From the Department of Microbiology, Pathology, and
Immunology, Division of Pathology, F46, Karolinska Institutet, Huddinge
University Hospital, SE-141 86 Stockholm, Sweden,
§ Södertörns Högskola (University
College), SE-141 04 Huddinge, Sweden, the
Department of
Biosciences at Novum, Center for Biotechnology, Karolinska Institutet,
SE-141 57 Huddinge, Sweden, and the ** Department of Medical
Biochemistry and Biophysics, Karolinska Institutet,
SE-171 77 Stockholm, Sweden
Received for publication, October 11, 2002
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ABSTRACT |
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The selenoprotein
thioredoxin reductase (TrxR1) is an essential antioxidant enzyme known
to reduce many compounds in addition to thioredoxin, its principle
protein substrate. Here we found that TrxR1 reduced ubiquinone-10 and
thereby regenerated the antioxidant ubiquinol-10 (Q10), which is
important for protection against lipid and protein peroxidation. The
reduction was time- and dose-dependent, with an apparent
Km of 22 µM and a maximal rate of
about 12 nmol of reduced Q10 per milligram of TrxR1 per minute. TrxR1 reduced ubiquinone maximally at a physiological pH of 7.5 at similar rates using either NADPH or NADH as cofactors. The reduction of Q10 by
mammalian TrxR1 was selenium dependent as revealed by comparison with
Escherichia coli TrxR or selenium-deprived mutant
and truncated mammalian TrxR forms. In addition, the rate of reduction
of ubiquinone was significantly higher in homogenates from human embryo
kidney 293 cells stably overexpressing thioredoxin reductase and was induced along with increasing cytosolic TrxR activity after the addition of selenite to the culture medium. These data demonstrate that
the selenoenzyme thioredoxin reductase is an important
selenium-dependent ubiquinone reductase and can explain how
selenium and ubiquinone, by a combined action, may protect the cell
from oxidative damage.
Thioredoxin reductase is part of a family of pyridine nucleotide
oxidoreductases to which glutathione reductase, lipoamide dehydrogenase, and mercuric ion reductase also belong (1). Mammalian
cytosolic thioredoxin reductase
(TrxR1)1 is a multifunctional
homodimeric selenoenzyme with a FAD, a functional disulfide/dithiol,
and a penultimate C-terminal selenocysteine residue in each subunit of
58 kDa (2, 3). This selenocysteine residue is essential for catalytic
activity and, together with the adjacent cysteine, forms a
selenenylsulfide as the active site that is kept reduced by the
conserved sequence Cys-Val-Asn-Val-Gly-Cys at the N-terminal of the
other subunit (2, 4, 5). TrxR1 has an exceptionally broad substrate
specificity, not only reducing thioredoxins from a variety of species
but many low molecular compounds as well; it is also a key enzyme in
selenium metabolism, reducing selenium compounds to the active form for
its own synthesis and for the synthesis of all other selenoproteins (6,
7). TrxR1 is a central enzyme for protection against oxidative
stress (8), both directly and linked to antioxidant functions of
thioredoxin and peroxiredoxins (8). The role of the enzyme is also
implicated in a variety of physiological and pathophysiological
processes (7, 9). Directly, TrxR1 may function as a peroxidase,
reducing organic hydroperoxides including lipid hydroperoxides, in
particular 15-S-HPETE, which is associated with
atherosclerosis and lipid peroxidation (10). It may also reduce
hydrogen peroxide, a reaction exclusively linked to the selenocysteine
residue (4), and also ascorbate (11, 12) and lipoic acid (13).
Ubiquinone is a widely distributed, redox-active quinoid compound
originally discovered as an essential part of the mitochondrial respiratory chain in mammals (14). The number of isoprenoid units of
the side chain of ubiquinone is specific in different species and is
reflected in the nomenclature, e.g. in rat ubiquinone-9 (Q9)
and in human ubiquinone-10 (Q10) with 9 and 10 isoprenoid units,
respectively. In humans, the reduced form of this molecule, ubiquinol-10, is the only endogenously synthesized lipid-soluble antioxidant. The antioxidant function is predominately protection against lipid and protein peroxidation (15, 16). The actions in
peroxidation are mainly to stop the initiation of the reaction by
reducing the perferryl radical and to terminate the propagation phase
by the regeneration of vitamin E necessary for the reduction of
lipidperoxyl radicals (16). Although the antioxidant function requires
that ubiquinol be continuously regenerated enzymatically, the
non-mitochondrial enzymatic systems involved are characterized only to
a limited extent.
