Vol. 135, No. 2
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 422-428 0021-9193/78/0135-0422$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Bacterial Xanthine Oxidase from Arthrobacter S-2 C. A. WOOLFOLK* AND J. S. DOWNARDt Department of Molecular Biology and Biochemistry* and Department of Medical Microbiology, University of California, Irvine, California 92717 Received for publication 13 February 1978
Arthrobacter S-2, originally isolated by enrichment on xanthine, produced high levels of xanthine oxidase activity, requiring as little as a 20-fold purification to approach homogeneity with some preparations. Molecular oxygen, ferricyanide, and 2,6-dichlorophenol-indophenol served as electron acceptors, but nicotinamide adenine dinucleotide did not. The enzyme was relatively specific when compared with previously studied xanthine-oxidizing enzymes, but at least one purine was observed to be oxidized at each of the three positions of the purine ring that have been subject to oxidation by this type of enzyme. The enzyme had a relatively high Km for xanthine (1.3 x 10' M), and substrate inhibition was not observed with this compound, in contrast to the enzyme from cow's milk. In fact, an opposite effect was observed, and double-reciprocal plots with xanthine as the variable substrate showed a concave downward deviation at high concentrations. At 2.5 mM xanthine the enzyme had a specific activity approximately 50 times that of the most active preparations of the milk enzyme. The spectrum of the Arthrobacter enzyme resembled that of milk xanthine oxidase, suggesting a similarity of the prosthetic centers of the two enzymes. The bacterial enzyme was relatively small and may be dimeric, with approximate native and subunit molecular weights of 146,000 and 79,000, respectively. As the result of a survey of some 47 diverse strains of bacteria containing xanthine-oxidizing enzymes, we observed that two strains of Arthrobacter gave by far the highest specific activities when ferricyanide was used as the electron acceptor (23). Furtherinore, the Arthrobacter activity was exceptional in that molecular oxygen was relatively efficiently utilized when the xanthine-oxidizing enzymes from the various bacteria were compared under certain standard assay conditions with several electron acceptors. Because pyridine nucleotides were not utilized by the Arthrobacter enzyme, the properties resemble those of milk xanthine oxidase (EC 1.2.3.2), which has been the subject of considerable previous investigation (5, 6). Bacterial xanthine-oxidizing enzymes which have been purified previously (4, 19, 20, 24) utilize molecular oxygen relatively slowly. Ferredoxin may serve as an electron acceptor with the enzymes from anaerobes (20), whereas nicotinamide adenine dinucleotide serves as an effective electron acceptor with the soluble xanthine dehydrogenases from aerobic pseudomonads (19, 23). All of these latter enzymes are appropriately referred to as xanthine dehydrogenases. This paper reports on the purification of a new oxidase and examines t Present address: Department of Microbiology, University of Washington, Seattle, WA 98105. 422
some of its basic features. The smaller size, exceptionally high activity, and relatively restricted specificity pattern heighten our interest in a more detailed comparison with the other bacterial xanthine-oxidizing enzymes and the ex-
tensively studied oxidase from milk. MATERIALS AND METHODS Growth of cells and preparation of extracts. Arthrobacter S-2 originally isolated on xanthine-containing media (23) was routinely grown on 0.1% xanthine-0.01% yeast extract in a 10-fold dilution of our previously described mineral salts base, 1XS (22). The final phosphate concentration of the medium was 0.1 M. Conditions of growth and harvesting, measurement of protein, and preparation of extracts were as reported earlier (22). Enzyme amsays. Xanthine oxidase was routinely assayed by a ferricyanide-linked assay described previously (22, 23). One unit of enzyme is that amount that will reduce 1 iLmol of ferricyanide per min under the conditions of this assay. Full activity was observed under anaerobic conditions (24). Thus, the assay does not depend on the use of molecular oxygen as an electron acceptor followed by nonenzymatic reaction of the uric acid produced with ferricyanide, as has been suggested previously (9). The latter reaction is relatively slow and incomplete at the neutral pH of our assay. The spectrophotometric assays with molecular oxygen as the acceptor and the use of other electron acceptors were also as previously described
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BACTERLAL XANTHINE OXIDASE
(22, 23). The 1XS mineral salts base was used as a buffer with all assays. Purification of Arthrobacter S-2 xanthine oxidase. A representative example of the preparation of the enzyme is shown in Table 1. The crude extract was first treated with streptomycin sulfate to give a 1% (wt/vol) solution; this was followed by centrifugation at 12,000 x g for 20 min. The supernatant was then heated at 50°C for 10 min and recentrifuged to give fraction 2 (Table 1). Precipitation with ammonium sulfate was achieved by addition of the appropriate volume of a saturated solution of ammonium sulfate at 40C. Ammonium sulfate precipitation was repeated several times by suspension of the centrifuged precipitate in 0.O1XS buffer without prior dialysis. For steps 4 and 5 (Table 1), the ammonium sulfate was first added very slowly until turbidity was barely discernible (usually about 33% saturation). The suspension was carefully equilibrated by stirring, and the precipitated material was removed by centrifugation and discarded. The enzyme activity was then precipitated by more rapid addition of ammonium sulfate to 43% saturation. After the ammonium sulfate fractionation, the enzyme was resuspended in 0.O1XS buffer and applied directly to a Sepharose 4B column. The column of Sepharose 4B was set up and eluted in a manner identical to that previously used with an extract of Escherichieae strain A (23). Ninety percent of the activity eluted from the column in the 260- to 305-nil fraction. This was concentrated fivefold by ultrafiltration through Amicon XM50 filters. The enzyme was then precipitated by addition of ammonium sulfate to 50% saturation, and the precipitate was redissolved and dialyzed against 0.O1XS buffer to give fraction 7 (Table 1). Gel electrophoresis. The procedure of Davis (7) was followed for electrophoresis using 7.5% gels. We have previously described details of the staining procedures, the method for visualization of enzyme activity using nitro blue tetrazolium, and the use of the densitometer employed in this laboratory (24). Sodium dodecyl sulfate (SDS) gel electrophoresis was by the method of Weber and Osborn (21). Gel permeation chromatography. Sephadex G150 was used to estimate the molecular weight of the native xanthine oxidase by comparison of its partition Step
1 2
423
coefficient [K.w (V. - V.)/(Vt - V.)] with a standard curve of such valueo obtained by measurement of the enzymatic activity of proteins of known molecular weights (18). The standards, their molecular weights, and the K.,, values obtained were as follows: milk xanthine oxidase, 300,000, Ka,, = 0.0; catalase, 250,000, K.,, = 0.0; yeast alcohol dehydrogenase, 151,000, K.,, = 0.19; hexokinase, 96,000, K,v = 0.37; and hemoglobin, 64,000, Kay = 0.48. =
RESULTS
Purification of the enzyme. A typical purification of the enzyme is shown in Table 1. When the purified enzyme was applied to gels and subjected to electrophoresis, only one main protein band was observed (Fig. 1A, curve 2), accounting for 90% or more of the stain taken up by the gel. This band coincided with the main band of enzyme activity which also could be exhibited on these gels (Fig. 1A, curve 3). The gels displayed a number of minor protein-staining bands (at Re = 0.16, 0.37, and 0.5) and a number of minor activity bands (at Re = 0.35 and 0.40) which did not coincide with the minor protein bands. Some preparations of the enzyme showed an additional activity band at Re = 0.27. These minor activity bands were not observed if xanthine was omitted from the developing reagent and became much more intense if the gels were incubated with the developing reagent for a longer time than the few seconds used to demonstrate the major activity band shown in Fig. 1A. The results of SDS electrophoresis also support the view that homogeneity was approached (Fig. 1B). We have prepared many extracts of Arthrobacter, grown as described, and have found that the starting specific activities are generally between 2.0 and 3.0, as originally reported (23). We have had several extracts with starting activities as high as 10.5 and, occasionally, lower specific activities approaching 1.0 are obtained. We exTABLE 1. Purification of xanthine oxidase from Arthrobacter S-2 Fold puriYield Total Vol (ml) Protein Fraction Sp act' (% fication units (mng)
12.8 460 15.5 2.32 Unfractionated extract 11.8 390 7.6 4.35 Supernatant from streptomycin and heat treatment 8.0 284 1.3 27.4 3 Ammonium sulfate precipitation (33 to 43%) 4.7 155 34.8 0.95 4 Ammonium sulfate precipitation (35 to 43%) 44.7 143 1.34 2.4 Ammonium sulfate precipitation 5 (33 to 43%) 135 45.0 85 0.014 6 Eluate from Sepharose 4B column 230 1.6 76 0.208 7 Supernatant after ultrafiltration, precipitation with 0 to 50% ammonium sulfate, and dialysis a Micromoles of ferricyanide disappearance per minute per milligram of protein.
