OXYGEN-FREE RADICALS ENHANCE THE NITRIC OXIDE-INDUCED COVALENT NAD(-LINKAGE TO NEURONAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE())

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 st udy, we show that 3-morpholino-sydnonimine (SIN-1) is much more effici ent than sodium nitroprusside (SNP) in stimulating the covalent labell ing of GAPDH from cultured striatal neurones in the presence of [adeny late-P-32]NAD(+) (877+/-110 and 266+/-33% increase in NAD(+)-labelling induced by maximally effective concentrations of SIN-1 and SNP respec tively). The difference in the efficacy of both NO-generating compound s 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 GAP DH. Catalase and selective scavengers of hydroxyl radicals, mannitol a nd dimethyl sulphoxide, did not alter the SIN-1-induced covalent modif ication of GAPDH, ruling out the involvement of hydroxyl radicals in t his phenomenon. Supporting further a role of oxygen free radicals in t he 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 superoxi de probably involves sulphydryl groups, since the radiolabelling of th e protein was reversed by exposure to HgCl2, and prevented by pretreat ment with the alkylating agent N-ethylmaleimide. Moreover, the NO-indu ced inhibition of GAPDH activity was enhanced by pyrogallol, which was ineffective alone. In conclusion, the present study indicates that su peroxide anions potentiate NO-induced covalent NAD(+)-linkage to GAPDH and enzyme inactivation.