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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.

SUBMITTER: Marin P

PROVIDER: S-EPMC1135715 | biostudies-other | 1995 Aug

REPOSITORIES: biostudies-other

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Project description:Previous studies have demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol when it comes into contact with .NO-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either .NO or .NO-related species, was indeed able to induce the NAD(H) linkage to GAPDH. We performed spin-trapping studies with purified apo-GAPDH to identify a putative thiol intermediate produced by AAPH as well as by .NO-related oxidants. As .NO sources we used .NO gas and two .NO-donors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydno-nimine hydrochloride (SIN-1). Because SIN-1 produces .NO and a superoxide radical simultaneously, we also tested the effects of peroxynitrite. All the .NO-related oxidants were able to induce the linkage of NAD(H) to GAPDH and the formation of a protein free-radical identified as a thiyl radical (inhibited by N-ethylmaleimide). .NO gas and the .NO-donors required molecular oxygen to induce the formation of the GAPDH thiyl radical, suggesting the possible involvement of higher nitrogen oxides. Thiyl radical formation was decreased by the reconstitution of GAPDH with NAD+. Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cysteine residues were alkylated or when it was reconstituted with NAD+. In addition, after treatment with AAPH, a thiyl radical of GAPDH was trapped at high enzyme concentrations. We suggest that the NAD(H) linkage to GAPDH is mediated by a thiyl radical intermediate not specific to .NO or .NO-related oxidants. The cysteine residue located at the active site of GAPDH (Cys-149) is oxidized by free radicals to a thiyl radical, which reacts with the neighbouring coenzyme to form Cys-NAD(H) linkages. Studies with the NAD+ molecule radio-labelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

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Oxygen free radicals enhance the nitric oxide-induced covalent NAD(+)-linkage to neuronal glyceraldehyde-3-phosphate dehydrogenase.

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