THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. Vol. 257, No. 7, Issue of April 10, pp. 3958-3962. 1982 Drosophila melanogaster ma4 Mutants Are Defective in the Sulfuration of Desulfo Mo Hydroxylases* (Received for publication, October 13, 1981) Robert C. Wahl$, Cynthia K. Warnerg, Victoria Finnertyg, and K. V. Rajagopalan$ From the +Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and §Department of Biology, Emory University, Atlanta, Georgia 30322 Xanthine dehydrogenase was purified more than 1500-fold from crude extracts of wild type Drosophila melanogaster. Like the bovine milk and chicken liver enzymes, the purified Drosophila enzyme was inacti- vated by cyanide, and the cyanide-inactivated desulfo enzyme was reactivated by anaerobic incubation with 1 ~ll~ sulfide and 1 mM dithionite. Application of the resulfuration procedure to crude extracts of Drosophila ma-1 flies which show pleiotropic deficiencies of xan- thine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase led to the emergence of xanthine dehydrogen- ase and aldehyde oxidase activities. Representatives of all the five known complementation groups of ma4 mutants were amenable to activation; 59-9596 of wild type xanthine dehydrogenase activity and 1-7% of wild type aldehyde oxidase activity were reconstituted. Ev- idence for the identity of in vitro reconstituted xanthine dehydrogenase from ma-1 mutants with wild type en- zyme is presented. Since the inactive xanthine dehy- drogenase and aldehyde oxidase proteins present in ma4 mutants are identical with the catalytically inac- tive desulfo forms obtained by cyanide treatment of active enzymes, these data constitute evidence for ge- netic control of the incorporation of the cyanolyzable sulfur of Mo hydroxylases. Genetic studies on molybdoenzymes in bacteria (l), fungi (a), and Drosophila (3) have revealed a complex set of events controlling the expression of these enzymatic activities. Of particular importance are the biochemical reactions leading to the formation of their active sites. With the exception of nitrogenase, the active sites of all Mo enzymes contain a pterin cofactor in association with Mo (4). The Mo sites of xanthine dehydrogenase and aldehyde oxidase also contain a unique sulfur moiety which is essential for enzyme activity and is quantitatively converted to thiocyanate in the presence of cyanide (5). This sulfur has been characterized to be an inorganic sulfide ion which is a terminal ligand of the Mo atom andis not linked either to the protein or to the pterin of the Mo cofactor (6). Spontaneous loss of the sulfur appears to occur under a variety of conditions generating a desulfo en- zyme in which the terminal sulfide is replaced by a terminal oxo ligand (7). The desulfo enzyme is unreactive with sub- strates or with inhibitors such as arsenite or alloxanthine, which interact with Lhe Mo center (8). The Mo center of sulfite oxidase, a Mo- and heme-contain- * This work was supported by Grants GM OOO91 and GM 23736 from the National Institutes of Health and by Emory University Research Committee Grant 4360. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. ing enzyme (9), lacks this particularsulfur, since the oxidized form of the enzyme is not subject to cyanide inactivation (10). Extended x-ray absorbance fine structure analysis of chicken liver sulfite oxidase has indicated that the Mo of active sulfite oxidase is liganded to two oxo groups but not to a terminal sulfur (7). The presence of terminal sulfide ligand in xanthine dehydrogenase but not in sulfite oxidase shows that the two enzymes have slightly different Mo centers. In Drosophila melanogaster mutations at three distinct nonstructural loci, ma-1, cin, and Zxd, affect molybdenum hydroxylase activities(3).Studies of the ma-Z locus have revealed several interesting features (11). In addition to xan- thine dehydrogenase, ma-l mutants are also deficient in two other molybdoenzymes, aldehyde oxidase (12) and pyridoxal oxidase (13). Interestingly, ma-l flies contain significant levels of functional molybdenum cofactor capable of reconstituting nitrate reductase activity in extracts of the Neurospora crassa mutant nit-1 (14). In addition, recent studies have shown that ma-Z mutants have wild type levels of sulfite oxidase activity whereas the cin and lxd mutants have severely reduced levels of this enzyme.’ This differential effect of the ma-1 mutation onthese enzyme activities suggested tous a relationship between the ma-l+ gene product and the cyanolyzable sulfur of the Mo hydroxylases. The presence of immunological cross- reacting materialfor xanthine dehydrogenase in ma-1 flies a t levels comparable to wild type hasbeen established (15). The CRM,2 isolated by immunoadsorption, has been reported to contain Mo, Fe, and FAD (16).Aldehyde oxidase CRM is either present in very low amounts or is defective in some manner (15, 16). In order to determine directly whether the inactive CRM in ma-l represents desulfo molecules, we have employed a procedure developed recently for incorporation of the sulfur into desulfo Mo hydroxylases (6). The data so obtained dem- onstrate that the enzymatically inactive xanthine dehydrogen- ase present in ma-l indeed represents the desulfoform of xanthine dehydrogenase. MATERIALS AND METHODS Xanthine Dehydrogenase Purification-Drosophila xanthine de- hydrogenase waspurified from a wild type strainby the procedureof Seybold (17) except that a protamine sulfate treatment was substi- tuted for Norit A treatment, and calcium phosphate gel (18) batch treatment was substituted for hydroxylapatite column chromatogra- Enzyme Assays-All assays were performed at 20-22 “C. An Aminco-Bowman SPF spectrofluorimeter was used for fluorimetric assays. Xanthine dehydrogenase activity was measured by monitoring isoxanthopterin formation (342 nm excitation and 410 nm emission) in a 1.5-ml mixture containing 0.1 M Tris buffer, pH 8, 6.67 p~ pterin, phy. ’ R. Premakumar and K. V. Rajagopalan, unpublished observa- ’’ The abbreviation used is: CRM, cross-reacting material. tions. 3958 by guest on April 12, 2018 http://www.jbc.org/ Downloaded from
Transcript
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Printed in U.S.A. Vol. 257, No. 7, Issue of April 10, pp. 3958-3962. 1982
Drosophila melanogaster ma4 Mutants Are Defective in the Sulfuration of Desulfo Mo Hydroxylases*
(Received for publication, October 13, 1981)
Robert C. Wahl$, Cynthia K. Warnerg, Victoria Finnertyg, and K. V. Rajagopalan$ From the +Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and §Department of Biology, Emory University, Atlanta, Georgia 30322
Xanthine dehydrogenase was purified more than 1500-fold from crude extracts of wild type Drosophila melanogaster. Like the bovine milk and chicken liver enzymes, the purified Drosophila enzyme was inacti- vated b y cyanide, and the cyanide-inactivated desulfo enzyme was reactivated by anaerobic incubation with 1 ~ l l ~ sulfide and 1 mM dithionite. Application of the resulfuration procedure to crude extracts of Drosophila ma-1 flies which show pleiotropic deficiencies of xan- thine dehydrogenase, aldehyde oxidase, and pyridoxal oxidase led to the emergence of xanthine dehydrogen- ase and aldehyde oxidase activities. Representatives of all the five known complementation groups of ma4 mutants were amenable to activation; 59-9596 of wild type xanthine dehydrogenase activity and 1-7% of wild type aldehyde oxidase activity were reconstituted. Ev- idence for the identity of in vitro reconstituted xanthine dehydrogenase from ma-1 mutants with wild type en- zyme is presented. Since the inactive xanthine dehy- drogenase and aldehyde oxidase proteins present in ma4 mutan t s are identical with the catalytically inac- tive desulfo forms obtained by cyanide treatment of active enzymes, these data constitute evidence fo r ge- netic control of the incorporation of the cyanolyzable sulfur of Mo hydroxylases.
