Schmidt Patrick, Youhnovski Nikolay, Daiber Andreas, Balan Alina, Arsic Momo, Bachschmid Markus, Przybylski Michael, Ullrich Volker
Laboratory of Biological Chemistry, Department of Biology, University of Konstanz, 78457 Konstanz, Germany.
J Biol Chem. 2003 Apr 11;278(15):12813-9. doi: 10.1074/jbc.M208080200. Epub 2003 Jan 31.
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.
用浓度不断增加的过氧亚硝酸盐（PN）处理含有活性前列环素合酶（PGI₂合酶）的牛主动脉微粒体，直至PN浓度达到250微摩尔，在免疫印迹上用抗3-硝基酪氨酸（3-NT）抗体对该酶进行特异性染色，而当PN浓度高于500微摩尔时，还观察到其他蛋白质的染色。用25微摩尔PN处理主动脉微粒体后，PGI₂合酶约有一半被最大程度地硝化，活性约被抑制一半。然后通过凝胶电泳分离该酶，并对其用几种蛋白酶进行蛋白水解消化。用嗜热菌蛋白酶消化24小时产生了一个单一的特异性肽段，通过高效液相色谱分离该肽段，并鉴定为对应于PGI₂合酶第427 - 430位的四肽Leu-Lys-Asn-Tyr(3-硝基)-COOH。通过使用傅里叶变换离子回旋共振-纳电喷雾质谱法进行精确质量测定和埃德曼微量测序确定其结构，并通过真实的酪氨酸硝化肽段的合成和质谱表征进行确认。只有在72小时后才能通过链霉蛋白酶完全消化为3-硝基酪氨酸，这表明硝化的Tyr-430残基可能被紧密折叠在血红素结合位点周围。这些结果为Tyr-430位点的硝化对PGI₂合酶的特异性抑制提供了证据，这种抑制可能在低水平的PN时就已发生，这是由于内皮细胞同时产生一氧化氮和超氧化物的结果。
