ROLE OF NITRIC OXIDE IN THE PATHOGENESIS OF LUNG DISEASE

超氧化物和一氧化氮是多种生物过程中重要的自由基介质。一氧化氮是支气管扩张和血管舒缩张力的重要调节剂。超氧化物是宿主防御的介质。这些药物的作用部分是由于蛋白质的翻译后修饰。NO是亲脂性的，容易通过细胞膜扩散，不仅与血浆和细胞外蛋白相互作用，还与细胞质蛋白相互作用。甘油醛-3-磷酸脱氢酶（GAPDH）是一种丰富的糖酵解酶，具有高活性的活性位点硫醇，可能是细胞内NO的靶标。它通过其活性部位半胱氨酸的亚硝基化被NO灭活，并在一氧化氮和硫醇存在下被NAD或NADH翻译后修饰。在SIN-1 （3- morpholinosydnon亚胺）和二硫苏糖醇存在下，NADH取代NAD使修饰度从~1%提高到25%。因此，可以得出结论，NADH是首选底物，硫醇通过亚硝化反应刺激NADH的共价附着。与这些报道相反，实验室观察表明，NADH与GAPDH的共价附着是在低分子量硫醇存在的情况下进行的，独立于NO。去除氧和过渡金属离子抑制改性，与活性氧的作用一致。超氧化物歧化酶的抑制作用，黄嘌呤氧化酶/次黄嘌呤的刺激作用，以及过氧化氢酶的缺乏作用支持了硫醇氧化产生的超氧化物的假设。电喷雾质谱分析显示NADH分子与GAPDH具有共价键。磷酸二酯酶裂解产物的表征表明，该连锁是通过NADH的烟酰胺发生的。GAPDH的Lys-C酶切、高效液相色谱分离肽段、基质辅助激光解吸电离飞行时间分析和Edman测序表明，NADH附着在活性位点巯基半胱氨酸-149上。该硫醇键对氯化汞稳定。因此，与NAD相反，GAPDH与NADH的连锁发生在硫醇存在的情况下，不依赖于NO，并由超氧化物介导。-一氧化氮，细胞因子，脂多糖-人类受试者

Superoxide and nitric oxide are important free radical mediators of diverse biological processes. NO is an important regulator of bronchodilation and vasomotor tone. Superoxide is a mediator in host defense. The effects of these agents, in part, result from post- translational modification of proteins. NO is lipophilic and diffuses readily through cellular membranes, interacting not only with plasma and extracellular proteins but with cytoplasmic proteins as well. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an abundant glycolytic enzyme with a highly reactive active site thiol, may represent an intracellular target of NO. It is inactivated by NO through nitrosylation of its active-site cysteine and post- translationally modified by NAD or NADH in the presence of nitric oxide species and thiols. Replacement of NAD by NADH in the presence of SIN-1 (3-morpholinosydnonimine) and dithiothreitol increased the degree of modification from ~1% to 25%. It had been concluded, therefore, that NADH was the preferred substrate and thiols stimulated covalent attachment of NADH via a transnitrosation reaction. In contrast to these reports, observations in the laboratory suggested that the covalent attachment of NADH to GAPDH proceeded in the presence of low molecular weight thiols, independent of NO. Removal of oxygen and transition metal ions inhibited modification, consistent with a role for reactive oxygen species. Inhibition by superoxide dismutase, stimulation by xanthine oxidase/hypoxanthine, and the lack of an effect of catalase supported the hypothesis that superoxide, generated from thiol oxidation, was involved. Electrospray mass spectrometry showed covalent linkage of the NADH molecule to GAPDH. Characterization of the product of phosphodiesterase cleavage demonstrated that the linkage occurred through the nicotinamide of NADH. Lys-C digestion of GAPDH, followed by peptide isolation by high performance liquid chromatography, matrix-assisted laser desorption ionization time-of- flight analysis, and Edman sequencing, demonstrated that NADH attachment occurred at cysteine-149, the active site thiol. This thiol linkage was stable to mercuric chloride. Thus, linkage of GAPDH to NADH, in contrast to NAD, occurs in the presence of thiol, is independent of NO and is mediated by superoxide. - nitric oxide, cytokines, lipopolysaccharide - Human Subjects