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
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