Peroxynitrite-mediated nitration of the stable free radical tyrosine residue of the ribonucleotide reductase small subunit

Ribonucleotide reductase activity is rate-limiting for DNA synthesis, and inhibition of this enzyme supports cytostatic antitumor effects of inducible NO synthase. The small R2 subunit of class I ribonucleotide reductases contains a stable free radical tyrosine residue required for activity. This radical is destroyed by peroxynitrite, which also inactivates the protein and induces nitration of tyrosine residues. In this report, nitrated residues in the E. coli R2 protein were identified by UV-visible spectroscopy, mass spectrometry (ESI-MS), and tryptic peptide sequencing. Mass analysis allowed the detection of protein R2 as a native dimer with two iron clusters per subunit. The measured mass was 87 032 Da, compared to a calculated value of 87 028 Da. Peroxynitrite treatment preserved the non-heme iron center and the dimeric form of the protein. A mean of two nitrotyrosines per E. coli protein R2 dimer were obtained at 400 microM peroxynitrite. Only 3 out of the 16 tyrosines were nitrated, including the free radical Tyr122. Despite its radical state, that should favor nitration, the buried Tyr122 was not nitrated with a high yield, probably owing to its restricted accessibility. Dose-response curves for Tyr122 nitration and loss of the free radical were superimposed. However, protein R2 inactivation was higher than nitration of Tyr122, suggesting that nitration of the nonconserved Tyr62 and Tyr289 might be also of importance for peroxynitrite-mediated inhibition of E. coli protein R2.     

Time course and site(s) of cytochrome c tyrosine nitration by peroxynitrite

Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.     

Nitration of the tyrosyl radical in ribonucleotide reductase by nitrogen dioxide: a gamma radiolysis study

Nitrogen dioxide is a product of peroxynitrite homolysis and peroxidase-catalyzed oxidation of nitrite. It is of great importance in protein tyrosine nitration because most nitration pathways end with the addition of *NO2 to a one-electron-oxidized tyrosine. The rate constant of this radical addition reaction is high with free tyrosine-derived radicals. However, little is known of tyrosine radicals in proteins. In this paper, we have used *NO2 generated by gamma radiolysis to study the nitration of the R2 subunit of ribonucleotide reductase, which contains a long-lived tyrosyl radical on Tyr122. Most of the nitration occurred on Tyr122, but nonradical tyrosines were also modified. In addition, peptidic bonds close to nitrated Tyr122 could be broken. Nitration at Tyr122 was not observed with a radical-free metR2 protein. The estimated rate constant of the Tyr122 radical reaction with *NO2 was of 3 x 10(4) M(-1) s(-1), thus several orders of magnitude lower than that of a radical on free tyrosine. Nitration rate of other tyrosine residues in R2 was even lower, with an estimated value of 900 M(-1) s(-1). This study shows that protein environment can significantly reduce the reactivity of a tyrosyl radical. In ribonucleotide reductase, the catalytically active radical residue is very efficiently protected against nitrogen oxide attack and subsequent nitration.     

Endogenously nitrated proteins in mouse brain: links to neurodegenerative disease

Increased abundance of nitrotyrosine modifications of proteins have been documented in multiple pathologies in a variety of tissue types and play a role in the redox regulation of normal metabolism. To identify proteins sensitive to nitrating conditions in vivo, a comprehensive proteomic data set identifying 7792 proteins from a whole mouse brain, generated by LC/LC-MS/MS analyses, was used to identify nitrated proteins. This analysis resulted in the identification of 31 unique nitrotyrosine sites within 29 different proteins. More than half of the nitrated proteins that have been identified are involved in Parkinson's disease, Alzheimer's disease, or other neurodegenerative disorders. Similarly, nitrotyrosine immunoblots of whole brain homogenates show that treatment of mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an experimental model of Parkinson's disease, induces an increased level of nitration of the same protein bands observed to be nitrated in brains of untreated animals. Comparing sequences and available high-resolution structures around nitrated tyrosines with those of unmodified sites indicates a preference of nitration in vivo for surface accessible tyrosines in loops, a characteristic consistent with peroxynitrite-induced tyrosine modification. In addition, most sequences contain cysteines or methionines proximal to nitrotyrosines, contrary to suggestions that these amino acid side chains prevent tyrosine nitration. More striking is the presence of a positively charged moiety near the sites of nitration, which is not observed for non-nitrated tyrosines. Together, these observations suggest a predictive tool of functionally important sites of nitration and that cellular nitrating conditions play a role in neurodegenerative changes in the brain.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

