; Protein Processing, Post-Translational; Protein Structure, Secondary; Ribonuclease, Pancreatic; Sequence Analysis, Protein; Superoxides; Tyrosine

Preparative electrooxidation of lysozyme at copper electrodes held at potentials around 1.2 V vs. a saturated calomel reference electrode induces the formation of a yellow chromophore with a concomitant decrease in the pI of the protein. Ion-exchange high-performance liquid chromatography revealed two new lysozyme species with pI values of 10.8 and 10.7 (lysozyme-11.0) which bear the chromophore. Sequence analysis of these two species showed that protein with lower pI was modified at both Tyr 23 and Tyr 20 and the other exclusively at Tyr 23. ribonuclease A, subtilisin BPN', and BSA were also found to produce the same chromophore using similar electrochemical reaction schemes. Characterization of the chromophore by a variety of techniques revealed that it is apparently 3-nitrotyrosine.

Topics: Alkalies; Amino Acid Sequence; Buffers; Cyanogen Bromide; Electrodes; Electrolysis; Fluorescein-5-isothiocyanate; Molecular Sequence Data; Muramidase; Oxidation-Reduction; Peptide Fragments; Peptide Mapping; Sequence Analysis; Tyrosine