INACTIVATION OF GLUTATHIONE-PEROXIDASE BY NITRIC-OXIDE - IMPLICATION FOR CYTOTOXICITY

S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, i nactivated bovine glutathione peroxidase (GPx) in a dose- and time-dep endent manner. The IC50 of SNAP for GPx was 2 mu M at 1 h of incubatio n and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-p hosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated, Incubation of the inactivated GPx with 5 mM dithio threitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibl y inactivated the enzyme, The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde -3-phosphate dehydrogenase, suggested that NO released from SNAP modif ied a cysteine-like essential residue on GPx, When U937 cells were inc ubated with 100 mu M SNAP for 1 h, a significant decrease in GPx activ ity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as ju dged by flow cytometric analysis using a peroxide-sensitive dye, Other major antioxidative enzymes, copper/zinc superoxide dismutase, mangan ese superoxide dismutase, and catalase, were not affected by SNAP, whi ch suggested that the increased accumulation of peroxides in SNAP-trea ted cells was due to inhibition of GPx activity by NO. Moreover, stimu lation with lipopolysaccharide significantly decreased intracellular G Px activity in RAW 264.7 cells, and this effect was blocked by NO synt hase inhibitor N-omega-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO, This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.