Uricase from soybean root nodules: Purification, properties, and comparison with the enzyme from cowpea

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 226, No. 1, October 1, pp. 190-197, 1933

Uricase from Soybean Root Nodules: Purification, Properties, Comparison with the Enzyme from Cowpea’ KAREN

LUCAS,

Department

MICHAEL

of Biochemistry, Received February

J. BOLAND,

Michisan

AND

State University,

KAREL

and

R. SCHUBERT

East Lansing,

Michigan

48824

24.1933, and in revised form May 13,1933

A 45-fold purification of uricase (urate:08 oxidoreductase, EC 1.7.3.3) from soybean root nodules by ammonium sulfate fractionation, gel filtration, and affinity chromatography is described. Electrophoresis on nondenaturing gels using an activity stain or on sodium dodecyl sulfate (SDS) gels demonstrated that the enzyme obtained was nearly homogeneous. The subunit molecular weight of uricase estimated from SDS gels was 32,000 -t 3000. Gel-filtration studies indicated that the native enzyme is a monomer at pH 7.5 which associates to form a dimer at pH 8.8. Enzyme activity was stabilized by the addition of dithiothreitol. The pH dependence of the enzyme showed an optimum of 9.5. Initial rate kinetics showed Km values of 10 and 31 PM for uric acid and oxygen, respectively, with an intersecting pattern of substrate dependence. Uricase activity was inhibited strongly by xanthine, which was competitive with respect to uric acid (Ki = 10 PM). No significant inhibition was observed in the presence of a variety of amino acids, ammonium, adenine, or allopurinol, in contrast with results reported for the cowpea enzyme. Gel-filtration chromatography and SDS-gel electrophoresis of uricase purified by the same method from cowpea nodules indicated that the native enzyme exists as a monomer of M, 50,000 at pH 7.5.

Those legumes which transport fixed nitrogen from the nodules in the form of ureides (allantoin and allantoic acid) synthesize the ureides by means of purine synthesis followed by oxidative degradation (l-4). The degradative pathway consists of the three enzymes, xanthine dehydrogenase, uricase (urate:Oz oxidore-

ductase, EC 1.7.3.3), and allantoinase (3,5, 6). The purification and properties of xanthine dehydrogenase from navy bean (Phmeolus vulgaris) (7) and soybean (Gigtine m) (8) have recently been described. Uricase has been partially purified from cowpea (fiw ungu&&ta) (9). We report here the purification and properties of uricase from soybean nodules, an enzyme which shows some similarities and a number of important differences from the enzymes from cowpea (9). A partial purification and some properties of the soybean nodule enzyme have been reported previously (10).

1 This research was supported by a grant to K.R.S. from the United States Department of Agriculture Science and Education Administration Competitive Research Grants Office (Grant 5901-0410-9-0948-O). M.J.B. was supported in part by a New Zealand Public Service Study Award and was on leave from the Applied Biochemistry Division, D.S.I.R., Palmerston North, New Zealand. This is journal article No. 10169 of the Michigan Agricultural Experiment Station. s To whom correspondence should be addressed: Plant Sciences Division-OBE, Monsanto Agricultural Products Co., 800 N. Lindbergh Boulevard, St. Louis, MO. 63166. 0003-9861/83

$3.00

Copyright Q 1983by AcademicPress.Inc. All rights of reproductionin any form reserved.

MATERIALS

AND

METHODS

Growth ofplants Soybean seeds (G. IL.1 Merr. cv. Amsoy 71) were inoculated with Rhizobium japonicum strain USDA 311b 110. Cowpea seeds (V. unguiculata [L.] Walp. cv. California Blackeye) were 190

