JXB Advance Access originally published online on August 27, 2004
Journal of Experimental Botany 2004 55(406):2191-2199; doi:10.1093/jxb/erh238
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RESEARCH PAPER |
Cloning and characterization of a 2-Cys peroxiredoxin from Pisum sativum
1Departamento de Bioquímica, Biología Molecular y Celular de Plantas, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, E-18008, Granada, Spain
2Departamento de Nutrición y Fisiología Vegetal, Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas, E-30080, Murcia, Spain
* To whom correspondence should be addressed. Fax: +34 958 129600. E-mail: jjlazaro{at}eez.csic.es
Received 7 April 2004; Accepted 29 June 2004
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Abstract |
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A cDNA sequence coding for a pea (Pisum sativum L.) 2-Cys peroxiredoxin (2-Cys Prx) has been cloned. The deduced amino acid sequence showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%), Arabidopsis thaliana (75%), and Spinacia oleracea (75%), and contained a chloroplast target sequence at its N-terminus. The mature enzyme, without the transit peptide, has a molecular mass of 22 kDa as well as two cysteine residues (Cys-53 and Cys-175) which are well conserved among proteins of this group. The protein was expressed in a heterologous system using the expression vector pET3d, and was purified to homogeneity by three sequential chromatographic steps. The enzyme exhibits peroxidase activity on hydrogen peroxide (H2O2) and t-butyl hydroperoxide (TBHP) with DTT as reducing agent. Although both pea Trxs f and m reduce oxidized 2-Cys Prx, Trx m is more efficient. The precise conditions for oligomerization of 2-Cys Prx through extensive gel filtration studies are also reported. The transition dimer–decamer produced in vitro between pH 7.5 and 8.0 and the influence of DTT suggest that a great change in the enzyme quaternary structure of 2-Cys Prx may take place in the chloroplast during the dark–light transition. In addition, the cyclophilin-dependent reduction of chloroplast 2-Cys Prx is shown.
Key words: Chloroplast, cyclophilin, decamer, hydrogen peroxide, peroxidase, peroxiredoxin, Pisum sativum, thioredoxin
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Peroxiredoxins (Prxs) are a special type of peroxidases recently identified that contain a conserved cysteine at the active site (Chae et al., 1994b
). They are present in organisms from all kingdoms and exist in multiple isoforms (Gonigle et al., 1998; Rhee et al., 2001; Dietz et al., 2002; Rouhier and Jacquot, 2002). All Prxs have one or two essential cysteines in conserved sequences and can be divided into four subgroups (Horling et al., 2003): 2-Cys Prx, Prx Q, and type II Prx, that contain two catalytic cysteines, as well as 1-Cys Prx with one conserved Cys.
2-Cys Prxs are enzymes that reduce H2O2 and alkyl hydroperoxide to water or alcohol, respectively (Netto et al., 1996). These proteins are homodimers and each subunit has the two conserved cysteines (Choi et al., 1998). The peroxide oxidizes the N-terminal cysteine of one subunit to sulphenic acid, which reacts with the C-terminal cysteine of the other subunit to form an intermolecular disulphide. To complete the catalytic cycle the enzyme is reduced via a thiol/disulphide redox interchange (Chae et al., 1994a).
2-Cys Prx was initially identified in yeast as a protein capable of preventing damage induced by the thiol oxidation system, but not by that induced by the ascorbate oxidation system (Kim et al., 1988). Because the activity was supported by thiols, the protein was named thiol-specific antioxidant (Chae et al., 1994b). Later this enzyme was shown to be a peroxidase that reduces peroxides with reduced Trx, thioredoxin reductase (TR), and NADPH. This protein was the first peroxidase identified that uses Trx as a hydrogen donor and was named thioredoxin peroxidase (Chae et al., 1994a); although Prxs were described recently, they now appear as a growing protein family and are considered to be involved in protection against oxidative stress (Kim et al., 1989; Chae et al., 1993).
The reactive oxygen species are produced as an unavoidable consequence of aerobic metabolism (Fridovich, 1978). They can damage some components of the cell (proteins, lipids, and DNA). This is because its production and removal must be strictly controlled (Sies, 1993). All cell compartments have antioxidant enzymes that can destroy these reactive oxygen species, such as, superoxide dismutase, catalase, and peroxidases. Now there is evidence that Prxs play an important role in the antioxidant defence of the cell (Chae et al., 1993; Baier and Dietz, 1999; Rabilloud et al., 2002).
