Báo cáo Y học: Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities pptx

Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase and thioredoxin peroxidase activities Ste ´ phane Herbette 1 , Catherine Lenne 1 , Nathalie Leblanc 1 , Jean-Louis Julien 1 , Joe¨ l R. Drevet 2 and Patricia Roeckel-Drevet 1 1 UMR 547-PIAF INRA/Universite ´ Blaise Pascal, Aubie ` re, France; 2 UMR 6547-GEEM CNRS, Universite ´ Blaise Pascal, Laboratoire Epididyme & Maturation des Game ` tes, Aubie ` re, France This study investigated the enzymatic function of two putative plant GPXs, GPXle1 from Lycopersicon esculentum and GPXha2 from Helianthus annuus, which show sequence identities with the mammalian phospholipid hydroperoxide glutathione peroxidase (PHGPX). Both purified recombin- ant proteins expressed in Escherichia coli show PHGPX activity by reducing alkyl, fatty acid and phospholipid hydroperoxides but not hydrogen peroxide in the presence of glutathione. Interestingly, both recombinant GPXle1 and GPXha2 proteins also reduce alkyl, fatty acid and phospholipid hydroperoxides as well as hydrogen peroxide using thioredoxin as reducing substrate. Moreover, thio- redoxin peroxidase (TPX) activities were found to be higher than PHGPX activities in terms of efficiency and substrate affinities, as revealed by their respective V max and K m values. We therefore conclude that these two plant GPX-like pro- teins are antioxidant enzymes showing PHGPX and TPX activities. Keywords: antioxidant; free radical scavenger; tomato; sun- flower. In all aerobic organisms, reactive oxygen species (ROS) originating from the metabolism of oxygen constitute a threat to virtually any cell constituent. In plants, it has been shown that environmental stresses can cause an increase in ROS levels [1–4]. Despite their noxious effects on proteins, lipids and nucleic acids, which could ulti- mately lead to cell death, ROS, in a more controlled manner, can participate in early signaling pathways in responses to both biotic and abiotic stresses [5,6]. To cope with elevated levels of ROS, plants have evolved different enzymatic and nonenzymatic mechanisms. In the latter are found reducing molecules such as carotene, tocopherol, ascorbate, Fe 2+ , glutathione, while the antioxidant enzy- matic equipment is composed of several enzymes including superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase, glutathione reductase (GR), glutathione peroxi- dase (GPX), glutathione-S-transferase (GST) or thiore- doxin peroxidase (TPX). In mammals, the GPX family of proteins can be divided into five clades according to their amino-acid sequence, substrate specificity and subcellular localization; the cyto- solic GPX (GPX1), the gastro-intestinal GPX (GPX2), the plasma GPX (GPX3), the phospholipid hydroperoxide GPX (GPX4) and selenoindependent epididymis GPX (GPX5) [7,8]. To date, cDNAs encoding proteins similar to animal GPX have been isolated from different plants and have been shown to be induced by biotic and abiotic stresses [9–11]. Plant GPX-like proteins exhibit the most identities to mammal selenium dependent GPX4. However, plant genes carry a codon for a cysteine residue instead of the opal codon UGA used for insertion of a selenocysteine in mammal GPXs. The selenocysteine residue is important for the catalytic activity of GPX as replacement of selenocysteine by cysteine greatly reduces the activity of the enzyme [12]. According to Eshdat et al. [13], this would result in a plant activity lower by three orders of magnitude when compared to the homologous animal GPX. However, replacement of the cysteine by a selenocysteine residue in the citrus GPX was not followed by a gain in activity comparable to that observed with selenium-dependent animal GPX [14]. Thus, the physiological role of plant GPXs is not yet clear. Furthermore, emerging reports on different living organisms display opposite results about the enzymatic functions of these GPX-like proteins [15–18]. These data prompted us to explore the enzymatic functions of these proteins in higher plants. In the present study, we characterized the expression in E. coli of two plant GPXs, GPXle1 and GPXha2, from Lycopersicon esculentum and Helianthus annuus, respect- ively. The purified recombinant proteins were obtained and used in enzymatic assays with various substrates in order to investigate their putative function. Correspondence