100 85
1 1.9
63
11.8
34
15.0
32
19.3
19 17
58 100
424
WOOLFOLK AND DOWNARD
c 0
-I
2
i0
LZ
Re
'
FIG. 1. Gel electrophoresis ofpurified Arthrobacter xanthine oxidase. (A) Densitometric tracings of gels (7). (Curve 1) Unstained gel. The densitometer was adjusted to read 5%, and this adjustment was used as a zero point for the other gels. There was some damping above 80% absorbance with the set-
tings used. (Curve 2) Enzyme (16 pg; specific activity, 78 U/mg ofprotein) stained with amido black. (Curve 3) Enzyme, as in curve 2 but stained for activity (24). (B) SDS electrophoresis of 32 Lg of enzyme stained with Coomassie briliant blue. (C) Subunit molecular weight estimation. The arrow shows the mobility of xanthine oxidase taken from (B). The standard proteins and their molecular weights are: 1, cytochrome 0.85 (not shown); 2, trypsin, 23,000; 3, c, 11,700, R. rabbit muscle lactic dehydrogenase, 36,000; 4, catalase, 58,000; 5, bovine serum albumin, 68,000; and 6, bovine serum albumin dimer, 136,00i. Re represents the distance from the origin relative to that traveled by the tracking dye. -
amined three purified preparations of the enThe starting specific activities of these preparations were 1.66, 2.32 (Table 1), and 10.5. The final specific activities after purification were 78, 230, and 214, with yields of 9, 17, and 40%, respectively. Because the preparations appeared to be equally homogeneous upon electrophoretic analysis, we do not feel that these differences in specific activity are accounted for by different levels of contaminating protein (see below). In fact, we have shown the electrophoresis of the preparation with the lowest specific zyme.
J. BACTERIOL.
activity (Fig. 1A) because the patterns with the other two preparations were similar but not superior. Precipitation of these purified enzymes with ammonium sulfate (Table 1, step 3) resulted in recovery of virtually all of the activity but no increase in the specific activity. This is another observation which we believe supports the electrophoretic evidence that the purified preparations with the lower specific activities are as free of foreign proteins as the preparation with the highest specific activity. When the ammonium sulfate step was applied carefully, as described, to partially purified preparations of the enzyme, there was invariably an increase in the specific activity. The failure to increase the specific activity of a given preparation by this step can serve as a convenient presumptive indication of a successful purification. Molecular properties of the enzyme. Native enzyme with a Kav of 0.2, corresponding to a molecular weight of approximately 146,000, was eluted from a Sephadex G-150 column. On the other hand, when the purified preparation was treated with SDS and examined by SDS electrophoresis, a major protein band plus one relatively minor band were observed (Fig. 1B). The position of the major band corresponded to a molecular weight of approximately 80,000 (Fig. 1C). A spectrum of the purified Arthrobacter xanthine oxidase is shown in Fig. 2. Specificity for purines. We have previously reported that 1-methylxanthine, but not 3-methylxanthine, serves as a substrate with the Arthrobacter enzyme (23). Figure 3 shows that saturation of the enzyme with xanthine was very imilar to that with 1-methylxanthine. The double-reciprocal plots were concave downward, and the same effect was observed when oxygen was the electron acceptor (Fig. 4). The quasilinear portion of the plot at lower concentrations of substrate extrapolated to an apparent Km value of 1.3 x 10-' M for both substrates (Fig. 3). 3-Methylxanthine was not an inhibitor at concentrations up to 30 times those used with the active substrates. Failure of the compound to bind effectively to the protein probably accounted for its nonreactivity. Similarly, purine, adenine, guanine, isoguanine, 2-aminopurine, and acetaldehyde did not serve as substrates, nor did they inhibit when they were added at 1 mM concentrations to the standard ferricyanide assay with xanthine. Hypoxanthine, 6,8-dioxypurine, 2-oxypurine, and allopurinol also served as substrates with the ferricyanide assay, giving rates of 27, 58, 23, and 5%, respectively, of that given by xanthine under the same conditions. The values with 6,8dioxypurine and 2-oxypurine were relatively
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425
ill
(nm)
FIG. 2. Spectrum of purified Arthrobacter xanthine oxidase. Purified xanthine oxidase (specific activity, 214 U/mg of protein) was concentrated and examined in microcuvettes in a total volume of 0.4 ml of O.OIXS buffer (0.825 mg ofprotein per ml). The absorbancy at 280 nm was 0.806.
Xantkne (mM)
FIG. 3. Effect of xanthine and 1-methylxanthine concentration on the activity of Arthrobacter xanthine oxidase using standard ferricyanide assay conditions. (A) Each assay contained 1.4 jig ofpurified xanthine oxidase (same preparation as that used in Fig. 2). Symbols: 0, xthine; 0, xanthine plus I mM 3-methylxanthine; A, 1-methylxanthine; A, 1-methylxanthine pls I mM 3-methylxanthine. (B) Doublereciprocal plot of data from (A).
high when compared with the values obtained when oxygen was used as the electron acceptor (23); this may be due to the relatively low concentration of substrates used in the latter assay.
