Vol. 253, No. 8, Issue of April 25, pp. 2604-2614, 1978 Printed in U.S.A.

Regulation, Purification, and Properties of Xanthine Dehydrogenase in Neurospora crassa*

(Received for publication, August 25, 1977)

ELLEN SUE LYON AND REGINALD H. GARRETT

From the Department of Biology, University of Virginia, Charlottesville, Virginia 22901

Xanthine dehydrogenase (EC 1.2.1.37) is the first enzyme in the degradative pathway by which fungi convert purines to ammonia. In uiuo, the activity is induced 6-fold by growth in uric acid. Hypoxanthine, xanthine, adenine, or guanine also induce enzyme activity but to a lesser degree. Immu- noelectrophoresis using monospecific antibodies prepared against Neurospora crassa xanthine dehydrogenase shows that the induced increase in enzyme activity results from increased numbers of xanthine dehydrogenase molecules, presumably arising from de nouo enzyme synthesis.

Xanthine dehydrogenase has been purified to homogene- ity by conventional methods followed by immunoabsorption to monospecific antibodies coupled to Sepharose 6B. Elec- trophoresis of purified xanthine dehydrogenase reveals a single protein band which also exhibits enzyme activity. The average specific activity of purified enzyme is 140 nmol of isoxanthopterine producedlminlmg. Xanthine dehydrogen- ase activity is substrate-inhibited by xanthine (0.14 mM), hypoxanthine (0.3 mM), and pterine (10 FM), is only slightly affected by metal binding agents such as KCN (6 mM), but is strongly inhibited by sulfhydryl reagents such as p-hydrox- ymercuribenzoate (2 pm). The molecular weight of xanthine dehydrogenase is 357,000 as calculated from a sedimentation coefficient of 11.8 S and a Stokes radius of 6.37 nm. Sodium dodecyl sulfate-gel electrophoresis of the enzyme reveals a single protein band having a molecular weight of 155,000. So the xanthine dehydrogenase protein appears to be a dimer. In contrast to xanthine dehydrogenases from animal sources which typically possess as prosthetic groups 2 FAD molecules, 2 molybdenum atoms, 8 atoms of iron, and 8 acid-labile sulfides, the Neurospora enzyme contains 2 FAD molecules, 1 molybdenum atom, 12 atoms of iron, and 14 eq of labile sulfide/molecule. The absorption spectrum of the enzyme shows maxima between 400 and 500 nm typical of a non-heme iron-containing flavoprotein.

The biodegradation of purines in numerous microorganisms results in the production of ammonia. In most fungi, this catabolic process is accomplished in a six-enzyme pathway as follows: hypoxanthine and xanthine + uric acid + allantoin + allantoic acid + ureidoglycollate + urea + ammonia (1, 2). In Aspergillus nidulans, Scazzocchio et al. have found that

* This research was supported in part by National Institute of Health Grants TI GM 01450 and GM 22738. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby &&ed “adue&ement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

purine degradation is regulated via the induction of the first four enzymes by uric acid (3). In Neurospora crassa, uricase and allantoicase, the second and fourth enzymes in the path- way, are known to be uric acid-inducible (4). Additional pathway control via ammonia repression has been proposed in A. nidulans and N. crassa on the basis of genetic evidence (5, 6).

Enzymes which catalyze the oxidation of xanthine have been studied in a variety of organisms, including bacteria, insects, birds, and mammals (7-10). The enzyme from bovine milk has been most extensively investigated (11, 12). Xan- thine-oxidizing enzymes exhibit broad substrate specificity. In addition to xanthine and hypoxanthine, other purines, pyrim- idines, pterins, aldehydes, and NADH are oxidized by the enzyme. The enzyme from different biological sources varies in its preferred electron acceptor. The xanthine-oxidizing enzyme from mammalian sources exists in two forms: one which utilizes NAD+ as a primary electron acceptor and the other which uses oxygen most efficiently. The NAD+-depend- ent form of the enzyme is thought to predominate in vivo (13, 14). Enzyme isolated from avian tissues utilizes NAD+ solely (9). The bacterial enzyme from Veillonella alcalescens reduces ferredoxin but xanthine-oxidizing enzymes from other bacte- ria utiiize oxygen or NAD+ as electron acceptors (7).

The protein from all sources reported is a complex contain- ing FAD, molybdenum, iron, and acid-labile sulfide as pros- thetic groups. In the enzyme from mammalian and avian sources, these groups are known to exist in a molar ratio of 1:1:4:4 (9, 12). Olson et al. (15) have suggested that the iron- sulfur centers of bovine milk xanthine oxidase serve as elec- tron sinks which connect the reductive molybdenum sites and the oxidative flavin sites within the enzyme. The relative reduction potentials of the electron carriers in the enzyme, FAD 2 Fe. S 2 MO, then dictate the transport of electrons within xanthine oxidase.

This paper presents studies on the xanthine dehydrogenase of N. crassa. The fungal enzyme has been purified to electro- phoretic homogeneity using conventional methods followed by immunoabsorption. Many physical and chemical parameters of this xanthine dehydrogenase have been characterized and compared to those of xanthine dehydrogenase from other biological sources. The regulation of xanthine dehydrogenase levels as a function of nitrogen source has been examined. Immunological techniques were used to determine that vary- ing levels of enzyme activity corresponded to varying amounts of enzyme present in the mycelia. A preliminary account of this work has been presented (16).

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Xanthine Dehydrogenase in Neurospora crassa 2605

EXPERIMENTAL PROCEDURES Prosthetic Group Determinations

Growth Conditions

Neurospora cmssa (wild type STA4) was cultured as previously described (17). Large scale growth for enzyme purification was carried out for 16 to 18 h following inoculation of 150 liters of liquid medium in a Biotec fermentor (capacity, 300 liters). The medium was modified so that 10 mr.r uric acid was the sole nitrogen source. The fermentor was maintained at 26°C under forced aeration of 8 cubic feet of airlmin. The mycelia were harvested by filtration, washed thoroughly with cold distilled water, squeezed dry in a cider press, and frozen in liquid nitrogen. Mycelia were stored at -70°C for up to 6 months before use. The average yield of a fermentor run was 2 kg mycelia wet weight.

Flauin Composition-The flavin composition of purified enzyme was determined fluorometrically using the method described by Faeder and Siegel (20). Samples contained 5 to 10 pg of protein.

Molybdenum and Iron Content-The molybdenum and iron con- tent of the protein was measured on a Perkins-Elmer atomic absorp- tion spectrophotometer (model 370) with a graphite furnace HGA 2100 attachment. A deuterium arc lamp was used for background correction. Protein samples containing 100 to 250 Fg of enzyme/ml were dialyzed overnight against deionized water. A sample volume of 20 pl was used for all determinations.

1. Molybdenum. A wavelength of 313.3 nm and a slit of 0.2 nm were used. All samples were dried for 20 s at llO”C, charred for 60 s at 17OO”C, and atomized for 20 s at 2800°C.

