Urate oxidase of Chlamydomonas reinhardii

PHYSIOL. PLANT. 62: 453-457. Copenhagen 1984

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Urate oxidase of Chlamydomonas reinhardii

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Manuel Pineda, Emiiio Fernandez and Jacobo Cardenas

Pineda, M., Fernandez, E. and Cardenas, J. 1984. Urate oxidase of Chlamydomonasreinhardii, - Physiol. Plant. 62: 453-457.

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines andpurine derivatives has been partially characterized. Crude enzyme preparations havea 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 wasparticularly sensitive to metal ion chelating agents like cyanide, cupferron, di-ethyidithiocarbamate and o-phenanthroline, and to structural analogues of urate likehypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on ade-nine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogensource, indicating that in this alga the standard pathway of aerobic degradation ofpurines of higher plants, animals and many microorganisms operates. As deducedfrom experiments in vivo, urate oxidase from Chlamydomonas is repressed in thepresence of ammonia or nitrate.

Additional key words - Enzyme repression, nitrogen metabolism, purine pathway.

M. Pineda, E. Fernandez and J, Cardenas (reprint requests), Departamento de Bio-quimica, Facultad de Ciencias, Avda. Medina Azahara s/n, Cordoba, Spain.

Introduction

The scanty reports on purine degradation in algae sug-gest that the catabolism of these compounds follows thepathway operating in yeasts and higher piants (Vogelsand Van der Drift 1976). Available data are restricted tophysiological studies on the use of purines and somedegradation products of purines as a nitrogen source forgrowth (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 en-zymes involved in the pathway (Villeret 1955, 1958,Fernandez and Cardenas 1981), but nothing is knownon the properties of these enzymes.

In the present work the partial characterization ofurate oxidase of the green alga Chlamydomonas re-inhardii is presented. In addition, the degradation stepsof purine oxidation in Chlamydomonas are establishedby means of physiological experiments.

Materials and methods

Cells of Chlamydomonas reinhardii 6145c (from the

collection of Dr Ruth Sager) were grown at 25°C, underconditions of light saturation, in the culture medium ofSueoka (1960). As the sole source of nitrogen purines ortheir 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 mid-exponential phase of growth, washed with distilledwater 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 gfor 10 min, and the resulting supernatant was used asthe source of enzyme. The kinetic studies were per-formed with enzyme preparations filtered through aSephadex G-25 column (1.5 X 7.5 cm) using 0.1 M Tris-HCl (pH 9.0) as eluent.

Growth was measured turbidimetricaily by followingthe absorbance of cell suspensions at 660 nm.

Urate oxidase (urate:02 oxidoreductase, EC 1.7,3.3)was assayed spectrophotometrically by the decrease inabsorbance at 292 nm due to enzymatic oxidation ofurate. The assay mixture contained in a total volume of1.0 ml: 100 mM Tris-HCl (pH 9.0), 50 pJW urate andenzyme extract. The assays were carried out aero-

Received 30 March, 1984; revised 26 June, 1984

Physiol. Piant. 52, 1984

bically. One unit of urate oxidase activity is defined asthe amount of enzyme which oxidizes 1 [i,mol of uratemin' under optimal conditions of assay. Specific ac-tivity is expressed in mU (mg protein)""'.

Protein was estimated by the method of Bradford(1976). Ammonia was determined colorimetrically bythe Conway microdiffusion technique (Conway 1957).Urate was measured directly by following its absor-bance at 292 nm (molar extinction coefficient 1.22 x10 ) or enzymatically by using the urate oxidase assay asdescribed above. Adenine, guanine, hypoxanthine, andxanthine were measured directly at 260, 274, 250, and268 nm, respectively, according to Eschmann and Kalt-wasser (1982). Allantoin and allantoate were estimatedby the alkaline formation of the chromophore of 2,4-dinitrophenylhydrazone of glyoxyiic acid (Borchers1977). All spectrophotometric assays and determina-tions were performed in a Bausch & Lomb Spectronic2000. Data presented in this work are mean values fromthree independent experiments.

Electrophoresis was performed on 7.5% (w/v) poiy-acrylamide gels according to Jovin et al. (1964).