Saturation plots similar to those of Fig. 3 with hypoxanthine as a substrate gave an apparent Km value of 2.9 x 10-5 M, a value significantly lower than that encountered with xanthine. Unlike the plots with xanthine, however, an optimum activity was observed at about 0.5 mM, and at 1.0 mM significant substrate inhibition was observed which was progressive and severe at higher concentrations. Allopurinol competitively inhibited the xanthine activity with molecular oxygen, giving a Ki of 6 x 10-6 M. Electron acceptors. In agreement with earlier studies using crude extracts (23), the Arthrobacter enzyme did not utilize nicotinamide adenine dinucleotide as a terminal electron acceptor, and, in contrast to milk xanthine oxidase, the purified enzyme did not possess reduced nicotinamide adenine dinucleotide oxidase activity. We have shown previously that the enzyme will utilize 2,6-dichlorophenol-indophenol but not benzyl viologen under conditions in which the milk enzyme utilizes both. The freshly purified Arthrobacter enzyme oxidized xanthine (1.2 x 10-4 M) at the expense of molecular oxygen at 64% the rate observed with the standard ferricyanide assay using 1 mM xanthine. The milk oxidase supported xanthine oxidation under these two conditions at approximately equivalent rates, but there was considerable substrate inhibition with the ferricyanide assay. The velocity for the bacterial enzyme with oxygen obtained by extrapolation of double-reciprocal plots using xanthine was 196% the value obtained by a similar extrapolation of ferricyanide data (compare Fig. 3 with Fig. 4). The
426
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WOOLFOLK AND DOWNARD
Xanthine
(mM)
r
(s)
FIG. 4. Effect of xanthine concentration on the activity of Arthrobacter xanthine oxidase with molecular oxygen as the electron acceptor. Standard spectrophotometric assay conditions were used except that each assay contained a total volume of 0.5 ml with 0.04 pg ofpurified enzyme (same preparation as that used in Fig. 2). (A) Initial rate as a function of xanthine concentration. (B) Double-reciprocal plot of data from (A). (C) Primary plot of initial rate data for the nine points of (A), in order.
specific activity of the enzyme with molecular geneity (14). The electrophoretic evaluation of oxygen at the highest concentration of xanthine our enzyme preparations and other data suggest used experimentally (Fig. 4) is 275 U/mg of that the large variation in specific activities enprotein. This is approximately 50 times that of countered with the different preparations may the most active preparations of pure milk xan- be due to differential modification of enzyme thine oxidase measured under optimal condi- activity during growth or processing and not to tions (13, 16, 17). gross contamination by other proteins. Such an The oxygen-linked activity of the purified en- explanation is consistent with the extensive litzyme has been found to be stable for more than erature relating to similar problems with the a year of storage at -75°C with alternate freez- milk enzyme involving molybdenum deficiency ing and thawing each time that the activity is (1, 3, 10, 11) and losses of an essential persulfide determined. However, during this period the group (8, 16). This last process has been consame preparation has shown significant losses of trolled somewhat by the addition of chelating ferricyanide-linked activity. Thus, the ratio of agents to milk xanthine oxidase preparations. these two activities may not be constant with all However, our enzyme seems more stable than preparations of the enzyme, and the possible the milk enzyme during purification as far as variation of this feature should be examined exposure to metals is concerned. We have added with future preparations of the enzyme. The magnesium and traces of iron to our buffers freshly purified enzyme gives the same ratio of because early experiments showed this to stabioxygen to ferricyanide electron acceptor activity lize the xanthine oxidase, and addition of ethylas the crude extracts (23). enediaminetetraacetic acid gave no measurable protective effect. We have noted that preparaDISCUSSION tions with relatively low specific activities tend Purification. The presence of minor activity to be obtained from extracts with low starting bands in the profiles of the polyacrylamide gels specific activities. Careful attention to the effect (Fig. 1A, curve 3) raises the possibility that the of growth conditions may reveal the source of contaminating bands may be derived from the variation with the extracts. main protein by aggregation or degradation. BeMolecular properties. Comparison of the cause these activity bands are somewhat varia- values obtained for the molecular weights of the ble and do not coincide directly with the detect- native enzyme and of the SDS-treated enzyme able minor protein-staining bands, it is possible suggests that the enzyme may be a dimer, as are that the enzyme tends to complex with contam- milk xanthine oxidase and related enzymes. inating proteins. One contaminant of milk xan- However, the Arthrobacter oxidase is approxithine oxidase which is difficult to remove has mately half the size of other xanthine-oxidizing been shown to be the result of aggregation of enzymes (6). inactive forms of the enzyme (6). Also, a proteA number of details in the spectrum of the olytic activity is known to copurify with milk Arthrobacter enzyme (Fig. 2) are remarkably xanthine oxidase which can give rise to hetero- similar to those of published spectra of milk
BACTERIAL XANTHINE OXIDASE
VOL. 135, 1978
xanthine oxidase and related enzymes (15-17); these include the shoulder at 550 nm, the minimum at 410 nm, the main peak at about 450 nm, and the shoulder at about 370 nm. This last feature may be somewhat less prominent in the spectrum of the bacterial enzyme. As is the case with the published spectra, the main peak of the bacterial enzyme is a composite of several peaks and shoulders at 450, 452, 464, and 482 nm. These detailed similarities suggest that the prosthetic content of the bacterial oxidase is very sinilar to that of the milk enzyme, but the new oxidase needs to be analyzed for prosthetic group content. With regard to the 550/450-nm absorption ratio, the Arthrobacter enzyme more closely resembles aldehyde oxidase and the xanthine dehydrogenases than milk xanthine oxidase (6). The 280/450-nm absorption ratio is approximately half that given by the milk enzyme (15). This low value supports the claim that the enzyme is homogeneous, or nearly so. On the assumption that each subunit of the bacterial enzyme is identical and that each contains a prosthetic center, as seems to be the case with the milk enzyme, the difference in this ratio may be a reflection of the fact that the bacterial oxidase has approximately one-half as much protein associated with each prosthetic center. Specificity and kinetics. This research was conducted as part of a general study of the specificity patterns of bacterial xanthine-oxidizing enzymes, and detailed studies are projected. However, the preliminary information presented in this paper, together with spectrophotometric determination of the sites of attack on oxygenated purines presented earlier (23), supports the view that the xanthine oxidase from Arthrobacter is more specific than previously studied xanthine-oxidizing enzymes. Noteworthy are the absence of activity with purine and the lack of inhibition by aminopurines. However, the oxidation of 2-oxypurine at position 6, of hypoxanthine and 6,8-dioxypurine at position 2, and of xanthine at position 8 (23) shows that the Arthrobacter oxidase is capable of oxidizing at least one purine substrate at each position of the purine ring subject to attack by this class of enzymes. The absence of 3-methylxanthine oxidase activity is like milk xanthine oxidase and the related enzymes from other gram-positive bacteria but unlike the enzyme from gram-negative bacteria (23). In the utilization of electron acceptors and the site of attack on 2-oxypurine, the Arthrobacter oxidase is similar to aldehyde oxidase (12), a second purine-oxidizing enzyme distributed in animal tissues. However, the exceptional activity with xanthine and the inactivity with purine and acetaldehyde clearly distinguish the Arthrobacter oxidase from the latter enzyme.