URICASE

FROM

inoculated with Rhizobium cowpea strain 32Hl. Plants were grown in Perlite and watered daily with a nitrogen-free nutrient solution (11). Nodules were harvested from plants 5 to 8 weeks old. Uricase assay. Uricase activity was assayed at room temperature (22°C) by following the disappearance of uric acid at 292.5 nm (3), using E = 12.2 mM-' cm-’ (10). A unit of activity is defined as 1 pmol uric acid oxidized min-‘. Assay solutions contained 0.1 M 2(Ncyclohexylamino)ethanesulfonic acid (Ches)S-KOH, pH 9.5, saturated with air, 0.1 rnhf uric acid, and lo100 ~1 of enzyme solution in a total volume of 1.5 ml. Because of a lag phase of l-2 min after addition of uric acid, assays were run for several minutes to allow maximum velocity to be reached. Kinetic studies. The pH dependence was measured using the standard assay method described above in different buffers. Buffers used were 0.1 M NTris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (Tes)-KOH for pH 6.5-8.4 and 0.1 M Ches-KOH for pH 8.4-11.1. Substrate-dependence experiments were run in mixtures of air-sparged and degassed, nitrogen-sparged buffer in anaerobic cuvettes with varying amounts of uric acid. Air-saturated buffer was used with varying concentrations of uric acid for xanthine inhibition studies. For other inhibitor studies, 18 pM uric acid in air-saturated buffer was used. Native gel electrophureeis. Native gels (9% total monomer concentration) were prepared in 7.5 mm X 13-cm glass tubes according to Davis (12) without a stacking gel and prerun for 30 min at 2 mA per gel before loading on the protein samples. Samples were subjected to electrophoresis at 2 mA per gel for 5 h. Gels were either stained for protein with 1.25g Coomassie blue R per liter 25% isopropanol:lO% acetic acid (v/v) or for enzyme activity. The activity stain contained (per 100 ml) 40 mg nitroblue tetrazolium, 0.8 mg phenazine methosulfate, and 20 mg uric acid in 50 mrd Tris-glycine, pH 8.3. Gels were incubated until activity hands were visible and then stored in 5% (v/v) acetic acid. SDS-gel electrophoresis. SDS-gel electrophoresis was carried out on 10% polyacrylamide slab gels (13). A l-ml aliquot of enzyme from the blue-Sepharose column was lyophilized and taken up in 0.2 ml 0.1 M Tris-HCl buffer, pH 6.8. Samples of enzymes that had not been concentrated and molecular weight standards were also used. All samples were boiled for 2 min with 2% SDS and 10% /3-mercaptoethanol before loading onto the gel. Bromphenol blue was used as

3 Abbreviations used: Ches, 2(N-cyclohexylamino)ethanesulfonic acid; Tes, N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid; SDS, sodium dodecyl sulfate; PVP, polyvinylpyrrolidone; Caps, cyclohexylaminopropanesulfonic acid.

SOYBEAN

191

NODULES

tracking dye and gels were electrophoresed at 2 mA cm-‘. Gels were stained with Coomassie blue. Protein &ternzination Protein was estimated by a modification of the method of Lowry et al as described by Bensadoun and Weinstein (14). Enzyme p-urificntia. All operations were carried out at 0-4’C. Nodules (5-50 g) were harvested and crushed in 2 ml of 25 mM Tes-KOH, pH 7.5, containing 5 mM dithiothreitol, 5% acid-washed insoluble polyvinylpyrrolidone (PVP), and 5.5% sorbitol per gram of nodules. PVP was added to prevent protein denaturation by phenolics (15). The brei was centrifuged for 10 min at 121g and the supernatant fluid was recentrifuged for 15 min at 27,OOOg.The supernatant fluid was taken to 35% saturation with ammonium sulfate (202 g liter-‘) and let stand for 30 min. The precipitate was removed by centrifugation at 27,OOOg for 20 min and redissolved in a minimum volume (416 ml) of 10 mM Tes-KOH, 2.5 mM dithiothreitol, pH 7.5, and loaded onto a column (2.5 X 75 cm) of Sepharose 6B. The column was developed with the same buffer at a flow rate of 48 ml h-l, and 8-ml fractions were collected. Fractions showing peak activity were pooled. If further purification was required, uricase was applied to a 0.25 X 5-cm column of reactive blueSepharose CL-6B which was then washed with 20 ml Tes-dithiothreitol buffer and developed with the same buffer saturated with uric acid. Fractions showing appreciable activity were pooled. Catakwe assay. The disappearance of HzOz was followed at 240 nm (16). Reaction mixtures contained 20 mM H202 in 0.1 M phosphate, pH 7.5.