to P. Roeckel-Drevet, UMR 547-PIAF INRA/ Universite ´ Blaise Pascal, 24 avenue des Landais, 63177 Aubie ` re, France. Fax:+33473407916,Tel.:+33473407912, E-mail: Patricia.DREVET@univ-bpclermont.fr Abbreviations: ROS, reactive oxygen species; GPX, glutathione peroxidase; GSH, glutathione; PHGPX, phospholipid hydroperoxide glutathione peroxidase; TPX, thioredoxin peroxidase. Enzymes: catalase (EC 1.11.1.6); glutathione peroxidase (EC 1.11.1.9); glutathione reductase (EC 1.6.4.2); glutathione-S-transferase (EC 2.5.1.18); L -ascorbate peroxidase (EC 1.11.1.11); thioredoxin reductase (EC 1.6.4.5); superoxide dismutase (EC 1.15.1.1). (Received 2 January 2002, revised 19 March 2002, accepted 26 March 2002) Eur. J. Biochem. 269, 2414–2420 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02905.x MATERIALS AND METHODS Plant materials and chemicals Tomato (Lycopersicon esculentum Mill. cv. VFN8) and sunflower plants (Helianthus annuus Hybrid EL64, kindly provided by F. Vear, INRA, Clermont-Ferrand, France) were raised from seeds in moist vermiculite in a controlled environment room: 16 h daylight at 60 lmolÆm )2 Æs )1 , photosynthetically active radiation provided by 40-W white daylight tubes (Mazda LDL, TF 40), 23 ± 1 °C(day)and 19 ± 1 °C (night), 60 ± 10% relative humidity. At the cotyledon stage, tomato plants were transferred to a mineral solution [19], while sunflower plants were grown in pots. Glutathione, Saccharomyces cerevisiae glutathione reduc- tase, b-NADPH, E. Coli thioredoxin, E. Coli thioredoxin reductase, Triton X-100 (peroxide free), t-butyl hydroper- oxide, cumene hydroperoxide, hydrogen peroxide, linoleic acid, L -a-phosphatidylcholine dilinoleoyl and soybean lipoxidase (type IV) were purchased from Sigma (Saint Quentin Fallavier, France). Linoleic acid and L -a-phos- phatidylcholine dilinoleoyl hydroperoxides were prepared using soybean lipoxidase as described previously [20]. Hydroperoxides formation was monitored by following the change in absorbance at 234 nm and their concentration calculated using an e-value of 25 000 M )1 Æcm )1 .The hydroperoxides were stored in ethanol at )20 °C. Heterologous expression and purification of recombinant GPXle1 and GPXha2 Total RNA was extracted from Lycopersicon esculentum internodes and Helianthus annuus leaves according to the method of Hall [21]. Full-length cDNAs encoding GPXle1 (GenBank accession number y14762) and GPXha2 (Gen- Bank accession number y14707) were amplified by reverse- transcription and PCR amplification as described by Drevet et al. [22] using total RNA as template. During amplifica- tion, the cDNAs were tagged with NdeI sites using appropriate primers. The sequence of the primers used in this study were 5¢-GAATTCGACATATGGCTACGC-3¢/ 5¢-GCTCTCCCATATGGTCG-3¢ and 5¢-CGATAAGCA TATGGCTACGC-3¢/5¢-GAATACTCAACATATGCAT CC-3¢ for each set of forward/reverse gpxle1 and gpxha2 primers, respectively. Amplified products were subsequently cloned into the NdeI linearized pET15b vector (Novagen, Fontenay-sous-bois, France) at the NdeI site to give in-frame fusion with a His 6 tag, and transformed in E. coli BL21 (DE3) pLysS (Promega, Charbonnieres, France). For both clones, sequence fidelity and proper insertion were checked out by automated dye terminator sequence analysis using the CEQ 2000 sequencer (Beckman-Coulter, Roissy Charles De Gaulle, France). Clones were grown in ampi- cillin (100 mgÆL )1 )-supplemented Luria–Bertani media at 37 °CuptoD 600 ¼ 0.6 and induced with 0.5 m M isopro- pyl thio-b- D -galactoside. Four hours after induction, cells were harvested by centrifugation (5000 g,10 min,4 °C) and resuspended in 0.05 M sodium phosphate, 0.3 M NaCl, 0.02 M imidazole at pH 7.5. The cells were then disrupted by sonication at 10 kHz for a total of 60 s with five intervals of 20 s each, and cell debris were sedimented by centrifu- gation (10 000 g,30min,4°C). The presence of the expected soluble recombinant protein was ascertained by SDS/PAGE. The His-tagged protein products of GPXle1 and GPXha2 were affinity purified from cell extracts on Ni 2+ -nitrilotriacetic acid matrix column according to the manufacturer’s instructions (Qiagen, Courtaboeuf, France). Protein concentrations in the eluted fractions were deter- mined using the Bradford assay [23] and fractions contain- ing the protein peaks were assayed immediately for enzymatic activity. As a control, cultures of