427
Of particular interest in comparing the present oxidase with the milk enzyme is the absence of substrate inhibition when xanthine is used as a substrate with the Arthrobacter enzyme. An opposite effect is observed, which may be due to a heterogeneity of enzyme forms. However, because this observation has been made with purified enzyme, the possibility is strengthened that it may be due to negative cooperativity (13). The finding of substrate inhibition when hypoxanthine is used as a substrate heightens interest in the effect of structure on these irregularities in any future substrate specificity studies. The combination of a relatively high Km with negative cooperativity prominent at the higher substrate concentrations seems consistent with the role of the enzyme as part of an inducible energysupplying system (22). Such an enzyme would be especially active under conditions where sufficient xanthine was present to support growth. The physiological role of milk xanthine oxidase is not clear. It is derived from a cellular form which possesses dehydrogenase activity, and specially purified preparations of the enzyme from milk can be converted to a dehydrogenase form by treatment with reducing agents (2). The mammalian enzyme seems to function in detoxification. The apparently subdued turnover, the low K., and the pattern of substrate inhibition may be antigout features selected for during the evolution of this form of the enzyme. It is possible that the Arthrobacter enzyme uses some as yet undetermined electron acceptor in the cell, but the effective utilization of molecular oxygen suggests that this may be the natural electron acceptor. Because of the kinetic features of the bacterial enzyme, a comparative examination of the activity by fast-reaction and electron spin resonance techniques would be of some interest. ACKNOWLEDGMENTS This research was supported by Public Health Service grants CA-08390 from the National Cancer Institute and GM22815 from the National Institute of General Medical Sciences.
LITERATURE CITED 1.
Avis, P. G., F. Bergel, and R. C. Bray. 1955. The
chemistry of xanthine oxidase. I. The properties of crystalline xanthine oxidase from cow's milk. J. Chem. Soc. 1955:1100-1105. 2. Batilli, M. G., E. Orenzoni, and F. Stirpe. 1973. Milk xanthine oxidase type D (dehydrogenase) and type 0 (oxidase). Biochem. J. 131:191-198. 3. Bergel, F., and R. C. Bray. 1957. The chemistry of xanthine oxidase. IV. The problems of enzyme inactivation and stabilization. Biochem. J. 73:182-192. 4. Bradshaw, W. EL, and H. A. Barker. 1960. Purification and properties of xanthine dehydrogenase from Clostridium cylindrosporum. J. Biol. Chem. 235:3620-3629. 5. Bray, R. C. 1963. Xanthine oxidase, p. 533-556. In P. D. Boyer, H. Lardy, and K. Myrback (ed.), The enzymes, vol. 7, 2nd ed. Academic Press Inc., New York.
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6. Bray, R. C. 1975. Molybdenum iron-sulfur flavin hydroxylases and related enzymes, p. 299-419. In P. D. Boyer (ed.), The enzymes, vol. 12B, 3rd ed. Academic Press Inc., New York. 7. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to serum proteins. Ann. N.Y. Acad. Sci. 121:404 427. 8. Edmondson, D., V. Massey, G. Palmer, L M. Beacham, and G. B. Elion. 1972. Resolution of active and inactive zanthine oxidase by affinity chromatography. J. Biol. Chem. 247:1597-1604. 9. Fridovich, I., and P. Handler. 1958. Xanthine oxidase. IV. Participation of iron in internal electron transport. J. Biol. Chem. 233:1581-1585. 10. Hart, L. I., and R. C. Bray. 1967. Improved xanthine oxidase purification. Biochim. Biophys. Acta 146: 611-613. 11. Hart, L I., M. A. McGartoll, H. R. Chapman, and R. C. Bray. 1970. The composition of milk xanthine oxidase. Biochem. J. 116:851-864. 12. Krenitsky, T. A., S. M. Neil, G. B. Elion, and G. H. Hitchings. 1972. A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch. Biochem. Biophys. 150:589-599. 13. Levitzki, A., and D. E. Koshland, Jr. 1969. Negative cooperativity in regulatory enzymes. Proc. Natl. Acad. Sci. U.S.A. 62:1121-1128. 14. Mangino, M. E., and J. R. Brunner. 1977. Isolation and partial characterization of xanthine oxidase associated with the milk fat globule membrane of cow's milk. J. Dairy Sci. 60:841-850. 15. Massey, V., P. E. Brumby, H. Komai, and G. Palmer.
J. BACTERIOL. 1969. Studies on milk zanthine ozidase. Some spectral and kinetic properties. J. Biol. Chem. 244:1682-1691. 16. Massey, V., and D. Edmondson. 1970. On the mechanismn of inactivation of zanthine oxidase by cyanide. J. Biol. Chem. 245:6595-6598. 17. Rajogopalan, K. V., and P. Handler. 1964. The absorption spectra of iron-flavoproteins. J. Biol. Chem. 239:1509-1514. 18. Reiland, J. L 1971. Gel filtration. Methods Enzymol. 22:287-321. 19. Sin, I. L 1975. Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans. Biochim. Biophys. Acta 410:12-20. 20. Smith, S. T., K. V. Rajagopalan, and P. Handler. 1967. Purification and properties of zanthine dehydrogenase from Micrococcus lactilyticus. J. Biol. Chem. 242:4108-4117. 21. Weber, K., and M. Osborm. 1969. The reliability of molecular weight determinations by dodecylsulfate polyacrylamide gels electrophoresis. J. Biol. Chem. 244:4406-4412. 22. Woolfolk, C. A. 1975. Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen. J. Bacteriol. 123:1088-1106. 23. Woolfolk, C. A., and J. S. Downard. 1977. Distribution of zanthine oxidase and zanthine dehydrogenase specificity types among bacteria. J. Bacteriol. 130: 1175-1191. 24. Woolfolk, C. A., B. S. Woolfolk, and H. R. Whiteley. 1970. 2-Oxypurine dehydrogenase from Micrococcus aerogenes. J. Biol. Chem. 245:3167-3178.
