Biochimicael BiophysicaArta, 1076(1991)203-208 © 1991 ElsevierSciencePublishersB.V.(BiomedicalDivision)0167-4838/91/$03.50 ADONIS 0167,0,8389100096Z
203
BBAPRO33824
Purification and molecular properties of urat¢ oxidase from Chlamydomonas reinhardtii Josefa M. Alamillo, Jacobo Cfirdenas a n d Manuel Pineda DepartwnenloBioqulmica,BiologlaMoleculary Fisiologla,Facultadde Ciencias,Uni~ersidadde C~rdoba.Cbrdoba(Spain) (Received19July1990) Keywords: Copperprotein;Enzymepurification;Uricase;Protein,copper;(Chlamydomonas) Urate oxidase (urate:oxygen oxidoreducta~ EC 1.7.3.3) from the unicellular green alga Chlam.vdomon~ rebdm~ii has been purified to elet~m~am¢~ and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fraetimmtion, gel filtration, ion exchange and xunlhlne-agarose affinity ehromatograFhy. The native enzyme has a relative molecular mass (Mr) of 124000 and consists of four identical or similar-sized submits of Mr 31000 each. The enzyme has a Stokes's radius of 3.87 rim, a sedimentation coe|ficient of 6.8 S and a n / / f o of 1.23, and exlu'bits its maximal absorption at 276 am. Optimum pH was 8,5 and maximum activity was shown at 40°C, with an activation energy of 53 kS-tool -I and a QIo of 1.96. Absorption spectrmn of native reduced enzyme showed two ~mnsient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the of cyanide. Inhibition by cyanide and neocupro~ but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic mate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 tool of copper per tool of enzyme has been found. Introduction Uricases (urate: oxygen oxidoreductase, EC 1.7.3.3) from animal, bacterial and fungal origin have been purified and characterized [1-5]. However, studies on uricase purification in higher plants are nearly restricted to roots and nodules, where it seems to play an important role in both symbiotic nitrogen fixation and ureides production [6-10]. Except for a paper of our group on the partial characterization of uricase of Chlamydomonas [11], not a single report exists on uricase purification or characterization in green algae or photosynthetic tissues of higher plants. In the present work, we report of the purification to homogeneity for the first time of the utate oxidase from a photosynthetic microorganism, the unicellular green alga Chlamydomonas reinhardtii. The purified enzyme has been characterized as a copper-protein and its most salient molecular properties are also presented. Materials and Methods Cell cullure and crude extracts preparation. Chlamydomonas reinhardtii 6145c cells (from the collection of Dr. IL Sager, Sidney Father Cancer Center, New York) Conespondence:J. C~denas,Departamentode Bioquimica,Faeullad de Ciencias.Universidadde Cbrdoba,14071C6rdoba,Spain.
were grown in the minimal liquid medium of Sueoka ¢t al. [12], under saturating light conditions (15-20 W. m-2), with 4 mM ammonium chloride as nitrogen source. Cells were harvested at the mid.logarithnfic phase of growth by centrifuging them at 4000 × g for 10 min, washing with distilled water and transferring them to a minimal medium with 1 mM urate as the sole nitrogen source. There, cells were allowed to grow until mid-exponential phase of growth, harvested and washed as above, and finally centrifuged at 20000 × g for 20 win. Cell pellet was broken by freezing at - 4 0 ° C and thawing with gentle stirring in 0.1 M Tris-glycine buffer (pH 8.5, 2 ml/g fresh weight). The suspension was then centrifuged at 27000 x g for 10 rain, and the resulting supernatam used as crude extract for enzyme purification. Enzyme purification procedure. Unless otherwise stated, all purification steps were carried out at 4°C in 0.1 M Tris-glycine buffer (pH 8.5). (1) Ammonium sulfate fractionation. Crude extract was made up, 25% (w/v) ammonium sulfate and, after 30 rain, centrifuged at 20000 × g for 10 rain. The supernatant was saturated with ammonium sulfate up to 45% and, after centrifugation, the resulting precipitate was dissolved in the minimal volume of buffer. The enzyme solution was centrifuged at 100000 x g for 60 rain and the supernatant was recovered.
