Proc. Nati. Acad. Sci. USA Vol. 80, pp. 1826-1829, April 1983 Biochemistry Reversible interconversion between sulfo and desulfo xanthine oxidase in a system containing rhodanese,.thiosilfate, and sulfhydryl reagent (sulfuration/desulfuration) TAKESHI NISHINO, CHIKAKO USAMI, AND KEIZO TSUSHIMA Department of Biochemistry, Yokohama City University School of Medicine, Minami-ku, Yokohama 232, Japan Communicated by David Shemin,. December 27, 1982 ABSTRACT The desulfo form of milk xanthine oxidase (xan- pendent loss of 50% of the activity. Evidence is given for the- thine:oxygen oxidoreductase, EC 1.2.3.2) was reactivated by in- reversible interconversion between -the sulfo and desulfo.-forms cubation with rhodanese (thiosulfate:cyanide sulfurtransferase, by the rhodanese system. EC 2.8.1.1), thiosulfate, and sulfhydryl reagent; 50% of full ae- tivity was recovered. No further reactivation occurred with ad- ditional incubation. It was also found that native enzyme in-the MATERIALS AND METHODS sulfo form with full activity was inactivated by incubation with. the Rhodanese was obtained from Sigma as a purified powder from same system, down' to half of full activity and no further inacti- bovine liver. Dithiothreitol and reduced glutathione were from vation occurred. After these incubations the enzyme was found to Boehringer Mannheim. [outer-35S]Thiosulfate was obtained from be a mixture of functional and nonfunctional enzymes based on Amersham and was diluted to appropriate concentrations (3,000- spectral changes with xanthine, on ['4C]oxipurinol equilibration, 5,000 cpm/nmol). ['4C]Oxipurinol'was synthesized according and on steady-state kinetics. The 35S of. [35S]thiosulfate was ins to the method of Elion et al. (13); specific activity was deter- corporated into desulfo xanthine oxidase in parallel with an in- mined to be 2,630 dpm/nmol by measurement of radioactivity crease in catalytic activity. Most of the 35S was cyanolysable but, and absorbance at 242 was protected from cyanolysis. by pretreatment with allopurinol. nm. The 'S was released from. asS-labeled reconstituted xanthine ox- Milk xanthine oxidase was purified by the method of Ball idase upon incubation with the rhodanese system containing un- (14) with some modifications. Removal of nonfunctional en- labeled 'thiosulfate. However, catalytic activity remained un, zyme was achieved by folate affinity chromatography according changed, indicating that the sulfur atom was exchanged during the to the method of Nishino et at (12). Desulfo enzyme was pre- incubation. pared essentially by the method of Massey et at (1); fully active enzyme was treated with 30 mM KCN for 10 min at 250C and The molybdenum-containing enzymes xanthine oxidase (xan- was dialyzed extensively against 0.1 M pyrophosphate, pH 8.5/ thine:oxygen oxidoreductase,. EC 1.2.3.2) (1), xanthine dehy- 0.2 mM EDTA. Xanthine oxidase activity was measured spec- drogenase (2, 3), and aldehyde oxidase (4) can be converted into trophotometrically at 295 nm in 0.1 mM xanthine/0.1 M py- inactive forms by treatment with cyanide, which results in the rophosphate, pH 8.5, at 250C (15). Enzyme concentration was release of an essential sulfur atom as thiocyanate. Recent ad- determined from absorbance at 450 nm by using a value of 37,800 vances in characterization of the desulfo enzyme show that. the. M-'cm-' for the molar absorptivity of enzyme-bound FAD (15). sulfur atom is present as molybdenum sulfide (5-8). The de- Radioactivity was measured in a Packard Tri-Carb liquid scin- sulfo enzyme is also known to be present in purified enzyme tillation counter (model 2660) and a counting solution of Aqua- preparations and has been considered to be a preparation or sol-2 (New England Nuclear). Absorbance spectra were re- storage artifact (9). However, the enzyme purified from the liv- corded on a Cary 17 or Aminco-Chance DW-2a spectro- ers of chickens fed a high-protein diet has a higher specific ac- photometer. tivity (10) and, furthermore, the molecular activity changes with The standard reaction 'conditions for activation of desulfo the diet (11). This suggests that a mechanism- exists in vivo xanthine oxidase or for inactivation of sulfo xanthine oxidase whereby interconversion between the sulfo and desulfo forms were as follows. Desulfo enzyme (final A4w, 0.2-0.4) with an may be effected. activity-to-flavin ratio (AFR) of 0-5 prepared by cyanide treat- The successful separation of sulfo and desulfo enzymes by ment or native sulfo enzyme (final A40, 0.2-0.4) with AFR 190- affinity chromatography (12) prompted us to investigate the in- 200 prepared by folate affinity chromatography was incubated terconversion between sulfo and desulfo forms by an enzymatic at 370C in 0.5-4 ml of 0.1 M pyrophosphate, pH 8.5/38 mM system. Na2S203/58 mM dithiothreitol containing 0.14 mg of rho- In the present study, we found that desulfo enzyme could be danese per ml. Samples were dialyzed overnight against 0. 