In animal cells, Prx are implicated in complex cellular processes including cell proliferation (Prosperi et al., 1993), differentiation (Yamamoto et al., 1989; Rabilloud et al., 1995), and apoptosis (Zhang et al., 1997; Zhou et al., 2000).
Its presence in plants was discovered by Baier and Dietz (1996); its localization in chloroplasts suggested that its reduction by Trx was coupled to the photosynthetic electron transport via ferredoxin and ferredoxin thioredoxin reductase (Baier and Dietz, 1997). Plant enzymes have been cloned from barley and spinach (Baier and Dietz, 1996), Arabidopsis thaliana (Baier and Dietz, 1997), chinese cabbage (Cheong et al., 1999), rye (Berberich et al., 1998), bean (Genot et al., 2001), and the liverwort Riccia fluitans (Horling et al., 2001). The nucleotide sequence of a 2-Cys Prx from pea has been reported (Bernier-Villamor et al., 2001).
Although more attention has been devoted to 2-Cys Prx, plants express other types of Prxs. Ten genes for the four subgroups of Prxs with different subcellular localization have recently been identified in the genome of Arabidopsis thaliana (Horling et al., 2002, 2003).
The identification of a new cDNA sequence of a 2-Cys Prx from pea, its heterologous expression, purification, and the biochemical characterization of the recombinant protein is described here. Since the formation of a decameric form of the enzyme could be relevant to the 2-Cys Prx regulation, the precise conditions for oligomerization of the enzyme are reported through extensive gel filtration studies. This analysis demonstrates that the reduction state and the pH (between 7.5 and 8.0), as well as the ionic strength, have a great influence in the transition dimer–decamer. Evidence of the reduction of 2-Cys Prx by chloroplast cyclophilin is also provided.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Plant material
Pea (Pisum sativum L. cv. Lincoln) seeds were germinated in moistened vermiculite arranged in plastic trays and grown for 14–21 d in a growth chamber.
RNA isolation and cDNA preparation
Total RNA was isolated from 3 g of young pea leaves using the phenol/SDS method (Sambrook et al., 1989). mRNA was isolated from 100 mg of young pea leaves using an mRNA isolation kit (Roche, Mannheim, Germany). cDNA was obtained from total RNA by reverse transcription for PCR and from mRNA for 5' RACE (Rapid Amplification of cDNA Ends) from young pea leaves using Superscript II reverse transcriptase (Invitrogen, Carlsbad, USA) and an oligo dT20 as a primer.
Isolation of a cDNA encoding a 2-Cys Prx from Pisum sativum
The cDNA encoding a 2-Cys Prx was isolated by RT-PCR. Two heterologous primers were designed from the primary structure of the 2-Cys Prx enzymes in higher eukaryotes: primer A, 5'-GACTTTACTTTCGTCTGCCCAACAGA-3', that encodes the peptide sequence DFTFVCPT, and primer B, 5'-ATTGTTGATGGTGGAATGTTGGATCAC-3', used as the reverse primer that encodes the peptide sequence VIQHSTINN. The 3' end was obtained with the primer 5'-GGGGTGATCCAACATTCCACCATC-3', which encodes the peptide sequence GVIQHSTI, and an oligo dT as the reverse primer. The 5' end was cloned by 5' RACE. Two PCR reactions were used: (i) primer B and an oligo dT with a sequence in the 5' end (5'-GTCCTAGTCGACGCGTGGCCA-3'); and (ii) a reverse primer complementary to this sequence and a homologue primer (5'-TGTTGGGCAAACGAACGTGAAGTC-3') which encodes the sequence DFTFVCPT.
The PCR was performed at 50 °C and its products were gel-purified, cloned in pGEM-T (Promega, Madison, USA) and sequenced.
Comparison of the deduced amino acid sequence of 2-Cys Prx and other typical Prx enzymes
The ProtParam program (prediction of physical characteristic) and ChloroP and PSORT programs (prediction of signal peptides and subcellular localization), provided by the ExPASy server, were used for the analysis of the deduced amino acid sequence. The FASTA 3 and Clustal W programs, provided by the EBI server, were used for comparison and alignment, respectively.
