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-morpholinosydnonimine 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 radiolabelled in the nicotinamide or adenine portion revealed that both portions of the NAD+ molecule are linked to GAPDH.

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