E. coli BL21 (DE3) pLysS transformed with pET15b vector alone were treated as indicated above in parallel experiments. Enzymatic assays Glutathione-dependent peroxidase activity was measured by monitoring NADPH oxidation with spectrophotometry at 340 nm [24]. A standard reaction mixture (1 mL), containing 100 m M Tris/HCl, pH 7.5, 5 m M EDTA, 0.2 m M b-NADPH, 3 m M GSH, 0.1% (v/v) triton X-100, 1.4 U of glutathione reductase and 50–100 lg of recom- binant protein, was incubated at 30 °Cfor5min.After 3 min of equilibration, the reaction was initiated by the addition of the peroxide substrate. The nonenzymatic activity due to auto-oxidation of GSH as well as the activity of any potentially co-purified E. coli proteins were also examined. Corrections were made to estimate the activity of recombinant proteins per se. Enzyme activities were calcu- lated using an e-value of 6220 M )1 Æcm )1 . For measurement of thioredoxin-dependent peroxidase activity, GSH and glutathione reductase in the above-mentioned mixture were replaced with E. coli thioredoxin (4 l M )andE. coli thiore- doxin reductase (0.3 UÆmL )1 ), respectively. NADPH- dependent peroxidase activity was assayed in a similar fashion to glutathione-dependent peroxidase activity, except that GSH and glutathione reductase were not added to the reaction mixture. RESULTS Heterologous expression of GPXle1 and GPXha2 E. coli BL21 (DE3) pLys cells transformed with the pET15b-derived expression plasmid efficiently produced GPXle1 or GPXha2, as indicated by the presence of a prominent band slightly greater than 20 kDa using SDS/ PAGE [Fig. 1]. This apparent molecular mass was in agreement with the expected molecular mass (2181 Da from the His 6 tag plus 18 847 Da from GPXle1 or 19 175 Da from GPXha2). The purification scheme using Ni-nitrilo- triacetic acid affinity matrix yielded a product of apparent electrophoretic homogeneity (Fig. 1). No product was purified from extracts from E. coli transformed with pET15b vector alone (data not shown). Enzymatic properties of GPXle1 and GPXha2 The glutathione peroxidase activities of GPXle1 and GPXha2 towards several physiological and nonphysiolog- ical hydroperoxides were monitored in the presence of glutathione and glutathione reductase. Assays were carried out using purified recombinant proteins or, as negative controls, using either extracts from E. coli transformed with the pET15b vector alone that had been affinity purified in parallel or elution buffer alone. Such controls accounted for Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2415 any nonenzymatic background due to auto-oxidation of GSHandalsoanyE. coli peroxidase activity that might have co-purified with the recombinant proteins. We found no difference between NADPH oxidation in the presence of affinity purified extracts from the E. coli control or in the presence of the elution buffer alone. These data suggested that no E. coli peroxidase activity copurified with our recombinant proteins. Under these conditions, the apparent K m and V max values for a variety of substrates were calculated for GPXle1 and GPXha2 (Table 1). Both proteins exhibited a higher affinity towards phospholipid hydroperoxides and a weaker affinity towards t-butyl hydroperoxide, as indicated by their respective apparent K m values. There was no detectable activity with hydrogen peroxide. Considering the replacement of the selenocysteine, one of the catalytic residues known to be critical for animal GPX activity, and considering the sequence identities with PHGPX (GPX4) that was reported to have no specificity towards GSH [25], we have investigated the electron donor requirements of GPXle1 and GPXha2. Three alternative physiological reducing substrates, GSH, thioredoxin and NADPH, were tested. Peroxidase activities were assayed with a fixed t-butyl hydroperoxide concentration (100 l M ) using four to five different reducing substrate concentra- tions. As carried out for GPX activity, control assays accounted for any nonenzymatic NADPH oxidation and also for any co-purified E. coli peroxidase activity. A thioredoxin-dependent peroxidase activity was found for both recombinant proteins in addition to the GPX activity. Double reciprocal plots of 1/activity against 1/[GSH] (Fig. 2A) or 1/[thioredoxin] (Fig. 2B) were linear and reproducible in each case. Under these conditions, apparent K m and V max values were calculated (Table 2). Neither GPXle1 nor GPXha2 were able to reduce t-butyl hydro- peroxide (Table 2) or others peroxides (data not shown) using NADPH as reducing substrate. Both plant enzymes showed higher affinity by three orders of magnitude towards E. coli thioredoxin than to GSH, as indicated by apparent K m values. Moreover, in reducing t-butyl hydroperoxide, apparent V max values revealed a thioredoxin- dependent peroxidase activity fivefold higher than glutathi- one-dependent peroxidase activity. For both proteins, the catalytic efficiencies (V max /K m ) in the presence of thiore- doxin are a lot higher than in the presence of glutathione [Table 2]. Thus, recombinant GPXle1 and GPXha2 pre- sented a TPX activity, albeit a slight GPX activity. Substrate specificities of the TPX activity was further investigated using a fixed concentration of E. coli thioredoxin (4 l M ) and four to five different substrate concentrations (Table 3). In agreement with the above data, whichever the tested substrate, TPX activity was found to be greater than the GPX activity in terms of efficiency and substrate affinity (Tables 1 and 3). Furthermore, both enzymes were able to reduce hydrogen peroxide, as well as linoleic acid, phos- phatidylcholine dilinoleoyl and t-butyl hydroperoxides, using thioredoxin as reducing substrate whereas such an activity was not detected in the presence of GSH. DISCUSSION An increasing number of proteins having at least two functions has been reported [26]. Among the GPX family, Fig. 1. Analysis by SDS/PAGE of the recombinant GPXle1 and GPXha2 proteins expressed in E. c oli cells and purified by Ni-nitrilo- triacetic acid affinity. Each crude extract (10 lgofprotein)andpurified recombinant enzyme (1 lgofprotein)wereanalyzedby15%SDS/ PAGE.Lane1,pET/GPXle1-transformedE. coli;lane2,purified recombinant GPXle1; lane 3, pET/GPXha2-transformed E. coli;lane 4, purified recombinant GPXha2. Proteins were stained with Coomassie brilliant blue. Positions and sizes of molecular mass protein markers are shown on the left side of the panel. Table 1. Glutathione peroxidase activities of GPXle1 and GPXha2 towards different substrates. Glutathione peroxidase assays were performed as described in Experimental procedures with a fixed concentration of GSH (3 m M ) using four or five different concentrations of peroxide. The data were analyzed by a Linewaever–Burk representation. Apparent maximum velocities (App. V max ), apparent maximum Michaelis constant (App. K m ) values (± SEM) and V max /K m ratios are shown as the average of three independent experiments. The Cit-sap protein values were taken from reference [32]. LA-OOH, linoleic acid hydroperoxide; PCdili-OOH, phosphatidylcholine dilinoleoyl hydroperoxide; t-butyl-OOH, ter-butyl hydroperoxide. Substrate GPXle1 GPXha2 Cit-Sap (citrus) App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m App. V max (nmolÆmin )1 Æmg )1 ) H 2 O 2 0––0 ––0 t-Butyl-OOH 37.7 ± 2.71 128 0.294 27.1 ± 1.45 95.3 ± 2.56 0.284 24 Cumene-OOH 57.5 ± 1.27 119.0 ± 1.39 3.03 38.9 ± 0.61 60.8 ± 1.37 0.640 50 LA-OOH 27.7 ± 0.04 39.3 ± 0.03 0.705 42.4 ± 0.11 82.7 ± 3.47 0.516 44 PCdili-OOH 19.0 ± 0.44 24.9 ± 0.82 0.763 15.8 ± 0.44 12.1 ± 0.55 1.31 40 2416 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the animal GPX4 (PHGPX) has been reported to be both a structural protein and an active enzyme in sperm cells [27]. In addition, the animal selenium-independent and epididy- mis-restricted GPX (GPX5) was also recently suspected to bear dual-function [8,28]. This report shows that the two previously reported plant GPX-like proteins [10,11] display a thioredoxin-dependent peroxidase activity as well as a glutathione peroxidase activity. Based on identities in their primary sequences with animal GPXs, they were found to be more related to GPX4, the phospholipid hydroperoxide glutathione peroxidase [10,11]. This is also the case for other characterized plant GPXs [13]. The mammalian GPX4 differs from the other animal GPXs in that the protein is monomeric due to deletions in regions thought to mediate tetramerization [25]. The small size and hydrophobic surface of these proteins can explain that PHGPXs (GPX4) are unique in their activity towards hydroperoxides integrated in membranes [29], suggesting that they may play a significant role in protecting membranes from oxidative damage. The sequence similarities led us to