Epitope motif of an anti‐nitrotyrosine antibody specific for tyrosine‐nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine‐specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3‐NT‐specific antibody, and have synthesized a series of tyrosine‐nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr‐430 had been previously identified upon reaction with peroxynitrite 17 . The determination of antibody‐binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr‐430, Tyr‐421, Tyr‐83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N‐terminal to the nitration site. The highest affinity to the anti‐3NT‐antibody was found for the PCS peptide comprising the Tyr‐430 nitration site with a K_D of 60 nM determined for the peptide, PCS(424‐436‐Tyr‐430NO₂); in contrast, PCS peptides nitrated at Tyr‐421 and Tyr‐83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody‐bound peptides and affinity‐MS analysis revealed highest affinity to the antibody for tyrosine‐nitrated peptides that contained positively charged amino acids in the N‐terminal sequence to the nitration site. Remarkably, similar N‐terminal sequences of tyrosine‐nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil‐cationic protein. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Carbon dioxide enhances nitration of surfactant protein A by activated alveolar macrophages

We assessed whether reactive oxygen-nitrogen intermediates generated by alveolar macrophages (AMs) oxidized and nitrated human surfactant protein (SP) A. SP-A was exposed to lipopolysaccharide (100 ng/ml)-activated AMs in 15 mM HEPES (pH 7.4) for 30 min in the presence and absence of 1.2 mM CO(2). In the presence of CO(2), lipopolysaccharide-stimulated AMs had significantly higher nitric oxide synthase (NOS) activity (as quantified by the conversion of L-[U-(14)C]arginine to L-[U-(14)C]citrulline) and secreted threefold higher levels of nitrate plus nitrite in the medium [28 +/- 3 vs. 6 +/- 1 (SE) nmol. 6.5 h(-1). 10(6) AMs(-1)]. Western blotting studies of immunoprecipitated SP-A indicated that CO(2) enhanced SP-A nitration by AMs and decreased carbonyl formation. CO(2) (0-1.2 mM) also augmented peroxynitrite (0.5 mM)-induced SP-A nitration in a dose-dependent fashion. Peroxynitrite decreased the ability of SP-A to aggregate lipids, and this inhibition was augmented by 1.2 mM CO(2). Mass spectrometry analysis of chymotryptic fragments of peroxynitrite-exposed SP-A showed nitration of two tyrosines (Tyr(164) and Tyr(166)) in the absence of CO(2) and three tyrosines (Tyr(164), Tyr(166), and Tyr(161)) in the presence of 1.2 mM CO(2). These findings indicate that physiological levels of peroxynitrite, produced by activated AMs, nitrate SP-A and that CO(2) increased nitration, at least partially, by enhancing enzymatic nitric oxide production.     

Light-dependent nitration of bacteriorhodopsin

Purple membranes were treated with tetranitromethane to modify tyrosine residues of bacteriorhodopsin. At pH 8.0, nitration is shown to be affected by illumination during the modification. Amino acid analysis revealed about 0.7 residues nitrated if reaction was in the dark while about 2.0 tyrosines were modified if illumination greater than 540 nm was provided. Tryptophan was unaffected under both conditions. Light-dependent nitration caused a blue shift of the absorbance maximum of bacteriorhodopsin from 568 to 530 nm while no chromophore shift was observed for the dark-modified preparation. Both preparations show an absorption band at 360 nm indicative of the presence of nitrotyrosines. Reduction by dithionite eliminated the pH-dependent changes associated with the 360-nm nitrotyrosine band. Circular dichroism spectra indicate that interactions between neighboring chromophores are altered concomitant with the blue shift of the absorbance maximum by nitration. These studies show that light is required for the nitration of the tyrosine residue, and that Tyr 26 (H. D. Lemke and D. Oesterhelt (1981) Eur. J. Biochem. 115, 595-604) is probably responsible for the blue shift of the absorbance maximum. The intrinsic fluorescence and photocycle kinetics of the tyrosine-modified preparation and reduction of nitrotyrosine by dithionite were studied. In dark modification, only pH-dependent dithionite-reducible nitrotyrosines were produced. It is concluded that surface tyrosines probably do not directly participate in the proton-translocation events coupled to the photocycle of bacteriorhodopsin.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Specific electrochemical nitration of horse heart myoglobin

Earlier findings on electronitration of hen egg-white lysozyme demonstrated a product which was mononitrated at Tyr23, by ion-exchange chromatography, absorbance at 430 nm, dithionite reduction, and Edman sequencing of a nitrated proteolytic peptide. However, the whole protein was not sequenced; therefore, although the enzyme remained active upon nitration, reaction at other residues could not be completely eliminated. This study has now been extended to the redox protein myoglobin. We demonstrate the novel electronitration (electrooxidation in the presence of nitrite) of a specific tyrosine residue in horse heart myoglobin and also in apomyoglobin. Production of the yellow chromophore, 3-nitrotyrosine (3-NT), was apparent in apomyoglobin from A430 but was masked in holomyoglobin by the Soret band. In both cases, the presence of 3-NT in the electronitrated samples was further indicated by the binding of antibody to 3-NT in Western blots. High-resolution electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry revealed a reaction product at [M + 45] (consistent with substitution of NO2 for H), indicating that the nitration reaction is the only reaction occurring which gives rise to a change in mass in the electrooxidation. Fragmentation mass spectrometry identified the nitration site as Tyr103, with no nitration at Tyr146. The procedure may be useful in preparing model nitrated proteins for the study of disease mechanisms.     