Genetic studies on molybdoenzymes in bacteria (l), fungi (a ) , and Drosophila (3) have revealed a complex set of events controlling the expression of these enzymatic activities. Of particular importance are the biochemical reactions leading to the formation of their active sites. With the exception of nitrogenase, the active sites of all Mo enzymes contain a pterin cofactor in association with Mo (4). The Mo sites of xanthine dehydrogenase and aldehyde oxidase also contain a unique sulfur moiety which is essential for enzyme activity and is quantitatively converted to thiocyanate in the presence of cyanide (5). This sulfur has been characterized to be an inorganic sulfide ion which is a terminal ligand of the Mo atom and is not linked either to the protein or to the pterin of the Mo cofactor (6). Spontaneous loss of the sulfur appears to occur under a variety of conditions generating a desulfo en- zyme in which the terminal sulfide is replaced by a terminal oxo ligand (7). The desulfo enzyme is unreactive with sub- strates or with inhibitors such as arsenite or alloxanthine, which interact with Lhe Mo center (8).
The Mo center of sulfite oxidase, a Mo- and heme-contain-
* This work was supported by Grants GM OOO91 and GM 23736 from the National Institutes of Health and by Emory University Research Committee Grant 4360. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.
ing enzyme (9), lacks this particular sulfur, since the oxidized form of the enzyme is not subject to cyanide inactivation (10). Extended x-ray absorbance fine structure analysis of chicken liver sulfite oxidase has indicated that the Mo of active sulfite oxidase is liganded to two oxo groups but not to a terminal sulfur (7). The presence of terminal sulfide ligand in xanthine dehydrogenase but not in sulfite oxidase shows that the two enzymes have slightly different Mo centers.
In Drosophila melanogaster mutations at three distinct nonstructural loci, ma-1, cin, and Zxd, affect molybdenum hydroxylase activities (3). Studies of the ma-Z locus have revealed several interesting features (11). In addition to xan- thine dehydrogenase, ma-l mutants are also deficient in two other molybdoenzymes, aldehyde oxidase (12) and pyridoxal oxidase (13). Interestingly, ma-l flies contain significant levels of functional molybdenum cofactor capable of reconstituting nitrate reductase activity in extracts of the Neurospora crassa mutant nit-1 (14). In addition, recent studies have shown that ma-Z mutants have wild type levels of sulfite oxidase activity whereas the cin and lxd mutants have severely reduced levels of this enzyme.’ This differential effect of the ma-1 mutation on these enzyme activities suggested to us a relationship between the ma-l+ gene product and the cyanolyzable sulfur of the Mo hydroxylases. The presence of immunological cross- reacting material for xanthine dehydrogenase in ma-1 flies a t levels comparable to wild type has been established (15). The CRM,2 isolated by immunoadsorption, has been reported to contain Mo, Fe, and FAD (16). Aldehyde oxidase CRM is either present in very low amounts or is defective in some manner (15, 16).
In order to determine directly whether the inactive CRM in ma-l represents desulfo molecules, we have employed a procedure developed recently for incorporation of the sulfur into desulfo Mo hydroxylases (6). The data so obtained dem- onstrate that the enzymatically inactive xanthine dehydrogen- ase present in ma-l indeed represents the desulfo form of xanthine dehydrogenase.
MATERIALS AND METHODS
Xanthine Dehydrogenase Purification-Drosophila xanthine de- hydrogenase was purified from a wild type strain by the procedure of Seybold (17) except that a protamine sulfate treatment was substi- tuted for Norit A treatment, and calcium phosphate gel (18) batch treatment was substituted for hydroxylapatite column chromatogra-
Enzyme Assays-All assays were performed a t 20-22 “C. An Aminco-Bowman SPF spectrofluorimeter was used for fluorimetric assays. Xanthine dehydrogenase activity was measured by monitoring isoxanthopterin formation (342 nm excitation and 410 nm emission) in a 1.5-ml mixture containing 0.1 M Tris buffer, pH 8, 6.67 p~ pterin,
phy.
’ R. Premakumar and K. V. Rajagopalan, unpublished observa-
’’ The abbreviation used is: CRM, cross-reacting material. tions.
1.33 mM NAD. and up to 10 pl of enzyme. Aldehyde oxidase activity was measured fluorimetrically by monitoringsalicylate formation (302 nm excitation and 410 nm emission) in 1.5 ml of 0.05 M potassium phosphate containing 10 pI of 10 mM salicylaldehyde in 958 ethanol and up to 10 pl of enzyme.