RESULTS

AND

DISCUSSION

Enzyme pur$catim Results of a typical enzyme purification are presented in Table I. The final purification was 45fold, with a recovery of 25%. Losses during purification were mostly due to the sacrifice of fractions showing less than maximum specific activity, particularly after gel-filtration chromatography. Dithiothreitol was required to maintain activity, and replacement with /3-mercaptoethanol caused a rapid and irreversible loss of activity. The enzyme was not stable upon freezing at -20 or -80°C but was relatively stable at 0-4°C (less than 10% loss of activity after storage for 3 months). The final specific activity was 6.8 pmol min-’ mg protein-’ at 22”C, fourfold higher than that obtained in an earlier preparation (10). The uv spectrum of the purified protein exhibited a maximum at 2’7’7nm and a 280/260 ratio

192

LUCAS,

BOLAND, TABLE

AND

SCHUBERT

I

PURIFICATION OF URICASE FROM SOYBEAN NODULES Volume (ml) Crude extract Ammonium sulfate Sepharose 6B Blue-Sepharose CL-6B a The activity each step.

Activity (pm01 min-‘)

Protein (mg)

Specific activity (U mg protein-‘)

86

49.9

16

41.3

45.1

32

21.0 5.4

3.4 0.8

12.7

of the crude extract

340

0.15 0.92

100 84

6.2 6.8

46 25

is defined as 100%; other values are corrected

of 1.15, which suggest that the enzyme was not contaminated with phenolics. The elution profile for both uricase and catalase from the gel-filtration step is shown in Fig. 1. The two activities were clearly resolved, in contrast to results obtained previously (9) for the enzyme purified from cowpea nodules. The small degree of purification in the final step suggests that the enzyme from the gel-filtration step was at a degree of purity approaching homogeneity. This is supported by results of gel electrophoresis on nondenaturing and SDS gels. The one band detected using an uricase activity stain corresponded to the only protein band visible on the gels (Fig. 2). SDS-gel electro-

Percentage recove@

for samples withheld

phoresis of the purified protein showed the presence of one major band, presumably uricase (Fig. 3). This band intensified throughout the purification (data not shown). The enzyme obtained was nearly homogeneous (greater than 95% pure) after the gel-filtration step, and affinity chromatography provided little additional purification. A second, weaker band of protein with a molecular weight twice that of the major band was tentatively identified as a dimer of uricase, covalently crosslinked. This material copurified with uricase, and was most noticeable in the eluate from the blue-Sepharose column. It is thought that hydrogen peroxide, produced in the uri-

24

IO

14

18

22

26

at

30

34

38

42

46

50

FRACTION FIG. 1. Elution profiles for uricase and catalase from soybean nodules from the Sepharose 6B column. The column was equilibrated with 25 rnM Tes, 5 rnrd dithiothreitol at pH 7.5. Catalase activity (A), uricase activity (0), and absorbance at 280 nm (0).

URICASE

FROM

SOYBEAN

193

NODULES

weight of 32,000 f 3000 was obtained (Fig. 5). A value of 68,000 was obtained for the second, weaker band. These results suggest that the native enzyme at pH 7.5 is a monomer with a molecular weight of about 32,000 to 35,000. At pH 8.8, the enzyme associates to form a dimer of about 68,000. This is an exceptionally low molecular weight for uricase. The enzyme from most other sources has a molecular weight of about 100,000 (17). pH dependence. The pH dependence of purified uricase from soybean nodules can be seen in Fig. 6. The maximum activity at pH 9.5 is in accord with a pH optimum of between 9 and 10 observed for the purified cowpea enzyme (9) and an early report on the soybean enzyme (10). Studies on the pH dependence of uricase in crude extracts showed a significant shoulder of

FIG. 2. Gel electrophoresis of uricase under nondenaturing conditions. Purified uricase (20 fig of protein from the pooled peak fractions off of a Sepharose 6B column) was subjected to electrophoresis for 5 h at 2 mA/gel. Gels were stained as follows: (A) activity stain minus uric acid, (B) activity stain with 0.2 mg/ ml uric acid, and (C) Coomassie blue.

