Drevet2 and Patricia Roeckel-Drevet1 1 UMR 547-PIAF INRA/Universite´ Blaise Pascal, Aubie`re, France;2UMR 6547-GEEM CNRS, Universite´ Blaise Pascal, Laboratoire Epididyme & Maturation de
Trang 1Two GPX-like proteins from Lycopersicon esculentum and Helianthus
glutathione peroxidase and thioredoxin peroxidase activities
Ste´phane Herbette1, Catherine Lenne1, Nathalie Leblanc1, Jean-Louis Julien1, Joe¨l R Drevet2
and Patricia Roeckel-Drevet1
1
UMR 547-PIAF INRA/Universite´ Blaise Pascal, Aubie`re, France;2UMR 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 Vmaxand Kmvalues
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, Fe2+, 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: + 33 4 73 40 79 16, Tel.: + 33 4 73 40 79 12,
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)
Trang 2M A T E R I A L S A N D M E T H O D S
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)2Æs)1,
photosynthetically active radiation provided by 40-W white
daylight tubes (Mazda LDL, TF 40), 23 ± 1C (day) and
19 ± 1C (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 hydroperhydroper-oxide, hydrogen perhydroper-oxide, 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 000M )1Æcm)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 His6tag, 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
37C up to D600 ¼ 0.6 and induced with 0.5 mM
isopro-pyl thio-b-D-galactoside Four hours after induction, cells
were harvested by centrifugation (5000 g, 10 min, 4C) and
resuspended in 0.05M sodium phosphate, 0.3M NaCl,
0.02Mimidazole at pH7.5 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, 30 min, 4C) 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
Ni2+-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 NADPHoxidation with spectrophotometry
at 340 nm [24] A standard reaction mixture (1 mL), containing 100 mM Tris/HCl, pH 7.5, 5 mM EDTA, 0.2 mMb-NADPH, 3 mMGSH, 0.1% (v/v) triton X-100, 1.4 U of glutathione reductase and 50–100 lg of recom-binant protein, was incubated at 30C for 5 min After
3 min of equilibration, the reaction was initiated by the addition of the peroxide substrate The nonenzymatic activity due to auto-oxidation of GSHas 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 6220M )1Æcm)1 For measurement
of thioredoxin-dependent peroxidase activity, GSHand glutathione reductase in the above-mentioned mixture were replaced with E coli thioredoxin (4 lM) and E 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 GSHand glutathione reductase were not added to the reaction mixture
R E S U L T S
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 His6 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
Trang 3any nonenzymatic background due to auto-oxidation of
GSH and also any E coli peroxidase activity that might
have co-purified with the recombinant proteins We found
no difference between NADPHoxidation 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
Km and Vmax 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
Kmvalues 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 lM) using four to five different reducing substrate concentra-tions As carried out for GPX activity, control assays accounted for any nonenzymatic NADPHoxidation 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
Km and Vmax values were calculated (Table 2) Neither GPXle1 nor GPXha2 were able to reduce t-butyl hydro-peroxide (Table 2) or others hydro-peroxides (data not shown) using NADPHas reducing substrate Both plant enzymes showed higher affinity by three orders of magnitude towards E coli thioredoxin than to GSH, as indicated
by apparent Km values Moreover, in reducing t-butyl hydroperoxide, apparent Vmaxvalues revealed a thioredoxin-dependent peroxidase activity fivefold higher than glutathi-one-dependent peroxidase activity For both proteins, the catalytic efficiencies (Vmax/Km) 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 lM) 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
D I S C U S S I O N
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 coli cells and purified by
Ni-nitrilo-triacetic acid affinity Each crude extract (10 lg of protein) and purified
recombinant enzyme (1 lg of protein) were analyzed by 15% SDS/
PAGE Lane 1, pET/GPXle1-transformed E coli; lane 2, 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
App V max
(nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
App V max (nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
App V max (nmolÆmin)1Æmg)1)
Trang 4the 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 GSHand present apparent maximum velocities with glutathione concentra-tions which are far above evaluated physiological values estimated to range from 1 to 4.5 mMin the chloroplast [31] Heterologous expressions of GPXle1 and GPXha2 in
E colido 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 annuusendogenous 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) and GPXha2 (s) are plotted against the reciprocal GSH
concentrations (1–10 m M ) or E 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 NADPHoxidized per min per mg of protein, and
GSHconcentrations are expressed in m M whereas thioredoxin
con-centrations are expressed in l M
Trang 5oxidative 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 GSHspecificity are not conserved in plasma
GPX (GPX3) and PHGPX (GPX4) groups (which include
GPXle1 and GPXha2), suggesting that GSHis 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 GSHand
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 Kmvalues for thioredoxin are of a
micromolar range, compatible with in vivo levels An in vivo
competition between GSHand thioredoxin for the plant
GPXs cannot be ruled out, because of the uncertainties
about the ratio between GSHand 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
App V max (nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
App V max (nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
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 ) and E 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) and V max /K m ratios are shown as the average of three independent experiments.
Substrate
App V max (nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
App V max (nmolÆmin)1Æmg)1)
App K m (l M ) V max /K m
Trang 6A C K N O W L E D G E M E N T S
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.
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