204
(2) Gel filtration. Supernatant from the above step was loaded onto a 2.5 × 70 cm column of Sephacril HR S-300. Filtration was carried out at a flow rate of 1 ml/min and 4-ml fractions were collected. This filtration step was also performed at 20°C with identical yield by coupling the column to a fast-protein liquid chromatography (FPLC) system. (3) Ion exchange chromatography. Fractions containing activity from step 2 were pooled and passed through a DEAE-Sephacel column (1.5 x 15 cm), previously equilibrated with buffer. After washing the column with buffer containing 0.1 M sodium chloride, uricase was eluted with 0.17 M NaCI in the same buffer. The flow rate was 0.33 ml/min and 2-ml fractions were collecte,~. (4) Affinity chromatography. Fractions with the highest activity were pooled, dialyzed against buffer, concentrated by uhrafiltration under vacuum and applied to a xanthine-agarose (Sigma) column (0.9 x 6 cm), previously equilibrated with the same buffer. Chromatography was carried out at a flow rate of 4 ml/h and fractions of 1 ml were collected. The column was first thoroughly washed with buffer and then with buffer containing 0.05 M NaCI. Uricase was eluted with 0.5 mM urate in buffer. Fractions containing purified uricase were pooled and, after dialysis against buffer, used as source of purified enzyme. Molecular parameters determination. Sedimentation coefficient of urate oxidase was determined according to Martin and Ames [13]. Enzyme and protein markers (30 pg in 0.1 ml) were layered on separate centrifuge tubes containing a linear 5-20~ sucrose density gradient in 0.1 M Tris-glycine buffer (pH 8.5). Bovine thyroglobulin (19.4 S), bovine liver catalase (11.3 S), rabbit muscle aldolase (7.2 S), bovine serum albumin (4.6 S), and egg lysozyme (1.8 S) were used as standards. Stokes's radius of urate oxidase was determined in a Biogel A 0.5 m column (2.5 x 64 cm), equilibrated with 0.1 M Tris-glycine buffer (pH 8.5) using horse spleen ferritin (7.8 nm), bovine liver catalase (5.22 nm), milk xanthine oxidase (4.7 nm), bovine serum albumin (3.7 nm) and ovalbumin (2.76 am), as standards [14]. Molecular weight determination under non-denaturing conditions was performea according to Hedrick and Smith [15]. As markers, the following standards (in kDa) were used: soybean urease (272), bovine lactalbumin (142), bovine serum albumin (66 and 132, monomer and dimer, respectively), ovalbumin (45) and erythrocytes carbonic anhydrase (29). Molecular mass under denaturing conditions was determined according to Laemmli [16]. As markers the following proteins (in kDa) were used: bovine serum albumin (66), ovalbumin (45), porcine stomach pepsin (34.7), bovine pancreas trypsinogen (24), bovine/]-Iactoglobulin (subunit, 18.4) and egg lysozyme (14.3). Enzyme assays. Urate oxidase activity was assayed spectrophotometrieally by following at 292 nm the de-
crease in absorbance of reaction mixture. Standard assay mixture contained, in a final volume of 1 ml: Tris-glycine buffer (pH 8.5), 100 pmol; uric acid, 50 nmol: and an adequate amount of enzyme. Routine assays were carried out aerobically at 30°C [tl]. In assays on polyacrylamide gels, uricase activity was visualized by dipping gels in 10 mI reaction mixture containing per ml: Tris-glycine buffer (pH 8.5), I00 pmol; 3,3-diaminobenzidine, 100 nmol; uric acid, 100 nmol, and 20 units of horseradish peroxidase. Reaction was performed at 30°C [11]. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the transformation of 1/zmol of substrate per rain under optimal assay conditions. Analytical determinations. Analytical electrophoresis in polyacrylamide gels 8~o in acrylamide was performed at pH 8.3 according to Jovin et al. [17]. Protein was determined as described by Bradford [I8], using bovine serum albumin as a standard. Copper was measured by a modification of the photokinetic m.=,,hod of Velasco et al. [19]. Enzyme samples were dri~ off and mineralized in a porcelain container at 750"~C for 2 h. Residues were redissolved in 0.01-0.1 .,:hiof an HNO3: HCI: H20 mixture (1 : 1 : 1) and then diluted 20-fold with distilled water up to an original protein concentration of 300 #g/ml. Aliquots of 0.2 ml were mixed with reagents up to 1 ml of assay reaction in which copper was determined. Antiserum preparation. Antibodies against Chlamydomonas uricase were raised by injecting New Zealand white rabbits with 125-150/~g of homogeneous protein emulsified in an equal volume of complete Freand's adjuvant. 