For in uivo regulation experiments, mycelia were grown in a 2.8- liter Fernbach flask containing 800 ml of Fries basal medium plus 80 rnM ammonium chloride, filtered in a Buchner funnel, and washed thoroughly with distilled water. Aliquots of mycelia weighing 1.5 g were placed in 500 ml Erlenmeyer flasks containing 100 ml of Fries basal medium plus an appropriate nitrogen source. The flasks were incubated in the dark at 25°C on a reciprocating shaker. After various time periods, the mycelia were harvested by filtration and thoroughly washed with distilled water. One-gram samples of these mycelia were frozen and stored in plastic bags at -70°C until used.

2. Iron. Samples were measured at a wavelength of 248.3 nm with a slit width of 0.2 nm. All samples were dried for 20 s at llO”C, charred for 60 s at llOo”C, and atomized for 15 s at 2600°C.

Labile Sulfide Content- The labile sulfide content of xanthine dehydrogenase was assayed by measuring methylene blue formation at 670 nm as described by King and Morris (21) and Siegel et al. (22). All protein samples were dialyzed overnight against deionized water. Samples contained 150 to 250 pg of protein.

Amino Acid Analysis

The frozen mycelia were homogenized in 3 volumes of 0.1 M phosphate buffer, pH 8 per g of mycelia with a Ten Broeck tissue grinder. This crude homogenate was centrifuged at 20,000 x g for 20 min and the supernatant, designated as the crude extract, was assayed for xanthine dehydrogenase activity by the fluorometric procedure (see below). When purines were used as the nitrogen source, the crude extracts were passed over Sephadex G-25 PD columns to remove any endogenous purine. Purines are effective in vitro inhibitors of xanthine dehydrogenase activity.

Amino acid analysis of purified xanthine dehydrogenase was performed on a Beckman automatic amino acid analyzer model 120C. Protein samples containing 100 pg of enzyme were hydrolyzed in equal volumes of 6 N hydrochloric acid for 24, 48, or 72 h. Twenty- microgram aliquots were applied to the columns for analysis. Cys- teine was determined as cysteic acid on the automatic analyzer following oxidation of the protein (50 pg) with performic acid. Tryptophan was measured spectrophotometrically according to the method of Edelhoch (23), following removal of interfering prosthetic groups.

Standard Enzyme Assay Procedures

Three procedures were used to assay xanthine dehydrogenase Immunological Techniques

activity. Serum Preparation

Xanthine Dehydrogenase Assay

The oxidation of xanthine or hypoxanthine can be assayed by measuring the concomitant reduction of NAD+ to NADH spectropho- tometrically (9). The assay was performed in 0.1 M phosphate buffer, pH 8, containing 20 PM NAD+ and 10 PM xanthine or hypoxanthine. A Gilford spectrophotometer (model 2400) was used to record the rate of absorbance increase at 340 nm. One unit of activity is defined as the reduction of 1 nmol of NAD+/min.

Fluorometric Xunthine Dehydrogenase Assay

The routine and most sensitive assay for xanthine dehydrogenase was an adaptation of the fluorometric assay described by Glassman and Mitchell (18) which measures the production of isoxanthopterin as 2-amino-4-hydroxypteridine is oxidized by the enzyme. The reac- tion mixture consisted of 1 ml of 0.1 M phosphate buffer, pH 8, containing 10 nmol of 2-amino-4-hydroxypteridine, 200 nmol of NAD+, and 5 to 100 pl of enzyme. The increase in fluorescence was measured with an Aminco-Bowman spectrofluorometer with the excitation wavelength set at 332 nm and the emission wavelength at 412 nm. One unit of activity is defined as the formation of 1 nmol of isoxanthopterin/min.

Antibodies against N. cmssa xanthine dehydrogenase were made by injecting each of two New Zealand White rabbits (3 kg) with 50 to 100 pg of xanthine dehydrogenase protein emulsified in 1 ml of complete Freund’s adjuvant. The protein was prepared by electro- phoresis of xanthine dehydrogenase preparations (Fraction 6, Seph- arose 4B eluatesl on 5% polyacrylamide gels, staining for enzyme activity, and slicing the gel to collect the active protein band. Three weeks later the rabbits received a booster containing 50 to 100 pg of protein emulsified in incomplete Freund’s adjuvant. Thirty millili- ters of blood were obtained from each rabbit 6 days later. The immunoglobulins were purified according to the procedure of Harboe and Ingild (24).

Preparation of Anti-xanthine Dehydrogenase Sepharose 6B

An immunoabsorption matrix was prepared by linking the puri- fied anti-xanthine dehydrogenase immunoglobulins to cyanogen bromide-activated Sepharose 6B (25). The purified immunoglobulins in 50 ml ofcoupling buffer (0.1 M phosphate (pH 7),0.5 M NaCl) were mixed with the activated gel (1 ml of gel/mg of purified immuno- globulin), gently stirred at 4°C overnight, and washed as described (2.9.

Diaphorase Xanthine Dehydrogenase Assay Immunoelectrophoresis

The diaphorase activity of xanthine dehydrogenase was measured by assaying the reduction of 2,6-dichloroindophenol in the presence of NADH (9). The assay was performed in 0.1 M phosphate buffer, pH 8, containing 0.25 mM NADH and 0.1 mM 2,6-dichloroindophenol. The reduction of 2,6-dichloroindophenol was followed at 600 nm using the Gilford spectrophotometer. One unit of activity is defined as the reduction of 1 pmol of 2,6-dichloroindophenol/min.

Other Assay Procedures

Protein Determination

The protein content of samples was measured by the method of Lowry et al. (19). Bovine serum albumin was used as the standard protein.

Immunoelectrophoresis was performed as described by Weeke (26) with a few modifications. The electrophoresis was done on glass plates (9 x 10 cm) layered with 0.8% agarose, 1 to 2 mm in thickness. A running buffer of 100 m&f Tris, 25 mM maleic acid, pH 8.6, was used. Paper wicks were used to bridge the plates to the buffer reservoir. When crude extracts of N. crassa were used as antigen, 1.0% Triton was added to the agarose. Antibody concentration varied from 1% when purified enzyme was used as the antigen to 0.02% when crude extracts were used. In general, plates were run over- night at less than 0.5 V/cm at 4°C. Gels were stained for protein with Coomassie blue R-250 and for xanthine dehydrogenase activity with a mixture of 10 mM hypoxanthine and 3 mM p-iodonitrotetra- zolium violet or nitro blue tetrazolium in 0.1 M phosphate buffer, pH 8. Enzymatic reduction of the tetrazolium dye produces a colored band where the enzyme is located.

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2606 Xanthine Dehydrogenase in Neurospora crassa

Materials

The following substances were purchased from the Sigma Chemi- cal Co.: NAD, NADH, pterine, hypoxanthine, xanthine, adenine, guanine, FAD, FMN, phosphorylase a, p-galactosidase, RNA po- lymerase, p-iodonitrotetrazolium violet, p-hydroxymercuribenzoate, iodoacetamide, blue dextran, streptomycin sulfate, and Z-mercapto- ethanol. Ammonium sulfate (special enzyme grade), ultrapure urea, and guanidine hydrochloride were products from Schwarz/Mann. Catalase was obtained from Worthington Biochemical Corp. Bio- Rad Laboratories supplied the reagents for the preparation of polyacrylamide gels, sodium dodecyl sulfate and agarose powder. Freund’s adiuvant (comolete and incomulete) was suuDlied bv Difco Laboratoriei. Salts’weri reagent grade’products of [hi J. T.“Baker Chemical Co. DEAE-cellulose (Whatman DE521 was supplied by H. Reeve Angel. Sepharose 4B, Sepharose 6B, and Se&adex were products of Pharmacia.