Expression of 2-Cys Prx
A fragment of cDNA encoding the mature protein (amino acids 65 to 263), without the chloroplast transit peptide, was obtained by RT-PCR. Two primers were designed; a primer (5'-CCAC/CATGGCTTCTGGTGAATTACC-3') with an NcoI restriction site (underlined; the / indicates the point of cleavage) and a reverse primer (5'-CGG/GATCCTACACAGCAGCAAAGTACTC-3') with a BamHI restriction site (underlined). PCR product was purified, then digested with NcoI and BamHI, and cloned in the pET3d expression vector (Novagen, Darmstadt, Germany). The resulting construction was used to transform BL21 E. coli (DE3). Transformed cells were cultured at 37 °C in Luria–Bertani medium supplemented with ampicillin (0.1 mg ml–1). When the optical density of the culture at 600 nm reached 0.6, IPTG was added to a final concentration of 0.1 mM. After induction for 6 h, cells were harvested by centrifugation and stored at –70 °C until use. Frozen cells were suspended in 50 mM TRIS-Cl (pH 7.5) and disrupted with French press.
Purification of recombinant 2-Cys Prx
Recombinant 2-Cys Prx was purified by three sequential chromatographic steps on FPLC (Amersham Biosciences, Uppsala, Sweden): Sephacryl S-200 gel filtration, Mono Q HR 5/5 ion exchange, and Phenyl Superose HR 5/5 hydrophobic chromatography columns. 2-Cys Prx was followed by a DTT-dependent peroxidase assay and SDS/PAGE analysis. After the last chromatographic step 2-Cys Prx eluted as two symmetric peaks at about 0.4 M ammonium sulphate and at the end of the gradient. Both peaks were pooled separately.
Gel filtration analysis
Analysis of oligomerization of 2-Cys Prx by gel filtration was performed at room temperature using the FPLC system with a Superdex-200 HR 10/30 column equilibrated with either 50 mM TRIS-Cl (pH 7.5, 8.0, or 8.8) or 50 mM phosphate (pH 6.5, 7.5, or 8.0) with and without 10 mM DTT containing the desired concentrations of NaCl (0.25, 0.5, 1.0, 1.5, or 2 M) at a flow of 0.5 ml min–1. In all cases, the column was equilibrated before the experiment with at least 2 vols of the elution buffer. Purified 2-Cys Prx was diluted about 1 h before the run to 1 mg ml–1 to obtain the conditions for the gel filtration experiments. The sample volume for each run was 50 µl. Calibration of the column was performed using proteins with known molecular-mass and Stokes radii supplied by Amersham Biosciences: chymotrypsinogen (25 kDa, 20.9 Å), ovalbumin (45 kDa, 30.5 Å), bovine serum albumin (67 kDa, 35.5 Å), catalase (240 kDa, 52.2 Å), and ferritin (440 kDa, 61 Å).
For each protein, Kav (the fraction of the stationary gel volume which is available for diffusion of a given solute species) was calculated from Equation 1, and a linear relationship was obtained by plotting Kav or (–log Kav)1/2 against the log molecular mass or the Stokes radius, respectively.
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Differences were not found in the elution volumes of the standard proteins when the column calibration was performed at concentrations of NaCl between 0.25 M and 1.5 M.
Preparation of rabbit 2-Cys Prx antiserum
The polyclonal antibody against mature 2-Cys Prx was raised in rabbits by five consecutive subcutaneous injections, at least 2 weeks apart, of 250 µg pure recombinant protein. The first injection contained complete Freund's adjuvant and subsequent injections contained incomplete Freund's adjuvant. The serum was taken 2 weeks after the last injection.
Polyacrylamide gel electrophoresis and western blot analysis
Denaturing SDS/PAGE was performed as described by Laemmli (1970) with acrylamide concentrations of 6% (stacking gel) and 12.5% (resolving gel). Gels were stained with silver nitrate or Coomassie brilliant blue R-250.
For western blot analysis, the proteins were transferred onto a nitrocellulose membrane by electroblotting. Immunoreaction was carried out by using rabbit serum against mature 2-Cys Prx diluted 1:5000 in PBS containing 5% dried milk. Horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma, St Louis, USA) was used as the secondary antibody. The antigen was detected by chemiluminescence using a Western Lighting chemiluminescence reagent plus (Perkin-Elmer, Boston, USA) following the manufacturer's protocol.