The aim of the present study was to characterize the reduction of
ubiquinone-10 by thioredoxin reductase. Additionally, we investigated
whether the reaction was specific for the mammalian enzyme and hence
selenium-dependent, using the smaller non-selenium-containing Escherichia coli enzyme as well as mutant human TrxR and
recombinant truncated rat TrxR lacking the last two Sec-Gly
residues. We have also probed the reaction in a cellular context using
transfected cell lines overexpressing thioredoxin reductase, which were
grown with and without the addition of selenite to the culture medium. Based on the results presented here, we conclude that TrxR1 is a major
cellular Q10 reductant.
Chemicals--
Tris-malate, Tris-HCl, EDTA, bovine serum
albumin, ubiquinone-10, ubiquinone-6, HEPES, glycerol, NADH, and NADPH
were obtained from Sigma. Methanol, petroleum ether (b.p.
40-60 °C), 2-propanol (analytical grade), n-hexane
(analytical grade), sucrose, CuSO4·5H2O, NaK-tartrate·4H2O, NaOH, Na2CO3,
NaCl, Folin-Ciocalteu's phenol reagent, sodium deoxycholate, and
ZnCl2 were purchased from Merck (Stockholm, Sweden).
Thioredoxin was obtained from Promega, and the thioredoxin reductase
from E. coli and calf thymus was from IMCO (Stockholm,
Sweden). 2',5'-ADP-Sepharose was purchased from Amersham Biosciences.
Dulbecco's modified Eagle's medium for cell culturing was purchased
from Invitrogen.
Reduction Assay of Ubiquinone-10--
The in vitro
incubation with Q10 was performed essentially as described previously
(17). In short, the reaction mixture (100 µl) was composed of 50 mM Tris-malate, pH 7.45, 0.4% sodium deoxycholate, 1 mM EDTA (only in the absence of zinc), 2 mM
NADH or NADPH, 50 µM ubiquinone-10, and 5 µg of enzyme,
and 40 µl of (0.3-1.7 mg of protein) homogenate or 200 µg of
cytosolic protein. To determine the effects of the amount of enzyme on
the reaction, 1-10 µg were added to the reaction mixture. The
effects of pH were investigated in the range of 5-8.5. Q10 was added
as a solution in ethanol, but the final concentration of ethanol never
exceeded 2%. The incubation time varied from 5-30 min. The
experimental tubes were flushed with nitrogen, covered, vortexed, and
placed in a water bath for incubation at 37 °C after the addition of all components. The reaction was terminated by the addition of methanol.
Extraction of Ubiquinone-10 and Ubiquinol-10--
Extraction of
ubiquinone-10 and ubiquinol-10 was performed essentially as described
by Olsson and co-workers (17). In short, at the end of the incubation
period, distilled water (400 µl), methanol (5 ml), the internal
standard Q6 (0.68 nmol), and petroleum ether (b.p. 40-60 °C) (3 ml)
were added. The samples were then mixed by shaking and centrifuged
(Labofuge 300, Labex, Helsingborg, Sweden) at 1200 rpm, for 3 min at
25 °C. The upper phase thus obtained was removed and evaporated
under nitrogen. The residue was subsequently dissolved in 25 µl of
methanol/n-hexane/2-propanol, 2:1:1 (solvent B, see below),
and injected directly into the HPLC system. This entire procedure was
completed within 10 min (18).
HPLC Analysis--
The extracts were analyzed by reversed-phase
HPLC as described by Olsson and co-workers. (17). In short,
a column (Hypersil ODS, 3 µm) was employed together with a gradient
system; Pump A contained methanol/H2O (9:1), and pump B
contained methanol/n-hexane/2-propanol (2:1:1). A linear
gradient of 10-50% of solvent B was applied for 15 min, followed by
50-55% of solvent B for 8 min and, finally, 55-100% of solvent B
for an additional 3 min, all at a flow rate of 1.5 ml/min and a
temperature of 14 °C. The absorbance of the eluate was monitored at
292 nm in a UV spectrophotometer. Ubiquinol-10 and ubiquinone-10 were
eluted in two well separated peaks with retention times of 19-20 and
25-26 min, respectively.