JOURNAL OF BACTERIOLOGY, Aug. 1978, p. 422-428 0021-9193/78/0135-0422$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Bacterial Xanthine Oxidase from Arthrobacter S-2 C. A. WOOLFOLK* AND J. S. DOWNARDt Department of Molecular Biology and Biochemistry* and Department of Medical Microbiology, University of California, Irvine, California 92717 Received for publication 13 February 1978
Arthrobacter S-2, originally isolated by enrichment on xanthine, produced high levels of xanthine oxidase activity, requiring as little as a 20-fold purification to approach homogeneity with some preparations. Molecular oxygen, ferricyanide, and 2,6-dichlorophenol-indophenol served as electron acceptors, but nicotinamide adenine dinucleotide did not. The enzyme was relatively specific when compared with previously studied xanthine-oxidizing enzymes, but at least one purine was observed to be oxidized at each of the three positions of the purine ring that have been subject to oxidation by this type of enzyme. The enzyme had a relatively high Km for xanthine (1.3 x 10' M), and substrate inhibition was not observed with this compound, in contrast to the enzyme from cow's milk. In fact, an opposite effect was observed, and double-reciprocal plots with xanthine as the variable substrate showed a concave downward deviation at high concentrations. At 2.5 mM xanthine the enzyme had a specific activity approximately 50 times that of the most active preparations of the milk enzyme. The spectrum of the Arthrobacter enzyme resembled that of milk xanthine oxidase, suggesting a similarity of the prosthetic centers of the two enzymes. The bacterial enzyme was relatively small and may be dimeric, with approximate native and subunit molecular weights of 146,000 and 79,000, respectively. As the result of a survey of some 47 diverse strains of bacteria containing xanthine-oxidizing enzymes, we observed that two strains of Arthrobacter gave by far the highest specific activities when ferricyanide was used as the electron acceptor (23). Furtherinore, the Arthrobacter activity was exceptional in that molecular oxygen was relatively efficiently utilized when the xanthine-oxidizing enzymes from the various bacteria were compared under certain standard assay conditions with several electron acceptors. Because pyridine nucleotides were not utilized by the Arthrobacter enzyme, the properties resemble those of milk xanthine oxidase (EC 1.2.3.2), which has been the subject of considerable previous investigation (5, 6). Bacterial xanthine-oxidizing enzymes which have been purified previously (4, 19, 20, 24) utilize molecular oxygen relatively slowly. Ferredoxin may serve as an electron acceptor with the enzymes from anaerobes (20), whereas nicotinamide adenine dinucleotide serves as an effective electron acceptor with the soluble xanthine dehydrogenases from aerobic pseudomonads (19, 23). All of these latter enzymes are appropriately referred to as xanthine dehydrogenases. This paper reports on the purification of a new oxidase and examines t Present address: Department of Microbiology, University of Washington, Seattle, WA 98105. 422
some of its basic features. The smaller size, exceptionally high activity, and relatively restricted specificity pattern heighten our interest in a more detailed comparison with the other bacterial xanthine-oxidizing enzymes and the ex-
tensively studied oxidase from milk. MATERIALS AND METHODS Growth of cells and preparation of extracts. Arthrobacter S-2 originally isolated on xanthine-containing media (23) was routinely grown on 0.1% xanthine-0.01% yeast extract in a 10-fold dilution of our previously described mineral salts base, 1XS (22). The final phosphate concentration of the medium was 0.1 M. Conditions of growth and harvesting, measurement of protein, and preparation of extracts were as reported earlier (22). Enzyme amsays. Xanthine oxidase was routinely assayed by a ferricyanide-linked assay described previously (22, 23). One unit of enzyme is that amount that will reduce 1 iLmol of ferricyanide per min under the conditions of this assay. Full activity was observed under anaerobic conditions (24). Thus, the assay does not depend on the use of molecular oxygen as an electron acceptor followed by nonenzymatic reaction of the uric acid produced with ferricyanide, as has been suggested previously (9). The latter reaction is relatively slow and incomplete at the neutral pH of our assay. The spectrophotometric assays with molecular oxygen as the acceptor and the use of other electron acceptors were also as previously described
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BACTERLAL XANTHINE OXIDASE
(22, 23). The 1XS mineral salts base was used as a buffer with all assays. Purification of Arthrobacter S-2 xanthine oxidase. A representative example of the preparation of the enzyme is shown in Table 1. The crude extract was first treated with streptomycin sulfate to give a 1% (wt/vol) solution; this was followed by centrifugation at 12,000 x g for 20 min. The supernatant was then heated at 50°C for 10 min and recentrifuged to give fraction 2 (Table 1). Precipitation with ammonium sulfate was achieved by addition of the appropriate volume of a saturated solution of ammonium sulfate at 40C. Ammonium sulfate precipitation was repeated several times by suspension of the centrifuged precipitate in 0.O1XS buffer without prior dialysis. For steps 4 and 5 (Table 1), the ammonium sulfate was first added very slowly until turbidity was barely discernible (usually about 33% saturation). The suspension was carefully equilibrated by stirring, and the precipitated material was removed by centrifugation and discarded. The enzyme activity was then precipitated by more rapid addition of ammonium sulfate to 43% saturation. After the ammonium sulfate fractionation, the enzyme was resuspended in 0.O1XS buffer and applied directly to a Sepharose 4B column. The column of Sepharose 4B was set up and eluted in a manner identical to that previously used with an extract of Escherichieae strain A (23). Ninety percent of the activity eluted from the column in the 260- to 305-nil fraction. This was concentrated fivefold by ultrafiltration through Amicon XM50 filters. The enzyme was then precipitated by addition of ammonium sulfate to 50% saturation, and the precipitate was redissolved and dialyzed against 0.O1XS buffer to give fraction 7 (Table 1). Gel electrophoresis. The procedure of Davis (7) was followed for electrophoresis using 7.5% gels. We have previously described details of the staining procedures, the method for visualization of enzyme activity using nitro blue tetrazolium, and the use of the densitometer employed in this laboratory (24). Sodium dodecyl sulfate (SDS) gel electrophoresis was by the method of Weber and Osborn (21). Gel permeation chromatography. Sephadex G150 was used to estimate the molecular weight of the native xanthine oxidase by comparison of its partition Step
1 2
423
coefficient [K.w (V. - V.)/(Vt - V.)] with a standard curve of such valueo obtained by measurement of the enzymatic activity of proteins of known molecular weights (18). The standards, their molecular weights, and the K.,, values obtained were as follows: milk xanthine oxidase, 300,000, Ka,, = 0.0; catalase, 250,000, K.,, = 0.0; yeast alcohol dehydrogenase, 151,000, K.,, = 0.19; hexokinase, 96,000, K,v = 0.37; and hemoglobin, 64,000, Kay = 0.48. =
RESULTS
Purification of the enzyme. A typical purification of the enzyme is shown in Table 1. When the purified enzyme was applied to gels and subjected to electrophoresis, only one main protein band was observed (Fig. 1A, curve 2), accounting for 90% or more of the stain taken up by the gel. This band coincided with the main band of enzyme activity which also could be exhibited on these gels (Fig. 1A, curve 3). The gels displayed a number of minor protein-staining bands (at Re = 0.16, 0.37, and 0.5) and a number of minor activity bands (at Re = 0.35 and 0.40) which did not coincide with the minor protein bands. Some preparations of the enzyme showed an additional activity band at Re = 0.27. These minor activity bands were not observed if xanthine was omitted from the developing reagent and became much more intense if the gels were incubated with the developing reagent for a longer time than the few seconds used to demonstrate the major activity band shown in Fig. 1A. The results of SDS electrophoresis also support the view that homogeneity was approached (Fig. 1B). We have prepared many extracts of Arthrobacter, grown as described, and have found that the starting specific activities are generally between 2.0 and 3.0, as originally reported (23). We have had several extracts with starting activities as high as 10.5 and, occasionally, lower specific activities approaching 1.0 are obtained. We exTABLE 1. Purification of xanthine oxidase from Arthrobacter S-2 Fold puriYield Total Vol (ml) Protein Fraction Sp act' (% fication units (mng)
12.8 460 15.5 2.32 Unfractionated extract 11.8 390 7.6 4.35 Supernatant from streptomycin and heat treatment 8.0 284 1.3 27.4 3 Ammonium sulfate precipitation (33 to 43%) 4.7 155 34.8 0.95 4 Ammonium sulfate precipitation (35 to 43%) 44.7 143 1.34 2.4 Ammonium sulfate precipitation 5 (33 to 43%) 135 45.0 85 0.014 6 Eluate from Sepharose 4B column 230 1.6 76 0.208 7 Supernatant after ultrafiltration, precipitation with 0 to 50% ammonium sulfate, and dialysis a Micromoles of ferricyanide disappearance per minute per milligram of protein.
