Xanthine oxidase reaction with nitric oxide and peroxynitrite.
Nitric oxide (.NO) and peroxynitrite (ONOO-) inhibit enzymes that depend on metal cofactors or oxidizable amino acids for activity. Since xanthine oxidase (XO) is a 2(2Fe2S) enzyme having essential sulfhydryl groups linked with Mo-pterin cofactor function, the influence of .NO and ONOO- on purified bovine XO was determined. Physiological (</=1 microM) and supraphysiological (</=100 microM) concentrations of dissolved .NO gas did not inhibit the catalytic activity or alter the spectral characteristics of XO at 25 degreesC and pH 7.0, differing from reports showing XO inhibition by .NO. The apparent decrease in XO activity observed previously was the result of depressed rates of uric acid accumulation in XO assay systems, due to ONOO--mediated oxidation of uric acid upon reaction of residual .NO with XO-derived superoxide (O*-2). Nitric oxide derived from S-nitrosoglutathione also did not inhibit cultured vascular endothelial cell XO activity. In contrast, purified and vascular endothelial cell catalase, a heme enzyme reversibly inhibited by .NO, was inhibited by similar concentrations and rates of production of . NO. In contrast to .NO, ONOO- inhibited XO (0.2 microM, 50 mU/ml) with an IC50 of 57 microM (for 3 microM/min infusion of ONOO-) or 120 microM (for bolus addition of ONOO-). Addition of 1% bovine serum albumin, 50 microM xanthine, or 10 microM uric acid protected XO from inactivation by ONOO-. Thus, in the presence of purine substrates and other more readily oxidized components of the biological milieu, XO should not be inhibited by either .NO or ONOO-. These observations reveal that .NO will not serve as an indirect antioxidant by inhibiting XO-derived production of reactive species and that the XO-derived products O*-2 and uric acid readily modify the reactivities of .NO and ONOO-.
Houston
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Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL 35233-6810, USA.
Chumley
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Freeman
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D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002374:Catalase / Q000037:antagonists & inhibitors; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology; D005609:Free Radicals / Q000378:metabolism; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D066298:In Vitro Techniques; D009566:Nitrates / Q000378:metabolism / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000494:pharmacology; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D026422:S-Nitrosoglutathione; D014527:Uric Acid / Q000378:metabolism; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
D001798:Blood Proteins; D005609:Free Radicals; D009566:Nitrates; D009603:Nitroso Compounds; D016877:Oxidants; D019820:Xanthine; C039642:peroxynitric acid; D014527:Uric Acid; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D002374:Catalase; D014969:Xanthine Oxidase; D005978:Glutathione