STIMULATION BY NITRIC-OXIDE OF AN NAD LINKAGE TO GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate deh ydrogenase (GAPDH) by [adenylate-P-32] NAD has been interpreted in rec ent reports as ADP-ribosylation. Incubations of GAPDH with the NO-rele asing agent sodium nitroprusside (SNP) and NAD resulted, however, in e ssentially equal incorporation of radiolabel from the adenine, phospha te, and nicotinamide moieties to the extent of almost-equal-to 0.02 mo l of NAD.mol of GAPDH-1. Modification of GAPDH by free adenosine 5'-di phosphoribose (ADP-ribose) was only 10% of that by NAD. Exposure of GA PDH modified by NAD in the presence of SNP to HgCl2, Which acts at thi ol linkages, released two products. Both contained nicotinamide and ad enylate but did not cochromatograph with NAD. GAPDH activity was inhib ited by SNP in a dose-dependent manner in the presence of NAD. When in hibition was 80%, with 1 mM SNP and 1 mM dithiothreitol, covalent modi fication with NAD was <2%. This result is consistent with the conclusi on that inhibition of GAPDH activity by SNP in the presence of NAD is due primarily to active-site nitrosylation, as reported by other worke rs, and is not due to the minor modification with NAD. These results d emonstrate that NO-stimulated modification of GAPDH with NAD is not AD P-ribosylation as previously reported but rather is covalent binding o f NAD through a NO-dependent thiol intermediate, possibly providing an example of an unexpected, altered reactivity of a nitrosylated protei n.