Specific nitration at tyrosine 430 revealed by high resolution mass spectrometry as basis for redox regulation of bovine prostacyclin synthase.
Treatment of bovine aortic microsomes containing active prostacyclin synthase (PGI(2) synthase) with increasing concentrations of peroxynitrite (PN) up to 250 microm of PN yielded specific staining of this enzyme on Western blots with antibodies against 3-nitrotyrosine (3-NT), whereas above 500 microm PN staining of additional proteins was also observed. Following treatment of aortic microsomes with 25 microm PN, PGI(2) synthase was about half-maximally 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-Asn-Tyr(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.
Schmidt
Patrick
P
Laboratory of Biological Chemistry, Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
Youhnovski
Nikolay
N
Daiber
Andreas
A
Balan
Alina
A
Arsic
Momo
M
Bachschmid
Markus
M
Przybylski
Michael
M
Ullrich
Volker
V
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000201:enzymology*; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009566:Nitrates / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D021241:Spectrometry, Mass, Electrospray Ionization; D013820:Thermolysin; D014357:Trypsin; D014443:Tyrosine
D009566:Nitrates; D010446:Peptide Fragments; D030421:Peroxynitrous Acid; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; D014357:Trypsin; D013820:Thermolysin; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase