title: Oxygen free radicals enhance the nitric oxide-induced covalent NAD(+)-linkage to neuronal glyceraldehyde-3-phosphate dehydrogenase.
abstract: Nitric oxide (NO) induces a covalent modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from various tissues. This phenomenon, which has previously been interpreted as an auto-ADP-ribosylation, is in fact a covalent binding of NAD+ to the enzyme. In the present study, we show that 3-morpholino-sydnonimine (SIN-1) is much more efficient than sodium nitroprusside (SNP) in stimulating the covalent labelling of GAPDH from cultured striatal neurones in the presence of [adenylate-32P]NAD+ (877 +/- 110 and 266 +/- 33% increase in NAD(+)-labelling induced by maximally effective concentrations of SIN-1 and SNP respectively). The difference in the efficacy of both NO-generating compounds could be due to the additional release of superoxide by SIN-1, since superoxide dismutase and the nitrone 5,5'-dimethyl pyrroline-1-oxide markedly inhibited the SIN-1-induced covalent binding of NAD+ to GAPDH. Catalase and selective scavengers of hydroxyl radicals, mannitol and dimethyl sulphoxide, did not alter the SIN-1-induced covalent modification of GAPDH, ruling out the involvement of hydroxyl radicals in this phenomenon. Supporting further a role of oxygen free radicals in the NAD+ linkage to GAPDH, pyrogallol, a superoxide generator, which alone was ineffective, potentiated the SNP-evoked response. The NAD+ linkage to neuronal GAPDH measured in the presence of NO and superoxide probably involves sulphydryl groups, since the radiolabelling of the protein was reversed by exposure to HgCl2 and prevented by pretreatment with the alkylating agent N-ethylmaleimide. Moreover, the NO-induced inhibition of GAPDH activity was enhanced by pyrogallol, which was ineffective alone. In conclusion, the present study indicates that superoxide anions potentiate NO-induced covalent NAD(+)-linkage to GAPDH and enzyme inactivation.
authors.affiliation: INSERM U114, Collège de France, Paris.
authors.forename: P
authors.initials: P
authors.lastname: Marin
authors.forename: M
authors.initials: M
authors.lastname: Maus
authors.forename: J
authors.initials: J
authors.lastname: Bockaert
authors.forename: J
authors.initials: J
authors.lastname: Glowinski
authors.forename: J
authors.initials: J
authors.lastname: Prémont
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D005609:Free Radicals; D005978:Glutathione / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009243:NAD / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology* / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D009599:Nitroprusside / Q000494:pharmacology; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D013481:Superoxides / Q000378:metabolism
chemical_list: D005609:Free Radicals; D009243:NAD; D013481:Superoxides; D009599:Nitroprusside; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D005978:Glutathione; D003545:Cysteine; D010100:Oxygen; D004229:Dithiothreitol