1 M reactivated by incubation with rhodanese (thiosulfate:cyanide pyrophosphate, pH 8.5/0.2 mM EDTA in preparation for ex- sulfurtransferase, EC 2.8.1.1), thiosulfate, and dithiothreitol; periments measuring spectral changes, for [14C]oxipurinol 50% of full activity was obtained, and no further reactivation equilibration, and for kinetic' analyses. Dialysis buffer, was occurred after additional' incubation with the system. Reacti- changed once. For measurement of 'S incorporation, 12 mM vation was. accompanied by the incorporation of a sulfur atom [outer-35S]Na2S203 was used in-the reaction system. After in- into the enzyme molecule. Incubation of fully active sulfo en- cubation, unbound [asS]Na2S2On and asS-labeled rhodanese were with the. same rhodanese resulted in a time-de- separated' from asS-labeled xanthine oxidase by dialysis against zyme system a 2:8 (vol/vol) mixture of 0.1 M pyrophosphate (pH 8.5) and 0.05 M Tris-HClI pH 8.5/0.2 mM EDTA followed by chro- The publication costs of this articlewere defrayed in part by page charge. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: AFR, activity-to-flavin ratio. 1826 Downloaded by guest on September 26, 2021 Biochemistry: Nishino et al. Proc. Natl. Acad. Sci. USA 80 (1983) 1827 matography on a folate affinity column (0.5 x 2 cm). [3S]Sulfo xanthine oxidase was eluted with 0.5 mM hypoxanthine as de- scribed (12) and then freed of hypoxanthine by gel filtration on Sephadex G-25 equilibrated with 0.1 M pyrophosphate, pH 8.5/ 0.2 mM EDTA. During these procedures, no pronounced changes of AFR values were observed. 0, RESULTS Go0.2 Reactivation of Desulfo Xanthine Oxidase and Inactivation of Sulfo Xanthine Oxidase by Incubation with Rhodanese, Thiosulfate, and Dithiothreitol. Desulfo xanthine oxidase pre- 300 400 500 600 pared by treatment of fully active enzyme with cyanide was Wavelength,nm reactivated by incubation with rhodanese, thiosulfate, and di- thiothreitol. In the absence of one of these components no FIG. 2. Absorbance spectra of various xanthine oxidase prepara- reactivation occurred, but dithiothreitol was replaceable by other tions before and after reduction by xanthine under anaerobic condi- sulfhydryl reagents such as mercaptoethanol, cysteine, or re- tions. Reactivated enzyme from desulfo enzyme and partially inacti- were incubation for 3 hr. duced glutathione. The apparent level of reactivation was only vated enzyme from sulfo enzyme prepared by Spectra were recorded before and immediately after mixing with 0.2 about 50% of full activity (AFR, 90-100) (Fig. 1). Further reac- mM xanthine under anaerobic conditions. Concentration differences tivation was not achieved by incubating longer than 3 hr. When were corrected for comparison of spectra of 2, 3, and 4. Curves: 1, reac- sulfo enzyme of AFR 190-200, prepared by folate affinity chro- tivated from desulfo-enzyme (AFR, 107); 2, reduced reactivated en- matography, was incubated with the same rhodanese system, zyme from desulfo enzyme (AFR, 107); 3, reduced inactivated enzyme enzyme activity decreased to about 50%o of full activity and stayed from sulfo enzyme (AFR, 112); 4, reduced native enzyme (AFR, 192). at that level. In the absence of any one of the components of the rhodanese system, no pronounced decrease of the enzyme tivation of sulfo enzyme with the rhodanese system. When these activity occurred during the incubation period. The limited half-active samples were mixed with xanthine under anaerobic reactivation and inactivation of desulfo and sulfo enzymes were conditions, the amount of rapid bleaching in the visible ab- not due to inactivation of rhodanese or to depletion of thio- sorbance spectrum was only half of that of native enzyme with sulfate or dithiothreitol. This was shown by the lack of any fur- full activity (Fig. 2). This result suggested that the low AFR ther pronounced effect when any or all of the system compo- value was due to a mixture of functional and nonfunctional en- nents were added after the activity level had reached its plateau. zymes (15, 16) and was not due to a decrease of molecular ac- Characterization of Rhodanese-Treated Enzyme. Xanthine tivity as in the inhibition of xanthine oxidase by fluorodinitro- oxidase samples with half the AFR value of fully active enzyme benzene (17). were prepared by reactivation of desulfo enzyme or by inac- This was further confirmed by equilibration of the reduced enzyme with [14C]oxipurinol which is known to bind tightly only 200 to functional enzyme (18). Only 0.53 mol of ['4C]oxipurinol per mol FAD of enzyme was bound to half-active enzyme, whereas almost 1 mol of ['4C]oxipurinol was bound to enzyme with AFR = 191 (Table 1). Kinetic analyses of native and rhodanese-treated enzymes, with xanthine as substrate and phenazine methosulfate-linked 150 reduction of cytochrome c as acceptor (18), also confirmed that rhodanese-treated enzymes were a mixture of functional and nonfunctional enzymes (data not shown).