Enzyme Assay--
TrxR1 activity in the homogenates and cytosol
was determined as described elsewhere (19). From each homogenate and
cytosol (HEK-IRES, HEK-TrxR11, HEK-TrxR15) 50 and 25 µg of protein,
respectively, was incubated with 80 mM HEPES (pH 7.5), 0.9 mg/ml NADPH, 6 mM EDTA, 2 mg/ml insulin, and 10 µM E. coli Trx (Promega) at 37 °C for 20 min in a final volume of 120 µl. A blank, containing everything except Trx, was incubated and treated in the same way as each sample.
The reaction was terminated by the addition of 500 µl of DTNB (0.4 mg/ml) in 6 M guanidine-HCl in 0.2 M Tris-HCl
(pH 8.0). The absorbance at 412 nm was measured, and the blank was subtracted from the corresponding absorbance of the sample. By comparison with a standard curve prepared by using purified calf thymus
TrxR1, the levels of TrxR1 could be determined.
Expression of Mutant Human TrxR (hTrxR5) and Recombinant
Full-length and Truncated Rat TrxR--
The hTrxR1 gene was PCR
amplified from the pGEMTeasy/hTrxR1 construct using the primers
hTrxR1-F2 (5'-CTACTAACCATGGACGGCCCTGAAG-3'), introducing an
NcoI restriction site, and hTrxR1-R2
(5'-TCAGCATCCGGACTGGAGGATGCTTGC-3'), introducing a BseA1
restriction site just upstream of the selenocysteine residue. This
product was cloned into the pET24d(+) vector (Novagen). The
pET-24d/hTrxR1a yields a mutant hTrxR1 (called hTrxR5) with a
translated C-terminal extension sequence of
Ser-Gly-Leu-Ala-Asn-Gly-Thr-Arg-Pro-Val-Ala-Ala-His in place of the
C-terminal amino acids Ala-Gly-Cys-Sec-Gly. The plasmid was transformed
into the E. coli strain BL21(DE3). 0.5 liters of the
bacteria was grown to an A600 of 0.5, and
then isopropyl-1-thio-
Recombinant rat TrxR1 containing selenocysteine was produced in
E. coli as described previously (20), and the two-amino acid
truncated form was produced in the same bacterial system by excluding
the selenocysteine insertion sequence element, as described (20).
Activity of rat TrxR1 was normalized for the selenocysteine-containing
fraction, i.e. 20% in the enzyme having 8 units/mg
(20).
Construction of Transfected Stable Overexpressing Human Embryo
Kidney Cell Lines (HEK293)--
It is well known that transfection for
overexpression of selenoproteins is difficult, and hTrxR1 is no
exception (21-24). To our surprise, we could nonetheless establish
stable cell lines overexpressing hTrxR1 using the following protocol.
A detailed molecular characterization of these cells is in
progress.2 The primers
hTrxR1-F1 (5'-GAATTCACCACCATGGACGGCCCTGAAGATCTTC-3') and hTrxR1-R1-3'
and hTrxR1-R1-R1-3'-UTR (5'-CCATTTCTTGAATTCGCCAAATGAGATGAGGACG-3') were used to amplify, by PCR, the hTrxrR1 cDNA from a human adrenal cDNA library and the introduction of EcoR1 restriction
sites for subsequent cloning. The construct includes the selenocysteine insertion sequence (SECIS element) and the first three of the six
AU-rich elements located in the 3'-UTR (21) and was cloned into the
pGEMTeasy vector (Promega) and sequenced. Stable cell lines
overexpressing hTrxR1 were subsequently generated using the pIRES
vector system (Clontech). The pGEMTasy/hTrxR1
mRNA was cleaved with EcoR1 and ligated into the pIRES
vector. 10 µg of the pIRES/hTrxR1 construct was then used to
transfect HEK293 cells using polyethyleneimine (PEI) (Aldrich). 0.5 µl of 0.1 M PEI was added to 0.1 µg/µl DNA in water.