Spectrophotometric assays measuring uric acid or NADH forma- tion were performed using a Cary 14 spectrophotometer as described (10). Acetaldehyde:2,6-dichlorophenolindophenol oxidoreductase ac- tivity was measured as described (12). except that bovine serum albumin was not added to the assay. Buffers contained 0.1 mM EDTA for protein purification and asays.
Preparation of I.:xtmcts-Crude extracts were prepared by ho- mogenizing frozen flies in 5 volumes of cold 0.05 M phosphate, pH 7.8, 0.1 mM EDTA with a motor-driven tissue grinder fitted with a Teflon pestle (Arthur H. Thomas) and centrifuging the homogenates at 27,000 X p for 20 min. The supernatants were chromatographed on Sephadex G-25 in homogenization buffer. The final protein concen- trations were 3-5 mg/ml.
Dithionite-Sulfide Treatment-One-half ml of extract (with 25 p~ methyl viologen, an indicator of reducing conditions) was made anaerobic in a 2-ml Becton-Dickinson Vacutainer by alternate evac- uation and flushing with anaerobic Ar through a 25-gauge needle inserted through the septum. 5 pI of anaerobic 0.1 M dithionite and/ or 1 pI of 0.5 M Na2S were added through the septum with a 10-pl Hamilton syringe, and the mixture was incubated at 37 "C for 30 min. Up to 10 pl was withdrawn for assay through the septum with a 10- pl Hamilton syringe.
Ofher Techniques-Protein was determined according to Lowry et al. (19) after trichloroacetic acid/deoxycholate precipitation of pro- tein (20). Electrophoresis was performed using 57 polyacrylamide gels (21). Gels were stained for xanthine dehydrogenase activity in 0.1 M Tris buffer, pH 8. 0.1 mM EDTA, 0.02'3 hypoxanthine. and 0.0257 nitroblue tetrazolium.
Drosophila Strains-The ma-/ (maroon-like. 1:64.R) mutants, ma- l"', ma-l ' , and ma-/"' have been previously described (12). The ma- /",' and ma-/"' alleles were the gift of Dr. J . Williamson. The ma-l alleles 29, C3. 1, F3, and C2 represent complementation groups I, 111, IV, V, and VI, respectively (15). The cin" strain (cinnamon, 1:O.O) and the /xd strain (leu, xanthine cfehvrfrogena,se, *'3:33) are also described elsewhere (22, 23) . The r y " ' mutant (rosy, 352.0) which contains a lesion in the xanthine dehydrogenase structural gene (24) and the N"' strain (24) which was used as the standard wild type strain for all experiments involving crude extracts were the gift of A. Chovnick. The ry' aldox" Ipo shd' strain (from Dr. W. Dickinson) contains structural gene mutations for xanthine dehydrogenase, al- dehyde oxidase, and pyridoxal oxidase and lacks cross-reacting ma- terial for each of the three enzymes (25). To eliminate the possibility that the presence of different wild tvpe isoalleles for the xanthine dehydrogenase and aldehyde oxidase structural genes would lead to differences in (reconstituted) enzymatic activities, the ma-/ strains, the l xd strain, and the standard wild type strain were made coisogenic (26) for the ry" and alrfox+ (358) loci. The wild type allele o f aldox' was simply that wild tvpe isoallele found on the ry" C ( 3 ) H chromo- some. The wild type flies used for purification of xanthine dehydro- genase (a 1'2 suhline of Oregon-H) were the gift of Dr. A. Mahowald.