RESULTS

Regulation in Vivo

Xanthine dehydrogenase activity was measured in crude extracts of N. crassa grown on Fries basal medium containing 80 mM ammonium chloride as a nitrogen source and then transferred to other nitrogen sources for various periods of time. When 7 mM uric acid was used as a nitrogen source, a 6- fold rise in activity was seen. The activity level typically peaked 10 to 11 h after transfer. The transfer of mycelia to medium containing 80 mM sodium nitrate or medium contain- ing no nitrogen source produced no such increase. A low level of xanthine dehydrogenase activity, approximately 0.15 unit/ mg, was present in all mycelia grown on these nitrogen sources and thus appears to define a constitutive level of this enzyme. The purines adenine, guanine, hypoxanthine, or xanthine at a concentration of 7 mM also induced a rise in xanthine dehydrogenase activity after 10 h of induction (Fig. 1). The Itivel of enzyme activity was 3-fold higher than uninduced levels, or about one-half the level of enzyme activ- ity found in uric acid-induced cells.

In order to determine that the increased xanthine dehydro- genase activity corresponded to increased numbers of enzyme molecules, immunoelectrophoresis was performed. Antibodies prepared against N. crassa xanthine dehydrogenase were shown to be monospecific by two-dimensional crossed immu- noelectrophoresis (26). The quantitative comparisons then were made employing the xanthine dehydrogenase-specific antibodies in a one-dimensional rocket immunoelectrophoresis system where the rocket height is directly proportional to the amount of antigen present (26). Fig. 2 shows that rocket height and specific activity of crude extracts from cells induced in various nitrogen sources do correspond.

The purine analogues 2- or 6-thioxanthine, Gazaxanthine, and &azahypoxanthine were tested for their ability to induce xanthine dehydrogenase. Although these analogues are oxi- dized by the enzyme, they cannot serve as nitrogen sources for growth of 1%‘. crassa. Therefore, 20 mM sodium nitrate was also added to the medium. These analogues did induce enzyme activity to an intermediate level as seen in Fig. 2B. Inclusion of 20 mM nitrate in 7 mM uric acid medium did not lessen the levels of enzyme activity seen in mycelia induced in uric acid alone. In all cells, the level of enzyme activity corresponded to the immunoprecipitable enzyme present. Thus, the increased levels of xanthine dehydrogenase enzyme activity observed upon induction are directly related to changes in the number of xanthine dehydrogenase molecules present.

The addition of cycloheximide (10 pg/ml) to the inducing uric acid medium at the time of transfer blocks the increase in xanthine dehydrogenase activity seen in uric acid-induced

I I I I I

I I 1 I I I 2 4 6 8 10

HOURS

FIG. 1. Induction of xanthine dehydrogenase activity by various nitrogen sources. Wild type cells were grown in NH,+/Fries medium for 36 h and transferred to medium containing 7 pM uric acid (O--O), 80 mM NOs- (X-X), 80 rnM NH,+ (LO), or 7 m&f xanthine, hypoxanthine, adenine, or guanine (m----m). Cells were harvested after 2, 4, 6, and 10 h. Crude extracts were prepared as described under “Experimental Procedures.” Enzyme activity was measured fluorometrically and is expressed as units/mg.

cells. When cycloheximide is added after 6 h of induction in uric acid medium, enzyme activity at 10 h after transfer was only 30% of the normal induced levels. Thus, the rise in xanthine dehydrogenase activity which results upon growth in uric acid medium is arr%sted when protein synthesis is blocked by cycloheximide.

Mycelia grown on 80 mM ammonium showed low levels of xanthine dehydrogenase activity. In addition, cells induced in medium containing 40 mM ammonium plus 7 mM uric acid showed enzyme levels only slightly above the uninduced levels (Fig. 2B). Transfer of uric acid-induced cells to ammonium- containing medium or to medium containing no nitrogen source produced a slow decrease in enzyme activity. However, 12 h after transfer, the level of xanthine dehydrogenase in the ammonium cultures was still 0.4 unitlmg, or almost four times the uninduced level. Induced mycelia transferred to medium containing other nitrogen sources, such as 10 mM glutamate or 80 mM nitrate, showed the same slow decrease. Enzyme levels in mycelia transferred to fresh uric acid me- dium remained high as long as 12 h. Once formed, the enzyme appears to be relatively stable in uivo and not dramatically affected by exposure of the organism to ammonium.

Purification of Xanthine Dehydrogenase

Frozen N. crassa mycelia (500 to 750 g) were homogenized with 3 liters of cold 0.1 M phosphate buffer, pH 8, containing 5 mM EDTA and 1500 g of acid-washed glass pavement marking beads (3M Company) in a 4-liter capacity stainless steel Waring Blendor for 5 min. (All operations were performed at 0-4°C unless otherwise noted.) The homogenate was centri- fuged at 20,000 x g for 20 min and the resulting supernatant fraction served as the crude extract (Fraction 1).

Streptomycin sulfate treatment was performed to remove nucleic acids by slowly adding 400 ml of 10% streptomycin sulfate to the crude extract. The mixture was stirred for 20 min and centrifuged as above. The streptomycin sulfate super-

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Xanthine Dehydrogenase in Neurospora crassa 2607

A

B

FIG. 2. Induction of xanthine dehydrogenase by various nitrogen sources. Wild type cells grown in NH,+/Fries basal medium for 36 h were transferred for 10 h incubation in medium containing the appropriate nitrogen source. In A, all nitrogen sources were 7 mM; in B, the concentrations were: 80 mM NH,+, 80 rnM NO,-, 1 rnM 6- thioxanthine + 20 rnM NO,-, 1 rnM 8-azaxanthine + 20 mM NO,-, 1 rnM uric acid + 20 rnM NO,-, and 1 mM uric acid + 20 rnM NH,+, respectively. Cells were harvested and crude extracts prepared as described under “Experimental Procedures.” Enzyme activity was assayed fluorometrically and is expressed as units/mg. Enzyme quantity was measured by rocket immunoelectrophoresis in 0.8% agarose containing 0.02% anti-xanthine dehydrogenase antibody and 1.0% Triton. Rocket plates were stained for xanthine dehydro- genase activity by incubation in a 10 rnM hypoxanthine, 3 mM p- iodonitrotetrazolium violet mixture in 0.1 M phosphate buffer, pH 8.

natant (Fraction 2) was brought to 40% ammonium sulfate saturation by the gradual addition of solid ammonium sulfate. The mixture was stirred for 30 min and then centrifuged. The ammonium sulfate concentration of the supernatant was raised to 55% by further addition of solid ammonium sulfate. The mixture was stirred for 30 min and centrifuged. The resulting pellet was resuspended in 150 ml of 0.1 M phosphate buffer, pH 8, containing 5 mM EDTA (Fraction 3) to give the 40 to 55% ammonium sulfate precipitate.