- Origin

case-catalyzed reaction, could cause oxidative crosslinking of enzyme molecules. This reaction would be most favorable when the reaction with uric acid occurs at high concentrations of enzyme and in the absence of catalase. Such conditions were present during the elution and in the eluate of the blue-Sepharose column. A third, very faint band which may be an impurity was sometimes visible at heavy loadings of protein. Molecular weight. The molecular weight of the enzyme was estimated by gel-filtration chromatography. At pH 7.5, the apparent molecular weight of uricase was 35,000 f 5000. The estimated molecular weight was twice that for the enzyme extracted and purified at pH 8.8 (Fig. 4). Based on the relative mobility after SDSgel electrophoresis, a subunit molecular

-Dimer ?

- Urateoxidase monomer -Dye + FIG. 3. SDS-gel electrophoresis of purified uricase from soybean nodules plus molecular weight markers. (A) 1.8 pg uricase after blue-Sepharose affinity chromatography; (B) molecular weight standards, BSA, aldolase, carbonic anhydrase; (C) 45 pg of purified uricase.

194

LUCAS,

T 2

Soybean

; .E E

2.0

u

1.5

a .z .> t

I.0

Soybean

uricase,

uricose,

BOLAND,

AND

SCHUBERT

pH 7

pH 8.8

a

u 0.5 % .-z 5 ’ 20

30

40

50 60 70 80 Fraction Number

90

100

FIG. 4. Elution profiles of soybean uricase and pig heart uricase at pH 7.5 and 8.8. Uricase was extracted and partially purified from 9-10 g of soybean nodules through the ammonium sulfate precipitation step in either 25 mM Tes, 5 mM dithioerythritol, pH 7.5, or 25 mM Tris-HCl, 5 mM dithioerythritol, pH 8.8. Samples were applied to a 1.5 X ‘75-cm Sepharose 6B column equilibrated with the same buffer as used in the initial extraction and purification. The elution profiles for soybean uricase at pH 7.5 (0) and pH 8.8 (0) and for pig heart uricase (IV, 100,000) at pH 7.5 are shown. The elution profile of pig heart uricase was unchanged at the higher pH. The peak of malic dehydrogenase (M, 70,000) activity was in Fraction 70.

PH

FIG. 6. Dependence of crude and purified uricase activity upon pH. Reaction mixtures contained 0.1 mM uric acid, 250 @M Oz, and 20 or 50 gl of crude (m) or purified (0) enzyme in a final volume of 1.5 ml. Buffers used were 50 mM Tes from pH 6.5 to 8.4 and 50 mM Ches from pH 8.4 to 11.1; 50 mM Caps buffer was used from pH 9.9 to 10.9 for the crude enzyme. Actual pH of reaction mixtures are presented.

activity at neutral pH. Part of this activity at neutral pH was lost during purification or with time in the crude extract. This ac-

0.8

0.5 F 4

0.4

kt 0.3 Y

0.2 i 44

4.5

4.6

4.7

4.6

4.9

LOG MW FIG. 5. Determination of molecular weight of uricase by SDS-gel electrophoresis. Standards used were bovine serum albumin (68,700); catalase (60,000); aldolase (40,000); carbonic anhydrase (29,000); and chymotrypsinogen (25,900). (+) Uricase.

I

I

I

IO 20 30 [ 02]-’ mM-’ FIG. 7. Substrate dependence of uricase activity at pH 9.5. Uric acid concentrations were 100 (O), 22 (0), and 14 PM (Cl). Inset: Replot of intercepts as a function of uric acid concentration.

URICASE

0

IO 20 [xanthine]

0

FROM

SOYBEAN

30 mM

I

I

I

I

I

I

IO

20

30

40

50

60

[Uric

acid]-’

mM-’

FIG. 8. Inhibition of uricase activity by xanthine at pH 9.5 in the presence of 250 qd oxygen. Concentrations of xanthine used were 0 (O), 10 (O), 20 (A), and 30 (0) pM. Inset: Replot of slopes as a function of xanthine concentrations.