100 85
1 1.9
63
11.8
34
15.0
32
19.3
19 17
58 100
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WOOLFOLK AND DOWNARD
c 0
-I
2
i0
LZ
Re
'
FIG. 1. Gel electrophoresis ofpurified Arthrobacter xanthine oxidase. (A) Densitometric tracings of gels (7). (Curve 1) Unstained gel. The densitometer was adjusted to read 5%, and this adjustment was used as a zero point for the other gels. There was some damping above 80% absorbance with the set-
tings used. (Curve 2) Enzyme (16 pg; specific activity, 78 U/mg ofprotein) stained with amido black. (Curve 3) Enzyme, as in curve 2 but stained for activity (24). (B) SDS electrophoresis of 32 Lg of enzyme stained with Coomassie briliant blue. (C) Subunit molecular weight estimation. The arrow shows the mobility of xanthine oxidase taken from (B). The standard proteins and their molecular weights are: 1, cytochrome 0.85 (not shown); 2, trypsin, 23,000; 3, c, 11,700, R. rabbit muscle lactic dehydrogenase, 36,000; 4, catalase, 58,000; 5, bovine serum albumin, 68,000; and 6, bovine serum albumin dimer, 136,00i. Re represents the distance from the origin relative to that traveled by the tracking dye. -
amined three purified preparations of the enThe starting specific activities of these preparations were 1.66, 2.32 (Table 1), and 10.5. The final specific activities after purification were 78, 230, and 214, with yields of 9, 17, and 40%, respectively. Because the preparations appeared to be equally homogeneous upon electrophoretic analysis, we do not feel that these differences in specific activity are accounted for by different levels of contaminating protein (see below). In fact, we have shown the electrophoresis of the preparation with the lowest specific zyme.
J. BACTERIOL.
activity (Fig. 1A) because the patterns with the other two preparations were similar but not superior. Precipitation of these purified enzymes with ammonium sulfate (Table 1, step 3) resulted in recovery of virtually all of the activity but no increase in the specific activity. This is another observation which we believe supports the electrophoretic evidence that the purified preparations with the lower specific activities are as free of foreign proteins as the preparation with the highest specific activity. When the ammonium sulfate step was applied carefully, as described, to partially purified preparations of the enzyme, there was invariably an increase in the specific activity. The failure to increase the specific activity of a given preparation by this step can serve as a convenient presumptive indication of a successful purification. Molecular properties of the enzyme. Native enzyme with a Kav of 0.2, corresponding to a molecular weight of approximately 146,000, was eluted from a Sephadex G-150 column. On the other hand, when the purified preparation was treated with SDS and examined by SDS electrophoresis, a major protein band plus one relatively minor band were observed (Fig. 1B). The position of the major band corresponded to a molecular weight of approximately 80,000 (Fig. 1C). A spectrum of the purified Arthrobacter xanthine oxidase is shown in Fig. 2. Specificity for purines. We have previously reported that 1-methylxanthine, but not 3-methylxanthine, serves as a substrate with the Arthrobacter enzyme (23). Figure 3 shows that saturation of the enzyme with xanthine was very imilar to that with 1-methylxanthine. The double-reciprocal plots were concave downward, and the same effect was observed when oxygen was the electron acceptor (Fig. 4). The quasilinear portion of the plot at lower concentrations of substrate extrapolated to an apparent Km value of 1.3 x 10-' M for both substrates (Fig. 3). 3-Methylxanthine was not an inhibitor at concentrations up to 30 times those used with the active substrates. Failure of the compound to bind effectively to the protein probably accounted for its nonreactivity. Similarly, purine, adenine, guanine, isoguanine, 2-aminopurine, and acetaldehyde did not serve as substrates, nor did they inhibit when they were added at 1 mM concentrations to the standard ferricyanide assay with xanthine. Hypoxanthine, 6,8-dioxypurine, 2-oxypurine, and allopurinol also served as substrates with the ferricyanide assay, giving rates of 27, 58, 23, and 5%, respectively, of that given by xanthine under the same conditions. The values with 6,8dioxypurine and 2-oxypurine were relatively
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425
ill
(nm)
FIG. 2. Spectrum of purified Arthrobacter xanthine oxidase. Purified xanthine oxidase (specific activity, 214 U/mg of protein) was concentrated and examined in microcuvettes in a total volume of 0.4 ml of O.OIXS buffer (0.825 mg ofprotein per ml). The absorbancy at 280 nm was 0.806.
Xantkne (mM)
FIG. 3. Effect of xanthine and 1-methylxanthine concentration on the activity of Arthrobacter xanthine oxidase using standard ferricyanide assay conditions. (A) Each assay contained 1.4 jig ofpurified xanthine oxidase (same preparation as that used in Fig. 2). Symbols: 0, xthine; 0, xanthine plus I mM 3-methylxanthine; A, 1-methylxanthine; A, 1-methylxanthine pls I mM 3-methylxanthine. (B) Doublereciprocal plot of data from (A).
high when compared with the values obtained when oxygen was used as the electron acceptor (23); this may be due to the relatively low concentration of substrates used in the latter assay.