Further removal of nucleic acid was achieved by the addi- tion of polyethylene glycol 6000 (30% w/w) in a proportion of 0.3 ml/ml of enzyme extract. The mixture was stirred for 30 min and centrifuged. The resulting supernatant was dialyzed for 4 h against 15 liters of 10 mM phosphate buffer, pH 8, plus 5 mM EDTA, and then clarified by centrifugation.

The resulting supernatant was applied to a DEAE-cellulose column (6.5 x 9 cm; column bed volume, 300 ml) which had been equilibrated with 10 mM phosphate buffer, pH 8, plus 5 mM EDTA. The column was washed with 500 ml of the equilibration buffer and then eluted with a linear phosphate gradient generated from 1 liter of 10 mM phosphate buffer, pH 8, plus 5 mM EDTA and 1 liter of 200 mM phosphate buffer, pH 8, plus 5 mM EDTA. The active fractions were combined to give Fraction 5 (the pooled DEAE-eluates). Peak activity eluted at approximately 40 mM phosphate.

Fraction 5 was brought to 80% saturation with ammonium sulfate, stirred for 30 min, and centrifuged, and the pellet was resuspended in 2 ml of 0.1 M phosphate buffer, pH 8, plus 5 mM EDTA. This concentrated DEAE-fraction was applied to a Sepharose 4B column (2.5 x 75 cm; column bed volume, 350 ml) which had been equilibrated with 0.1 M phosphate buffer, pH 8, plus 5 mM EDTA. The column was eluted with the same buffer and the active fractions were pooled (Fraction 6).

The pooled Sepharose 4B fractions were treated with am- monium sulfate to 80% saturation, and after 30 min, the resultant precipitate was collected by centrifugation and re- suspended in 1 to 2 ml of 10 mM phosphate buffer, pH 8, plus 5 mM EDTA. An equal volume of 10 mM sodium sulfide in 10 mM phosphate buffer, pH 8, plus 5 mM EDTA was added to this concentrated Sepharose fraction. The mixture was heated at 55°C for 4 min, cooled immediately on ice, and centrifuged for 30 min at 25,000 x g. The resulting supernatant was designated Fraction 7. This heat treatment of the enzyme in the presence of sodium sulfide increases its activity 2- to 3- fold. This increase is presumably due to a restoration of an active persulfide group on the enzyme molecule (27). In six similar purification experiments, the specific activity of Frac- tion 7 ranged from 80 to 140 units/mg.

Fraction 7 was dialyzed against 10 mM phosphate buffer, pH 8, plus 5 mM EDTA for 3 h, and 0.5 ml (4 mg/ml) of the dialyzed enzyme was applied to an anti-xanthine dehydrogen- ase/Sepharose 6B column (0.5 x 11 cm; column bed volume, 2 ml) previously equilibrated with 0.1 M phosphate buffer, pH 8, plus 5 mM EDTA. The column was then closed for at least 1 h to enhance antibody-antigen binding, and then washed with 40 ml of 0.1 M phosphate buffer, pH 8, plus 5 mM EDTA. The enzyme was then eluted from the column with 30 ml of 3 M

potassium thiocyanate in 10 mM phosphate buffer, pH 8, plus 5 mM EDTA. The column was then washed with 10 mM phosphate buffer, pH 8, plus 5 mM EDTA plus 0.02% sodium azide to return it to conditions optimal for storage. The immunoabsorption column was used 15 times over 4 months with no apparent decrease in binding strength.

The enzyme fractions eluted from the immunoabsorption

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2608 Xanthine Dehydrogenase in Neurospora crassa

column were immediately dialyzed against 10 mM phosphate buffer, pH 8, plus 5 mM EDTA since 3 M potassium thiocya- nate inactivates the enzyme. The enzyme was then concen- trated by dialysis against 0.1 M phosphate buffer, pH 8, plus 5 mM EDTA saturated with ammonium sulfate. The dialyzed enzyme was centrifuged and the pellet was resuspended in 10 mM phosphate buffer, pH 8, plus 5 mM EDTA (Fraction 81. The specific activity of Fraction 8 ranged from 90 to 250 units/ mg. A summary of the data from a typical purification procedure is given in Table I.

acrylamide concentrations ranging from 5 to 10%. Only one protein band was visible on all gels. The molecular weight of the xanthine dehydrogenase subunit protein band was deter- mined by co-electrophoresis of xanthine dehydrogenase with standard proteins of known subunit molecular weight. The relative mobility of xanthine dehydrogenase and several stan- dard proteins is seen in Fig. 4. Xanthine dehydrogenase is apparently a dimer of subunits of molecular weight 155,000 gl mol.

The purity of the xanthine dehydrogenase preparation (Fraction 8) was determined by electrophoresis on 5% poly- acrylamide gels at pH 8.3 (28). The gels showed one protein band which also stained for xanthine dehydrogenase activity, as seen in Fig. 3.

Molecular Weight Determinations

Sucrose density gradient centrifugation was performed ac- cording to the method of Martin and Ames (30) to determine a sedimentation coefficient for xanthine dehydrogenase. A lin- ear 15.5% to 33% w/v sucrose density gradient containing 0.1 M phosphate, pH 8, plus 5 mM EDTA was layered with 0.2 ml of xanthine dehydrogenase (concentrated Fraction 6) and 800 units of catalase as a marker. The gradients were centrifuged at 170,000 x g for 24 h in a Beckman ultracentrifuge using a SW 50 rotor. Gradients were fractionated and assayed for enzyme activity. The xanthine dehydrogenase peak migrated slightly ahead of the catalase peak. An average sedimentation coefficient for N. crassa xanthine dehydrogenase is 11.8 S.

The Stokes radius of xanthine dehydrogenase was deter- mined by gel filtration on Sepharose 4B as described by Ackers (31). Gel filtration data are summarized in Table II. The Stokes radius for xanthine dehydrogenase was determined to be 6.37 nm.

The following formula was used to calculate the molecular weight of xanthine dehydrogenase (32):

M = 6vNaS r (1 - Op)

where Avogadro’s number, N = 6 x 10z3 mol-i; viscosity, n = 1.152 (5”C, H20)g/cm-s(102); partial specific volume, fi = 0.725 cm3/g; and density, p = 1 g/ml. The resulting molecular weight for xanthine dehydrogenase is 357,000.

Subunit Structure of Xanthine Dehydrogenase

Homogeneous xanthine dehydrogenase was dissociated by treatment with sodium dodecyl sulfate or guanidine hydro- chloride. The dissociated protein was electrophoresed on poly- acrylamide gels containing sodium dodecyl sulfate and various

A B FIG. 3. Polyacrylamide gel electrophoresis of purified xanthine

dehydrogenase. 5% polyacrylamide gels were prepared as described by Shuster (28). Twenty-five micrograms of Fraction 8 in a volume of 100 pl were applied to each gel. Bromphenol blue, 0.1% w/v, in 10% sucrose was used as a tracker dye. Electrophoresis was per- formed at 4°C for 5 to 7 h at a current of 1 to 5 mA/gel. Gel A was stained for xanthine dehydrogenase activity with 10 mM hypoxan- thine and 3 mM p-iodonitrotetrazolium violet in 0.1 M phosphate buffer, pH 8. Gel B was stained for protein with Coomassie blue G- 250 in 3.5% perchloric acid (29). The lower band in both gels is the bromphenol blue tracker dye.