2 weeks later animals received a second injection and after another 2 weeks they were injected with a booster containing 80-100 lag of protein emulsified in incomplete Freund's adjuvant. Blood (20-30 ml) was obtained 10 days later and, after centrifngation (5000 x g, 5 rain), immunoglobulins were purified by ammonium sulfate fractionation and DEAE chromatography. Results and Discussiml Urate oxidase from Chlamydomonas reinhardtii has been purified to eleetrophoretic homogeneity for the first time. The main steps of the purification procedure are listed in Table I. Affinity chromatography on xanthine-agarose was the most efficient step, since it had a purification factor of 8 and most of the uricase activity could be eluted in only two 1-ml fractions (Fig. 1). This step could not be replaced by other affinity chromatographies such as chelating Sepharose 6B-Cu 2+ or -Ni 2+, from which uricase activity could never be recovered, even though different buffers at several pH values were used. This was probably due to the in-
205 I'~aC= 50raM =
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UP,ATE O5mM tt
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80 _~
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R:IACTK~NNUMBER
Fig. I. Xanthine-agarosechromatographyof uricase from C. reinhar='tii. Details of the purification steps are given in Materiats and Mclhods. Where indicated by the arrows, elution buffer was made either 50 mM in NaC[ or 0.5 mM in uric acid. (o). protein: and (@), uricaseactivity. activating effect of bivalent cations on uricases from Chlamydomonas (see Table Ill) and other origins [1]. A similar affinity chromatography on 8-aminoxanthinebound Sepharose has been successfully used to purify rat fiver uricase [20]. The purification procedure had a 24% yield and a purification factor of 350, and the specific activity of purified uricase was 13.3 U/mR protein (Table I). These data indicate that urate oxidase in Chlamydomonas only represents 0.28% of the total soluble protein, ,,hercas it is about 2% in extracts from plant nodules [8,10] and 50% in those from B. fastidiosus [2]. Specific activity of uricase from Chlamydomonas was similar to that reported for uricases from other origins (10.0-15.6 U/mR) [1,3-5,7,9,20,21], although much higher values have been found for the enzyme from B. fastidiosus (75.5 U/mg; [2]) and soybeam (15600 U/mg; [8]) and pea (6400 U/mR; [10]) nodules. The native enzyme appeared homogeneous according to electrophoretic criteria, and under non-denaturing conditions in 8% polyacrylamide gels two protein bands, both of them exhibiting uricase activity, were observed (Fig. 2). The slower weak band is probably an aggregation artifact, since under denaturing conditions a single protein band was detected. Such a band was only ob-
served after xanthine-agarose chromatography but not in crude or partially purified extracts. A similar aggregation probably caused by oxidative crosslinking of enzyme molecules has been seen in uricase from soybean
root nodules [9]. Homogeneity of the preparation was confirmed by using specific rabbit antisera raisedagainst pure uricase (results now shown). In Table II, some molecular properties of purified uricase from Chlamydomonas reinhardtii are shown. Native uricase is a tetramer of about 124 kDa consisting of four identical or similar-sized subunits of 31 kDa. Values from 100 to 150 kDa have been reported for animal [22,23], fungal [3,5] and bacterial [2,4,2t] uricases. Most of them have been found to be tetramers with subunits of 30-35 kDa, although plant nodule uricases have been reported as monomers of 32000 (soybean) or 50000
o O
0.75
~ 0.511 2 < QI n~[ ]
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20
.o/°
•
/
~_ ~o g
\ •
15
i -~
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0 L j:
P
!
v t,5
TEMPERATURE ('CI
t 60
12[
\
0 0
20
40
6'0
TIME (rain }
Fig. 3. Effectof temperatureon activityand stabilityof C reinhardtii uricase.(A) Activitydata were obtained by measuringthe urate oxidized during the first2 tin of reactionat the indicatedtemperatures.(B) Enzymesolutionswereincubatedat the indicatedtemperaturesand activitywas measuredunderoptimalassayconditionsat the indicatedtimes.
