From the Laboratory of Cell Signaling and
Laboratory of Biochemistry, NHLBI, National Institutes of
Health, Bethesda, Maryland 20892
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ABSTRACT |
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A thioredoxin reductase (TrxR), named here TrxR2,
that did not react with antibodies to the previously identified TrxR
(now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a
dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids
with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame
TGA codon encodes Secys. The 3'-untranslated region of the cDNA
contains a canonical Secys insertion sequence element. The deduced
amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and
contained 36 additional residues upstream of the experimentally
determined NH2-terminal sequence. The sequence of
this 36-residue region is typical of that of a mitochondrial leader
peptide. Immunoblot analysis confirmed that TrxR2 is localized almost
exclusively in mitochondria, whereas TrxR1 is a cytosolic protein.
Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 µg/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 µg/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in
mitochondria, together with the previous identification of
mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of
defense against H2O2 produced by the
mitochondrial respiratory chain.
Thioredoxin (Trx)1 is a
widely expressed 12-kDa protein that performs pleiotropic cellular
functions (1, 2). The active site of Trx contains the sequence
-Cys-Gly-Pro-Cys-, and the reduced form of the protein serves as a
hydrogen donor for ribonucleotide reductase (3), protein methionine
sulfoxide reductase (4), thioredoxin-dependent peroxidase
(5), and protein tyrosine phosphatase (6) as well as contributes to the
up-regulation of various transcription factors (7-11). In addition,
the reduced form, but not the oxidized form, of Trx binds to and
inhibits the catalytic activity of apoptosis signal-regulating kinase, also known as mitogen-activated protein kinase kinase kinase (12). Furthermore, Trx serves as a growth factor that stimulates the proliferation of T lymphocytes (13).
Oxidized Trx is converted back to the reduced form by thioredoxin
reductase (TrxR) with the use of electrons from NADPH (14, 15). TrxR is
a homodimeric enzyme with a redox-active disulfide and contains one FAD
molecule per subunit (14, 15). It belongs to a superfamily of
flavoprotein disulfide oxidoreductases that includes glutathione
reductase (GR), dihydrolipoamide reductase, mercuric reductase, and
alkylhydroperoxide reductase (16, 17). Mammalian TrxR is distinct from
those of prokaryotes and yeast. The mammalian enzyme exhibits a broader
substrate specificity, having the ability to reduce chemically
unrelated compounds such as selenite and 5,5'-dithiobis(2-nitrobenzoic
acid) in the presence of NADPH (18, 19), is larger in subunit size (58 kDa, compared with 35 kDa for prokaryote and yeast enzymes), and
contains a much longer COOH-terminal region (18, 20). In addition,
mammalian TrxR is a selenoprotein that contains a penultimate
selenocysteine (Secys) residue in the sequence -Gly-Cys-Secys-Gly (14,
21-23), which can serve as a redox center (24).
Mammalian cells contain two distinct forms of Trx: Trx1 is located in
the cytosol and nucleus and is also secreted (10), whereas Trx2 is
restricted to mitochondria (25). However, only one isoform of TrxR has
previously been identified in mammalian cells, and it has not been
known whether this protein is expressed in mitochondria. We now
describe the purification and cloning of a second type of TrxR, named
TrxR2, from rat liver and demonstrate that this protein is specifically
expressed in mitochondria.
Materials--
Rat liver was obtained from Bioproducts for
Science, Inc. (Indianapolis, IN). Rabbit antiserum to rat TrxR1 was
produced by immunization with purified enzyme according to standard
procedures. Rabbit antiserum to TrxR2 was prepared by injection with a
hemocyanin-conjugated peptide (RSGLDPTVTGCCG) corresponding to the
COOH-terminal sequence of rat TrxR2, with the exception that the
penultimate residue (Secys) was changed to Cys. Horseradish
peroxidase-conjugated antibodies to rabbit immunoglobulin G and the
enhanced chemiluminescence (ECL) immunoblot detection system were
from Amersham; biotin-conjugated iodoacetamide,
N-(biotinoyl)-N'-(iodoacetyl)ethylenediamine
(BIAM), was from Molecular Probes; horseradish
peroxidase-conjugated streptavidin, 3,3',5,5'-tetramethyl benzidine,
and Neutravidin beads were from Pierce; and yeast GR was from
Boehringer-Mannheim. Recombinant rat Trx was prepared as described
(26).