Trx-dependent reduction of 2-Cys Prx
Purified recombinant 2-Cys Prx (1 µM) was incubated in phosphate buffer 50 mM pH 7.5 with and without 1 µM Trx f or m in the presence of DTT 100 µM. After 15 min at 37 °C the reduction of 2-Cys Prx was analysed by SDS/PAGE and western blot with an antibody against 2-Cys Prx. Recombinant pea Trx f and m were purified from cell lysates after expression in E. coli (Hodges et al., 1994; López-Jaramillo et al., 1997).
Peroxide assay
Peroxidase activity of recombinant protein was measured as described by Thurman et al. (1972). The kinetic parameters, Km and Vmax were calculated with a 2-Cys Prx concentration of 0.5 µM and varying concentrations of H2O2. The remaining substrate was measured by the thiocyanate method (Thurman et al., 1972).
Preparation of chloroplast cyclophilin (CyP) from pea leaves
300 g of pea leaves were homogenized (2:1 w/v) in 50 mM HEPES NaOH buffer (pH 7.0). The homogenate was filtered through four layers of nylon cloth, and the extract centrifuged at 16 300 g for 20 min. CyP was purified by ammonium sulphate fractionation (40–90% saturation) and three sequential steps on FPLC: HiPrep Q XL ion exchange, Sephacryl S-200 gel filtration and Phenyl Superose hydrophobic chromatography columns. CyP was followed by peptidylprolyl cis, trans-isomerase assay (Fischer et al., 1989). The molecular size and purity were confirmed by 15% SDS/PAGE analysis. For N-terminal sequencing, the purified protein was separated by SDS/PAGE and transferred to poly(vinylidene difluoride) (PVDF) membrane (Millipore, Billerica, MA, USA) using a Bio-Rad Trans-Blot Cell. After Coomassie blue staining the spot was sequenced using an automatic protein peptide microsequencer (Procise 494; Applied Biosystem, Foster city, CA USA).
CyP-dependent reduction of 2-Cys Prx
The CyP-dependent reduction of recombinant 2-Cys Prx was tested in a DNA-cleavage assay. Reduction of Fe(III) was initiated by incubation of 3 µM FeCl3 for 2 h at 37 °C in the presence of 10 mM DTT. Plasmid DNA (2 µg) was then added and incubated for 5 h at 37 °C under different conditions. The reaction contained 2-Cys Prx (6 µg) and CyP (3 µg). The reaction products were then electrophoresed in a 1.0% agarose gel and stained with ethidium bromide.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Cloning of a 2-Cys Prx and analysis of its amino acid sequence
The cloning of a cDNA sequence encoding a pea 2-Cys Prx is reported. The primary structure of the known plant 2-Cys Prx enzymes was used to design heterologous oligonucleotides to obtain a cDNA fragment encoding a pea 2-Cys Prx. A sequence of 300 bp was obtained that showed more than 50% homology to known Prx enzymes. With this sequence it was possible to design homologous oligonucleotides to obtain the complete sequence.
The cDNA sequence comprised 1044 bp (Bernier-Villamor et al., 2001). The start codon was found at position 1 and the stop codon at position 789. A typical polyadenylation signal was found at position 1010. The open reading frame comprised 263 amino acids with a molecular mass of 28 864 Da and a pI of 5.98. Plant 2-Cys Prxs are located in chloroplasts but are nuclear-encoded (Baier and Dietz, 1997; Cheong et al., 1999; Genot et al., 2001), and contain a signal peptide characteristic of proteins that are imported and processed in the chloroplast (Karlin-Neumann and Tobin, 1986). This signal peptide contains 64 amino acids at the N-terminus, which targets the pre-form of 2-Cys Prx to the chloroplast stroma, where it is cleaved to the mature form of 21 913 Da and a pI of 4.77. The protein presented two conserved cysteine residues in positions 53 and 175 of the mature protein, and showed the highly conserved peptide regions around the conserved cysteine residues. The protein showed a high sequence homology to the 2-Cys Prx enzymes of Phaseolus vulgaris (86%) (Genot et al., 2001), Arabidopsis thaliana (75%), and Spinacia oleracea (75%) (Baier and Dietz, 1997).