TABLE

195

NODULES

tivity may be due to an unstable isozyme of uricase, such as that described from radicles of developing soybean seedlings (10). Even the purified enzyme exhibited a shoulder of activity at neutral pH. This may be due to the reduced catalytic activity of monomeric uricase. Alternatively, small amounts of the dimer may be in equilibrium with the monomer at neutral pH and account for the activity. Substrate dependent. The dependence of the reaction rate on substrate concentration is given in Fig. 7, which indicates an intersecting pattern of lines, i.e., the reaction involves a ternary complex. Lines were fitted using a weighted least-squares method (18). Each point is the mean of three replicates, and the degree of scatter is mostly due to difficulty in maintaining the required oxygen concentrations. The intercept replot gives a K, for uric acid of 10 f 1 PM. When these results were plotted as a function of uric acid concentration (not shown), a K, for oxygen of 31 & 5 PM was obtained from the intercept replot. The K, values found here do not differ markedly from values of 18 PM for uric acid and 29 PM for oxygen reported for the cowpea enzyme (9). A Km for uric acid of 8.1 PM has been reported for the soybean

II

COMPARISON BETWEEN PURIFIED SOYBEAN AND COWPEA URICASE Property pH optimum

Km (Uric acid) Km (0,) Specific activityb Molecular weight Catalase activity Xanthine inhibition Inhibition by NH:, glutamine, adenine, allantoin

Soybean

Cowpea”

9.5 10 PM 31 &LM 10.0 gmol-’ mg-’ 32,000 (pH 7.5) 68,000 (pH 8.8) No Strong

9-10 18 /.LM 29 pM 5.7 pm01 min-’ mg-’ 50,000 (pH7.5) 190,000 (pH 8.8) Yesd Strong

Weak, if at all

“Data from Ref. (9). * Corrected for temperature of assays in (9) (30°C). ‘Results obtained in this work. d Not confirmed in this work.

Strong

196

LUCAS,

BOLAND,

AND

I

I

40 FRACTION FIG. 9. Elution profiles for catalase and uricase from cowpea nodules after chromatography on a Sepharose 6B column. Catalase (0) and uricase (0) activities in 8.6-ml fractions.

enzyme (10). Rainbird et al. (9) suggested that the relatively high Km for oxygen may limit uricase activity in nodules where the concentration of dissolved Oz in bacteroidcontaining cells is believed to be lo-200 nM. This has now been largely circumvented. Organelle fractionation studies (19) have indicated that uricase is located in the peroxisomes, and electron microscopy studies (20) demonstrate that these organelles are largely located in the uninfected cells of the nodule. These cells contain no leghemoglobin and, since air is believed to circulate freely throughout the nodule (21), the availability of oxygen at concentrations in equilibrium with atmospheric concentrations (250 PM) should not represent a factor limiting uricase activity and, therefore, ureide biogenesis. Inhibition studies. Uricase from cowpea TABLE

SCHUBERT

has been reported to be inhibited by a variety of nitrogenous compounds (9). Results of inhibition studies with the purified soybean enzyme were substantially different. Other than xanthine, the only significant inhibition was caused by ammonium, which inhibited the reaction by 16%. This occurred at an unphysiologically high concentration (100 mM). The strong reported inhibition of the cowpea enzyme by 0.5 mM adenine, 0.5 mM allantoin, 0.006 mM glutamine, or 0.006 mM ammonium (9) was not observed with the soybean enzyme. Glutamate (10 mM), glutamine (5 mM), aspartate (5 mM), asparagine (5 InM), allantoin (5 mM), allopurinol (0.1 mM), adenine (0.5 IrIM), and serine (10 mM) did not inhibit the uricase reaction significantly (less than 10%). Xanthine, however, was found to be a strong inhibitor, competitive with respect to uric acid (Fig. 8). From these results a K1 of 10 I.~M was obtained using a slope replot (Fig. 8, inset). This KI is somewhat lower than a value of 45 PM previously reported for the soybean enzyme (10). Com~+m with urkase from cowpea. Soybean uricase has many kinetic properties similar to those of the cowpea enzyme; however, some properties are markedly different (Table II). A partial purification of uricase was carried out according to the soybean protocol for 20.4 g of cowpea nodules. Most of the enzyme (93%) was recovered in the O-35% ammonium sulfate pellet. The elution profile for the Sepharose 6B column is shown in Fig. 9. Uricase and catalase were clearly separated. The estimated molecular weight of cowpea uricase was 50,000 + 20,006 at pH 7.5, in contrast with the value of 100,000 reported III