Purification of TrxR1--
Rat livers (1 kg) were homogenized in
4 liters of 20 mM Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM dithiothreitol (DTT), 0.05 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF), pepstatin (0.5 µg/ml), leupeptin (0.5 µg/ml), and
aprotinin (0.5 µg/ml). The homogenate was centrifuged at 70,000 × g for 30 min, and the resulting supernatant was adjusted
to pH 5.0 with 1 M acetic acid and then centrifuged again
at 70,000 × g for 30 min. The resulting pellet and
supernatant were subjected to immunoblot analysis with antibodies to
TrxR1 and TrxR2 (see Fig. 6A). TrxR1 was detected only in
the supernatant, whereas TrxR2 was present mostly in the pellet. Thus,
the supernatant and pellet served as the sources for purification of
TrxR1 and TrxR2, respectively.
For purification of TrxR1 (elution profiles of column chromatographies
are not shown), the supernatant (40 g of protein) from the pH 5 precipitation step was adjusted to pH 7.8 with 1 M ammonium hydroxide and then applied to a DEAE-Sephacel (Pharmacia) column (10 × 16 cm) that had been equilibrated with 20 mM
Tris-HCl (pH 7.8) containing 1 mM EDTA, 1 mM
DTT, and 0.01 mM AEBSF. The column was washed consecutively
with 2.5 liters of equilibration buffer and 2.5 liters of equilibration
buffer containing 100 mM NaCl. Proteins were eluted from
the column with a linear gradient of 100 to 400 mM NaCl in
5 liters of equilibration buffer, and fractions (25 ml) were collected
and assayed for TrxR1 by immunoblot analysis.
The peak fractions (10.4 g of protein), corresponding to 300 to 380 mM NaCl on the gradient, were pooled, dialyzed overnight against 20 mM Tris-HCl (pH 7.5) containing 1 mM
EDTA, 1 mM DTT, and 0.01 mM AEBSF, and then
applied to a 2'5'-ADP-agarose column (2 × 7 cm) that had been
equilibrated with 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The column was washed with 100 ml of equilibration
buffer, and proteins were then eluted stepwise with 100 ml each of
equilibration buffer containing 200 mM KCl, equilibration buffer containing 200 mM sodium phosphate and 200 mM KCl, and equilibration buffer containing 1 M
NaCl and 200 mM KCl. The 58-kDa TrxR1 was present almost
exclusively in the fractions eluted by the buffer containing 200 mM KCl as revealed both by SDS-polyacrylamide gel
electrophoresis (PAGE) and Coomassie Blue staining and by immunoblot analysis.
Peak fractions (19.8 mg of protein) were pooled and then adjusted to
1.2 M ammonium sulfate by addition of 4 M
ammonium sulfate. After removal of the resulting precipitate by
centrifugation, the supernatant was applied to a Phenyl-5PW
high-performance liquid chromatography (HPLC) column (0.75 × 7.5 cm) that had been equilibrated with 20 mM Hepes-NaOH (pH
7.5) containing 1 mM DTT, 1 mM EDTA, and 1.2 M ammonium sulfate. The column was washed with 60 ml of equilibration buffer, and proteins were then eluted with a decreasing linear gradient of 1.2 to 0 M ammonium sulfate in 120 ml of
20 mM Hepes-NaOH (pH 7.5) containing 1 mM DTT
and 1 mM EDTA. Peak fractions, corresponding to 0.8 to 0.64 M ammonium sulfate on the gradient, were pooled,
concentrated, dialyzed against 20 mM Hepes-NaOH (pH 7.5)
containing 1 mM DTT and 1 mM EDTA, divided into
portions, and stored at Purification of TrxR2--
The pellet derived from the pH 5 precipitation step described for the purification of TrxR1 was
dissolved in 2 liters of 20 mM Tris-HCl (pH 7.8) containing
1 mM EDTA, 1 mM DTT, 0.05 mM AEBSF, leupeptin (0.5 µg/ml), and aprotinin (0.5 µg/ml). The pH of the suspension was adjusted to 7.8 with 1 M ammonium hydroxide.