Saturation plots similar to those of Fig. 3 with hypoxanthine as a substrate gave an apparent Km value of 2.9 x 10-5 M, a value significantly lower than that encountered with xanthine. Unlike the plots with xanthine, however, an optimum activity was observed at about 0.5 mM, and at 1.0 mM significant substrate inhibition was observed which was progressive and severe at higher concentrations. Allopurinol competitively inhibited the xanthine activity with molecular oxygen, giving a Ki of 6 x 10-6 M. Electron acceptors. In agreement with earlier studies using crude extracts (23), the Arthrobacter enzyme did not utilize nicotinamide adenine dinucleotide as a terminal electron acceptor, and, in contrast to milk xanthine oxidase, the purified enzyme did not possess reduced nicotinamide adenine dinucleotide oxidase activity. We have shown previously that the enzyme will utilize 2,6-dichlorophenol-indophenol but not benzyl viologen under conditions in which the milk enzyme utilizes both. The freshly purified Arthrobacter enzyme oxidized xanthine (1.2 x 10-4 M) at the expense of molecular oxygen at 64% the rate observed with the standard ferricyanide assay using 1 mM xanthine. The milk oxidase supported xanthine oxidation under these two conditions at approximately equivalent rates, but there was considerable substrate inhibition with the ferricyanide assay. The velocity for the bacterial enzyme with oxygen obtained by extrapolation of double-reciprocal plots using xanthine was 196% the value obtained by a similar extrapolation of ferricyanide data (compare Fig. 3 with Fig. 4). The
426
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WOOLFOLK AND DOWNARD
Xanthine
(mM)
r
(s)
FIG. 4. Effect of xanthine concentration on the activity of Arthrobacter xanthine oxidase with molecular oxygen as the electron acceptor. Standard spectrophotometric assay conditions were used except that each assay contained a total volume of 0.5 ml with 0.04 pg ofpurified enzyme (same preparation as that used in Fig. 2). (A) Initial rate as a function of xanthine concentration. (B) Double-reciprocal plot of data from (A). (C) Primary plot of initial rate data for the nine points of (A), in order.
specific activity of the enzyme with molecular geneity (14). The electrophoretic evaluation of oxygen at the highest concentration of xanthine our enzyme preparations and other data suggest used experimentally (Fig. 4) is 275 U/mg of that the large variation in specific activities enprotein. This is approximately 50 times that of countered with the different preparations may the most active preparations of pure milk xan- be due to differential modification of enzyme thine oxidase measured under optimal condi- activity during growth or processing and not to tions (13, 16, 17). gross contamination by other proteins. Such an The oxygen-linked activity of the purified en- explanation is consistent with the extensive litzyme has been found to be stable for more than erature relating to similar problems with the a year of storage at -75°C with alternate freez- milk enzyme involving molybdenum deficiency ing and thawing each time that the activity is (1, 3, 10, 11) and losses of an essential persulfide determined. However, during this period the group (8, 16). This last process has been consame preparation has shown significant losses of trolled somewhat by the addition of chelating ferricyanide-linked activity. Thus, the ratio of agents to milk xanthine oxidase preparations. these two activities may not be constant with all However, our enzyme seems more stable than preparations of the enzyme, and the possible the milk enzyme during purification as far as variation of this feature should be examined exposure to metals is concerned. We have added with future preparations of the enzyme. The magnesium and traces of iron to our buffers freshly purified enzyme gives the same ratio of because early experiments showed this to stabioxygen to ferricyanide electron acceptor activity lize the xanthine oxidase, and addition of ethylas the crude extracts (23). enediaminetetraacetic acid gave no measurable protective effect. We have noted that preparaDISCUSSION tions with relatively low specific activities tend Purification. The presence of minor activity to be obtained from extracts with low starting bands in the profiles of the polyacrylamide gels specific activities. Careful attention to the effect (Fig. 1A, curve 3) raises the possibility that the of growth conditions may reveal the source of contaminating bands may be derived from the variation with the extracts. main protein by aggregation or degradation. BeMolecular properties. Comparison of the cause these activity bands are somewhat varia- values obtained for the molecular weights of the ble and do not coincide directly with the detect- native enzyme and of the SDS-treated enzyme able minor protein-staining bands, it is possible suggests that the enzyme may be a dimer, as are that the enzyme tends to complex with contam- milk xanthine oxidase and related enzymes. inating proteins. One contaminant of milk xan- However, the Arthrobacter oxidase is approxithine oxidase which is difficult to remove has mately half the size of other xanthine-oxidizing been shown to be the result of aggregation of enzymes (6). inactive forms of the enzyme (6). Also, a proteA number of details in the spectrum of the olytic activity is known to copurify with milk Arthrobacter enzyme (Fig. 2) are remarkably xanthine oxidase which can give rise to hetero- similar to those of published spectra of milk
BACTERIAL XANTHINE OXIDASE
VOL. 135, 1978
xanthine oxidase and related enzymes (15-17); these include the shoulder at 550 nm, the minimum at 410 nm, the main peak at about 450 nm, and the shoulder at about 370 nm. This last feature may be somewhat less prominent in the spectrum of the bacterial enzyme. As is the case with the published spectra, the main peak of the bacterial enzyme is a composite of several peaks and shoulders at 450, 452, 464, and 482 nm. These detailed similarities suggest that the prosthetic content of the bacterial oxidase is very sinilar to that of the milk enzyme, but the new oxidase needs to be analyzed for prosthetic group content. With regard to the 550/450-nm absorption ratio, the Arthrobacter enzyme more closely resembles aldehyde oxidase and the xanthine dehydrogenases than milk xanthine oxidase (6). The 280/450-nm absorption ratio is approximately half that given by the milk enzyme (15). This low value supports the claim that the enzyme is homogeneous, or nearly so. On the assumption that each subunit of the bacterial enzyme is identical and that each contains a prosthetic center, as seems to be the case with the milk enzyme, the difference in this ratio may be a reflection of the fact that the bacterial oxidase has approximately one-half as much protein associated with each prosthetic center. Specificity and kinetics. This research was conducted as part of a general study of the specificity patterns of bacterial xanthine-oxidizing enzymes, and detailed studies are projected. However, the preliminary information presented in this paper, together with spectrophotometric determination of the sites of attack on oxygenated purines presented earlier (23), supports the view that the xanthine oxidase from Arthrobacter is more specific than previously studied xanthine-oxidizing enzymes. Noteworthy are the absence of activity with purine and the lack of inhibition by aminopurines. However, the oxidation of 2-oxypurine at position 6, of hypoxanthine and 6,8-dioxypurine at position 2, and of xanthine at position 8 (23) shows that the Arthrobacter oxidase is capable of oxidizing at least one purine substrate at each position of the purine ring subject to attack by this class of enzymes. The absence of 3-methylxanthine oxidase activity is like milk xanthine oxidase and the related enzymes from other gram-positive bacteria but unlike the enzyme from gram-negative bacteria (23). In the utilization of electron acceptors and the site of attack on 2-oxypurine, the Arthrobacter oxidase is similar to aldehyde oxidase (12), a second purine-oxidizing enzyme distributed in animal tissues. However, the exceptional activity with xanthine and the inactivity with purine and acetaldehyde clearly distinguish the Arthrobacter oxidase from the latter enzyme.