The mixture was vortexed and incubated for 10 min at room temperature
and then added to the cells. The cells were allowed to grow for 2 days
without supplementation of sodium selenite and were then passaged.
Resistant colonies were selected with 1 mg/ml (HEK-TrxR11) or 1.5 mg/ml
(HEK-TrxR15) G418 (Calbiochem). Control cells were prepared by
transfecting HEK293 cells with the empty pIRES vector and then selected
as above (HEK-IRES). Single resistant colonies were picked, expanded, and analyzed for overexpression by activity measurements and Western blot analysis.
Reselection and Maintenance of Cells--
The HEK293 cells were
grown until confluence at 37 °C and 5% CO2 in
Dulbecco's modified Eagle's medium (containing 1 mg/ml glucose) and
F-12 nutrient mixture (ratio 1:1) supplemented with 10% fetal calf
serum (Invitrogen). The medium was changed every other day. The
cells were reselected every 4 weeks with one passage of medium as above
containing 1 mg/ml G418. No supplementation of sodium selenite was used
in the medium during the maintenance of the cells.
Culturing Cells for Ubiquinone-10 Reduction
Experiments--
Each experiment was started with 0.07 × 106 cells per culture dish (100 mm, Sarstedt). The cells
were left to adhere for 6 h before adding sodium selenite, where
indicated, in different concentrations. 10-12 cell culture dishes were
used per concentration. After 72 h the cells were harvested, and
homogenates and cytosol were prepared. For the preparation of cytosol,
cells were sonicated in 50 mM phosphate buffer, pH 7.0, and
centrifuged at 105,000 × g for 60 min at 4 °C. The
protein concentrations were measured according to Lowry (25).
Characterization of the Reduction of Ubiquinone-10 by
Purified Bovine TrxR1, Rat TrxR1, E. coli TrxR, Human
Mutant TrxR, and Truncated Rat TrxR--
The reduction of
ubiquinone-10 by commercially available bovine thioredoxin
reductase was characterized (Fig. 1).
There was a linear relation between the reduction of Q10 and the amount of enzyme added to the reaction mixture (Fig. 1a) as well as
to the time of incubation in the presence of NADPH as cofactor (Fig. 1b). The half-maximal rate of reduction was achieved at a
substrate concentration of 22 µM (apparent
Km) (Fig. 1c). The maximal rate of
reduction in this assay was ~12 nmol of Q10 reduced per milligram of
selenoprotein TrxR1 per minute, and similar results were obtained by
recombinant rat selenocysteine-containing TrxR1 but not with TrxR forms
lacking selenocysteine (Fig. 2). Zinc, which is known to be a potent inhibitor of thiol oxidoreductases (26)
with a number of known
substrates,3 also inhibited
the reduction of Q10 by TrxR1; at a concentration of 50 µM zinc the activity was decreased to 50%, and at 100 µM the activity was only ~15% (Fig. 1d).
Furthermore, the pH dependence of the reaction was measured in the
interval 5-8.5, and the reaction had a sharp physiological pH
optimum of ~7.5 (Fig. 1e).
Fig. 2 shows that there was no difference in the rate of Q10 reduction
when NADH was used instead of NADPH as the electron donor. This finding
was surprising, because no other known reaction catalyzed by mammalian
TrxR1 uses NADH as cofactor with similar rates as NADPH; for
example. using lipoamide as substrate for TrxR1, NADH
instead of NADPH gives only 5% activity, and with lipoic acid NADH
cannot function at all as a reductant (13). Thioredoxin reductase from
E. coli did not essentially reduce Q10. The reduction of Q10
was specific for mammalian selenocysteine-containing TrxR1, as shown in
Fig. 2. A major difference between E. coli and mammalian
TrxR1 is the presence of a redox-active selenolthiol motif formed by a
cysteine and a selenocysteine residue in the C-terminal of the
mammalian enzyme (2). To further study the importance of the selenium
moiety of mammalian TrxR1 for Q10 reduction, mutant human TrxR (hTrxR5)
in which the penultimate C-terminal selenocysteine residue was
substituted for alanine along with additional amino acids (see
"Experimental Procedures") as well as a truncated recombinant rat
TrxR lacking the last two amino acids, including the selenocysteine
residue (4, 20), were also investigated. These forms of the mammalian
enzyme did not essentially reduce Q10, suggesting that the presence of
selenocysteine was essential for TrxR1-mediated reduction of Q10 (Fig.