Fraction

TABLE I

Purification summary for xanthine dehydrogenase Volume Total protein Total activity

ml w units Specific activity

unitslmg Recovery

w

1. Crude extract 2. Streptomycin sulfate supernatant 3. 40 to 55% (NH&SO, precipitate 4. Polyethylene glycol supernatant 5. Pooled DEAE-eluates 6. Pooled Sepharose 4B eluates 7. Heat-treated concentrated Fraction 6

+ 10 mM Na,S

8. Immunoabsorption eluates

3,800 22,800 2,460 0.108 100

4,100 20,500 2,085 0.102 85

165 2,800 1,190 0.425 48

195 2,600 1,120 0.431 46

65 100 720 1.2 29

40 14.5 555 38.3 23

3 12 1,540 128 63

6 1.8 275 152 11

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TABLE II

Calculation of xanthine dehydrogenase stokes radius (a) from Sepharose 4B gel filtration data

The Stokes radius of xanthine dehydrogenase was determined by gel filtration on Sepharose 4B. All samples were chromatographed on a Sepharose 4B column (2.5 x 75 cm; bed volume, 350 ml) equilibrated and eluted with 0.1 M phosphate buffer, pH 8, plus 5 rnM EDTA. The flow rate was 20 ml/h. The void volume of the

column was 125 ml as determined by the elution volume for blue dextran. Blue dextran was chromatographed separately since xan- thine dehydrogenase will bind to it. Blue dextran will bind proteins possessing dinucleotide folds. Catalase and ferritin were used as marker proteins.

Substance and Distribution experiment num- coefficient a:r ratio Known a Calcu- C&U-

ber (K,) (Ref. 31) (Ref. 32) lated r kited a

Xanthine de- hydrogen- ase

1 2 3

Catalase 1 2 3

Ferritin 1 2

3

0.459 0.166 6.27

0.449 0.170 6.44

0.482 0.157 6.40

0.510 0.146 5.2 35.6

0.515 0.144 5.2 36.1

0.550 0.130 5.2 40.0

0.492 0.150 6.0 40.0 0.496 0.151 6.0 39.7

0.499 0.144 6.0 41.6

nm nm nm

Absorption Spectrum

A visible absorption spectrum of homogeneous xanthine dehydrogenase (500 pg/ml) was obtained on a Cary model 14 spectrophotometer equipped with a 0 to 0.1, 0.1 to 0.2 A slide- wire. The resulting spectrum is shown in Fig. 5. Maxima are seen in the region of flavin and non-heme iron absorbance, i.e. from 400 to 460 nm. Absorbance in this region is significantly

bleached upon addition of sodium dithionite (not shown). The ratio of absorbance at 280 and 450 nm was 5.6. The ratio of absorbance at 450 nm to that at 550 nm was 1.8.

Amino Acid Composition of Xanthine Dehydrogenase

The amino acid composition of xanthine dehydrogenase is given in Table III. The partial specific volume of the enzyme was calculated from the amino acid composition by the method of Cohn and Edsall (34) and found to be 0.725 cm” g-‘.

Prosthetic Group Content of Xanthine Dehydrogenase

Flavin - The quantitative fluorescence method of Faeder

and Siegel (201, by taking advantage of the markedly different fluorescent behaviors of FMN and FAD as a function of pH, allows for the separate determination of FMN and FAD concentrations in solutions where both flavins might be found. Using this procedure, measurements of the flavin content of supernatants from boiled homogeneous xanthine dehydrogen- ase yielded a value of 2.0 mol of FAD/m01 of enzyme (Table IV). No FMN was present. Addition of up to 10 mM FAD to the enzyme assay mixtures produced no increase in enzyme activity.

Molybdenum - Atomic absorption measurements on en- zyme dialyzed against distilled water and enzyme previously digested with nitric acid gave an average value of 1.01 mol of molybdenum/mol of enzyme (Table IV). The values obtained with four different preparations were 0.81, 1.47, 0.76, and 0.96

5 .A 5 6 ‘/ 5 RELATIVE MOBILITY

FIG. 4. Subunit molecular weight of xanthine dehydrogenase. Five per cent polyacrylamide gels containing 0.1% sodium dodecyl sulfate were prepared and electrophoresed according to Maize1 (33). Xanthine dehydrogenase (Fraction 81 was co-electrophoresed with myosin, /3-galactosidase, and phosphorylase a. Proteins were disso- ciated by a modification of the method of Maize1 (33) using 1 to 2% sodium dodecyl sulfate and 1% 2-mercaptoethanol. Bromphenol blue, O.l%, in 10% sucrose solution was used as a tracker dye. Gels were stained with Coomassie blue R-250 in 10% acetic acid and 50% methanol and destained electrophoretically in 7.5% acetic acid and 5% methanol solution.

0.07 -

0.06 -

0.05 -

c

2 8 0.04-

2

0.03 -

0.02 -

O.Ol-

0’ I I I I 350 450 550 650

WAVE LENGTH IN nm

FIG. 5. Visible absorption spectrum of purified xanthine dehydro- genase (0.5 mg/ml). The spectrum was recorded using a Cary model 14 spectrophotometer applied with a 0 to 0.1, 0.1 to 0.2 A slidewire.

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2610 Xanthine Dehydrogenase in Neurospora crassa

TABLE III

Amino acid composition of xanthine dehydrogenase

Each value represents the average of three samples hydrolyzed for 24, 48, or 72 h with the exception of serine and threonine, where the values were extrapolated to zero time. Cysteine was determined as cysteic acid following performic oxidation of the protein. Trypto- phan was measured spectrophotometrically (23) in protein samples treated to remove interfering prosthetic groups by boiling the sample for 3 min and chromatographing it on a Sephadex G25 column. The absence of absorption at 450 nm indicated that flavins had been removed from the sample.

Number of resi- dues/hfF = 357,000 % total residues

Alanine 334 9.3 Arginine 128 3.6 Aspartic acid + asparagine 342 9.5 Cysteic acid 140 3.9 Glutamic acid + glutamine 395 11.0

Glycine 381 10.6

Histidine 68 1.9

Isoleucine 164 4.5 Leucine 261 7.2 Lysine 229 6.4 Methionine 77 2.2 Phenylalanine 101 2.8 Proline 159 4.4 Serine 233 6.5 Threonine 253 7.0 Tryptophan 63 1.7 Tyrosine 15 2.1 Valine 202 5.6

Acidic 10.3 Basic 11.9 Aromatic 6.6 Hydrophobic 22.3

TABLE IV

Prosthetic group content of xanthine dehydrogenase Number of enzyme

comwnent Molhol of enzyme preparations exam- . . ined

FAD” 2.00 f 0.10 4 Molybdenumb 1.01 f 0.20 4 Ironb 12.1 f 0.82 3 Sulfide” 14.4 rt 0.82 3

” Measured fluorometrically by method of Faeder and Siegel (20). b Measured on a Perkins-Elmer atomic absorption spectrophotom-

eter (model 370) with a graphite furnace HGA 2100 attachment. c Measured by methylene blue formation as described by King

and Morris (21) and Siegel et al. (22).

mol of molybdenum/mol of enzyme. Standard curves measured with sample addition showed the same slope and gave the same molybdenum concentration as standard curves done without added enzyme samples, indicating a lack of matrix interference by the protein.