After centrifugation of the suspension, the resulting supernatant (7.3 g of protein) was applied to a DEAE-Sephacel column (10 × 16 cm) that had been equilibrated with 20 mM Tris-HCl (pH 7.8)
containing 1 mM EDTA, 1 mM DTT, and 0.01 mM AEBSF. The column was washed with 1.5 liters of
equilibration buffer and then with 2.5 liters of equilibration buffer
containing 50 mM NaCl. Proteins were eluted with a
linear gradient of 50 to 800 mM NaCl in 5 liters of
equilibration buffer, and fractions (20 ml) were collected. TrxR2 was
assayed by immunoblot analysis (see Fig. 6B).
Peak fractions (numbers 80 to 120, containing 1.8 g of protein)
were pooled, dialyzed overnight against equilibration buffer, and then
applied to a 2'5'-ADP-agarose column (2 × 7 cm) that had been
equilibrated with 20 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The column was washed with 100 ml of equilibration buffer, and proteins were then eluted consecutively with 60 ml of
equilibration buffer containing 20 mM KCl, a linear
gradient of 20 to 200 mM KCl in 260 ml of equilibration
buffer, 60 ml of equilibration buffer containing 200 mM
KCl, 100 ml of equilibration buffer containing 200 mM
sodium phosphate and 200 mM KCl, and 120 ml of
equilibration buffer containing 1 M NaCl and 200 mM KCl. Fractions were assayed for TrxR2 by immunoblot
analysis (see Fig. 6C).
Peak fractions eluted between 340 and 352 min were pooled and then
adjusted to 1.2 M ammonium sulfate by adding 4 M ammonium sulfate. After removal of the resulting
precipitate by centrifugation, the supernatant (6.1 mg of protein)
was applied to a Phenyl-5PW HPLC column (0.75 × 7.5 cm) that had
been equilibrated with 20 mM Hepes-NaOH (pH 7.5) containing
1 mM DTT, 1 mM EDTA, and 1.2 M
ammonium sulfate. The column was washed with 60 ml of equilibration buffer, and proteins were then eluted with a decreasing linear gradient
of 1.2 to 0 M ammonium sulfate in 120 ml of 20 mM Hepes-NaOH (pH 7.5) containing 1 mM DTT and
1 mM EDTA. Fractions (2 ml) were collected and assayed for
TrxR2 by immunoblot analysis (see Fig. 6D). Peak fractions
eluted between 32 and 42 min (2.1 mg of protein) were pooled,
concentrated, dialyzed against 20 mM Hepes-NaOH (pH 7.5)
containing 1 mM EDTA and 1 mM DTT, divided into
portions, and stored at Determination of Protein Concentration--
The concentrations
of recombinant Trx and TrxR1, TrxR2, and GR were determined
spectrophotometrically, with A280 values for 0.1% solutions of 0.738, 0.938, 1.081, and 1.091, respectively, which
were calculated based on their amino acid composition. The concentrations of other proteins were determined with the BCA protein
assay reagent (Pierce), with bovine serum albumin as a standard.
