Urate oxidase of Chlamydomonas reinhardii

September 25, 2017 | Autor: Manuel Pineda | Categoria: Metabolism, Plant Biology, Nitrogen metabolism, Biochemistry and cell biology, Thallophyta
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PHYSIOL. PLANT. 62: 453-457. Copenhagen 1984 •i-.






Urate oxidase of Chlamydomonas reinhardii Manuel Pineda, Emiiio Fernandez and Jacobo Cardenas

Pineda, M., Fernandez, E. and Cardenas, J. 1984. Urate oxidase of Chlamydomonas reinhardii, - Physiol. Plant. 62: 453-457. Urate oxidase (EC of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O^ for activity, have an apparent K^ of 12 jiA/ for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyidithiocarbamate and o-phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo, urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate. Additional key words - Enzyme repression, nitrogen metabolism, purine pathway. M. Pineda, E. Fernandez and J, Cardenas (reprint requests), Departamento de Bioquimica, Facultad de Ciencias, Avda. Medina Azahara s/n, Cordoba, Spain.


The scanty reports on purine degradation in algae suggest that the catabolism of these compounds follows the pathway operating in yeasts and higher piants (Vogels and Van der Drift 1976). Available data are restricted to physiological studies on the use of purines and some degradation products of purines as a nitrogen source for growth (Miller and Fogg 1958, Birdsey and Lynch 1962, Ammann and Lynch 1964, Cain 1965, Antia ei al. 1980, Devi Prasad 1983) and to the occurrence of some enzymes involved in the pathway (Villeret 1955, 1958, Fernandez and Cardenas 1981), but nothing is known on the properties of these enzymes. In the present work the partial characterization of urate oxidase of the green alga Chlamydomonas reinhardii is presented. In addition, the degradation steps of purine oxidation in Chlamydomonas are established by means of physiological experiments. Materials and methods

Cells of Chlamydomonas reinhardii 6145c (from the Received 30 March, 1984; revised 26 June, 1984 Physiol. Piant. 52, 1984

collection of Dr Ruth Sager) were grown at 25°C, under conditions of light saturation, in the culture medium of Sueoka (1960). As the sole source of nitrogen purines or their derivatives were used at a eoncentration of 1 mM, or urea and ammonia at 2 and 4 mM, respectively. Unless otherwise stated cells were harvested at midexponential phase of growth, washed with distilled water and, after centrifugation, broken by freezing at -40°C and thawing with gentle stirring in 0.1 M Tris-HCl (pH 9.0). The homogenate was centrifuged at 27000 g for 10 min, and the resulting supernatant was used as the source of enzyme. The kinetic studies were performed with enzyme preparations filtered through a Sephadex G-25 column (1.5 X 7.5 cm) using 0.1 M TrisHCl (pH 9.0) as eluent. Growth was measured turbidimetricaily by following the absorbance of cell suspensions at 660 nm. Urate oxidase (urate:02 oxidoreductase, EC 1.7,3.3) was assayed spectrophotometrically by the decrease in absorbance at 292 nm due to enzymatic oxidation of urate. The assay mixture contained in a total volume of 1.0 ml: 100 mM Tris-HCl (pH 9.0), 50 pJW urate and enzyme extract. The assays were carried out aero-

bically. One unit of urate oxidase activity is defined as the amount of enzyme which oxidizes 1 [i,mol of urate min' under optimal conditions of assay. Specific activity is expressed in mU (mg protein)""'. Protein was estimated by the method of Bradford (1976). Ammonia was determined colorimetrically by the Conway microdiffusion technique (Conway 1957). Urate was measured directly by following its absorbance at 292 nm (molar extinction coefficient 1.22 x 10^) or enzymatically by using the urate oxidase assay as described above. Adenine, guanine, hypoxanthine, and xanthine were measured directly at 260, 274, 250, and 268 nm, respectively, according to Eschmann and Kaltwasser (1982). Allantoin and allantoate were estimated by the alkaline formation of the chromophore of 2,4dinitrophenylhydrazone of glyoxyiic acid (Borchers 1977). All spectrophotometric assays and determinations were performed in a Bausch & Lomb Spectronic 2000. Data presented in this work are mean values from three independent experiments. Electrophoresis was performed on 7.5% (w/v) poiyacrylamide gels according to Jovin et al. (1964). Adenine, guanine, hypoxanthine, xanthine, uric acid, aliantoin, atlantoic acid, Coomassie Brilliant Blue G-250 and /7-hydroxymercuribenzoate were purchased from Sigma, St. Louis, MO, USA. Sephadex G-25 was from Pharmacia, Uppsala, Sweden. All other chemicals used were of analytical grade. Results