Sarcalumenin plays a critical role in age-related cardiac dysfunction due to decreases in SERCA2a expression and activity

Impaired Ca(2+) reuptake into the sarcoplasmic reticulum (SR) underlies a primary pathogenesis of heart failure in the aging heart. Sarcalumenin (SAR), a Ca(2+)-binding glycoprotein located in the longitudinal SR, regulates Ca(2+) reuptake by interacting with SR Ca(2+)-ATPase (SERCA). Here we found that the expression levels of both SAR and SERCA2 proteins were significantly downregulated in senescent wild-type mice (18-month old) and that downregulation of SAR protein preceded downregulation of SERCA2 protein. The downregulation of SERCA2 protein was greater in senescent SARKO mice than in age-matched senescent wild-type mice, which was at least in part due to progressive degradation of SERCA2 protein in SARKO mice. Senescent SARKO mice exhibited typical findings of heart failure such as increased sympathetic activity, impaired exercise tolerance, and upregulation of biomarkers of cardiac stress. Consequently, cardiac function was progressively decreased in senescent SARKO. We also found that the expression levels of endoplasmic reticulum (ER) stress-related genes such as x-box binding protein 1 (XBP1) were significantly increased in senescent SARKO mice, indicating that senescent SARKO mice exhibited ER stress. Thus we uncovered the important role of SAR in maintaining Ca(2+) transport activity of SERCA2a and cardiac function in the senescent population.     

Peroxynitrite-induced nitration of tyrosine hydroxylase: identification of tyrosines 423, 428, and 432 as sites of modification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tyrosine-scanning mutagenesis

Tyrosine hydroxylase (TH), the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter dopamine, is inactivated by peroxynitrite. The sites of peroxynitrite-induced tyrosine nitration in TH have been identified by matrix-assisted laser desorption time-of-flight mass spectrometry and tyrosine-scanning mutagenesis. V8 proteolytic fragments of nitrated TH were analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry. A peptide of 3135.4 daltons, corresponding to residues V410-E436 of TH, showed peroxynitrite-induced mass shifts of +45, +90, and +135 daltons, reflecting nitration of one, two, or three tyrosines, respectively. These modifications were not evident in untreated TH. The tyrosine residues (positions 423, 428, and 432) within this peptide were mutated to phenylalanine to confirm the site(s) of nitration and assess the effects of mutation on TH activity. Single mutants expressed wild-type levels of TH catalytic activity and were inactivated by peroxynitrite while showing reduced (30-60%) levels of nitration. The double mutants Y423F,Y428F, Y423F,Y432F, and Y428F,Y432F showed trace amounts of tyrosine nitration (7-30% of control) after exposure to peroxynitrite, and the triple mutant Y423F,Y428F,Y432F was not a substrate for nitration, yet peroxynitrite significantly reduced the activity of each. When all tyrosine mutants were probed with PEO-maleimide activated biotin, a thiol-reactive reagent that specifically labels reduced cysteine residues in proteins, it was evident that peroxynitrite resulted in cysteine oxidation. These studies identify residues Tyr(423), Tyr(428), and Tyr(432) as the sites of peroxynitrite-induced nitration in TH. No single tyrosine residue appears to be critical for TH catalytic function, and tyrosine nitration is neither necessary nor sufficient for peroxynitrite-induced inactivation. The loss of TH catalytic activity caused by peroxynitrite is associated instead with oxidation of cysteine residues.     