Labeling of TrxR2 with BIAM--
All procedures were performed
in an anaerobic chamber with solutions that were free of oxygen. TrxR2
(130 µg/ml) in 1 ml of 20 mM Hepes-NaOH (pH 7.5)
containing 1 mM EDTA was reduced by incubation for 10 min
at room temperature first with 54 µM NADPH and then with
100 µM DTT. The reduced enzyme was dialyzed on ice against 50 mM Bis-Tris-HCl (pH 6.5) containing 1 mM EDTA. The dialyzed protein (100 µg) was then incubated
at room temperature and in the dark for 10 min in 4 ml of 50 mM Bis-Tris-HCl (pH 6.5) containing 0.5% Triton X-100, 5%
glycerol, 150 mM NaCl, 1 mM EDTA, and 10 µM BIAM. The biotinylation reaction was stopped by the addition of iodoacetamide to a final concentration of 20 mM, and the pH of the reaction mixture was adjusted to 7.5. After 10 min, the mixture was dialyzed twice against 50 mM
Tris-HCl (pH 8.0) containing 1 mM EDTA.
Purification of Tryptic Peptides Derived from BIAM-labeled
TrxR2--
Dialyzed BIAM-labeled TrxR2 (80 µg) was digested by
incubation overnight at room temperature with 4 µg of trypsin.
One-fourth of the resulting digest was analyzed by HPLC on a
C18 column; peptides were eluted with a linear gradient (0 to 60%, v/v) of acetonitrile in 0.1% trifluoroacetic acid at a flow
rate of 1 ml/min over 60 min. Fractions (500 µl) were collected, and
a portion (2 µl) of each was analyzed for BIAM-labeled peptide.
Peptides were immobilized on maleic anhydride-activated microplates
(Pierce) and labeled peptide was detected with horseradish
peroxidase-conjugated streptavidin and the peroxidase substrate
3,3',5,5'-tetramethyl benzidine, the oxidation of which was monitored
spectrophotometrically at 405 nm. A major BIAM-labeled peptide eluted
at 28.5 min. Two nonlabeled peptides that eluted at 38.3 and 40.1 min
were also collected and subjected to sequence analysis.
The remaining three-fourths of the BIAM-labeled TrxR2 digest were
incubated with 15 µl of Neutravidin beads (30% slurry) for 50 min at
room temperature. The beads were separated by brief centrifugation,
washed twice with 1 ml of phosphate-buffered saline containing 0.01%
Lubrol, and then incubated for 30 min at 37 °C with 6 M
guanidine hydrochloride in 500 mM potassium phosphate (pH
2.5). The released peptides were further purified by HPLC on a
C18 column as described above, and the BIAM-labeled peptide that eluted at 28.5 min was collected for sequence analysis.
Mass and Peptide Analyses--
The purity and mass of the
isolated BIAM-labeled peptide were assessed by matrix-assisted laser
desorption ionization with time of flight detection (MALDI-TOF) mass
spectroscopy (Hewlett-Packard model G2025A), using sinapinic acid as
the matrix (21). Electrospray mass spectroscopy was performed with a
Hewlett-Packard model G1946A instrument interfaced to a model 1100 HPLC
system equipped with a Vydac 218TP narrow bore C18 column.
The effluent from the column (200 µl/min) was mixed in a tee with
acetic acid, pumped by another 1100 series pump (100 µl/min), and the
mixture was introduced into the mass spectrometer (27). Sequences were
determined by automated Edman degradation with a Hewlett-Packard model
G1005 sequencer running version 3.5 of the manufacturer's chemistry program.
Assay of TrxR and GR Activities--
Reduction of oxidized Trx
was measured in a mixture (1 ml) containing 50 mM potassium
phosphate (pH 7.0), 50 mM KCl, 1 mM EDTA, 0.25 mM NADPH, and 120 µM oxidized Trx. After
addition of the TrxR source, the oxidation of NADPH was monitored
spectrophotometrically at 340 nm and 25 °C.
The GR assay mixture contained 50 mM potassium phosphate
(pH 7.0), 1 mM EDTA, 0.25 mM NADPH, 1 mM GSSG, and enzyme source in a final volume of 0.5 ml, and
the reaction was monitored spectrophotometrically at 340 nm and
25 °C. For both TrxR and GR assays, activity was calculated as
micromoles of NADPH oxidized per minute at 25 °C from the relation
A340 × 0.5/6.22. Assay mixtures lacking enzyme served as controls.
