Reversible interconversion between sulfo and desulfo xanthine oxidase in a system containing rhodanese, thiosulfate, and sulfhydryl reagent.

Abstract

The desulfo form of milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2) was reactivated by incubation with rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate, and sulfhydryl reagent; 50% of full aetivity was recovered. No further reactivation occurred with additional incubation. It was also found that native enzyme in-the sulfo form with full activity was inactivated by incubation with. the same system, down' to half of full activity and no further inactivation occurred. After these incubations the enzyme was found to be a mixture of functional and nonfunctional enzymes based on spectral changes with xanthine, on [ The molybdenum-containing enzymes xanthine oxidase (xanthine:oxygen oxidoreductase,. EC 1.2.3.2) (1), xanthine dehydrogenase (2, 3), and aldehyde oxidase (4) can be converted into inactive forms by treatment with cyanide, which results in the release of an essential sulfur atom as thiocyanate. Recent advances in characterization of the desulfo enzyme show that. the. sulfur atom is present as molybdenum sulfide (5-8). The desulfo enzyme is also known to be present in purified enzyme preparations and has been considered to be a preparation or storage artifact (9). However, the enzyme purified from the livers of chickens fed a high-protein diet has a higher specific activity (10) and, furthermore, the molecular activity changes with the diet (11). This suggests that a mechanism-exists in vivo whereby interconversion between the sulfo and desulfo forms may be effected.The successful separation of sulfo and desulfo enzymes by affinity chromatography (12) (13); specific activity was determined to be 2,630 dpm/nmol by measurement of radioactivity and absorbance at 242 nm.Milk xanthine oxidase was purified by the method of Ball (14) with some modifications. Removal of nonfunctional enzyme was achieved by folate affinity chromatography according to the method of Nishino et at (12). Desulfo enzyme was prepared essentially by the method of Massey et at (1); fully active enzyme was treated with 30 mM KCN for 10 min at 250C and was dialyzed extensively against 0.1 M pyrophosphate, pH 8.5/ 0.2 mM EDTA. Xanthine oxidase activity was measured spectrophotometrically at 295 nm in 0.1 mM xanthine/0.1 M pyrophosphate, pH 8.5, at 250C (15). Enzyme concentration was determined from absorbance at 450 nm by using a value of 37,800 M-'cm-' for the molar absorptivity of enzyme-bound FAD (15). Radioactivity was measured in a Packard Tri-Carb liquid scintillation counter (model 2660) and a counting solution of Aquasol-2 (New England Nuclear). Absorbance spectra were recorded on a Cary 17 or Aminco-Chance DW-2a spectrophotometer.The standard reaction 'conditions for activation of desulfo xanthine oxidase or for inactivation of sulfo xanthine oxidase were as follows. Desulfo enzyme (final A4w, 0.2-0.4) with an activity-to-flavin ratio (AFR) of 0-5 prepared by cyanide treatment or native sulfo enzyme (final A40, 0.2-0.4) prepared by folate affinity chromatography was incubated at 370...

Posttranslational inactivation of human xanthine oxidoreductase by oxygen under standard cell culture conditions

“…Molybdopterin forms part of the binding site of the purine substrate, and the essential sulfur atom attached to the molybdenum atom can be replaced by oxygen as a result of treatment of XOR with cyanide (25) or nitric oxide (15). The resulting desulfo-XOR can be reactivated in vitro (25), and molybdenum cofactor sulfurase, the enzyme responsible for reinsertion of the sulfur atom at the active site in vivo, has recently been cloned and characterized in human tissues (14). Modification of the cyanolyzable sulfur at the molybdenum center has been shown to accompany the inactivation of chicken liver XOR by H 2 O 2 (2).…”

The Crystal Structure of Leishmania major 3-Mercaptopyruvate Sulfurtransferase

“…These enzymes are widely distributed in plants, animals, and bacteria (1-3) and have been implicated in a wide range of biological processes. For example, sulfurtransferases may be involved in the formation and maintenance of iron-sulfur clusters in protein (4,5), detoxification of cyanide (6,7), degradation of cysteine (8), biosynthesis of the molybdopterin cofactor of xanthine oxidase (9), selenium metabolism (2,10), and thiamine and 4-thiouridine biosynthesis (11,12). The expression of specific sulfurtransferases is up-regulated under conditions of peroxide or hypo-sulfur stress, osmotic shock, and phage infection (13), suggesting that such enzyme activity is protective of the cell and/or involved in repair processes.…”

Inhibition of Xanthine Oxidase and Xanthine Dehydrogenase by Nitric Oxide

“…Reactivation of NO-treated XO and Desulfo-XO-Both desulfo-XO (AFR ϭ 0) and NO-treated XO (AFR ϭ 4.6) (4.6 M) were reactivated using sodium thiosulfate and rhodanese as previously reported (34).…”