RESULTS
Purified Xanthine Dehydrogenase-The absorption spec- t rum of purified Drosophila xanthine dehydrogenase (Fig. 1) is similar to those of Mo hydroxylases from other sources, suggesting the presence of FAD and Fe/S in a 1:4 ratio (26). Acrylamide gel electrophoresis showed a single protein hand (Fig. 1, inset), indicating that xanthine dehydrogenase was the major protein component in the preparation. With NAD as acceptor the purified enzyme showed specific activity of 3.5 pmol min I mg ' for the oxidation of pterin, under the stan- dard conditions, a value near1.v twice that reported by Seybold (17). Molecular oxygen in aerobic buffer was only 5'2 as active as electron acceptor with either pterin or salicylaldehyde as the reducing substrate. The stable nature of the NAD reduc- tase activity of the Drosophila xanthine dehydrogenase is similar to that of the avian enzyme and unlike that of mam- malian enzymes which rapidly undergo dehydrogenase to oxidase conversion (28) . In contrast, the aldehyde oxidase and
3959
FIG. 1. Absorption spectrum of purified Drosophila xan- thine dehydrogenase. A Cary 219 spectrophotometer was used to obtain the spectrum of the enzyme at 50 pg/ml. Inset. polyacrylamide gel electrophoresis o f purified L)rosophi/o xanthine dehydrogenase. The enzyme. 27 pg, was stained with Coomassie brilliant blue.
pyridoxal oxidase of Drosophila appear to he hona fide O2 reductases, since their activities are unaffected by the presence of NAD even in crude extracts of flies.
Allopurinol is a potent inhibitor of xanthine-oxidizing en- zymes (29). Preincubation of the purified Drosophila enzyme with 80 p~ allopurinol completely inhibited the oxidation of all reducing substrates including pterin, salicylaldehyde (6.7 pmol min-l mg" a t 0.17 mM salicylaldehyde), and acetalde- hyde (5.0 pmol min- ' mg I a t 0.05 M acetaldeh.vde). Since aldehyde oxidase also oxidizes these compounds, differential sensitivity of the two enzymes to allopurinol allows quantita- tion of these enzymes in crude extracts when both activities are present. Allopurinol has no measurable effect on the salicylaldehyde to 0, activity of ry or wild type extracts because the salicylaldehyde to 0 2 activity of xanthine dehv- drogenase is much lower than that of aldehyde oxidase.'' However, when the ratio of xanthine dehydrogenase to alde- hyde oxidase is large, as in the reconstituted ma-l mutants, both enzymes contribute to the measured salicylaldehyde to O2 activity, and allopurinol treatment is necessary to accu- rately assay aldehyde oxidase by salicylaldehyde oxidation. Purified Drosophila xanthine dehydrogenase has an activity of less than 10 units/mg in the acetaldehyde to dichloroindo- phenol assay described by Courtright (12), whereas aldehyde oxidase is reported to have an activity of 6250 units/mg (16). This assay is thus quite specific for aldehyde oxidase.
Cyanide Inactivation and Resulfitration o f Xanthine De- hydrogenase and Aldehyde Oxidase in Vitro-Purified I h - sophila xanthine dehydrogenase was progressively inactivated by incubation with 2.5 mM cyanide in 0.1 M Tris-HC1, pH 8, a t room temperature, the half-time of inactivation being 20 min. After cyanide removal, reactivation of the inactivated enzyme requires both sulfide and dithionite (Fig. 2) as previouslv observed with the bovine milk and chicken liver enzymes. The activity of enzyme not treated with CN could also he en- hanced by this treatment, indicating that the purified prepa- ration contained desulfo molecules. The limited ability of dithionite to activate the Mo hydroxylases without the addi- tion of sulfide is ascribed to sulfide generation from the added dithionite (6).
Cyanide inactivation and subsequent activation by dithio- nite/sulfide treatment can also be performed in crude extracts if the extracts are desalted to remove substances which could reduce the Mo enzymes (Table I) . Reduced Mo hydroxylases are refractory to sulfur abstraction by cyanide and instead undergo reversible inactivation by a modification of the Mo center (10). Enhancement of xanthine dehydrogenase and aldehyde oxidase activity upon treatment of Drosophila wild type extract with dithionite and sulfide is ascribable to the presence of desulfo enzymes in the extract. It can be seen that there is essentially complete reconstitution of both xanthine dehydrogenase and aldehyde oxidase activity of cyanide- treated extracts by dithionite/sulfide treatment. Moreover, both sulfide and dithionite are required for optimum activa- tion of xanthine dehydrogenase and aldehyde oxidase in cya- nide-inactivated wild type extracts.
