Specific Nitration at Tyrosine 430 Revealed by High Resolution Mass Spectrometry as Basis for Redox Regulation of Bovine Prostacyclin Synthase

Abstract

Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI 2 synthase) with increasing concentrations of peroxynitrite (PN) up to 250 M of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 M PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 M PN, PGI 2 synthase was about halfmaximally nitrated and about half-inhibited. It was then isolated by gel electrophoresis and subjected to proteolytic digestion with several proteases. Digestion with thermolysin for 24 h provided a single specific peptide that was isolated by high performance liquid chromatography and identified as a tetrapeptide Leu-Lys-AsnTyr(3-nitro)-COOH corresponding to positions 427-430 of PGI 2 synthase. Its structure was established by precise mass determination using Fourier transform-ion cyclotron resonance-nanoelectrospray mass spectrometry and Edman microsequencing and ascertained by synthesis and mass spectrometric characterization of the authentic Tyr-nitrated peptide. Complete digestion by Pronase to 3-nitrotyrosine was obtained only after 72 h, suggesting that the nitrated Tyr-430 residue may be embedded in a tight fold around the heme binding site. These results provide evidence for the specific inhibition of PGI 2 synthase by nitration at Tyr-430 that may occur already at low levels of PN as a consequence of endothelial co-generation of nitric oxide and superoxide.The nitration of tyrosine residues in proteins has become a well recognized reaction, but has been heavily disputed with regard to the mechanisms involved and its physiological and/or pathophysiological significance (1)(2)(3)(4)(5) . from SIN-1 (18). In cellular systems the inhibition of nitration by a NO synthase inhibitor and polyethylene-glycolated superoxide dismutase provided evidence for the involvement of PN, whereas nitrite was ineffective (18). Because NO and PGI 2 are important for the endothelial barrier function the formation of PN and the nitration of PGI 2 synthase could play a role in the process of endothelial activation for adhesion and emigration of white blood cells into the tissue (19). Interestingly, PGI 2 synthase was found localized to the caveolae-like endothelial NO synthase (20) and hence PN formation may occur in close vicinity to PGI 2 synthase. This localization in a "quasiextracellular" compartment may be a further important factor for efficient nitration by low concentrations of PN. Beyond this physiological background no proof for the molecular basis of enzyme inhibition has been hitherto obtained by identification of nitrated tyrosine. Substrate analogs of prostaglandin-endoperoxide have been recently shown to inhibit the nitration (17), which suggested a proximity to the heme attached to the protein by the Cys-441 residue (21-23); however, previous attempts have been unsuccessful to detect and identify the nitrated tyrosine. In this study we present molecular evidence for the specific nitration ...

Postischemic hyperoxia reduces hippocampal pyruvate dehydrogenase activity

“…Whereas many enzymes are subject to inhibition by peroxynitrite, the contribution of tyrosine nitration, S-nitrosation, and other mechanisms to inactivation varies considerably [67][68][69][70]. DTT only partially protected against PDHC inhibition, suggesting that tyrosine nitration is the primary mechanism of inactivation.…”

CD40 Ligand–Dependent Tyrosine Nitration of Prostacyclin Synthase In Vivo

“…32,33 Our previous studies 20,21 demonstrated that ONOO Ϫ selectively nitrates tyrosine 450, a residue situated in the active site of PGIS. To assay whether CD40L inhibits PGIS by tyrosine nitration of the enzyme, PGIS proteins were first immunoprecipitated with a polyclonal antibody against PGIS and were further stained for a monoclonal antibody against 3-NT in Western blots.…”

“…15,16 Using normal aortic vessel strips, we have subsequently shown that ONOO Ϫ exposure leads to nitration of PGIS, associated with impaired PGI 2 production, and a defective vasorelaxation. 19 The underlying mechanism for PGIS blockade has been suggested as the nitration of tyrosine 430 at the active site 20 through a reaction catalyzed by the ferric iron of this heme-thiolate (P450) protein. 21 In spite of the cellular antioxidant potential, ONOO Ϫ causes PGIS nitration in whole cells 13,22 and in intact coronary arteries.…”

Statistical evaluation of electrospray tandem mass spectra for optimized peptide fragmentation

“…Tandem mass spectra acquired on quadrupole-time of flight instruments were averaged for at least one minute, whereas at least five scans were averaged on the triple quadrupole instrument, depending on the peptide concentration. FT-ICR tandem mass spectra were obtained by collecting 16 single scans using experimental conditions as described [26]. For the calibration plot, potential differences were stepped by 2 V with an acquisition time of 15 s per increment for each of the 22 precursor ions.…”