suggest that GPX4-like plant GPXs could be involved in membrane protection. Indeed, in our experiments, GPXle1 and GPXha2 were found to display glutathione-dependent peroxidase activity towards organic peroxides such as phospholipid hydroperoxides, but not towards hydrogen peroxide, thus behaving as expected for a GPX4-like GPX. However, these in vitro activities remain low. This can be explained by the lack of the rare selenocysteine residue replaced by a cysteine in the catalytic site of plant GPXs [13]. To date, low activities [15,17,18], or no activity [16,30], were recorded for all seleno-independent GPXs that have been investigated. In addition, GPXle1 and GPXha2 exhibit a low affinity towards GSH and present apparent maximum velocities with glutathione concentra- tions which are far above evaluated physiological values estimatedtorangefrom1to4.5m M in the chloroplast [31]. Heterologous expressions of GPXle1 and GPXha2 in E. coli do not seem to affect their activity, because in vitro values were found to be similar to those obtained from a plant purified citrus GPX [32]. The low PHGPX activity of GPXle1 and GPXha2 recorded in vitro does not necessarily reflect the in vivo situation and does not rule out the possibility that these proteins are indeed involved in phospholipid hydroperoxides detoxification in the cell. In yeast, it has been reported that PHGPX deletion mutants were sensitive to induced lipid peroxidation, suggesting that this seleno-independent protein protects membranes from oxidative stress [17]. Our in vitro analysis of GPXle1 and GPXha2 enzymatic functions strongly suggests that these two GPXs can also function as thioredoxin peroxidases (TPX). Such a finding was recently reported for a previously characterized GPX from Plasmodium falciparum, which as a consequence has been reclassified as a TPX [18]. In addition, it has been very recently shown that a protein from chinese cabbage, which is highly homologous to PHGPX, functions also as a TPX [33]. Dual function for an antioxidant enzyme has also been recently reported for a human 1-cys peroxiredoxin, which exhibits glutathione peroxidase activity [34], and a bovine eye protein showing homologies to TPXs but acting as a seleno-independent GPX [35]. Our TPX assays rely on the use of exogenous thioredoxin and thioredoxin reductase from E. coli, instead of Lycopersicon esculentum and Helianthus annuus endogenous ones. This bacterial thiore- doxin system has successfully been used with the plasmo- dium TPX protein [18]. As it was the case with the TPX from Plasmodium falciparum, one could expect that GPXle1 and GPXha2 react faster with endogenous thioredoxins from their respective plant species. However, thioredoxin systems are probably not markedly different among living organisms as proved by the fact that an E. coli thioredoxin has been shown to enhance recovery of human cells after Fig. 2. Analysis of GPXle1- and GPXha2-catalyzed reduction of t-butyl hydroperoxide (100 l M ) with different concentrations of GSH (A) and thioredoxin (B). The reciprocal apparent maximum velocities of GPXle1 (d)andGPXha2(s) are plotted against the reciprocal GSH concentrations (1–10 m M )orE. coli thioredoxin concentrations (1–6 l M ) as a Linewaever–Burk representation. Each value (± SEM) is representative of three experiments. GPX and TPX activities are expressed as nmol of NADPH oxidized per min per mg of protein, and GSH concentrations are expressed in m M whereas thioredoxin con- centrations are expressed in l M . Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2417 oxidative stress [36]. Thus, it is likely that the TPX activities recorded in the present study reflect the activity in plants as well. Supporting further this dual GPX/TPX function, sequence alignments have shown that amino-acid residues necessary for GSH specificity are not conserved in plasma GPX (GPX3) and PHGPX (GPX4) groups (which include GPXle1 and GPXha2), suggesting that GSH is unlikely to be the sole physiological electron donor under all circum- stances. For example, GPX3 can use thioredoxin as a reducing substrate [37], and GPX4 exhibits an alternate enzymatic thiol oxidase activity towards thiols contained in various proteins [38]. Nevertheless, GPXle1 and GPXha2 do not accept all reducing substrates, as indicated by the lack of activity when NADPH (Table 2) or NADH (data not shown) were used. This