Adenine, guanine, hypoxanthine, xanthine, uric acid,aliantoin, atlantoic acid, Coomassie Brilliant BlueG-250 and /7-hydroxymercuribenzoate were purchasedfrom Sigma, St. Louis, MO, USA. Sephadex G-25 wasfrom Pharmacia, Uppsala, Sweden. All other chemicalsused were of analytical grade.

Results

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

Tab. 1. Urate oxidation by cell extracts of Chlamydomonasreinhardii. The complete system included in a final volume of 1mi: 0.1 ml (0.2 mg protein) enzyme extract, 100 mM Trls-HCl,pH 9.0 and 50 \iM urate. The assay was carried out aerobicallyin open cuvettes. Where indicated 0.1 ml of boiled extract wasadded. Cells were grown on urate as nitrogen source.

System Specific activity(mU • mg-')

Completeminus urateminus O2 ' . • • '•minus cell extract

Complete, cell extract heated5 min at lOO C

Complete plus boiled extract

41200

034

1

Fig. 1. Visualization of urate oxidase on polyacrylamide gels.Extracts from cells grown on urate were run on polyacrylamidegels under conditions described in Materials and methods. Theurate oxidase band (arrows) was located by immersing the gelsin a solution containing 0.1 mM urate, 2 mM ^-nitro bluetetrazolium and 0.03 mM phenazine methosulfate in 0.1 MTris-HCl, pH 9.0 (gel 2), or 0.1 mM urate, 2.8 mM di-aminobenzidine and 3.3 U of horseradish peroxidase in thesame buffer (gel 4). Gels 1 and 3 are the corresponding con-trols without urate. The colorless bands on gels 1 and 2 corre-spond to superoxide dismutase present in the extracts.

centrations, the activity did not increase correspon-dingly (results not shown), which corroborates the pre-sence of an inhibitor in the crude enzyme extract. Afterdialysis urate oxidase activity was proportional to theamount of enzyme extract used.

Extracts obtained by different celi disruption tech-niques showed great differences in activity. The highestactivity was found after freezing and thawing [41 mU(mg protein)"']. Lower activities were found aftersonication (90 W, 2 min) (33 mU mg'), or Frenchpress (8.5 MPa) treatment (18 mU - mg'). After filter-ing the extracts through a Sephadex G-25 column thespecific activities increased 1.2-2.8 times, indicating thepresence of an inhibitor in the extracts. This inhibitor isthermostable, since crude extracts heated at 100°C for 5min still inhibited the urate oxidase reaction (Tab. 1).

The enzyme activity could also be detected on 7.5%polyacrylamide gels as one band (Rp = 0.27) either byurate oxidation with /j-nitro blue tetrazolium or by spe-cific reaction of H2O2, one of the products of the en-zymatic oxidation of urate, with diaminobenzidine inthe presence of peroxidase (Fig. 1).

The pH optimum for the urate oxidase reaction was9.0 using Tris-HC! and borate buffers. A double re-ciprocal plot yielded an apparent K^ for urate of 12 ^iM

Physiol. Plant. 62,19S4

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Tab. 3. Effect of purines and purine derivatives on urate ox-idase activity. Compounds were preincubated with the enzymefor 5 min before the addition of urate. Other experimentalconditions were as described in the caption of Tab. 1.

50 100 150U R A T E ( ; J M )

200

Fig. 2. The dependency of the initial velocity of urate oxidaseon urate concentration. Assays were carried out with 100 ilaliquots of filtered extracts freed from inhibitor. Inset: Doublereciprocal plot of initial velocity and substrate concentration.

(Fig. 2). The reaction was inhibited by higher con-centrations of urate.

The effect of several chelating agents on urate oxi-dase activity is shown in Tab. 2. At low concentrationsthe most effective inhibitors were cyanide, cupferron,diethyidithiocarbamate and o-phenanthroline. Cyanate,L-ascorbate, 8-hydroxyquinoline, thiosemicarbazideand hydroxylamine were also inhibitory but at higherconcentrations,

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. 1except that reagents were preincubated with the enzyme for 5min before starting the reaction by addition of urate.

Agent added

Sodiuni cyanide

Cupferron

Diethyidithiocarbamateo-Phenanthroline

Sodium cyanate

Ascorbate . -.