Iron - Measurements of the iron content of xanthine dehy- drogenase by atomic absorption spectroscopy on enzyme sam- ples dialyzed against glass-distilled water gave 12.1 mol of ironlmol of enzyme (Table IV). As with the molybdenum measurements, no matrix interference from the sample was found.

Labile Sulfide - Quantitative measurement of labile sulfide in the enzyme gave a value of 14.4 mol of sulfide/m01 of enzyme (Table IV). Labile sulfide and iron are present in the enzyme in approximately a 1:l ratio.

Substrate Affinities and Molecular Activity

The enzymatic rate of NAD+ reduction in the presence of xanthine or hypoxanthine was dependent on the concentration of xanthine or hypoxanthine. The kinetics of this dependence are shown in Fig. 6 as a double reciprocal Lineweaver-Burk plot. Both substances exhibited substrate inhibition. Xanthine inhibited enzyme activity at a concentration greater than 0.13 mM, while hypoxanthine inhibited activity at a concentration greater than 0.3 mM. Substrate inhibition of the enzymatic reaction was also observed in the fluorescent assay when 2 amino-4-hydroxypteridine concentrations were above 10 pM.

Typical Michaelis-Menten kinetics were seen with respect to the other substrates used, including NADH. The apparent K, for NAD+ is 2.2 PM. Table V summarizes the apparent K, values for the different xanthine dehydrogenase activities. The molecular activity of the xanthine dehydrogenase for xanthine oxidation was calculated to be 473 mol of NAD+ reduced/m01 of enzymelmin, or 1 mol of xanthine dehydrogen- ase has an activity equivalent to 7.1 katals. Under optimal conditions, N. crassa thus contains approximately 0.14 nmol of xanthine dehydrogenaselg wet weight or about 5 pg of enzyme/g of total protein.

Inhibitors of Enzyme Activity

Sulfiydryl Reagents - Several sulfhydryl agents were tested for inhibitory effects on xanthine dehydrogenase activ- ity. Arsenite effectively inhibits the oxidation of pterine or xanthine at a concentration of 0.3 mM. Diaphorase activity was unaffected by arsenite up to a concentration of 20 mM. Sodium p-hydroxymercuribenzoate and iodosobenzoate both effectively inhibited pterine and xanthine oxidation. Both inhibitors also affected 2,6-dichloroindophenol reduction, but only at more than lo-fold higher concentrations. The effects of sullhydryl agents are summarized in Table VI.

Metal Binding Agents - Various metal binding agents were tested for their inhibitory effects on xanthine dehydrogenase activity. Sodium azide and thiourea had relatively little effect

10

1

-.l v .l (10‘OM ‘s r’ ‘3

FIG. 6. Lineweaver-Burk plot of the effect of hypoxanthine or xanthine concentration on xanthine dehydrogenase activity. Xan- thine dehydrogenase activity was measured spectrophotometrically by following the change in absorbance at 340 nm. Assay mixtures contained 20 yg of enzyme (concentrated Fraction 6).

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Xanthine Dehydrogenase in Neurospora crassa 2611

TABLE V

Summary of substrate affinities of xanthine dehydrogenase

The Km values were derived from Lineweaver-Burk plots of the data. The data was obtained using an enzyme assay system which contained the second substrate at the fixed concentrations indicated in the footnotes.

Apparent K, values

Substrate Xanthine de- Fluorescent hydrogenase xanthine dehy- Diaphorase

activity drogenase ac- activity tivitv

N-f PJf Electron acceptor

NAD+ 28” 2.26 2,6-Dichloroindophenol 21 /.LM’

Electron donor Hypoxanthine 21” Xanthine 8.2” Pterine 3.5e NADH 6 mMf

u 0.20 rn~ hypoxanthine. b 5.0 mM pterine. c 0.25 rn~ NADH. d 0.15 rn~ NAD+. e 0.1 rn~ NAD+. ’ 70 +LM 2,6-dichloroindophenol.

TABLE VI

Effect of inhibitors on xanthine dehydrogenase activity

Enzymatic activity was measured using the standard assay pro- tocols (see “Experimental Procedures”) with addition of the inhibitor at the concentration shown. Data are expressed as percentage of uninhibited rates. Fraction 6 was used; each assay contained from 2 to 25 pg of enzyme protein. - .-

% inhibition

Inhibitor andti:Tl concentra- Xanthine de- Fluorometric hydrogenase xanthine dehy- Diaphorase

%Stly drogenase as- activity say

p-Hydroxymercuribenzoate 1 PM 73 20 0 2 W 62 0

Iodosobenzoate 5 PM 41 10 ,LM 100 28 0

Sodium azide, 20 rn~ 25 45 0 Thiourea, 20 rn~ 65 54 0 Potassium cvanide. 5 rn~ 35 60

on the oxidation of pterine or xanthine, and did not affect diaphorase activity at all. Relatively high concentrations (5 to 15 mM) of potassium cyanide and o-phenanthroline were also needed to inhibit enzyme activity by 50%. The effects of these inhibitors are also shown in Table VI.

Other Znhibitors - Uric acid, the immediate end product of xanthine oxidation, inhibits fluorometric xanthine dehydro- genase activity at a concentration of 60 PM. Subsequent products of the purine degradation pathway, including allan- toin, urea, and ammonium at a final concentration of 10 mM had no effect on enzyme activity, nor did other nitrogenous compounds such as nitrate, L-glutamine, L-aspartic acid, and L-glutamic acid at this final concentration. The purines, adenine and guanine, were effective enzyme inhibitors at a concentration of 1 PM. Allopurinol, a specific inhibitor of xanthine oxidase activity, inhibited pteridine oxidation at a concentration of 0.3 PM.

NADP+ was tested as an electron acceptor for xanthine, but no reduction was observed. Further, when both NAD+ and NADP+ were present in the assay mixture at a concentration of 0.5 mM, no inhibition of enzyme activity was observed.

DISCUSSION

Regulation of the level of xanthine dehydrogenase activity in uivo is determined by the nitrogen source present in the growth medium. When uric acid is provided enzyme activity levels are maximal; other purines elicit activity to a lesser extent. Enzyme levels in mycelia grown in media containing ammonium, nitrate, or various other non-purine nitrogen sources such as allantoin are only 15% of maximal. This basal level of enzyme activity is seen in all mycelia grown on medium containing a noninducing nitrogen source and so it is assumed to represent constitutive expression. Upon transfer of mycelia from noninducing medium to medium containing purines, a relatively slow rise in xanthine dehydrogenase activity occurs, and maximum levels of enzyme activity are not reached until after 10 h of induction. In contrast, in Aspergillus nidulans, maximal induction of xanthine dehy- drogenase was achieved after 5 h of growth in inducing medium (35).