Cloning and Sequencing of TrxR2 cDNA from a Rat Liver
cDNA Library--
Complementary DNA encoding TrxR2 was amplified
from Marathon rat liver cDNA () by the
polymerase chain reaction (PCR) with the 5' primer
5'-CA(G/A)CA(G/A)AA(C/T)TT(C/T)GA and 3' primer 5'-GTNACNGGCTGCTGAGG,
corresponding to the determined NH2-terminal (QQNFDLLVIGGGS) and COOH-terminal SGLDPTVTGCUG (where U represents Secys) sequences, respectively, of the purified protein. The PCR products were separated on a 1% agarose gel, and the amplified 1.5-kilobase molecule was eluted from the gel with a Qiagen gel extraction kit. After ligation of the eluted DNA into the pCR3.1 vector
(Invitrogen) and transformation of Escherichia coli,
positive clones were identified by nested-PCR with the internal forward primer 5'-GA(C/T)GA(C/T)ATNTT(C/T)TGG or the internal reverse primer
5'-CCA(G/A)AANAT(G/A)TC(G/A)TC, both of which were derived from the
internal amino acid sequence HGITSDDIFWLK of TrxR2. Plasmid DNA was
purified from the positive E. coli clones with a Qiagen miniplasmid preparation kit, and was sequenced with the T7 primer and
pCR3.1 reverse primer on an ABI sequencer. To extend the 5' sequence,
we perfomed 5'-rapid amplification of cDNA ends (5'-RACE) by PCR
with Marathon-Ready rat liver cDNA as the template, the adapter
primer 1 () as the forward primer, and a
reverse primer complementary to the sequence 5'-CTGTGGCTGACTATGTGGAA. 5'-RACE was also performed with nested adapter primer 2 () as the forward primer and a reverse
primer complementary to the sequence 5'-GCAGCAGAACTTCGATCTC. PCR
products were cloned into the TA vector and sequenced. The 5'-extended
sequences determined from the two independent 5'-RACE experiments were
identical. Similarily, the sequence of the 3'-untranslated region was
determined by two independent 3'-RACE experiments with two different
sense primers (5'-GCTTCATACGCACAGGTGATGCAG and 5'-TGGTTAAGCTGCACATCTCC)
and the adapter primer 1 as the antisense primer.
Analytical Ultracentrifugation--
The Beckman Optima model
XL-A analytical ultracentrifuge equipped with a four-place An-Ti rotor
was used for sedimentation velocity experiments at 20.0 °C. The
density ( Atomic Absorption Spectrometry--
The selenium content of
TrxR1, TrxR2, and the BIAM-labeled peptide derived from BIAM-labeled
TrxR2 was determined with a Perkin-Elmer model 4100 ZL atomic
absorption spectrometer with the use of a palladium-magnesium nitrate
modifier and temperature conditions as described (32). Calibration
solutions were prepared by diluting the selenium stock solution (1 g/liter) with a solution containing 1.16 mM
Na2HPO4, 0.31 mM
KH2PO4, 10.26 µM NADPH, and bovine serum albumin (0.120 mg/ml) to give final concentrations of 0, 10, 30, and 90 µg/liter. Various concentrations of TrxR1 and TrxR2 were prepared in
the same solution devoid of albumin; the BIAM-labeled peptide solution
was prepared in double-distilled water.
Distribution of TrxR Isozymes in Rat Tissues and in Subcellular
Fractions of Rat Liver--
Frozen rat tissues (Pel-Freeze
Biologicals) were sonicated in a solution containing 20 mM
Tris-HCl (pH 7.5), 1 mM EDTA, aprotinin (2.5 µg/ml), and
leupeptin (5 µg/ml). The sonicates were centrifuged at 100,000 × g for 15 min, and the resulting supernatants were subjected to immunoblot analysis with antibodies specific for TrxR1 or
TrxR2. Rat liver homogenates were prepared, and cytosolic and
mitochondrial fractions were separated by ultracentrifugation as
described (33, 34).