Purification of recombinant 2-Cys protein
The recombinant protein was purified by three sequential chromatographic steps: Sephacryl S-200, Mono Q, and Phenyl Superose. In the last step of the purification (Phenyl Superose column) two peaks appeared, when denaturing polyacrylamide gel electrophoresis showed only one band corresponding to the recombinant protein (Fig. 1A). However, when both peaks were processed separately in Superose 12 (Fig. 1B, C) the result was different. The first peak behaved as a heterogeneous sample: in addition to the 44 kDa form, a peak with a molecular mass higher than 240 kDa appeared (Fig. 1B). It has been described that 2-Cys Prx forms decamers (Schröder et al., 2000; Alphey et al., 2000; Wood et al., 2002, 2003). These results point out that this form corresponds to the decamer. The second peak of Phenyl Superose behaved in Superose 12 as a homogeneous form with a molecular mass of about 44 kDa (Fig. 1C). Since the dimer–dimer interactions to form the decamer are mainly hydrophobic (Alphey et al., 2000; König et al., 2002; Schröder et al., 2000; Wood et al., 2003), it is possible that, given the high ionic strength (1 M (NH4)2SO4) in the hydrophobic column, 2-Cys Prx partially forms decamers that eluted before the dimers. The interaction dimer–phenyl group in the column is probably stronger than the interaction decamer–phenyl group because hydrophobic groups of the protein are involved in the dimer–dimer interface to form the decamer. When the first peak, corresponding to the decameric form and obtained by chromatography through Phenyl Superose (Fig. 1A), was subjected to chromatography through Superose 12, not only did the decameric form elute but the dimeric form as well. This is probably due to the lower ionic strength of the elution buffer used in the chromatography through Superose 12, which could weaken the dimer–dimer interactions.
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The oligomeric properties of 2-Cys Prxs have been considered as an important regulatory mechanism in erythrocytes (Schröder et al., 2000
), trypanosomes (Alphey et al., 2000), and barley (König et al., 2002, 2003).
Determination of 2-Cys Prx molecular mass and Stokes radius
Since an approximated value of the molecular mass of the decamer obtained on a Superose 12 column was higher than the theoretical value, the molecular mass of both the decamer and the dimer was accurately determined. Their Stokes radii were also determined in order to compare them with the reported values from crystallographic studies of 2-Cys Prx from other origins (Schröder et al., 2000; Alphey et al., 2000). A Superdex 200 HR 10/30 column was equilibrated with 50 mM TRIS-Cl pH 7.5, 1 M NaCl and calibrated by injecting proteins with known molecular mass and Stokes radii. Calibration plots were obtained (see Materials and methods) (Fig. 2). The estimated molecular mass for the dimer (49 kDa) is close to the theoretical value (44 kDa). However, the estimated molecular mass of the decamer (388 kDa) is higher than the theoretical value (220 kDa) (Fig. 2A). This apparent molecular mass relates to the toroidal nature of the decameric structure: five dimers form a decameric ring with a large central hole which increases its overall effective diameter (Alphey et al., 2000; Wood et al., 2002). In fact, when the Stokes radii were determined for the dimer and the decamer (Fig. 2B), values comparable with those obtained from the structure of 2-Cys Prx from other origins were found (Alphey et al., 2000; Gourlay et al., 2003).