2).
The effects of lowering concentrations of NADPH and NADH on the
reduction of Q10 by the recombinant selenocysteine-containing rat TrxR1
showed that the similar rates of reduction using both cofactors were
also maintained at lower concentrations (Fig.
3). Thus, the reduction of Q10 differs in
this respect compared with the reduction of other known
substrates, by TrxR1.
Reduction of Ubiquinone-10 in Homogenates from Stable Human Kidney
Cell Lines Overexpressing TrxR1--
The identification of TrxR1 as an
enzyme regenerating ubiquinol-10 was confirmed by analysis of the
relative Q10 reduction rates in homogenates from two selected stable
cell lines overexpressing thioredoxin reductase, HEK-TrxR11 and
HEK-TrxR15 (Fig. 4). The amounts of
active TrxR1 in those cell lines were analyzed using a well established
enzyme assay wherein only active enzyme exclusively is measured based
upon thioredoxin-linked insulin reduction (Fig. 4a). The
amount of endogenous TrxR1 activity of the mock control cell lines
(corresponding to 70 ng/mg protein) was set as 100%. The increased
TrxR1 activities in the overexpressing cell lines mirrored closely
their total capacity to reduce Q10 (Fig. 4b), indicating
that TrxR1 indeed reduces Q10 to a major extent also in a cellular
context.
The addition of selenite in increasing concentrations revealed that the
activity increased dose dependently in the overexpressing cells but
only marginally in the control cells2 as was also shown
previously in COS cells with a transient-expression system (27).
Optimal activity was achieved at a selenite concentration of 0.1 µM and did not increase further at a concentration of 2 µM. At these concentrations, the activity of TrxR1 was
increased 3- and 2-fold in cytosols from HEK-TrxR11 and HEK-TrxR15,
respectively, whereas the activity in the mock control, HEK-IRES, was
essentially unchanged (Fig.
5a). There was also a
corresponding increase in cytosolic ubiquinone-10 reductase activity
upon exposure to increasing concentrations of selenite (Fig.
5b).
This paper demonstrates an important novel function for the
selenoenzyme TrxR1 as an ubiquinone-10 reductase implicating a vital
function in the protection against oxidative stress. At physiological
conditions, the reduction of ubiquinone-10 by mammalian TrxR1 was shown
to be the most efficient described to date. Our data using cells
overexpressing TrxR1 strongly support the belief that TrxR1 is an
important enzyme in the extramitochondrial ubiquinone-10/ubiquinol-10 redox cycle.
The molecular basis for cellular non-mitochondrial Q10 reduction has
previously been known only partly. However, the characteristics of the
reaction by TrxR1 as found here are nearly identical to the kinetic
parameters found in the cytosol from normal rat liver and rat liver
nodules.4 For example, in the
cytosol >90% of the reductase activity was found in the protein
fraction with a molecular mass above 100 kDa (TrxR1; 116 kDa)
and the reaction had a pH optimum of 7.5. Also, the reduction of Q10 in
rat liver cytosol was inhibited by the addition of zinc to the reaction
mixture. The observation that the rate of Q10 reduction was similar
with both NADH and NADPH as cofactors using purified enzyme
preparations as well as cytosolic protein is surprising and intriguing.