1 Fig. 1. Visualization of urate oxidase on polyacrylamide gels. Extracts from cells grown on urate were run on polyacrylamide gels under conditions described in Materials and methods. The urate oxidase band (arrows) was located by immersing the gels in a solution containing 0.1 mM urate, 2 mM ^-nitro blue tetrazolium and 0.03 mM phenazine methosulfate in 0.1 M Tris-HCl, pH 9.0 (gel 2), or 0.1 mM urate, 2.8 mM diaminobenzidine and 3.3 U of horseradish peroxidase in the same buffer (gel 4). Gels 1 and 3 are the corresponding controls without urate. The colorless bands on gels 1 and 2 correspond to superoxide dismutase present in the extracts.

Extracts of Chlamydomonas reinhardii cells oxidized urate enzymatically (Tab. 1). Cell extract, urate and Oj were required for oxidation, whereas no oxidizing activity was detected after heating of the extract. Dialysis of the extract enhanced the activity, which suggests the presence of an inhibitor of low molecular weight in the enzyme preparation (results not shown). The amount of urate oxidized was linear with time for at least 5 min, but it was proportional to the amount of ceil extract only at low enzyme concentrations. At high con-

centrations, the activity did not increase correspondingly (results not shown), which corroborates the presence of an inhibitor in the crude enzyme extract. After dialysis urate oxidase activity was proportional to the amount of enzyme extract used. Extracts obtained by different celi disruption techniques showed great differences in activity. The highest activity was found after freezing and thawing [41 mU (mg protein)"']. Lower activities were found after sonication (90 W, 2 min) (33 mU mg'), or French Tab. 1. Urate oxidation by cell extracts of Chlamydomonas press (8.5 MPa) treatment (18 mU - mg'). After filterreinhardii. The complete system included in a final volume of 1 ing the extracts through a Sephadex G-25 column the mi: 0.1 ml (0.2 mg protein) enzyme extract, 100 mM Trls-HCl, specific activities increased 1.2-2.8 times, indicating the pH 9.0 and 50 \iM urate. The assay was carried out aerobically presence of an inhibitor in the extracts. This inhibitor is in open cuvettes. Where indicated 0.1 ml of boiled extract was thermostable, since crude extracts heated at 100°C for 5 added. Cells were grown on urate as nitrogen source. min still inhibited the urate oxidase reaction (Tab. 1). Specific activity System The enzyme activity could also be detected on 7.5% (mU • mg-') polyacrylamide gels as one band (Rp = 0.27) either by urate oxidation with /j-nitro blue tetrazolium or by spe41 Complete cific reaction of H2O2, one of the products of the en2 minus urate zymatic oxidation of urate, with diaminobenzidine in 0 minus O2 ' . • • '• the presence of peroxidase (Fig. 1). 0 minus cell extract The pH optimum for the urate oxidase reaction was Complete, cell extract heated 0 5 min at lOO^C 9.0 using Tris-HC! and borate buffers. A double reComplete plus boiled extract 34 ciprocal plot yielded an apparent K^ for urate of 12 ^iM Physiol. Plant. 62,19S4







Tab. 3. Effect of purines and purine derivatives on urate oxidase activity. Compounds were preincubated with the enzyme for 5 min before the addition of urate. Other experimental conditions were as described in the caption of Tab. 1.

1 V



Compound added





^ 0.5




0.2 -



Adenine Guanine Hypoxanthine Xanthine


Allantoin Allantoate Urea NH+,

LJ •







0 0 48 44 100 11 7 0 0

0.05 0.5 2.0 2.0 2.0 2.0

-• . -



Fig. 2. The dependency of the initial velocity of urate oxidase on urate concentration. Assays were carried out with 100 ^il aliquots of filtered extracts freed from inhibitor. Inset: Double reciprocal plot of initial velocity and substrate concentration.