ml in 0.1 M Tris buffer, pH 8,O.l mM EDTA, was reconstituted with sulfide and/or dithionite as described under "Materials and Methods." 0, native enzyme, treated with sulfide and dithio- nite. 0, cyanide-inactivated enzyme. treated with sulfide and dithio- nite. 0, cyanide-inactivated enzyme, treated with sulfide for 20 min; at the arrow, dithionite was added. m. cyanide-inactivated enzyme, treated with dithionite for 20 min; at the arrow, sulfide was added. Cyanide was removed from cyanide-inactivated enzyme by gel filtra- tion before these experiments.
TABLE I Requirement of sulfide and dithionite for Mo hydroxylase activation in ma-1 and cyanide-inactivated wild type crude
extracts Extracts of wild type flies were incubated with 20 mM KCN for 3
h at room temperature. This completely destroyed Mo hydroxylase activity. The cyanide was removed by Sephadex G-25 chromatogra- phy. Wild type. cyanide-treated wild type, and ma-1' extracts were then treated with dithionite and/or sulfide as described under "Materials and Methods."
FIG. 3. Activity stain of xanthine dehydrogenase after 5% polyacrylamide slab gel electrophoresis of wild type, ry, and ma-1 crude extracts. Extracts were prepared and dithionite/sulfide treatment was performed as described under "Materials and Meth- ods." Ilithionite and sulfide were removed by gel filtration before electrophoresis. Lanes are counted from the left. All lanes show the bromphenol blue band at the bottom of the gel. The band at RF = 0.53 is active xanthine dehydrogenase. All extracts were dithionite/ sulfide treated except for the extract electrophoresed in lane 5, which contained untreated ma-1''' extract. The lanes contained: 1, ma-l'.''; 2, ma-1'"; 3, wild type; 4, ry2"; 5, ma-1':' (untreated); 6, ma-1'"; 7, ma-1'; 8, m a - P .
The xanthine dehydrogenase and aldehyde oxidase activi- ties of extracts of various Drosophila Mo hydroxylase mutants were measured in duplicate before and after dithionite/sulfide treatment (Table 11). In accordance with earlier data (3), xanthine dehydrogenase and aldehyde oxidase activities were undetectable in untreated extracts of ma-1, cin, and the ry' aldox double mutant and were present in attenuated levels in lxd. As expected, r y 2 contained normal levels of aldehyde oxidase but no detectable xanthine dehydrogenase. Dithio- nite/sulfide treatment activated xanthine dehydrogenase in ma4 extracts to 59-95% of the levels of dithionite/sulfide- treated wild type extracts, with representatives of all five ma- l complementation groups (15) being amenable to activation. This reconstituted activity was stable even after removal of
FIG. 4. Heat inactivation of xanthine dehydrogenase activ- ity in dithionite/sulfide-treated crude extracts of wild type and ma-1’. Extract preparation and dithionite/sulfide treatment were performed as described under “Materials and Methods.” Dithi- onite and sulfide were removed by gel filtration in phosphate/EDTA buffer. The final protein concentrations of the ma-1 and wild type extracts were 2.6 and 2.5 mg/ml, respectively. 100% activity was 1.5 nmol of pterin:NAD oxidoreductase/min/mg. The extracts were in- cubated at 55 “C, and 10-pl aliquots were removed and immediately assayed at the indicated times. The lines drawn are the linear least squares fit. 0, is ma-1’ extract; 0, is wild type extract.
dithionite and sulfide by Sephadex G-25 chromatography. The activity of the reconstituted dialyzed enzyme was abol- ished by treatment with allopurinol, cyanide, or arsenite. The electrophoretic mobility of reconstituted xanthine dehydro- genase was identical with that of the wild type enzyme on 5% acrylamide gels (Fig. 3). The heat lability of dithionite/sulfide- treated wild type and ma-1‘ xanthine dehydrogenase at 55 “C is shown to be identical in Fig. 4. By these criteria, the in vitro reconstituted xanthine dehydrogenase in ma-1 extracts is iden- tical with wild type enzyme. Xanthine dehydrogenase activity could not be detected in ry or cin fly extracts after dithionite/ sulfide treatment. Since the cin flies have a t least 50% of the wild type levels of xanthine dehydrogenase CRM,4 this dem- onstrates that the cin lesion is different than the ma-1 lesion.