implies that GSH and thioredoxin affinities are somehow specific. Altogether, these data on plant and animal GPXs suggest a putative link existing between the glutathione-based antioxidant system and the thioredoxin-based one. TPX activities monitored here can be considered physio- logical. Indeed, apparent K m values for thioredoxin are of a micromolar range, compatible with in vivo levels. An in vivo competition between GSH and thioredoxin for the plant GPXs cannot be ruled out, because of the uncertainties about the ratio between GSH and thioredoxin concentra- tions in many tissues and physiological circumstances. In line with these considerations, we can assume that the electron donor and therefore the enzymatic function of the proteins would depend on this ratio. Although there is no sequence homologies between our plant GPXs and classical TPXs, some similarities can be found with the PHCC-TPx from chinese cabbage [33]. In particular, there are several Cys residues that can be found at roughly equivalent positions in these proteins. Interestingly, Jung et al.[33] have put forward a putative role played by Cys residues in the dual GPX/TPX catalytic process (i.e. exchange of disulfide bonds) in the chinese cabbage PHCC-TPx. In any case, the efficient TPX activity of GPXle1 and GPXha2 do not exclude a PHGPX function but rather points to other unknown biological roles for plant PHGPXs. Another interesting trait of our results is that both GPXle1 and GPXha2 can reduce hydrogen peroxide in the presence of thioredoxin but not in the presence of GSH. Such data are in agreement with the literature, as classical TPXs [39] are known to metabolize hydrogen peroxide while plant GPX- like enzymes do not [29]. This report shows that in plants, GPXle1 and GPXha2 can behave in vitro both as a GPX or/and as a TPX, provided that the proper substrate and electron donor are available. Considering the various subcellular localizations of plant PHGPX-like proteins [10,11,40,41], the variations in the tissue and the subcellular concentrations of substrates and reducing substrates, dual catalytic activities for a given enzyme might constitute an economical way plant cells have evolved in order to cope with various physiological stresses or situations. Indeed, we have previously shown that both biotic and abiotic stresses were able to increase GPXha2 expression at the mRNA level [11]. Further in vivo investigations such as mutant analysis or modifications of expression in transgenic plants will be necessary to clarify this dual physiological role of plant PHGPXs. Table 3. Thioredoxin-dependent peroxidase activities of GPXle1 and GPXha2 towards different substrates. Thioredoxin-dependent peroxidase assays were performed as described in Experimental procedures with a fixed concentration of E. coli thioredoxin (4 l M ) using four different peroxide concentrations. The data were analyzed by a Linewaever–Burk representation. Apparent maximum velocities (App. V max ), apparent maximum Michaelis constant (App. K m ) values (± SEM) and V max /K m ratios are shown as the average of three independent experiments. LA-OOH, linoleic acid hydroperoxide; Pcdili-OOH, phosphatidylcholine dilinoleoyl hydroperoxide; t-butyl-OOH, tert-butyl hydroperoxide. Substrate GPXle1 GPXha2 App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m H 2 O 2 153.8 ± 1.79 13.7 ± 0.02 11.2 147.1 ± 2.12 13.9 ± 0.20 10.6 t-Butyl-OOH 147.2 ± 1.34 16.6 ± 0.28 8.87 161.3 ± 0.96 14.1 ± 0.35 11.4 LA-OOH 147.1 ± 1.11 8.60 ± 0.50 17.1 169.5 ± 1.46 16.2 ± 0.35 10.5 PCdili-OOH 108.7 ± 0.56 14.4 ± 0.12 7.55 126.7 ± 0.05 9.44 ± 0.31 13.4 Table 2. Reducing substrate specificities of GPXle1 and GPXha2 in catalyzed reduction of t-butyl hydroperoxide (100 l M ). Peroxidase assays were performed as described in experimental procedures with a fixed concentration of t-butyl hydroperoxide (100 l M ) using four or five different reducing substrate concentrations. The reducing substrates tested are GSH (1–10 m M ), NADPH (100–200 l M )andE. coli thioredoxin (1–6 l M ). The data were analyzed by a Linewaever–Burk representation as illustrated in Fig. 2. Apparent maximum velocities (App. V max ), apparent maximum Michaelis constant (App. K m )values(±SEM)andV max /K m ratios are shown as the average of three independent experiments. Substrate GPXle1 GPXha2 App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m App. V