207 TABLEIII Effect of iron, copperand metal chelatingagents on Chlamydomonas andporcineliver~ate oxidases Unlessotherwisestated,compoundswereincubatedwithhomogenous uricase preparationsat the indicatedconcentrationfor 5 rain befo,e starting the reactionby additionof 50 ixM urate. Both iron (It and II!) and copper(I!) wereaddedas theirchlorideand sulfatesaltswith no differettcein the results. Compound
Concentration Relativeactivityof uricasefrom (M) Chlamydomonasporcineliver
None
-
100
Iron
1-10-7
97 125 a
98
96
100
1-10 -~
100
117 a Copper
102 82 18
102 90 14
2-10-4
87 51
86 54
Salicylhy&ox1-10-4 amicacid 5-10-4
103
10Q
103
98
1-10-a 1.10 -~
1.10-2 Neocuproin
1.10-4
Enzymeafter ion exchangechromatography. not appear (Fig. 4D), which reinforces the involvement of metals in the enzymatic oxidation of urate. Purines form complexes with heavy metal ions [27]. ~,~len spectra for metal-urate complexes were recorded, the two transient maxima at 392 and 570 nm were only observed with Cu but not with Fe, Zn, Co or Ni, although most of them exhibited an absorption peak in the 380-400 nm region (Results not shown). This suggests that copper is present in mate oxidase from C reinhardtii cells. Umte oxidases from different sources have been reported to be metailoenzymes containing either Fe or Cu, although the identity of the metal remains uncertain [1]. In Table III, the effects of several metals and chelating agents on uricase activities from Chlamydomonas and porcine fiver, a typical Cu-enzyme, are compared. Iron slightly activated the partially purified Chiamydomonas uficase, but did not affect either algal or porcine liver purified enzymes. Salicylhydroxamic acid, a chelating agent for non-heine iron, inhibited nei[~er Chlamydomonas nor porcine fiver uricase even at higher concentrations than that inhibiting urate oxidase from nitrogen-fixing nodules of cowpea [7]. These results suggest that uriease from Chlamydomonas does not contain any iron e~sential for activity, in contrast to what has been proposed for the enzymes of bacteria [28,29], cowpea nodules [7], and soybean roots and nodules [6]. Iron has been found in partially purified preparations of porcine fiver uricases, but not in the highly purified ones [30].
Both Chlamydonomas and porcine fiver udcases were similarly inhibited by copper ions and neoeuproin (2,9dimethyi-l,10-phenanthroline) (Table Ill). Copper inhibition is a very common feature for uricases [1,4,7] and has been observed even in enzymes lacking any metal [2]. Inhibition by neocuproin, a copper chelating reagent [29], suggests that uricase from Chlamydomonas contains copper as it had been previously indicated on the basis of spectral data. Finally, both irot, and copper were assayed in pure enzyme preparations from porcine liver and Chlamydomonas. No iron was found in either enzyme using o-phenanthroline, whereas the photokinetic method revealed the presence of 0.7 and 4.3 tool of copper per tool of uricase from porcine liver and Chlamydomonas, respectively. The Chlamydomonas er~-,yme is the first described to have 4 mol of copper per mol of enzyme, which suggests that each tool of subunit contains 1 tool of the metal. However, the functional role of copper needs further investigation, since uricases lacking metaLs have been descdbed [2,3] and urate can be oxidized by a number of chemical and biological systems [1], the cupric ion inclusive [31], other than urieases.
Acknowledgements This work was supported by Grant No. PB86-0167CO3-01 from CAICYT (Spain) and Grant No. 5160.040 from Junta de Andahicia. The skillful secretarial assistance of C. Santos and I. Molina is acknowledged. References
1 Vogels, G.D. and Van der Drift, C. (1976) Bacteriol. Rev. 40, 403--468. 2 Bongaerts, G.P.A., Vitzetter, J., Bronns, R. and Vogels, C.D. (1978) Biochim.Biophys.Acta 527, 348-358. 3 Conley,T.G. and Priest, D.G. (1980) Bioehem.J. 187,727-732. 4 Machida, Y. and Nakanishi,T. (1980) Agric. Biol. Chem. 44. 2811-2815. 5 Wang, LC. and Marzluf,G.A. (1980) Arch, Biochem.Biophys. 201, 185-193. 6 Tajima, S. and Yamamoto, Y. (I975) Plant Cell Ph~'siol. 16, 271-282. 7 Rainbird,R.M. and Atkins,C,A. (1981) Bioehim.Biophys.Acta 659, 132-140. 8 Bergmann,H., Preddie,E and Verma,D.ES. (1983) EMBOJ, 2, 2333-2339, 9 Lucas,K., Boland,M.J. and Schubert,K.R.(1983)Arch.Biochcm. Biophys.226, 190-197. 10 SkncheT.,F., Campos, F., Padilla,J., Bonneville,J.-M., Enriquez, C. and Caput, D. (1987) Plant Physiol.8a, i143-1147, I1 Pineda,M., Fernfindez,E, and Cbxdenas.J. (1984) Physiol.Plant. 62, 453-457. 12 $ueoka,N.. Chiang,K.S.and Kates,J.R. (1976) J. Mol, Biol,25. 47-66. I3 Martin, R.G. and Ames, B.N. (1961) J. Biol.Chem. 236, 13721379. 14 Siegel,LM. and Monly,K.J. (1966) Biochim.Biophys.Acta 112, 346-362,
208 15 HedricL A.U and Smith, AJ. (1968) Arch. Biochem. Biophys. 126, 155-164. 16 Laemmli, U.K. (1970) Nature 27"/, 680-685. 17 JoAn, T.. Chrambach, A. and Nanghton, M.A. (1964) Anal. Biochem. 9, 351-364. 18 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 19 Velascn, A, Silva, M. and Val¢iircel,M. (1990) Anal. Chim. Acta 229, 107-114. 20 Watanabe, T. and Suga, 1". (19"/8) Anal. Biochern. 89, 343-M7. 21 Simonyam A,L, Tatikyan, S.Sh. and Kulis, Y.Y. (1985) Biochemistry (USSR), Engl. transl. 50, 657-660. 22 Mahler, HJL (1963) in The Enzymes, 2rid Edn. (P.D. Boyer, ed.), Vol. 8, pp. 285-296, Academic Press, New York. 23 Pitts, O,M., Priest, D.G. and Fish, W.W. (1974) Biochemistry 13, 888-892.
24 Theimer~ R_P,. and Beevers, H. (1971) P!~nt Physiol. 47, 246-251. 25 Malder, H.R., Baum, H. and HUbscber, G. (1956) Scicn~ 124, 705-708. 26 Baum, H~ Hfibscher, G. and Mahler, H.R. (1956) Biochim. Biophys. Acta 22, 514-S~7. 27 Albert, A. (1953) Biochem. J. 54, 646-654. 28 Nose, K. and Afima, K. (1968) Biochim. Biophys. Acta 151, 63-69. 29 Arima, K. and Nose, K. (1968) Biochim. Biophys. Acta 151, 54-62. 30 Mahler, HJL, Hnbseher, G. and Baum, H. (1955) J. Biol. Chem. 216. 6?.5-64L 31 Schein, HA. and Kunin, S.A. (1969) FEBS Lett. 2, 339-341.
203
BBAPRO33824
Purification and molecular properties of urat¢ oxidase from Chlamydomonas reinhardtii Josefa M. Alamillo, Jacobo Cfirdenas a n d Manuel Pineda DepartwnenloBioqulmica,BiologlaMoleculary Fisiologla,Facultadde Ciencias,Uni~ersidadde C~rdoba.Cbrdoba(Spain) (Received19July1990) Keywords: Copperprotein;Enzymepurification;Uricase;Protein,copper;(Chlamydomonas) Urate oxidase (urate:oxygen oxidoreducta~ EC 1.7.3.3) from the unicellular green alga Chlam.vdomon~ rebdm~ii has been purified to elet~m~am¢~ and immunological homogeneity by a procedure which includes as main steps ammonium sulfate fraetimmtion, gel filtration, ion exchange and xunlhlne-agarose affinity ehromatograFhy. The native enzyme has a relative molecular mass (Mr) of 124000 and consists of four identical or similar-sized submits of Mr 31000 each. The enzyme has a Stokes's radius of 3.87 rim, a sedimentation coe|ficient of 6.8 S and a n / / f o of 1.23, and exlu'bits its maximal absorption at 276 am. Optimum pH was 8,5 and maximum activity was shown at 40°C, with an activation energy of 53 kS-tool -I and a QIo of 1.96. Absorption spectrmn of native reduced enzyme showed two ~mnsient maxima at 392 and 570 nm, very similar to those of metal-urate complexes, which disappeared in the of cyanide. Inhibition by cyanide and neocupro~ but not by salicylhydroxamic acid, strongly suggests that copper is the metal involved in enzymatic mate oxidation. By using a sensitive photokinetic method for copper determination, a content of 4 tool of copper per tool of enzyme has been found. Introduction Uricases (urate: oxygen oxidoreductase, EC 1.7.3.3) from animal, bacterial and fungal origin have been purified and characterized [1-5]. However, studies on uricase purification in higher plants are nearly restricted to roots and nodules, where it seems to play an important role in both symbiotic nitrogen fixation and ureides production [6-10]. Except for a paper of our group on the partial characterization of uricase of Chlamydomonas [11], not a single report exists on uricase purification or characterization in green algae or photosynthetic tissues of higher plants. In the present work, we report of the purification to homogeneity for the first time of the utate oxidase from a photosynthetic microorganism, the unicellular green alga Chlamydomonas reinhardtii. The purified enzyme has been characterized as a copper-protein and its most salient molecular properties are also presented. Materials and Methods Cell cullure and crude extracts preparation. Chlamydomonas reinhardtii 6145c cells (from the collection of Dr. IL Sager, Sidney Father Cancer Center, New York) Conespondence:J. C~denas,Departamentode Bioquimica,Faeullad de Ciencias.Universidadde Cbrdoba,14071C6rdoba,Spain.