Mycelia grown in medium containing nitrate and any of several nonmetabolizable purine analogues such as &azaxan- thine or 64hioxanthine show high levels of enzyme activity. Xanthine dehydrogenase in A. nidulans is also induced by these purine analogues (35). Induction by these analogues suggests that it is principally the purine moiety that is recognized by the regulatory elements which govern xanthine dehydrogenase levels.

The rise in enzyme activity in vivo when purines are present in the medium is a direct result of an increase in the amount of enzyme present in the mycelia as shown by immunoelectro- phoresis. Further evidence of the role of protein synthesis in the control of enzyme levels comes from the fact that when cycloheximide is added to the inducing medium, no rise in enzyme activity is seen.

The alternative possibility of activation of enzyme previ- ously synthesized but present in the cells in an inactive form seems unlikely on the basis of immunoelectrophoretic studies. These studies show that xanthine dehydrogenase (Fraction 5) contains the same amount of immunoprecipitable protein when the enzyme is untreated, activated with sodium sulfide, or denatured by boiling. Since inactive and active enzyme are equally antigenic, no increase in immunoprecipitable enzyme would be expected if increased xanthine dehydrogenase activ- ity resulted from the activation of previously synthesized enzyme. Thus, rise in enzyme activity when purines, espe- cially uric acid, are present in the growth media appears to result from de nouo synthesis of the enzyme.

The role of ammonium as a regulatory agent in the purine degradation pathway is complex. Growth of either N. cmssa or A. nidulans on medium containing both uric acid and ammonium fails to fully induce xanthine dehydrogenase activ- ity. This phenomenon could result from an antagonism be- tween ammonium and uric acid transport as well as from ammonium repression of enzyme synthesis. Xanthine dehy- drogenase levels in N. crassa mycelia fully induced in uric acid and then transferred to ammonium medium were rela- tively stable. After 12 h in ammonium medium, the enzyme level was still one-half the fully induced level. Transfer of induced mycelia to medium containing various other nitrogen sources such as nitrate produced the same slow decrease in

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2612 Xanthine Dehydrogenase in Neurospora crassa

activity. Thus, ammonium appears to have no significant influence on xanthine dehydrogenase levels once the enzyme is formed. Induction would appear to be the major regulatory mechanism to control the levels of this enzyme. The constitu- tive level of xanthine dehydrogenase present even in ammo- nia-grown mycelia indicates that negative control of the system if it exists is never total. That is, ammonium does not repress enzyme levels below this constitutive expression. The organism must require a certain amount of the enzyme at all times, suggesting that the xanthine dehydrogenase does not function solely to provide an alternative source of ammoniacal nitrogen. Xanthine dehydrogenase probably plays a role in the balance of the nucleotide pool also.

Uric acid is a better inducer of xanthine dehydrogenase activity in N. crassa than are the other purines, suggesting that it may be the physiological signal for increased synthesis of the enzyme. Scazzocchio et al. (3) have presented genetic evidence to support the idea that uric acid is the true inducer of xanthine dehydrogenase in A. nidulans. The fact that uric acid, the end product of the enzyme reaction, acts as the inducer might be explained through the role of xanthine dehydrogenase in purine salvage. If the enzyme and subse- quently the remainder of the pathway were induced by hypo- xanthine or xanthine, there would be little opportunity for purine salvage. If no purine salvage is needed, large amounts of uric acid will accumulate triggering increased pathway activity. In order for this regulation mechanism to be effective, the conversion of hypoxanthine to uric acid in vivo must be essentially irreversible. The high K,,, for NADH (6 mM) renders the reverse reaction unlikely under physiological conditions.

Scazzocchio et al. (3) have presented evidence for the exis- tence of t,wo immunologically related but distinct xanthine dehydrogenases in A. nidulans. Xanthine dehydrogenase I is inducible by uric acid, whereas xanthine dehydrogenase II is inducible by nicotinate and will use nicotinate as well as xanthine as substrate. Polyacrylamide gel electrophoresis at pH 8.3 and pH 7.0 and immunoelectrophoresis of N. crassa crude extracts from mycelia induced in various nitrogen sources revealed only one species of xanthine dehydrogenase. NO enzyme activity was observed when nicotinate was used as a substrate. The xanthine dehydrogenase of N. crassa dis- cussed here exhibits the same characteristics as the xanthine dehydrogenase I described by Darlington et al. (35). No protein similar to xanthine dehydrogenase II was seen in N. crassa by any techniques. N. crassa would not grow on media contain- ing 10 mM nicotinate and xanthine dehydrogenase activity was not induced by growth on media containing 1 mM nicotin- ate plus 20 mM sodium nitrate.

This xanthine dehydrogenase from N. crassa has been purified to homogeneity, marking the first time that the enzyme has been obtained in a pure form from a eukaryotic organism capable of subsisting on purines as a sole nitrogen source. Conventional procedures, such as ammonium sulfate precipitation, DEAE-ion exchange chromatography, and Sepharose 4B gel filtration chromatography, were used to purify the enzyme approximately 350-fold (Fraction 7). Frac- tion 7 is approximately 90% pure; however, polyacrylamide gel electrophoresis reveals the presence of three to six protein bands. An immunoabsorption matrix, made by linking mono- specific anti-xanthine dehydrogenase antibodies to Sepharose 6B, was used to further purify Fraction 7. Although this final step in the purification procedure sacrificed much in terms of recovery, it produced homogeneous protein. Other affinity

matrices produced by linking blue dextran or purines to Sepharose 6B were not as effective. Although the enzyme used for these studies was made by the procedure outlined in Table I, it is now possible to make homogeneous enzyme from Fraction 3 by immunoabsorption, thus shortening the prepa- ration time to 1 day. Ten per cent of the initial xanthine dehydrogenase activity is recovered in the pure fractions. The average specific activity of purified preparations of N. crassa xanthine dehydrogenase was 150 units/mg when assayed fluorometrically.

The molecular weight of N. crassa xanthine dehydrogenase as calculated from a sedimentation coefficient of 11.8 S, a Stokes radius of 6.37 nm, and a partial specific volume of 0.725 ml/g was 357,000 g/mol. This value is similar to the reported molecular weight of 300,000 glmol for both bovine milk xan- thine oxidase (12) and chicken liver xanthine dehydrogenase (9).

The subunit composition of N. crassa xanthine dehydrogen- ase was determined by polyacrylamide gel electrophoresis under dissociating conditions. Only one protein subunit, hav- ing a molecular weight of 155,000 glmol, was seen. So, the xanthine dehydrogenase apparently exists as a dimer. The purified enzyme from Drosophila melanogaster was shown by Andres (36) to be composed of two subunits of 150,000 g/m01 each.