Tyrosine nitration impairs mammalian aldolase A activity

Protein tyrosine nitration increases in vivo as a result of oxidative stress and is elevated in numerous inflammatory-associated diseases. Mammalian fructose-1,6-bisphosphate aldolases are tyrosine nitrated in lung epithelial cells and liver, as well as in retina under different inflammatory conditions. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we now show that aldolase A is nitrated in human skin fibroblasts. To reveal the consequences of tyrosine nitration, we studied the impact of peroxynitrite on the glycolytic functions of aldolase A. A peroxynitrite concentration-dependent decrease in fructose-1,6-bisphosphate cleavage activity was observed with a concomitant increase in nitrotyrosine immunoreactivity. Both V(max) and the K(m) for fructose-1,6-bisphosphate decreased after incubation with peroxynitrite. Aldolase nitrotyrosine immunoreactivity diminished following carboxypeptidase Y digestion, demonstrating that tyrosine residues in the carboxyl-terminal region of aldolase are major targets of nitration. Aldolase A contains a carboxyl-terminal tyrosine residue, Tyr(363), that is critical for its catalytic activity. Indeed, tandem mass spectrometric analysis of trypsin-digested aldolase showed that Tyr(363) is the most susceptible to nitration, with a modification of Tyr(342) occurring only after nitration of Tyr(363). These tyrosine nitrations likely result in altered interactions between the carboxyl-terminal region and enzyme substrate or reaction intermediates causing the decline in activity. The results suggest that tyrosine nitration of aldolase A can contribute to an impaired cellular glycolytic activity.     

Exposed tyrosine residues of lambda cro repressor protein evidenced by nitration and photo CIDNP experiments

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).     

Proteomic analysis of protein nitration in aging skeletal muscle and identification of nitrotyrosine-containing sequences in vivo by nanoelectrospray ionization tandem mass spectrometry

The nitration of protein tyrosine residues represents an important post-translational modification during development, oxidative stress, and biological aging. To rationalize any physiological changes with such modifications, the actual protein targets of nitration must be identified by proteomic methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most of these studies have failed to prove nitration unambiguously through the actual localization of 3-nitrotyrosine to specific sequences by mass spectrometry. In this paper we have applied sequential solution isoelectric focusing and SDS-PAGE for the proteomic characterization of specific 3-nitrotyrosine-containing sequences of nitrated target proteins in vivo using nanoelectrospray ionization-tandem mass spectrometry. Specifically, we analyzed proteins from the skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well accepted animal model for biological aging. We identified the 3-nitrotyrosine-containing sequences of 11 proteins, including cytosolic creatine kinase, tropomyosin 1, glyceraldehyde-3-phosphate dehydrogenase, myosin light chain, aldolase A, pyruvate kinase, glycogen phosphorylase, actinin, gamma-actin, ryanodine receptor 3, and neurogenic locus notch homolog. For creatine kinase and neurogenic locus notch homolog, two 3-nitrotyrosine-containing sequences were identified, i.e. at positions 14 and 20 for creatine kinase and at positions 1175 and 1205 for the neurogenic locus notch homolog. The selectivity of the in vivo nitration of creatine kinase at Tyr14 and Tyr20 does not correspond to the product selectivity in vitro, where exclusively Tyr82 was nitrated when creatine kinase was exposed to peroxynitrite. The latter experiments demonstrate that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo.     

Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting

We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inflammatory stimulated rat abdominal, liver and lung tissue homogenates caused no immunoreactivity, in contrast to a polyclonal nitrotyrosine antibody applied in fixed and non-fixed tissues. Western blot studies using both mono- and polyclonal antibodies showed a temperature- and heme group-dependent reduction of NO(2)Tyr in nitrated rat and bovine serum albumin incubated with dithiothreitol. Mass spectrometric analyses of a nitrated peptide angiotensin II revealed under similar conditions a positive temperature effect between 56 and 70 degrees C on reduction of NO(2)Tyr to 3-aminotyrosine which is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins.     

Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.     

Tyrosine Nitration is a Novel Post-translational Modification Occurring on the Neural Intermediate Filament Protein Peripherin

The biological implication of protein tyrosine nitration in signaling pathways triggered by nitric oxide is recently emerging. Here we report for the first time that nitrotyrosination occurs in the neural intermediate filament protein peripherin. In neuron-like PC12 cells, nitrated peripherin is associated with the cytoskeleton fraction, its level increases during the progression of NGF-induced differentiation and the nitrated protein remains closely associated with stable microtubules. Tyr 17 and Tyr 376 were identified by MALDI-TOF analyses as two specific residues endogenously nitrated. Finally, peripherin nitration is not restricted to PC12 cells but it is also present in vivo in rat brain. Gabriella Tedeschi and Graziella Cappelletti contributed equally to this work.     

Tyrosine nitration moderates the peptidase activity of human methionyl aminopeptidase 2

Methionyl aminopeptidase 2 (MetAP2) plays an important role in the regulation of angiogenesis. This study examined whether nitration of MetAP2 alters its enzymatic activity in vitro. The activity of unmodified, nitrated and oxidised MetAP2 was assessed and it was found that nitration significantly reduced its ability to cleave a chromogenic substrate. Mass spectrometry analysis identified Tyr336 as a nitrated residue in MetAP2. Structural and evolutionary analysis indicate that this is an important residue for MetAP2 activity. Combined, the results show that the activity of MetAP2 is reduced by nitration and raise the possibility that nitration of MetAP2 is a mechanism contributing to endothelial dysfunction.