were grown in the minimal liquid medium of Sueoka ¢t al. [12], under saturating light conditions (15-20 W. m-2), with 4 mM ammonium chloride as nitrogen source. Cells were harvested at the mid.logarithnfic phase of growth by centrifuging them at 4000 × g for 10 min, washing with distilled water and transferring them to a minimal medium with 1 mM urate as the sole nitrogen source. There, cells were allowed to grow until mid-exponential phase of growth, harvested and washed as above, and finally centrifuged at 20000 × g for 20 win. Cell pellet was broken by freezing at - 4 0 ° C and thawing with gentle stirring in 0.1 M Tris-glycine buffer (pH 8.5, 2 ml/g fresh weight). The suspension was then centrifuged at 27000 x g for 10 rain, and the resulting supernatam used as crude extract for enzyme purification. Enzyme purification procedure. Unless otherwise stated, all purification steps were carried out at 4°C in 0.1 M Tris-glycine buffer (pH 8.5). (1) Ammonium sulfate fractionation. Crude extract was made up, 25% (w/v) ammonium sulfate and, after 30 rain, centrifuged at 20000 × g for 10 rain. The supernatant was saturated with ammonium sulfate up to 45% and, after centrifugation, the resulting precipitate was dissolved in the minimal volume of buffer. The enzyme solution was centrifuged at 100000 x g for 60 rain and the supernatant was recovered.
204
(2) Gel filtration. Supernatant from the above step was loaded onto a 2.5 × 70 cm column of Sephacril HR S-300. Filtration was carried out at a flow rate of 1 ml/min and 4-ml fractions were collected. This filtration step was also performed at 20°C with identical yield by coupling the column to a fast-protein liquid chromatography (FPLC) system. (3) Ion exchange chromatography. Fractions containing activity from step 2 were pooled and passed through a DEAE-Sephacel column (1.5 x 15 cm), previously equilibrated with buffer. After washing the column with buffer containing 0.1 M sodium chloride, uricase was eluted with 0.17 M NaCI in the same buffer. The flow rate was 0.33 ml/min and 2-ml fractions were collecte,~. (4) Affinity chromatography. Fractions with the highest activity were pooled, dialyzed against buffer, concentrated by uhrafiltration under vacuum and applied to a xanthine-agarose (Sigma) column (0.9 x 6 cm), previously equilibrated with the same buffer. Chromatography was carried out at a flow rate of 4 ml/h and fractions of 1 ml were collected. The column was first thoroughly washed with buffer and then with buffer containing 0.05 M NaCI. Uricase was eluted with 0.5 mM urate in buffer. Fractions containing purified uricase were pooled and, after dialysis against buffer, used as source of purified enzyme. Molecular parameters determination. Sedimentation coefficient of urate oxidase was determined according to Martin and Ames [13]. Enzyme and protein markers (30 pg in 0.1 ml) were layered on separate centrifuge tubes containing a linear 5-20~ sucrose density gradient in 0.1 M Tris-glycine buffer (pH 8.5). Bovine thyroglobulin (19.4 S), bovine liver catalase (11.3 S), rabbit muscle aldolase (7.2 S), bovine serum albumin (4.6 S), and egg lysozyme (1.8 S) were used as standards. Stokes's radius of urate oxidase was determined in a Biogel A 0.5 m column (2.5 x 64 cm), equilibrated with 0.1 M Tris-glycine buffer (pH 8.5) using horse spleen ferritin (7.8 nm), bovine liver catalase (5.22 nm), milk xanthine oxidase (4.7 nm), bovine serum albumin (3.7 nm) and ovalbumin (2.76 am), as standards [14]. Molecular weight determination under non-denaturing conditions was performea according to Hedrick and Smith [15]. As markers, the following standards (in kDa) were used: soybean urease (272), bovine lactalbumin (142), bovine serum albumin (66 and 132, monomer and dimer, respectively), ovalbumin (45) and erythrocytes carbonic anhydrase (29). Molecular mass under denaturing conditions was determined according to Laemmli [16]. As markers the following proteins (in kDa) were used: bovine serum albumin (66), ovalbumin (45), porcine stomach pepsin (34.7), bovine pancreas trypsinogen (24), bovine/]-Iactoglobulin (subunit, 18.4) and egg lysozyme (14.3). Enzyme assays. Urate oxidase activity was assayed spectrophotometrieally by following at 292 nm the de-