Amino acid analysis of N. crassa xanthine dehydrogenase reveals that the enzyme is similar in amino acid composition to other dehydrogenases such as horse alcohol dehydrogenase and pig glycerol-3-phosphate dehydrogenase (37). Approxi- mately one-fourth of the amino acids in xanthine dehydrogen- ase are hydrophobic, with another one-fourth of the residues being equally split between the acidic and basic amino acids.

The absorption spectrum of N. crassa xanthine dehydrogen- ase is similar to those recorded for milk xanthine oxidase and chicken liver xanthine dehydrogenase (9,12). All three spectra show maxima between 400 and 460 nm indicative of flavin and non-heme iron components. These absorbances are all typi- cally bleached upon reduction of the enzyme by sodium dithionite.

All xanthine dehydrogenase proteins described so far have contained flavin as a prosthetic group, and the xanthine dehydrogenase of N. crassa is no exception. Fluorometric measurements reveal 2 mol of FAD present/m01 of enzyme. Both milk xanthine oxidase and chicken liver xanthine dehy- drogenase are also reported to contain 2 mol of flavinlmol of enzyme (9, 11).

In addition to flavin, N. crassa xanthine dehydrogenase contains molybdenum, iron, and labile sulfide as prosthetic groups. Atomic absorption measurements revealed an average of 1 g atom molybdenum present/m01 of enzyme, a result which contrasts with the 2 g atoms of molybdenum/mol of enzyme reported for the enzyme from milk and chicken liver (9, 12). Nitrate reductase, the other molybdoflavoprotein in N. crassa, also contains only 1 g atom of molybdenum/mol of enzyme (38).

Measurements of the iron content of the N. crassa xanthine dehydrogenase show 12 g atoms of iron to be present/m01 of enzyme. This is assumed to be present as non-heme iron, since no heme-like chromophore is seen in the visible absorption spectrum, either prior to or following dithionite reduction. Acid-labile sulfide, which is often associated with non-heme iron, was found in the enzyme. Approximately 14 mol of sulfide was measured/m01 of enzyme. It is most likely that the iron and sulfide are associated in iron-sulfur centers each containing 4 iron and 4 sulfur atoms. The enzyme would then

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Xanthine Dehydrogenase in Neurospora crassa 2613

TABLE VII

Source

Properties of zanthine dehydrogenase from various biological sources

Molecular activity Inhibitor (50% I) MOlC!CUlar Physiological Prosthetic groups

weight electron ac- Ct?PtoT NAD+ Pterine FAD

Molybde- ll”lIl Iron Sulfur Arsenite CN-

N. crassa 357,000 NAD+ 413 89 Chicken liver (9) 300,000 NAD+ 1,000 Bovine milk (12) 300,000 O,/NAD+ D. melanogaster (36) 300,000 NAD+ 80 V. alcalescens (40) 250,000 Fd

contain three such iron-sulfur centers. Measurements from milk xanthine oxidase and chicken liver xanthine dehydro- genase show the presence of 8 g atoms of iron and 8 mol of sulIide/mol of enzyme (9, 12). The milk enzyme has been shown to contain two (Fe,.&) centers (39). The remaining 2 mol of sulfide measured can be attributed to the destruction of a persulfide group on the enzyme. The presence of such a group on the enzyme was established by the sulfide activation of partially purified enzyme. A similar situation was found to exist in milk xanthine oxidase (12). It is tempting to speculate that the N. crassa xanthine dehydrogenase possesses a third iron-sulfur center to compensate for its complement of only a single molybdenum atom.

N. crassa xanthine dehydrogenase displays a high affinity for the substrates involved in xanthine oxidation. The appar- ent K,, for NAD+ in this reaction is 28 PM. The enzyme shows no strong preference for xanthine over hypoxanthine, the K, values being 8.2 and 21 PM, respectively.

Xanthine dehydrogenase activity is inhibited by the purine- related substrates at low concentrations. Hypoxanthine and xanthine inhibit activity when present at a concentration of 10 PM. Substrate inhibition by purines and aldehydes was observed with milk xanthine oxidase by Bray (111, but no such inhibition was reported for the chicken liver enzyme (9). An accelerated disappearance of xanthine at high concentrations of xanthine has been reported to occur with the bacterial enzyme (40); however, here xanthine is converted by dismu- tation to both uric acid and hypoxanthine.

Metal-binding agents, such as azide, thiourea, and potas- sium cyanide, only slightly inhibit xanthine dehydrogenase activities in N. crassa, and only at relatively high concentra- tions (5 to 10 mM). Milk xanthine oxidase and chicken liver xanthine dehydrogenase respond to treatment with metal- binding agents in much the same way, with inhibition being observed only at high concentrations (9, 11). Other molybdo- proteins are strongly inhibited by these agents. The nitrate reductase of N. crassa is inhibited effectively by these agents at a concentration of 0.1 mM (17). This difference suggests that the metal moieties of xanthine dehydrogenase are less acces- sible or more completely liganded.

Xanthine dehydrogenase in N. crassa is effectively in- hibited by sulfhydryl reagents such as p-hydroxymercuriben- zoate and iodosobenzoate at concentrations of 5 to 10 PM.

Sulfhydryl reagents also inhibit diaphorase activity but only at more than lo-fold higher concentrations. Sulfhydryl re- agents have been reported to inhibit the enzyme from milk and chicken liver (9, 11).

Uric acid, the immediate end product of the xanthine dehydrogenase reaction, does inhibit enzyme activity 50% at a concentration of 35 PM. Allantoin, urea, and ammonium, further products of purine degradation, have no effect on xanthine dehydrogenase activity. Of the other nitrogenous

2 1 12 14 0.3 5 2 2 8 8 0.1 5 2 2 8 8 1 1

0.5 0.54 0.69 2 >1.5 8 8 0 0

compounds tested, including various amino acids and nitrate, only the purines adenine and guanine inhibited activity.

The purified xanthine dehydrogenase from N. crassa is similar to xanthine oxidizing enzymes from other biological sources in its essential properties (Table VII). The N. crassa enzyme is a large protein having a molecular weight of 357,000 g/mol and exists as a dimer whose subunits have a molecular weight of 155,000 g/mol. The purified enzyme contains 2 mol of FAD, 1 mol of molybdenum, 12 mol of iron, and 14 mol of sulfide/mol of enzyme as prosthetic groups. If the subunits are identical and therefore the enzyme molecule symmetrical, the distribution of the odd numbers of prosthetic groups (one molybdenum and three iron-sulfur centers) must somehow be shared, which would indicate interesting structure and func- tion relationships within this enzyme. In addition to the other analogies, the inhibitory patterns of N. crassa xanthine de- hydrogenase are similar to those found in the enzyme from other biological sources. These observations suggest that the electron transport sequence proposed by Olson et al. (15) for xanthine oxidase would also apply to the N. crassa enzyme. The xanthine dehydrogenase proteins from widely divergent phylogenetic origins are functionally very similar and share structural resemblances also; thus this enzyme appears to have assumed a design so effective as to merit maintenance throughout the evolutionary hierarchy.

Acknowledgment -One of us (E. S. L.) notes with pride the participation of Benjamin B. Lyon in the latter stages of this work.

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E S Lyon and R H GarrettNeurospora crassa.

Regulation, purification, and properties of xanthine dehydrogenase in

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