427
Of particular interest in comparing the present oxidase with the milk enzyme is the absence of substrate inhibition when xanthine is used as a substrate with the Arthrobacter enzyme. An opposite effect is observed, which may be due to a heterogeneity of enzyme forms. However, because this observation has been made with purified enzyme, the possibility is strengthened that it may be due to negative cooperativity (13). The finding of substrate inhibition when hypoxanthine is used as a substrate heightens interest in the effect of structure on these irregularities in any future substrate specificity studies. The combination of a relatively high Km with negative cooperativity prominent at the higher substrate concentrations seems consistent with the role of the enzyme as part of an inducible energysupplying system (22). Such an enzyme would be especially active under conditions where sufficient xanthine was present to support growth. The physiological role of milk xanthine oxidase is not clear. It is derived from a cellular form which possesses dehydrogenase activity, and specially purified preparations of the enzyme from milk can be converted to a dehydrogenase form by treatment with reducing agents (2). The mammalian enzyme seems to function in detoxification. The apparently subdued turnover, the low K., and the pattern of substrate inhibition may be antigout features selected for during the evolution of this form of the enzyme. It is possible that the Arthrobacter enzyme uses some as yet undetermined electron acceptor in the cell, but the effective utilization of molecular oxygen suggests that this may be the natural electron acceptor. Because of the kinetic features of the bacterial enzyme, a comparative examination of the activity by fast-reaction and electron spin resonance techniques would be of some interest. ACKNOWLEDGMENTS This research was supported by Public Health Service grants CA-08390 from the National Cancer Institute and GM22815 from the National Institute of General Medical Sciences.
LITERATURE CITED 1.
Avis, P. G., F. Bergel, and R. C. Bray. 1955. The
chemistry of xanthine oxidase. I. The properties of crystalline xanthine oxidase from cow's milk. J. Chem. Soc. 1955:1100-1105. 2. Batilli, M. G., E. Orenzoni, and F. Stirpe. 1973. Milk xanthine oxidase type D (dehydrogenase) and type 0 (oxidase). Biochem. J. 131:191-198. 3. Bergel, F., and R. C. Bray. 1957. The chemistry of xanthine oxidase. IV. The problems of enzyme inactivation and stabilization. Biochem. J. 73:182-192. 4. Bradshaw, W. EL, and H. A. Barker. 1960. Purification and properties of xanthine dehydrogenase from Clostridium cylindrosporum. J. Biol. Chem. 235:3620-3629. 5. Bray, R. C. 1963. Xanthine oxidase, p. 533-556. In P. D. Boyer, H. Lardy, and K. Myrback (ed.), The enzymes, vol. 7, 2nd ed. Academic Press Inc., New York.
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6. Bray, R. C. 1975. Molybdenum iron-sulfur flavin hydroxylases and related enzymes, p. 299-419. In P. D. Boyer (ed.), The enzymes, vol. 12B, 3rd ed. Academic Press Inc., New York. 7. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to serum proteins. Ann. N.Y. Acad. Sci. 121:404 427. 8. Edmondson, D., V. Massey, G. Palmer, L M. Beacham, and G. B. Elion. 1972. Resolution of active and inactive zanthine oxidase by affinity chromatography. J. Biol. Chem. 247:1597-1604. 9. Fridovich, I., and P. Handler. 1958. Xanthine oxidase. IV. Participation of iron in internal electron transport. J. Biol. Chem. 233:1581-1585. 10. Hart, L. I., and R. C. Bray. 1967. Improved xanthine oxidase purification. Biochim. Biophys. Acta 146: 611-613. 11. Hart, L I., M. A. McGartoll, H. R. Chapman, and R. C. Bray. 1970. The composition of milk xanthine oxidase. Biochem. J. 116:851-864. 12. Krenitsky, T. A., S. M. Neil, G. B. Elion, and G. H. Hitchings. 1972. A comparison of the specificities of xanthine oxidase and aldehyde oxidase. Arch. Biochem. Biophys. 150:589-599. 13. Levitzki, A., and D. E. Koshland, Jr. 1969. Negative cooperativity in regulatory enzymes. Proc. Natl. Acad. Sci. U.S.A. 62:1121-1128. 14. Mangino, M. E., and J. R. Brunner. 1977. Isolation and partial characterization of xanthine oxidase associated with the milk fat globule membrane of cow's milk. J. Dairy Sci. 60:841-850. 15. Massey, V., P. E. Brumby, H. Komai, and G. Palmer.
J. BACTERIOL. 1969. Studies on milk zanthine ozidase. Some spectral and kinetic properties. J. Biol. Chem. 244:1682-1691. 16. Massey, V., and D. Edmondson. 1970. On the mechanismn of inactivation of zanthine oxidase by cyanide. J. Biol. Chem. 245:6595-6598. 17. Rajogopalan, K. V., and P. Handler. 1964. The absorption spectra of iron-flavoproteins. J. Biol. Chem. 239:1509-1514. 18. Reiland, J. L 1971. Gel filtration. Methods Enzymol. 22:287-321. 19. Sin, I. L 1975. Purification and properties of xanthine dehydrogenase from Pseudomonas acidovorans. Biochim. Biophys. Acta 410:12-20. 20. Smith, S. T., K. V. Rajagopalan, and P. Handler. 1967. Purification and properties of zanthine dehydrogenase from Micrococcus lactilyticus. J. Biol. Chem. 242:4108-4117. 21. Weber, K., and M. Osborm. 1969. The reliability of molecular weight determinations by dodecylsulfate polyacrylamide gels electrophoresis. J. Biol. Chem. 244:4406-4412. 22. Woolfolk, C. A. 1975. Metabolism of N-methylpurines by a Pseudomonas putida strain isolated by enrichment on caffeine as the sole source of carbon and nitrogen. J. Bacteriol. 123:1088-1106. 23. Woolfolk, C. A., and J. S. Downard. 1977. Distribution of zanthine oxidase and zanthine dehydrogenase specificity types among bacteria. J. Bacteriol. 130: 1175-1191. 24. Woolfolk, C. A., B. S. Woolfolk, and H. R. Whiteley. 1970. 2-Oxypurine dehydrogenase from Micrococcus aerogenes. J. Biol. Chem. 245:3167-3178.