Hydroxylamine8-HydroxyquinolineThiosemicarbazidep-Hydroxymercuribenzoatea,a'-DipyridylSodium azide ,.SemicarbazideSodium thiocyanateEDTA

Physiol. Ptant, 62, J9S4

Concentration(M)

2x10^2x10-52x10-*2x10-^2X1CH2x10-'

' 2x10-3, 1x10-3

2x10-31x10-32xl{h3

• 2x10-32x10-32x10-32x10-32x10-34x10-3

•• 2 x 1 0 - 3

2x10-3' 2x10-3

Inhibition(%)

5210011

1003653

10074

10058

10052323323150000

Compound added

AdenineGuanineHypoxanthineXanthine

AllantoinAllantoateUreaNH+,

Concentration(mM)

2.00.12.00.050.52.02.02.02.0

Inhibition(%)

0 -• .0

4844 -

100 -11700

Tab. 4. Growth rate and urate oxidase activity ofChlamydomonas reinhardii cells cultured with different sourcesof nitrogen. Cells grown on ammonia were transferred to me-dia containing the indicated N-sources (4-5 mM in N) andharvested at mid-exponential phase of growth. Activity wasdetermined in crude extracts as described in Materials andmethods and in the caption of Tab. 1.

Nitrogensource

AdenineGuanineHypoxanthineXanthineUrateAllantoinAllantoateUreaAmmoniaNitrate

Doubling time(h)

18.08.0

11.511.08.0

12.012.07.58.59.0

Urate oxidaseactivity

(mU • mg-')

212331313916141555

oxidase activity, whereas the enzyme was unaffected oronly slightly inhibited by adenine, guanine, allantoin,allantoate, urea, or ammonia (Tab. 3).

Chlamydomonas cells could grow phototrophicallyon purines and purine derivatives as sole source ofnitrogen (Tab. 4). Similar rates of growth were achievedwith urea, urate, guanine, ammonia and nitrate. Hypo-xanthine, xanthine, allantoin and aliantoate were alsogood nitrogen sources but yielded lower growth rates,whereas adenine showed the lowest growth rate of allnitrogen sources tested. The course of uptake of somenitrogen sources is presented in Fig. 3. Hypoxanthine,allantoin and allantoate exhibited longer lag periodsthan urate or xanthine before being taken up.

The specific activity of urate oxidase was highest incells grown on urate (Tab. 4). High levels of enzymewere also detected in cells grown on xanthine, hypox-anthine, guanine and adenine, whereas cells grown onallantoin, aliantoate and urea had less than 50% of theactivity found in ceils grown on urate. The enzyme ievelwas low in cells cultured with nitrate or ammonia, or

0 15TIME (hours)

Fig. 3. The uptake of purine and purine derivatives by Chlamy-domonas reinhardii cells. Cells grown on ammonia werewashed and transferred to media containing the indicated ni-trogenous compounds: • , hypoxanthine; • , xanthine; • ,urate; O. aliantoin; D, allantoate; A, ammonia.

0.510

TIME (hours)

whenever ammonia was present in the culture medium(TJib. 4, Fig. 4B). After exhaustion of ammonia, urateoxidase activity increased (Fig. 4B). When ammoniaand urate were present together in the medium, am-monia was taken up immediately, whereas urate wasused simultaneously with ammonia only after a lagperiod of 8 h. In this case, urate oxidase activity re-mained low even after urate accumulation within thecells until ammonia disappeared from the medium (re-sults not shown). When cells grown on ammonia weretransferred to a medium with urate, an increase of urateoxidase activity parallel to growth was observed (Fig.4A). Depletion of urate was always accompanied by anenhancement of the enzyme levels.

Discussion

In the present paper the urate oxidase characterizationof a green alga is reported for the first time. Previousstudies on purine degradation in algae indicated thatsome Chlorella (Birdsey and Lynch 1962) andChlamydomonas (Cain 1965) species were able to useuric acid as nitrogen source for growth. Similar types ofgrowth in response to uric acid had been found in someXanthophyceae (Miller and Fogg 1958) and marineblue-green algae (Van Baalen and Marler 1963), butnothing had been reported on enzymatic urate degrada-tion in green algae.