PARTIAL PURIFICATION OF URICASE FROM COWPEA NODULES Total activity” (pm01 min-‘) Crude extract Ammonium sulfate Pooled gel filtration

pellet fractions*

19.1 (12.3) 19.4 (4.2) 11.1 (1.1)

Specific activity (U mg protein-‘) 0.15 (0.03) 1.1 (0.17) 5.6 (0.21)

o From 20.4-g nodules. Values in parentheses are those reported in (9) from 26-g nodules. b Corrected for volume of resuspended pellet which was applied to the column.

Percentage recovery 100 (100) 101 (34) 53 (9)

URICASE

FROM

SOYBEAN

previously for the enzyme at pH 8.8 (9). The estimated subunit molecular weight was 50,000 (data not shown). The formation of a dimer at pH 8.8, as observed for the soybean enzyme, would account for this apparent anomaly. Our purification is summarized in Table III. Results reported previously are given in parentheses. The yields and specific activity for cowpea uricase purified according to the procedure developed for soybeans were several-fold higher than those reported previously (9). Three significant differences exist between the two methods of purification: (i) the inclusion of PVP in the extraction medium which helps prevent denaturation of proteins by phenolic compounds in the nodule tissue; (ii) the use of dithiothreitol in buffers throughout purification which stabilizes enzyme activity; and (iii) the difference in pH used during the isolation and purification of uricase. ACKNOWLEDGMENTS We would like to thank Dr. N. E. Tolbert and Dr. C. Sueiter for their review of this manuscript. REFERENCES 1. ATKINS, C. A., RAINBIRD, R. M., AND PATE, J. S. (1980) 2. PJEanzenphysioL 97,249-260. 2. FUJIHARA, S., AND YAMAGUCHI, V. (1978) Plant PhysioL 62, 495-498.

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3. SCHUBERT, K. R. (1981) Plant PhysioL 68, 11151122. 4. BOLAND, M. J., AND SCHUBERT, K. R. (1981) Arch. Biochem Biophys. 209,524-533. 5. TRIPLEXT, E. W., BLEVINS, D. E., AND RANDALL, D. D. (1980) Plant PhysioL 65, 1203-1206. 6. ATKINS, C. A. (1981) FEBS ktt. 125, 89-93. 7. BOLAND, M. J. (1981) Biochem. Int. 2,567-574. 8. TRIPLETT, E. W., BLEVINS, D. G., AND RANDALL, D. D. (1981) Plant PhysioL 675, 27. 9. RAINBIRD, R. M., AND ATKINS, C. A. (1981) B&him Biophys. Actu 659, 132-140. 10. TAJIMA, S., AND YAMAMOTO, Y. (1975) Plant Cell PhysioL 16,271-282. 11. FISHBECK, K., EVANS, H. J., AND BOERSMA, L. L. (1973) Apm J. 65,429-433. 12. DAVIS, B. J. (1964) Ann N. Y. Acud. Sci 121,404427. 13. LAEMMLI, U. K., ANDFAVRE, M. (1973)J. MoL BioL 80,575-599. 14. BENSADOLJN,A., AND WEINSTEIN, D. (1976) Anal Biochem. 70,241-250. 15. LOOMIS, W. D. (1974) in Methods in Enzymology (Fleischer, S., and Packer, L., eds.), Vol. 31, pp. 528-544, Academic Press, New York. 16. LUCK, H. (1965) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd ed., pp. 885-894, Academic Press, London/New York. 17. VOGELS, G. D., AND VAN DER DRIFT, C. (1976) BacterioL Rev. 40,403-468. 18. WILKINSON, G. N. (1969) Biodwm. J. 80,324. 19. HANKS, J. F., TOLBERT, N. E., AND SCHUBERT, K. R. (1981) Plant PhysioL 68, 65-69. 20. NEWCOMB, E. H., ANDTANDON, S. I. (1981) Science 212, 1394-1396. 21. BERGERSEN, F. J., AND GOODCHILD, D. J. (1973) Amt. J. BioL Sci 26, 729-740.