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Oligomeric assembly of recombinant 2-Cys Prx
In order to determine the precise conditions for oligomerization of 2-Cys Prx, an extensive gel filtration study was performed. To study the effect of ionic strength on the oligomerization state of the enzyme, purified 2-Cys Prx was injected into a Superdex 200 HR 10/30 column, previously calibrated and equilibrated with 50 mM TRIS-Cl pH 7.5, at different concentrations of NaCl. Figure 3A shows the relative amounts of dimers and decamers based on the NaCl concentration. Two forms of 2-Cys Prx appeared depending on the NaCl concentration. Without NaCl and at 0.25 M there are only dimers. From 0.5 to 2 M the amount of dimer decreases at the expense of an increase in the decamer. At 1.5 M there is about the same amount of dimers and decamers. This confirms that the dimer–dimer interaction mainly has a hydrophobic character. König et al. (2002
, 2003) observed under different conditions the positive influence of the ionic strength in the aggregation state. In the presence of 10 mM DTT, at pH 7.5 (Fig. 3A), the decameric form appeared without NaCl, and at 0.25 M only a minimal amount of dimer appeared. The enzyme in reduced state does not form monomers, but stabilizes the decameric form. The monomer–monomer interaction, in the reduced enzyme, can be stabilized by hydrogen bonds, salt bridges, and hydrophobic interactions (Alphey et al. 2000). It is possible that the disulphide bonds between the Cys-53 of one subunit and the Cys-175 of the other subunit that form the dimer provide strictness to the dimeric molecule, obstructing its association with other dimers to form the decamer. The reduction of the disulphide bonds may produce a conformational change in the dimeric structure facilitating the dimer–dimer interaction. König et al. (2003) have postulated that in vivo, 2-Cys Prx exists as reduced decamer associated with the thylakoid membrane in the chloroplast. In the oxidized state, the ionic strength to form the decamer has to be higher, since it has to overcome the strictness of the disulphide bonds. At pH 8.0 and 8.8, in the presence of a high ionic strength (1.5 M NaCl), only the dimer appeared (Fig. 3A). The difference between pH 7.5 and 8.0 indicates that a group which titrates in this interval of pH affects the oligomerization state, facilitating in its protonated state the decamer formation. At pH 8.8 with 10 mM DTT the behaviour is the same as at pH 7.5 (Fig. 3A). The conformational change produced in the reduction of the 2-Cys Prx minimizes the influence of this group. In order to extend the pH to the acidic zone, a 50 mM phosphate buffer was used at pH 6.5, 7.5, and 8.0 with two NaCl concentrations (0.5 and 1.5 M). When these results were compared with those obtained with TRIS-Cl at pH 7.5 and 8.0, the effect of an acidic pH and the specific effect of phosphate (Fig. 3B) could be seen. At pH 6.5 only the decameric form of the enzyme was present. At both pH 7.5 and 8.0 there was a great difference between TRIS and phosphate. Whereas in phosphate at pH 7.5 and 1.5 M NaCl, the decamer was the predominant species, in TRIS the amount of dimer and decamer was similar. By contrast, at pH 8.0 in TRIS there was only the dimer, whereas in phosphate the amount of both species was similar. However, the phosphate buffer minimizes the presence of NaCl and there was nearly no difference between 0.5 and 1.5 M NaCl. The differences observed between both buffers can be explained by both the higher ionic strength provided by the phosphate ion and the effectiveness of this ion in increasing hydrophobic interactions. König et al. (2002, 2003) have analysed the effect of different phosphate concentrations in the transition of 2-Cys Prx from dimer to decamer. The effect of one concentration of TRIS and phosphate at two different NaCl concentrations is compared here (Fig. 3B). The influence of the ionic strength, the reduction state and the pH in the oligomerization of 2-Cys Prx has been studied independently in other origins with contradictory results (Chauhan and Mande, 2001; Wood et al., 2003). The influence that these factors have and the interaction among them has been systematically studied here. The relative amount of dimer and decamer of recombinant 2-Cys Prx from pea at four pH values and with two different buffers that exist in oxidized and reduced state and at various NaCl concentrations is also shown.
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The dark–light transition induces a rise in the stromal pH (from 7.0 to about 8.0) and in the chloroplast redox state in vivo. The main change in the oligomerization state takes place between pH 7.5 and 8.0 (Fig. 3B) in vitro. This, along with the influence of the reduction state on the dimer–decamer transition (Fig. 3A), suggests that a great change in the enzyme quaternary structure takes place at the dark–light transition.
Biochemical characterization of recombinant 2-Cys Prx
It has been investigated whether higher plants chloroplast Trx f or m reduces 2-Cys Prx. The enzyme was incubated in the presence of Trx f or m with and without 100 µM DTT before SDS/PAGE. The oxidized form of 2-Cys Prx was partially reduced by incubation with Trx f and m in the presence of a low concentration of DTT (100 µM), although Trx m was more efficient (Fig. 4). This reduction was not observed only with this concentration of DTT (Fig. 4, line 4). Trx f appears after SDS/PAGE as a monomer and as a dimer, both in the presence and absence of DTT. The dimer showed an apparent molecular mass similar to the Prx monomer (Fig. 4, left). This might be because the visualization with silver nitrate did not confirm the reduction of 2-Cys Prx by Trx f. However, the western blot developed with antibodies against Prx (Fig. 4, right) indicated that Trx f reduces the enzyme, although to a lower degree than Trx m. König et al. (2002) in a different system (complete electron transfer system from NADPH to Trx) found that both Trxs reduced oxidized enzyme with similar efficiency, and Goyer et al. (2002) reported that Trx f was more efficient in the Trx-dependent peroxidase activity of Prx from Chlamydomonas. However, Motohashi et al. (2001) reported that 2-Cys Prx is a target for Trx m in spinach chloroplasts. Collin et al. (2003) reported that a new chloroplastic Trx, Trx x, reduces the enzyme with a higher specificity than Trx f or m. As König et al. (2002) did, the presence of a decameric form of the enzyme could be observed; in this system, however, rather than increasing, reduction decreased this form. This discrepancy might be due to the different methods used to reduce Prx.