Urate oxidase activity from Chlamydomonas reinhar-dii cells could be detected either by following the en-zymatic oxidation of urate spectrophotometrically or byspecific staining reactions on polyacrylamide gels.Crude extracts obtained by freezing and thawing,sonication or disruption of cells in a French press con-tained a thermostable inhibitor of low molecular weightsince, after filtration through Sephadex G-25, the spe-cific activity increased 1.2 to 2.8-fold. The pH optimum(9.0) for in vitro assay is similar to that of the enzyme ofsoybean nodules infected by Rhizobium (Tajima andYamamoto 1975, Lucas et al. 1983), glyoxysomes ofcastor bean endosperm (Theimer and Beevers 1971) ornitrogen-fixing nodules of cowpea (Rainbird and Atkins1981). Urate oxidase of Chlamydomonas has a low ap-parent Kn, for urate (12 \iM), is inhibited by high con-centrations of urate and shows a high sensitivity tometal chelating agents. Similar properties have beenreported for urate oxidases from different sources(Theimer and Beevers 1971, Tajima and Yamamoto1975, Vogels and Van der Drift 1976, Rainbird andAtkins 1981, Lucas et al. 1983). The inhibition bymetal-chelating agents suggests that Chlamydomonasurate oxidase is also a metallo-enzyme. . . -: . •.

Fig. 4. Growth and urate oxidase levels of Chlamydomonasreinhardii cells cultured with urate (A) and ammonia (B) assole nitrogen source. Cells grown on 10 mM ammonia werewashed and transferred to the indicated media.

Physiol. Plant. 62, 1584

Hypoxanthine and xanthine inhibited urate oxidaseof Chlamydomonas very efficiently. A similar inhibitionhas been reported for the enzyme from nodules of someleguminous plants (Tajima and Yamamoto 1975, Lucaset al. 1983) and mammalian tissues (Mahler et at. 1956).The strong inhibition of the cowpea nodule enzyme byadenine, aliantoin and ammonia (Rainbird and Atkins1981) was not observed for urate oxidase fromChlamydomonas.

Chlamydomonas cells could use adenine, guanine,hypoxanthine, xanthine, urate, allantoin and allantoateas sole nitrogen source for phototrophic growth. Ade-nine and urate have been reported as nitrogen sourcefor some Chlorophyceae (Birdsey and Lynch 1962, Cain1965) and Xanthophyceae (Miller and Fogg 1958). Chlo-rella pyrenoidosa grows with hypoxanthine andxanthine but cannot use allantoin, presumably due to itsinability to transport this compound (Ammann andLynch 1964). In contrast, allantoin serves as sole nitro-gen source for growth of some marine benthic micro-algae (Antia et al. 1980) and freshwater green algae(Devi Prasad 1983). Chlamydomonas reinhardii usedeasily not only allantoin but also allantoate as sole nitro-gen source (Tab. 4, Fig. 3) after a lag period probablyneeded for the synthesis of the uptake system. Ourresults strongly suggest that the standard pathway ofaerobic degradation of purines (adenine —* hypoxan-thine -* xanthine —* urate -^ allantoin -^ allantoate -^end products), as is found in higher plants., animals andmany microorganisms (Vogels and Van der Drift 1976,Thomas and Schrader 1981), also operates inChlamydomonas, In fact, we have detected xanthinedehydrogenase (Fernandez and Cardenas 1981), urateoxidase and allantoinase activities in Chlamydomonas(M. Pineda, E. Fernandez and J. Cardenas, un-published results) which corroborates the existence ofthis degradative pathway.

Ammonia depressed urate oxidase levels and, afterammonia exhaustion, activities similar to those existingin cells grown on urate were reached. Since ammoniadid not prevent the entry of urate into the cells, weconclude that ammonia represses the synthesis of theenzyme, as reported for uricase of Neurospora crassa(Wang and Marzluf 1979). On the other hand, uratedoes not seem to be essential for the induction of en-zyme since, after urate exhaustion from the medium,activity levels higher than in the presence of urate werefound, and the rate of enzyme synthesis was very similarin both urate and ammonia-depleted media (Fig. 4A,B). The enzyme repression caused by nitrate is probablydue to the formation of ammonia since the cells haveactive nitrate and nitrite reductases.

Acknowledgements - This work has been supported by theGrant no. 1834-82 of Comision Asesora de Investigacion Cien-ti'fica y Tecnica (Spain).

Edited by B. Jergil

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Physio!. Plant. 62, 1984