(Fig. 2). The reaction was inhibited by higher concentrations of urate. The effect of several chelating agents on urate oxidase activity is shown in Tab. 2. At low concentrations the most effective inhibitors were cyanide, cupferron, diethyidithiocarbamate and o-phenanthroline. Cyanate, L-ascorbate, 8-hydroxyquinoline, thiosemicarbazide and hydroxylamine were also inhibitory but at higher concentrations, Hypoxanthine and xanthine markedly inhibited urate Tab. 2. Effect of chelating agents on urate oxidase activity. Assay conditions were as described in the caption of Tab. 1 except that reagents were preincubated with the enzyme for 5 min before starting the reaction by addition of urate. Agent added

Concentration (M)


2x10^ 2x10-5 2x10-* 2x10-^ 2X1CH 2x10-' 2x10-3 1x10-3 2x10-3 1x10-3 2xl{h3 2x10-3

52 100 11 100 36 53 100 74 100 58 100 52 32 33 23 15

Sodiuni cyanide Cupferron Diethyidithiocarbamate o-Phenanthroline

' ,

Sodium cyanate . -.

Hydroxylamine 8-Hydroxyquinoline Thiosemicarbazide p-Hydroxymercuribenzoate a,a'-Dipyridyl Sodium azide ,. Semicarbazide Sodium thiocyanate EDTA Physiol. Ptant, 62, J9S4

2.0 0.1 2.0









' •

Concentration (mM)

2x10-3 2x10-3 2x10-3 2x10-3 4x10-3 ••



2x10-3 2x10-3



0 0 0

Tab. 4. Growth rate and urate oxidase activity of Chlamydomonas reinhardii cells cultured with different sources of nitrogen. Cells grown on ammonia were transferred to media containing the indicated N-sources (4-5 mM in N) and harvested at mid-exponential phase of growth. Activity was determined in crude extracts as described in Materials and methods and in the caption of Tab. 1. Nitrogen source Adenine Guanine Hypoxanthine Xanthine Urate Allantoin Allantoate Urea Ammonia Nitrate

Doubling time Urate oxidase (h) activity (mU • mg-') 18.0 8.0

11.5 11.0 8.0

12.0 12.0 7.5 8.5 9.0

21 23 31 31 39 16 14 15 5 5

oxidase activity, whereas the enzyme was unaffected or only slightly inhibited by adenine, guanine, allantoin, allantoate, urea, or ammonia (Tab. 3). Chlamydomonas cells could grow phototrophically on purines and purine derivatives as sole source of nitrogen (Tab. 4). Similar rates of growth were achieved with urea, urate, guanine, ammonia and nitrate. Hypoxanthine, xanthine, allantoin and aliantoate were also good nitrogen sources but yielded lower growth rates, whereas adenine showed the lowest growth rate of all nitrogen sources tested. The course of uptake of some nitrogen sources is presented in Fig. 3. Hypoxanthine, allantoin and allantoate exhibited longer lag periods than urate or xanthine before being taken up. The specific activity of urate oxidase was highest in cells grown on urate (Tab. 4). High levels of enzyme were also detected in cells grown on xanthine, hypoxanthine, guanine and adenine, whereas cells grown on allantoin, aliantoate and urea had less than 50% of the activity found in ceils grown on urate. The enzyme ievel was low in cells cultured with nitrate or ammonia, or