max (nmolÆmin )1 Æmg )1 ) App. K m (l M ) V max /K m Glutathione 48.8 ± 4.56 9300 ± 209 5.24 · 10 )3 46.7 ± 3.89 4900 ± 120 9.53 · 10 )3 NADPH 0 – – 0 – – Thioredoxin (E. coli) 263.2 ± 0.36 2.2 ± 0.30 119.6 243.9 ± 0.50 1.5 ± 0.06 162.6 2418 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ACKNOWLEDGEMENTS S. H. is a recipient of a french pre-doctoral fellowship (Ministe ` re de la Recherche et de l’Enseignment Supe ´ rieur). We thank G. Pe ´ riot for technical assistance and Dr E. Mare ´ chal (Laboratoire de Physiologie Cellulaire Ve ´ ge ´ tale, CEA, Grenoble, France) for the gift of the pET15b vector. REFERENCES 1. Alsher, R.G. & Hess, J.L. (1993) Antioxydants in Higher Plants. CRC Press, Boca Raton, FL. 2. Foyer, C.H. & Mullineaux, P. (1994) Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. CRC Press, Boca Raton, FL. 3. Dangl, J.L., Dietrich, R.A. & Richberg, M.S. (1996) Death don’t have no mercy: cell death programs in plant–microbe interactions. Plant Cell 8, 1793–1807. 4. Hammond-Kosack, K.E. & Jones, J.D. (1996) Resistance gene- dependent plant defence responses. Plant Cell 8, 1773–1791. 5. Lamb, C. & Dixon, R.A. (1997) The oxidative burst in plant disease resistance. Annu.Rev.PlantMol.Biol.48, 89–108. 6. Levine, A., Tenhaken, R., Dixon, R. & Lamb, C. (1994) H 2 O 2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583–593. 7. Chu, F.F. (1994) The human glutathione peroxidase genes GPX2, GPX3 and GPX4 map to chromosome 14, 5 and 19, respectively. Cytogenet. Cell. Genet. 66, 96–98. 8. Drevet, J.R. (2000) Glutathione peroxidases expression in the mammalian epididymis and vas deferens. In Proceedings of the 1st European Congress of Andrology (Francavilla, F., Francavilla, S. & Forti, G., eds), pp. 427–461. Andrology 2000, Aquila, Italy. 9. Holland, D., Ben-Hayyim, G., Faltin, Z., Camoin, L., Strosberg, A.D. & Eshdat, Y. (1993) Molecular characterization of salt- stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases. Plant Mol. Biol. 21, 923–927. 10. Depege, N., Drevet, J. & Boyer, N. (1998) Molecular cloning and characterization of tomato cDNAs encoding glutathione perox- idase-like proteins. Eur. J. Biochem. 253, 445–451. 11. Roeckel-Drevet, P., Gagne, G., Tourvieille de Labrouhe, D., Dufaure, J.P., Nicolas, P. & Drevet, J.R. (1998) Molecular char- acterization, organ distribution and stress-mediated induction of two glutathione peroxidase-encoding mRNAs in sunflower (Helianthus annuus). Physiol. Plant 103, 385–394. 12. Maiorino, M., Aumann, K.D., Brigelius-Flohe, R., Doria, D., van denHeuvel,J.,McCarthy,J.,Roveri,A.,Ursini,F.&Flohe,L. (1995) Probing the presumed catalytic triad of selenium-contain- ing peroxidases by mutational analysis of phospholipid hydro- peroxide glutathione peroxidase (PHGPx). Biol. Chem. Hoppe Seyler 376, 651–660. 13. Eshdat, Y., Holland, D., Faltin, Z. & Ben Hayyim, G. (1997) Plant glutathione peroxidases. Physiol. Plant 100, 234–240. 14. Hazebrouck, S., Camoin, L., Faltin, Z., Strosberg, A.D. & Eshdat, Y. (2000) Substituting selenocysteine for catalytic cysteine 41 enhances enzymatic activity of plant phospholipid hydroperoxide glutathione peroxidase expressed in Escherichia coli. J. Biol. Chem. 275, 28715–28721. 15. Wilkinson, S.R., Meyer, D.J. & Kelly, J.M. (2000) Biochemical characterization of a trypanosome enzyme with glutathione- dependent peroxidase activity. Biochem. J. 352, 755–761. 16. Gaber, A., Tamoi, M., Takeda, T., Nakano, Y. & Shigeoka, S. (2001) NADPH-dependent glutathione peroxidase-like proteins (Gpx-1, Gpx-2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803. FEBS Lett. 499, 32–36. 17. Avery, A.M. & Avery, S.V. (2001) Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione perox- idases. J. Biol. Chem. 276, 33730–33735. 18. Sztajer, H., Gamain, B., Aumann, K.D., Slomianny, C., Becker, K., Brigelius-Flohe, R. & Flohe, L. (2001) The putative glu- tathione peroxidase gene of Plasmodium falciparum codes for a thioredoxin peroxidase. J. Biol. Chem. 276, 7397–7403. 19. Morizet, J. & Mingeau, M. (1976) Influence des facteurs du milieu sur l’absorption hydrique. Etude effectue ´ e sur la tomate de ´ capite ´ e en exsudation. Ann. Agron. (Paris) 27, 183–205. 20. Ursini, F., Maiorino, M. & Gregolin, C. (1985) The selenoenzyme phospholipid hydroperoxide glutathione peroxidase. Biochim. Biophys. Acta 839, 62–70. 