Inactivation of glutathione peroxidase by nitric oxide. Implication for cytotoxicity.
S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC50 of SNAP for GPx was 2 microM at 1 h of incubation and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly 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 modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 microM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase 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.
Asahi
M
M
Department of Biochemistry, Osaka University Medical School, Japan.
Fujii
J
J
Suzuki
K
K
Seo
H G
HG
Kuzuya
T
T
Hori
M
M
Tada
M
M
Fujii
S
S
Taniguchi
N
N
D000818:Animals; D002417:Cattle; D002460:Cell Line; D002470:Cell Survival; D003600:Cytosol / Q000201:enzymology / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D005979:Glutathione Peroxidase / Q000037:antagonists & inhibitors*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D009569:Nitric Oxide / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D010545:Peroxides / Q000378:metabolism; D026423:S-Nitroso-N-Acetylpenicillamine; D014407:Tumor Cells, Cultured
D008070:Lipopolysaccharides; D010545:Peroxides; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D005979:Glutathione Peroxidase; D010396:Penicillamine; D004229:Dithiothreitol