NITRIC OXIDE-DEPENDENT NAD LINKAGE TO GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE - POSSIBLE INVOLVEMENT OF A CYSTEINE THIYL RADICAL INTERMEDIATE

Previous studies have demonstrated that glyceraldehyde-3-phosphate deh ydrogenase (GAPDH) undergoes NAD(H) linkage to an active site thiol wh en it comes into contact with (NO)-N-.-related oxidants. We found that a free-radical generator 2,2'-azobis-(2-amidinopropane) hydrochloride (AAPH), which does not release either (NO)-N-. or (NO)-N-.-related sp ecies, was indeed able to induce the NAD(H) linkage to GAPDH. We perfo rmed spin-trapping studies with purified apo-GAPDH to identify a putat ive thiol intermediate produced by AAPH as well as by (NO)-N-.-related oxidants. As (NO)-N-. sources we used (NO)-N-. gas and two (NO)-N-.-d onors, S-nitroso-N-acetyl-D,L-penicillamine and 3-morpholinosydnonimin e hydrochloride (SIN-1). Because SIN-1 produces (NO)-N-. and a superox ide radical simultaneously, we also tested the effects of peroxynitrit e. All the (NO)-N-.-related oxidants were able to induce the linkage o f NAD(H) to GAPDH and the formation of a protein free-radical identifi ed as a thiyl radical (inhibited by N-ethylmaleimide). (NO)-N-. gas an d the (NO)-N-.-donors required molecular oxygen to induce the formatio n of the GAPDH thiyl radical, suggesting the possible involvement of h igher nitrogen oxides. Thiyl radical formation was decreased by the re constitution of GAPDH with NAD(+). Apo-GAPDH was a strong scavenger of AAPH radicals, but its scavenging ability was decreased when its cyst eine 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) link age to GAPDH is mediated by a thiyl radical intermediate not specific to (NO)-N-. or (NO)-N-.-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 th e NAD(+) molecule are linked to GAPDH.