21. Hall, T.C., Buchinder, M.Y., Pyres, J.W., Sun, S.M. & Bliss, F.A. (1978) Messenger RNA for G1 protein of french bean seeds: cell free translation and product characterization. Proc. Natl Acad. Sci. USA 75, 3196–3200. 22. Drevet, J.R., Swevers, L. & Iatrou, K. (1995) Developmental regulation of a silkworm gene encoding multiple GATA-type transcription factors by alternative splicing. J. Mol. Biol. 246, 43–53. 23. Bradford, M.N. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. 24. Maiorino, M., Gregolin, C. & Ursini, F. (1990) Phospholipid hydroperoxide glutathione peroxidase. Methods Enzymol. 186, 448–457. 25. Brigelius-Flohe, R., Aumann, K.D., Blocker, H., Gross, G., Kiess, M., Kloppel, K.D., Maiorino, M., Roveri, A., Schuckelt, R., Usani, F. et al. (1994) Phospholipid-hydroperoxide glutathione peroxidase. Genomic DNA, cDNA, and deduced amino acid sequence. J. Biol. Chem. 269, 7342–7348. 26. Jeffery, C.J. (1999) Moonlighting proteins. Trends Biochem. Sci. 24, 8–11. 27. Ursini, F., Heim, S., Kiess, M., Maiorino, M., Roveri, A., Wissing, J. & Flohe, L. (1999) Dual function of the selenoprotein PHGPX during sperm maturation. Science 285, 1393–1396. 28. Drevet, J.R. (2001) Regulation of gene expression in epididymis. In Proceedings of the Vii th International Congress of Andrology (Robaire, B., Chernes, H. & Morales, C., eds), pp. 199–213. Medimond Medical Publications, Montre ´ al, Quebec, Canada. 29. Ursini, F. & Bindoli, A. (1987) The role of selenium peroxidases in the protection against oxidative damage of membranes. Chem. Phys. Lipids 44, 255–276. 30. Okamura, N., Iwaki, Y., Hiramoto, S., Tamba, M., Bannai, S., Sugita, Y., Synti, P., Dacheux, F. & Dacheux, J.L. (1997) Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epidi- dymal fluid. Biochim. Biophys. Acta 1336, 99–109. 31. Noctor, G. & Foyer, C.H. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279. 32. Beeor-Tzahar, T., Ben-Hayyim, G., Holland, D., Faltin, Z. & Eshdat, Y. (1995) A stress-associated citrus protein is a distinct plant phospholipid hydroperoxide glutathione peroxidase. FEBS Lett. 366, 151–155. 33. Jung, B.G., Lee, K.O., Lee, S.S., Chi, Y.H., Jang, H.H., Kang, S.S., Lee, K., Lim, D., Yoon, S.C., Yoon, D.J., Inoue, Y., Cho, M.J. & Lee, S.Y. (2002) A Chinese cabbage cDNA with high sequence identity to phospholipid hydroperoxide glutathione peroxidases encodes a novel isoform of thioredoxin-dependent peroxidase. J. Biol. Chem. 31, DOI: 10.1074/jbc.M110791200. 34. Chen, J.W., Dodia, C., Feinstein, S.I., Jain, M.K. & Fisher, A.B. (2000) 1-Cys peroxiredoxin, a bifunctional enzyme with glu- tathione peroxidase and phospholipase A2 activities. J. Biol. Chem. 275, 28421–28427. Ó FEBS 2002 Dual activities for plant antioxidant enzymes (Eur. J. Biochem. 269) 2419 35. Singh, A.K. & Shichi, H. (1998) A novel glutathione peroxidase in bovine eye. Sequence analysis, mRNA level, and translation. J. Biol. Chem. 273, 26171–26178. 36. Spector, A., Yan, G.Z., Huang, R.R., McDermott, M.J., Gasco- yne, P.R. & Pigiet, V. (1988) The effect of H 2 O 2 upon thioredoxin- enriched lens epithelial cells. J. Biol. Chem. 263, 4984–4990. 37. Bjo ¨ rnstedt, M., Xue, J., Huang, W., Akesson, B. & Holmgren, A. (1994) The thioredoxin and glutaredoxin systems are efficient electron donors to human plasma glutathione peroxidase. J. Biol. Chem. 269, 29382–29384. 38. Godeas, C., Tramer, F., Micali, F., Soranzo, M., Sandri, G. & Panfili, E. (1997) Distribution and possible novel role of phos- pholipid hydroperoxide glutathione peroxidase in rat epididymal spermatozoa. Biol. Reprod. 57, 1502–1508. 39. Chae, H.Z., Chung, S.J. & Rhee, S.G. (1994) Thioredoxin- dependent peroxide reductase from yeast. J. Biol. Chem. 269, 27670–27678. 40. Churin, Y., Schilling, S. & Borner, T. (1999) A gene family encoding glutathione peroxidase homologues in Hordeum vulgare (barley). FEBS Lett. 459, 33–38. 41. Mullineaux, P.M., Karpinski, S., Jimenez, A., Cleary, S.P., Robinson, C. & Creissen, G.P. (1998) Identification of cDNAS encoding plastid-targeted glutathione peroxidase. Plant J. 13,375– 379. 2420 S. Herbette et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Two GPX-like proteins from Lycopersicon esculentum and Helianthus annuus are antioxidant enzymes with phospholipid hydroperoxide glutathione peroxidase. two plant GPXs, GPXle1 and GPXha2, from Lycopersicon esculentum and Helianthus annuus, respect- ively. The purified recombinant proteins were obtained and used