whenever ammonia was present in the culture medium (TJib. 4, Fig. 4B). After exhaustion of ammonia, urate oxidase activity increased (Fig. 4B). When ammonia and urate were present together in the medium, ammonia was taken up immediately, whereas urate was used simultaneously with ammonia only after a lag period of 8 h. In this case, urate oxidase activity remained low even after urate accumulation within the cells until ammonia disappeared from the medium (results not shown). When cells grown on ammonia were transferred to a medium with urate, an increase of urate oxidase activity parallel to growth was observed (Fig. 4A). Depletion of urate was always accompanied by an enhancement of the enzyme levels. Discussion In the present paper the urate oxidase characterization of a green alga is reported for the first time. Previous studies on purine degradation in algae indicated that Fig. 3. The uptake of purine and purine derivatives by Chlamy- some Chlorella (Birdsey and Lynch 1962) and domonas reinhardii cells. Cells grown on ammonia were Chlamydomonas (Cain 1965) species were able to use washed and transferred to media containing the indicated ni- uric acid as nitrogen source for growth. Similar types of trogenous compounds: • , hypoxanthine; • , xanthine; • , growth in response to uric acid had been found in some urate; O. aliantoin; D, allantoate; A, ammonia. Xanthophyceae (Miller and Fogg 1958) and marine blue-green algae (Van Baalen and Marler 1963), but nothing had been reported on enzymatic urate degradation in green algae. Urate oxidase activity from Chlamydomonas reinhardii cells could be detected either by following the enzymatic oxidation of urate spectrophotometrically or by specific staining reactions on polyacrylamide gels. Crude extracts obtained by freezing and thawing, sonication or disruption of cells in a French press contained a thermostable inhibitor of low molecular weight since, after filtration through Sephadex G-25, the specific activity increased 1.2 to 2.8-fold. The pH optimum (9.0) for in vitro assay is similar to that of the enzyme of soybean nodules infected by Rhizobium (Tajima and Yamamoto 1975, Lucas et al. 1983), glyoxysomes of castor bean endosperm (Theimer and Beevers 1971) or nitrogen-fixing nodules of cowpea (Rainbird and Atkins 1981). Urate oxidase of Chlamydomonas has a low apparent Kn, for urate (12 \iM), is inhibited by high concentrations of urate and shows a high sensitivity to metal chelating agents. Similar properties have been reported for urate oxidases from different sources (Theimer and Beevers 1971, Tajima and Yamamoto 1975, Vogels and Van der Drift 1976, Rainbird and Atkins 1981, Lucas et al. 1983). The inhibition by metal-chelating agents suggests that Chlamydomonas urate oxidase is also a metallo-enzyme. . . -: . •.



15 TIME (hours)

10 TIME (hours)

Fig. 4. Growth and urate oxidase levels of Chlamydomonas reinhardii cells cultured with urate (A) and ammonia (B) as sole nitrogen source. Cells grown on 10 mM ammonia were washed and transferred to the indicated media. Physiol. Plant. 62, 1584

Hypoxanthine and xanthine inhibited urate oxidase of Chlamydomonas very efficiently. A similar inhibition has been reported for the enzyme from nodules of some leguminous plants (Tajima and Yamamoto 1975, Lucas et al. 1983) and mammalian tissues (Mahler et at. 1956). The strong inhibition of the cowpea nodule enzyme by adenine, aliantoin and ammonia (Rainbird and Atkins 1981) was not observed for urate oxidase from Chlamydomonas. Chlamydomonas cells could use adenine, guanine, hypoxanthine, xanthine, urate, allantoin and allantoate as sole nitrogen source for phototrophic growth. Adenine and urate have been reported as nitrogen source for some Chlorophyceae (Birdsey and Lynch 1962, Cain 1965) and Xanthophyceae (Miller and Fogg 1958). Chlorella pyrenoidosa grows with hypoxanthine and xanthine but cannot use allantoin, presumably due to its inability to transport this compound (Ammann and Lynch 1964). In contrast, allantoin serves as sole nitrogen source for growth of some marine benthic microalgae (Antia et al. 1980) and freshwater green algae (Devi Prasad 1983). Chlamydomonas reinhardii used easily not only allantoin but also allantoate as sole nitrogen source (Tab. 4, Fig. 3) after a lag period probably needed for the synthesis of the uptake system. Our results strongly suggest that the standard pathway of aerobic degradation of purines (adenine —* hypoxanthine -* xanthine —* urate -^ allantoin -^ allantoate -^ end products), as is found in higher plants., animals and many microorganisms (Vogels and Van der Drift 1976, Thomas and Schrader 1981), also operates in Chlamydomonas, In fact, we have detected xanthine dehydrogenase (Fernandez and Cardenas 1981), urate oxidase and allantoinase activities in Chlamydomonas (M. Pineda, E. Fernandez and J. Cardenas, unpublished results) which corroborates the existence of this degradative pathway. Ammonia depressed urate oxidase levels and, after ammonia exhaustion, activities similar to those existing in cells grown on urate were reached. Since ammonia did not prevent the entry of urate into the cells, we conclude that ammonia represses the synthesis of the enzyme, as reported for uricase of Neurospora crassa (Wang and Marzluf 1979). On the other hand, urate does not seem to be essential for the induction of enzyme since, after urate exhaustion from the medium, activity levels higher than in the presence of urate were found, and the rate of enzyme synthesis was very similar in both urate and ammonia-depleted media (Fig. 4A, B). The enzyme repression caused by nitrate is probably due to the formation of ammonia since the cells have active nitrate and nitrite reductases.