The results for aldehyde oxidase reconstitution were anal- ogous to those for xanthine dehydrogenase except that much lower levels of aldehyde oxidase relative to wild type were reconstituted in the ma-1 mutants. The use of allopurinol allows us to state with confidence that the low levels of aldehyde oxidase observed with dithionite/sulfide-treated ma- l extracts is indeed activation of aldehyde oxidase and not an artifact of assay due to the overlapping substrate specificites of the Mo hydroxylases. Also, the acetaldehyde:dichloro- indophenol assay of aldehyde oxidase was in close agreement with the salicylaldehyde assays.
DISCUSSION
The experiments described here demonstrate that dithio- nite/suKlde treatment restores normal levels of xanthine de- hydrogenase activity in Drosophila ma-l extracts. Several criteria, including heat stability and electrophoretic mobility, show that the reconstituted xanthine dehydrogenase of ma-1 is identical with the wild type enzyme. It is clear from these data that ma4 extracts contain relatively high levels of the inactive desulfo form of xanthine dehydrogenase. Immunolog- ical studies have characterized the CRM in ma4 flies to be indistinguishable from the wild type enzyme whereas the aldehyde oxidase CRM is reported to be defective. These studies provide an explanation for our observation of relatively high levels of xanthine dehydrogenase resulfuration compared to the modest levels found for aldehyde oxidase. This dem- onstration of the generation of enzymatically active xanthine
C. K. Warner, unpublished observations.
dehydrogenase and aldehyde oxidase in ma-1 extracts indi- cates that the mad+- gene product functions in the acquisition of the cyanolyzable sulfur moiety of the Mo centers of xan- thine dehydrogenase and aldehyde oxidase. The absence of this sulfur moiety in sulfite oxidase explains the presence of active sulfite oxidase in ma-1 flies.
Two alternative hypotheses can be put forward for the ma- l’ function. The first possibility assumes that the ma-P locus directs the synthesis of a S = Mo-cofactor complex, which is subsequently inserted into the apoproteins. The existence of desulfo xanthine dehydrogenase in ma-1 mutants is explained by postulating that 0 = Mo-cofactor can be efficiently inserted into the apoprotein of xanthine dehydrogenase in the absence of the S = Mo-cofactor complex. The low levels of aldehyde oxidase CRM could be due to the inability of apoaldehyde oxidase to accept the 0 = Mo-cofactor, the apoprotein being degraded at a much faster rate than cofactor-containing en- zyme.
The second possibility is that the addition of the terminal sulfide atom to desulfo Mo hydroxylase may be the last step in Mo hydroxylase biosynthesis, and desulfo Mo hydroxylase would then be a normal biosynthetic intermediate. The ma-1 mutant would thus mimic a classic metabolic mutant, in that desulfo Mo hydroxylase, the intermediate preceding the de- fective step, is expected to accumulate. This hypothesis sug- gests that all the Mo enzymes (except nitrogenase) could share steps in the incorporation of Mo and the pterin-based Mo cofactor. The biosynthesis of di-oxo Mo enzymes such as sulfite oxidase and nitrate reductase would be complete with the incorporation of Mo and Mo cofactor as a di-oxo complex, but the Mo hydroxylases would require the subsequent addi- tion of the cyanolyzable sulfur. I t is conceivable that a single protein, a sulfur source:cyanolyzable sulfur transferase, could be the product of the ma-l+ gene and could constitute the entire Mo sulfide biosynthetic pathway. The presence of low levels aldehyde oxidase CRM in ma-1 flies is difficult to explain by this hypothesis. In any case, a distinction between these alternatives must await further studies.
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