Possibly, Q10 slightly affects the conformation of the TrxR1 enzyme so
that NADH becomes better accepted as an electron donor or,
alternatively and more likely, the relatively low turnover compared
with other substrates such as thioredoxin or DTNB, which is probably
determined by the rate of reduction of Q10 by the selenolthiol motif,
obscures the differences in rates between NADH and NADPH in the first
reductive half-reaction. Nonetheless, the increased activity in TrxR1
and the corresponding increase in Q10 reductase activity after the addition of selenite to the transfected cells implicate a direct relation between the reduction of Q10 and the specific activity of
TrxR1. In addition, both the expression of Q10 reductase and TrxR1 as
well as the levels of ubiquinone-9 were elsewhere found to be increased
in the cytosol isolated from neoplastic rat liver nodules4
(28, 29). Taken together, these data support the results presented in this study and strongly suggest that TrxR1 is an important
mammalian cytosolic Q10 reductase in normal as well as neoplastic
cells. The related enzymes lipoamide dehydrogenase (LipDH) and
glutathione reductase (GR) can also reduce ubiquinone, but apparently
at lower rates compared with
TrxR15 (17) under normal
physiological conditions. Moreover, the reduction of Q10 performed by
these two enzymes is stimulated by zinc, contrary to TrxR1, which is
inhibited by zinc. In addition, the pH optimum for LipDH- and
GR-mediated reduction of ubiquinone is acidic5 (30)
and not physiological as is the case for the reduction monitored in
cytosol extracts and also for the reduction by TrxR1. Although we
suggest TrxR1 to be a predominant Q10 reductase in cytosol LipDH, GR
and also the previously suggested ubiquinone reductase DT-diaphorase
(31) could constitute important systems under other conditions and at
other intracellular sites. As all those enzymes are up-regulated in
neoplastic rat liver nodules, it might be an important mechanism for
increasing ubiquinol regeneration and thereby increasing the
antioxidant properties of this lipid in cells highly resistant to
oxidative stress.
The thioredoxin system may regulate the activity of several receptors
and transcription factors through the reduction of conserved cysteine
residues leading to conformational changes with resulting activation/deactivation of gene expression (7, 8). This process is
known as thiol redox control and may be especially important during
situations of oxidative stress. For instance, reduced Trx that mirrors
the activity of TrxR1 prevents apoptosis by inhibition of ASK-1 (32).
In addition, several important antioxidant reactions have been directly
described for mammalian TrxR1, e.g. the reduction of
hydrogen peroxide (4), lipid hydroperoxides (10), ascorbate (11),
lipoic acid, and lipoamide (13). The results reported in this
paper provide additional evidence that TrxR1 plays an important role in
the cellular defense against oxidative stress, because reduction of Q10
could prevent and terminate peroxidation.
The results that showed that the E. coli TrxR1, mutant
mammalian TrxR, and truncated rat TrxR forms lacking the penultimate C-terminal selenocysteine residue failed to reduce Q10 demonstrates the
specificity for the mammalian enzyme and an essential function for
selenocysteine in this reaction. This is in agreement with previous reports of a vital role for selenocysteine in the reduction of
other substrates and the participation of a unique
selenolthiol/selenenylsulfide motif in the catalytic cycle (2). The
background activities detected with the E. coli TrxR and the
selenium-deficient recombinant mammalian enzymes were in the same order
of magnitude as the direct chemical reaction between FAD and ubiquinone
described previously (30). Because all TrxRs contain a FAD in each
subunit, we suggest that the background reaction was mainly caused by
such a chemical reaction between protein-bound FAD and Q10. However, a
slow reaction between Q10 and the thiols of the N-terminal domain part
of the active center may not be excluded either (3).
Selenium in low to moderate concentrations is known to induce the
expression of several selenoenzymes, including thioredoxin reductases
and glutathione peroxidases (33). Several reports have shown a relation
between the levels of ubiquinone and selenium (34, 35). The molecular
basis for this relation has not, however, been established. Our data
now provide an explanation; TrxR1-mediated reduction of Q10 is
dependent on its selenocysteine. We therefore conclude that the
cytosolic cellular selenoenzyme TrxR1 is a link between the function of
the antioxidant compound ubiquinol-10 and the trace element selenium.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (IPTG) was
added to a final concentration of 0.2 mM to induce the
overexpression of the protein. The cells were harvested after 20 h
at 25 °C by centrifugation. The pellet was resuspended in 50 mM HEPES-NaOH, pH = 7.5, 0.5 M NaCl, 1%
(v/v) Triton X-100 and 1% sarcosyl (w/v), and lysis of the cells was
performed by the addition of 300 ml of lysozyme (50 mg/ml) and
incubation on ice for 30 min with stirring. Then, 300 ml each of 1 M MgCl2, 0.1 M MnCl2, 1 mg/ml DNase, and 1 mg/ml RNase were added to the lysate, which was
further incubated for 45 min. The lysate was subsequently centrifuged,
applied to a 2-ml 2',5'-ADP-Sepharose column equilibrated with 10 mM Tris-HCl, pH 8.0 buffer with 1 mM
EDTA and purified as described (20).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Characterization of thioredoxin
reductase-mediated reduction of ubiquinone-10 using purified bovine
enzyme and NADPH as cofactor. The effects of enzyme concentration
(a), time of incubation (b), substrate
concentration (c), increasing concentrations of zinc
(d), and pH (e) are shown. All data represent the
mean value of two to four individual experiments.