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The thiol peroxidase activity of pea recombinant 2-Cys Prx was demonstrated in the presence of 4 mM DTT. The enzyme catalysed the reduction of H2O2 or TBHP both in a time-dependent (Fig. 5A) and in a concentration-dependent (Fig. 5B) fashion. Whereas it can be observed that H2O2 is partially reduced by DTT in the absence of Prx, TBHP did not change its reduction state (Fig. 5B). However, the rate of reduction was similar with both peroxides in the presence of Prx.
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The kinetic parameters obtained for pea recombinant 2-Cys Prx (Km, 27.6 µM; kcat, 0.69 s–1; and kcat/Km, 0.25x105 M–1 s–1) show a lower efficiency than that found for the enzyme from barley (König et al. 2003
).
Reduction of 2-Cys Prx by chloroplast CyP
Recently, Lee et al. (2001) have reported that mammalian CyP activates all human Prx isotypes (2-Cys and 1-Cys Prxs). Motohashi et al. (2001, 2003) also found that chloroplast CyP is an actual target protein for Trx. In order to see whether chloroplast CyP can reduce pea recombinant 2-Cys Prx, CyP was isolated from pea leaf extracts, as described in the Materials and methods. It was verified that the purified protein was CyP by analysing the N-terminal sequence which was AQGDVALQAKVTSKXFFDV. Alignment of this amino acid sequence with sequences of other chloroplast CyP homologues from fava bean (Luan et al., 1994), Arabidopsis thaliana (Lippuner et al., 1994), and spinach (Motohashi et al. 2001), revealed that pea CyP shares a high degree of similarity. In addition, its molecular mass was checked (about 20 kDa) as well as the presence of an intramolecular disulphide bond by SDS/PAGE, with and without DTT, because the intramolecular bond between Cys in the oxidized state could form a more compact molecule with a faster mobility on SDS/PAGE that the protein in the reduced state (Fig. 6A). To see whether CyP could reduce 2-Cys Prx, the ability of both proteins to protect DNA from the DTT/Fe3+ was investigated. It is shown in Fig. 6B that CyP does not protect DNA against degradation, but with 2-Cys Prx there is complete protection. It is reported here for the first time that there is a direct interaction between chloroplast 2-Cys Prx and CyP. However, the physiological role of this interaction awaits further studies.
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One proposed function of the 2-Cys Prx in higher plants is the reduction of reactive oxygen species, which are produced by photosynthetic electron transport. 2-Cys Prx can reduce TBHP as efficiently as H2O2, suggesting that the enzyme in the chloroplast can protect not only against H2O2 but also against alkyl hydroperoxides via electron flow through the ferredoxin-thioredoxin system. On the other hand, the results obtained after gel filtration chromatography suggest that the dimer–decamer transition can play an important role in the enzyme regulation in the chloroplast during the dark–light transition in vivo.
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Acknowledgements |
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We would like to thank Dr Olga Martinez-Augustin and Dr Victor Puerta for their invaluable help with the preparation of the antibodies against 2-Cys Prx, and Dr Javier López-Jaramillo and Dr Matilde Barón for helpful discussion. We also thank Francisca Castro for her excellent technical assistance. This work was supported by Dirección General de Enseñanza Superior e Investigación Científica (Ministerio de Educación y Cultura) (project PB98-0476).
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Footnotes |
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Abbreviations: 2-Cys Prx, 2-Cys peroxiredoxin; Trx, thioredoxin; TR, thioredoxin reductase; TBHP, t-butyl hydroperoxide; DTT, dithiothreitol; CyP, cyclophilin.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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