; .-,^^4'.

Ammann, E. C. B. & Lynch, V. H. 1964. Purine metabolism by unicellular algae. II. Adenine, hypoxanthine, and xanthine degradation by Chlorella pyrenoidosa. - Biochim. Biophys. Acta 87: 370-379. Antia, N. J., Berland, B. R., Bonin, D. J. & Maestrini, S. Y. 1980. Ailantoin as nitrogen source for growth of marine benthic microalgae, - Phycologia 19: 103-109. Birdsey, E. C. & Lynch, V. H. 1962. Utilization of nitrogen compounds by unicellular algae. - Science 137: 763-764. Borchers, R. 1977. Allantoin determination - Anal. Biochem. 79: 612-613. Bradford, M. M. 1976. A rapid and sensitive method for the quEintitation of microgram quantities of protein utilizing the principle of protein-dye binding - Anal. Biochem. 72: 248254. Cain, J. 1965. Nitrogen utilization in 38 freshwater chlamydomonad algae - Can. J. Bot. 43: 1367-1378. Conway, D. J. 1957. Microdiffusion Analysis and Volumetric Error - pp. 90-132. Crosby Lockwood, London. Devi Prasad, P. V. 1983. Hypoxanthine and allantoin as nitrogen sources for the growth of some freshwater green algae New Phytol. 93: 575-580. Eschmann, K. & Kaltwasser, H. 1982. Purine uptake by Acaligenes eutrophus H 16 - Arch. Microbiol. 131; 191196, Fernandez, E. & CTardenas, J. 1981. Occurrence of xanthine dehydrogenase in Chlamydomonas reinhardii: A common cofactor shared by xanthine dehydrogenase and nitrate reductase - Planta 153: 254-257. Jovin, T., Chrambach, A. & Naughton, M. A. 1964. Apparatus for preparative temperature regulated polyacrylamide gel electrophoresis - Anal. Biochem. 9: 351-364. Lucas, K., Boland, M. J. & Schubert, K. R. 1983. Uricase from soybean root nodules: Purification, properties, and comparison with the enzyme from cowpea - Arch. Biochem. Biophys. 226: 190-197. Mahler, H. R., Baum, H, M. & Hubscher, G. 1956. Enzymatic oxidation of urate - Science 124: 705-708. Miller, J. D. A. & Fogg, G. E. 1958. Studies on the growth of Xanthophyceae in pure culture. II. The relations of Monodus subterraneus to organic substances - Arch. Microbiol. 30: 1-16. Rainbird, R. M. & Atkins, C. A. 1981. Purification and some properties of urate oxidase from nitrogen-fixing nodules of cowpea - Biochim. Biophys. Acta 659: 132-140. Sueoka, N. 1960. Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardii-Vroc. Natl. Acad. Sci. USA 46: 83-91. Tajima, S. & Yamamoto, Y. 1975. Enzymes of purine catabolism in soybean plants - Plant Ceil Physiol. 16: 271-282. Theimer, R. R. & Beevers, H. I97L Uricase and allantoinase in glyoxysomes - Plant Physiol. 47: 246-251. Thomas, R. J. & Schrader, L. E. 1981. Ureide metabolism in higher plants - Phytochemistry 20: 361-371. Van Baalen, C. & Marler, J. E. 1963. Characteristics of marine blue-green algae with uric acid as nitrogen souree - J. Gen. Microbiol. 32: 457-463. Villeret, S, 1955. Sur la presence des enzymes des ur^ifdes glyoxyliques chez les algues d'eau douce - C. R. Acad. Sci. (Paris) 241: 90-92. - 1958. Recherches sur la presence des ureides giyoxyliques chez les algues marines - C. R. Acad. Sci. (Paris) 2461452-1454. Vogels, G. D. & Van der Drift, C. 1976. Degradation of and pyrimidines by microorganisms - Bacteriol Acknowledgements - This work has been supported by the purines Rev. 40: 403-468. Grant no. 1834-82 of Comision Asesora de Investigacion Cien- Wang, L. W. C. & Marzluf, G. A. 1979. Nitrogen regulation of ti'fica y Tecnica (Spain). uricase synthesis in Neurospora crassa - Molec. Gen Genet. 176: 385-392.

Edited by B. Jergil Physio!. Plant. 62, 1984

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