crease in absorbance of reaction mixture. Standard assay mixture contained, in a final volume of 1 ml: Tris-glycine buffer (pH 8.5), 100 pmol; uric acid, 50 nmol: and an adequate amount of enzyme. Routine assays were carried out aerobically at 30°C [tl]. In assays on polyacrylamide gels, uricase activity was visualized by dipping gels in 10 mI reaction mixture containing per ml: Tris-glycine buffer (pH 8.5), I00 pmol; 3,3-diaminobenzidine, 100 nmol; uric acid, 100 nmol, and 20 units of horseradish peroxidase. Reaction was performed at 30°C [11]. One unit of enzymatic activity is defined as the amount of enzyme that catalyzes the transformation of 1/zmol of substrate per rain under optimal assay conditions. Analytical determinations. Analytical electrophoresis in polyacrylamide gels 8~o in acrylamide was performed at pH 8.3 according to Jovin et al. [17]. Protein was determined as described by Bradford [I8], using bovine serum albumin as a standard. Copper was measured by a modification of the photokinetic m.=,,hod of Velasco et al. [19]. Enzyme samples were dri~ off and mineralized in a porcelain container at 750"~C for 2 h. Residues were redissolved in 0.01-0.1 .,:hiof an HNO3: HCI: H20 mixture (1 : 1 : 1) and then diluted 20-fold with distilled water up to an original protein concentration of 300 #g/ml. Aliquots of 0.2 ml were mixed with reagents up to 1 ml of assay reaction in which copper was determined. Antiserum preparation. Antibodies against Chlamydomonas uricase were raised by injecting New Zealand white rabbits with 125-150/~g of homogeneous protein emulsified in an equal volume of complete Freand's adjuvant. 2 weeks later animals received a second injection and after another 2 weeks they were injected with a booster containing 80-100 lag of protein emulsified in incomplete Freund's adjuvant. Blood (20-30 ml) was obtained 10 days later and, after centrifngation (5000 x g, 5 rain), immunoglobulins were purified by ammonium sulfate fractionation and DEAE chromatography. Results and Discussiml Urate oxidase from Chlamydomonas reinhardtii has been purified to eleetrophoretic homogeneity for the first time. The main steps of the purification procedure are listed in Table I. Affinity chromatography on xanthine-agarose was the most efficient step, since it had a purification factor of 8 and most of the uricase activity could be eluted in only two 1-ml fractions (Fig. 1). This step could not be replaced by other affinity chromatographies such as chelating Sepharose 6B-Cu 2+ or -Ni 2+, from which uricase activity could never be recovered, even though different buffers at several pH values were used. This was probably due to the in-
205 I'~aC= 50raM =
°i
04
g b
UP,ATE O5mM tt
1
1
120 E
80 _~
z_ 0
~o.2
0
/
40 _u
oo. Io ~.~..~.o
0
10
20
R:IACTK~NNUMBER
Fig. I. Xanthine-agarosechromatographyof uricase from C. reinhar='tii. Details of the purification steps are given in Materiats and Mclhods. Where indicated by the arrows, elution buffer was made either 50 mM in NaC[ or 0.5 mM in uric acid. (o). protein: and (@), uricaseactivity. activating effect of bivalent cations on uricases from Chlamydomonas (see Table Ill) and other origins [1]. A similar affinity chromatography on 8-aminoxanthinebound Sepharose has been successfully used to purify rat fiver uricase [20]. The purification procedure had a 24% yield and a purification factor of 350, and the specific activity of purified uricase was 13.3 U/mR protein (Table I). These data indicate that urate oxidase in Chlamydomonas only represents 0.28% of the total soluble protein, ,,hercas it is about 2% in extracts from plant nodules [8,10] and 50% in those from B. fastidiosus [2]. Specific activity of uricase from Chlamydomonas was similar to that reported for uricases from other origins (10.0-15.6 U/mR) [1,3-5,7,9,20,21], although much higher values have been found for the enzyme from B. fastidiosus (75.5 U/mg; [2]) and soybeam (15600 U/mg; [8]) and pea (6400 U/mR; [10]) nodules. The native enzyme appeared homogeneous according to electrophoretic criteria, and under non-denaturing conditions in 8% polyacrylamide gels two protein bands, both of them exhibiting uricase activity, were observed (Fig. 2). The slower weak band is probably an aggregation artifact, since under denaturing conditions a single protein band was detected. Such a band was only ob-
served after xanthine-agarose chromatography but not in crude or partially purified extracts. A similar aggregation probably caused by oxidative crosslinking of enzyme molecules has been seen in uricase from soybean
root nodules [9]. Homogeneity of the preparation was confirmed by using specific rabbit antisera raisedagainst pure uricase (results now shown). In Table II, some molecular properties of purified uricase from Chlamydomonas reinhardtii are shown. Native uricase is a tetramer of about 124 kDa consisting of four identical or similar-sized subunits of 31 kDa. Values from 100 to 150 kDa have been reported for animal [22,23], fungal [3,5] and bacterial [2,4,2t] uricases. Most of them have been found to be tetramers with subunits of 30-35 kDa, although plant nodule uricases have been reported as monomers of 32000 (soybean) or 50000
o O
0.75
~ 0.511 2 < QI n~[ ]
0.25
;>
20
.o/°
•
/
~_ ~o g
\ •
15
i -~
ef °
0 L j:
P
!