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[in a new window]
Fig. 2.
Reduction of ubiquinone-10 by recombinant rat
thioredoxin reductase (Rat), bovine thioredoxin
reductase (Bovine), E. coli
thioredoxin reductase (E.
coli), human selenocysteine to alanine mutant TrxR
(hTrxR5), and truncated rat thioredoxin reductase
lacking selenocystein (tTrxR) with NADPH
(gray bar) and NADH (striped
bar) as cofactors. ***, p < 0.001 compared with bovine and rat TrxR1 using Student's t
test.
View larger version (15K):
[in a new window]
Fig. 3.
Reduction of Trx and insulin disulfides
(a) and Q10 (b) by TrxR1 in the
presence of increasing concentrations of NADPH (gray
bar) and NADH (striped
bar).
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[in a new window]
Fig. 4.
TrxR1 activity in homogenates from stable
transfected human kidney cell lines overexpressing TrxR1 (HEK-TrxR15
and HEK-TrxR11) and mock transfected control cells (HEK-IRES)
(a) and the reduction of Q10 in
overexpressing and control cell homogenates (b).
The results are expressed as percentage of control where the
controls are 100%. *, p < 0.05; ***,
p < 0.001 compared with HEK-IRES using Student's
t test.
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[in a new window]
Fig. 5.
The effect of incubation with increased
concentrations of selenite on the TrxR1-activity and the reduction of
Q10 in cytosol from mock control cells, HEK-IRES (gray
bar) cell lines overexpressing TrxR1, HEK-TrxR15
(white bar), and HEK-TrxR11 (striped
bar). Cells were grown for 72 h in minimum
Eagle's medium/F12 (10% fetal calf serum) supplemented with selenite
in indicated concentrations prior to cell harvesting. Data represent
the mean of three experiments performed at three different occasions.
***, p < 0.001 (Student's t test) as
compared with the mock control (HEK-IRES).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This investigation was supported by grants from the Swedish Cancer Society, the Swedish Medical Association, the Karolinska Institutet, and Medical Research Council Grant 13X-10370.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Microbiology, Pathology and Immunology, Karolinska Inst., Division of Pathology, F46, Huddinge University Hospital, SE-141 86 Stockholm, Sweden. Tel.: 46-8-58583809; Fax: 46-8-58581020; E-mail:
mikael.bjornstedt@impi.ki.se.
Published, JBC Papers in Press, November 14, 2002, DOI 10.1074/jbc.M210456200
2 I. Nalvarte, A. Damdimopoulos, T. Nordman, J. M. Olsson, M. Björnstedt, E. S. J. Arnér, and G. Spyrou, manuscript in preparation.
3 L. Xia, T. Nordman, J. M. Olsson, and M. Björnstedt, unpublished observations.
4 L. Xia, M. Björnstedt, T. Nordman, L. Björkhem-Bergman, L. Eriksson, and J. M. Olsson, submitted for publication.
5 T. Nordman, M. Björnstedt, and J. M. Olsson, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Trx, thioredoxin; TrxR1, Trx reductase 1; Sec, selenocysteine; Q10, ubiquinone-10; HEK293, human embryo kidney cells; PEI, polyethyleneimine; IRES, internal ribosome entry sequence; DTNB, 5,5'-dithiobis(nitrobenzoic acid); HPLC, high performance liquid chromatography.
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