v t,5
TEMPERATURE ('CI
t 60
12[
\
0 0
20
40
6'0
TIME (rain }
Fig. 3. Effectof temperatureon activityand stabilityof C reinhardtii uricase.(A) Activitydata were obtained by measuringthe urate oxidized during the first2 tin of reactionat the indicatedtemperatures.(B) Enzymesolutionswereincubatedat the indicatedtemperaturesand activitywas measuredunderoptimalassayconditionsat the indicatedtimes.
207 TABLEIII Effect of iron, copperand metal chelatingagents on Chlamydomonas andporcineliver~ate oxidases Unlessotherwisestated,compoundswereincubatedwithhomogenous uricase preparationsat the indicatedconcentrationfor 5 rain befo,e starting the reactionby additionof 50 ixM urate. Both iron (It and II!) and copper(I!) wereaddedas theirchlorideand sulfatesaltswith no differettcein the results. Compound
Concentration Relativeactivityof uricasefrom (M) Chlamydomonasporcineliver
None
-
100
Iron
1-10-7
97 125 a
98
96
100
1-10 -~
100
117 a Copper
102 82 18
102 90 14
2-10-4
87 51
86 54
Salicylhy&ox1-10-4 amicacid 5-10-4
103
10Q
103
98
1-10-a 1.10 -~
1.10-2 Neocuproin
1.10-4
Enzymeafter ion exchangechromatography. not appear (Fig. 4D), which reinforces the involvement of metals in the enzymatic oxidation of urate. Purines form complexes with heavy metal ions [27]. ~,~len spectra for metal-urate complexes were recorded, the two transient maxima at 392 and 570 nm were only observed with Cu but not with Fe, Zn, Co or Ni, although most of them exhibited an absorption peak in the 380-400 nm region (Results not shown). This suggests that copper is present in mate oxidase from C reinhardtii cells. Umte oxidases from different sources have been reported to be metailoenzymes containing either Fe or Cu, although the identity of the metal remains uncertain [1]. In Table III, the effects of several metals and chelating agents on uricase activities from Chlamydomonas and porcine fiver, a typical Cu-enzyme, are compared. Iron slightly activated the partially purified Chiamydomonas uficase, but did not affect either algal or porcine liver purified enzymes. Salicylhydroxamic acid, a chelating agent for non-heine iron, inhibited nei[~er Chlamydomonas nor porcine fiver uricase even at higher concentrations than that inhibiting urate oxidase from nitrogen-fixing nodules of cowpea [7]. These results suggest that uriease from Chlamydomonas does not contain any iron e~sential for activity, in contrast to what has been proposed for the enzymes of bacteria [28,29], cowpea nodules [7], and soybean roots and nodules [6]. Iron has been found in partially purified preparations of porcine fiver uricases, but not in the highly purified ones [30].
Both Chlamydonomas and porcine fiver udcases were similarly inhibited by copper ions and neoeuproin (2,9dimethyi-l,10-phenanthroline) (Table Ill). Copper inhibition is a very common feature for uricases [1,4,7] and has been observed even in enzymes lacking any metal [2]. Inhibition by neocuproin, a copper chelating reagent [29], suggests that uricase from Chlamydomonas contains copper as it had been previously indicated on the basis of spectral data. Finally, both irot, and copper were assayed in pure enzyme preparations from porcine liver and Chlamydomonas. No iron was found in either enzyme using o-phenanthroline, whereas the photokinetic method revealed the presence of 0.7 and 4.3 tool of copper per tool of uricase from porcine liver and Chlamydomonas, respectively. The Chlamydomonas er~-,yme is the first described to have 4 mol of copper per mol of enzyme, which suggests that each tool of subunit contains 1 tool of the metal. However, the functional role of copper needs further investigation, since uricases lacking metaLs have been descdbed [2,3] and urate can be oxidized by a number of chemical and biological systems [1], the cupric ion inclusive [31], other than urieases.
Acknowledgements This work was supported by Grant No. PB86-0167CO3-01 from CAICYT (Spain) and Grant No. 5160.040 from Junta de Andahicia. The skillful secretarial assistance of C. Santos and I. Molina is acknowledged. References
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