Purification of TrxR1 and TrxR2--
In previous studies, we have
used the Trx system as the electron donor for reduction of
H2O2 by peroxiredoxins (26) and for
reactivation of H2O2-inactivated protein
tyrosine phosphatase (6). For these experiments, the 58-kDa TrxR,
designated here as TrxR1, was routinely purified from rat liver by a
procedure that included acidification of tissue homogenate to pH 5 and
sequential chromatography of the acid-soluble proteins on DEAE
Sephacel, 2',5'-ADP-agarose affinity matrix, and Phenyl-5PW.
A purification in which the pH 5 precipitation step was inadvertently
omitted yielded two peaks of flavoprotein after the 2',5'-ADP-agarose
column step, as judged from the ratio
A280/A460. The first peak
eluted with the buffer containing 200 mM KCl and contained
a protein with an apparent molecular mass of 58-kDa, whereas the second
peak eluted with the buffer containing 1 M NaCl and 200 mM KCl and comprised predominantly a 55-kDa protein. Further purification of these peak fractions by phenyl-Sepharose column
chromatography yielded highly purified preparations of the 58- and
55-kDa proteins (Fig. 1A).
Whereas both preparations exhibited TrxR activity, polyclonal
antibodies to 58-kDa TrxR1 recognized the 58-kDa protein from the first
peak but not the 55-kDa protein from the second peak (Fig.
1B), suggesting that the 55-kDa protein was not derived from
TrxR1. The 55-kDa protein was thus named TrxR2. Like TrxR1, TrxR2 was
demonstrated to be a flavin-containing protein by its absorption
spectrum, which showed maxima at 280, 352, and 446 nm in a ratio of 1, 0.12, and 0.11, respectively (Fig. 1C).
Sequences of Peptides Derived from TrxR2--
TrxR1 contains a
penultimate Secys residue at its COOH terminus that can be selectively
labeled with an alkylating agent (35). To determine whether TrxR2 also
contains such a residue, the purified protein was labeled with 10 µM BIAM at pH 6.5 and subsequently incubated with 2 mM iodoacetamide at pH 7.5. The labeled protein was cleaved
with trypsin, and a BIAM-labeled peptide was purified with Neutravidin
affinity matrix and a C18 column (Fig.
2). Edman degradation of the labeled
peptide yielded the sequence SGLDPTVTGCXG; the residue
corresponding to cycle 10 was identified as carboxymethylated cysteine,
and the residue corresponding to cycle 11 was unknown. Determination of
the molecular mass of the purified peptide by MALDI-TOF mass
spectrometry yielded a mass of 1540.8 (data not shown), which is
virtually identical to the value of 1539.8 calculated for the
dodecameric peptide with a carboxymethylated Cys and BIAM-labeled Secys. These data suggest that the residue corresponding to cycle 11 was a Secys that had been selectively labeled with BIAM at the lower
pH, whereas the Cys residue adjacent to the Secys was alkylated with
iodoacetamide after the pH and the concentration of alkylating reagent
were increased. The presence of selenium in the BIAM-labeled peptide
was confirmed by atomic absorption spectrometry (see below).
Two nonlabeled TrxR2 peptides were also purified (Fig. 2A)
and yielded the sequences IIVDAQEATSVPHIYAIGDV and HGITSDDIFWLK. In
addition, TrxR2 was subjected to automated Edman sequencing for 20 cycles, with the major sequence being readable for 19 cycles: GGQQNFDLLVIGGGSGGLA. A minor sequence was consistent with loss of the
first glycine residue. Two preparations of TrxR2 were analyzed by
electrospray mass spectroscopy, giving a mass of 53,037 ± 2, in
excellent agreement with the 53,036 calculated from the sequence deduced from the cDNA, and confirming the amino-terminal site as
well as the presence of selenocysteine.
TrxR1 was similarily labeled with BIAM, and the BIAM-labeled peptide
generated by digestion with endoproteinase Lys-C was purified.
Sequencing and MALDI-TOF analysis of the labeled peptide (data not
shown) yielded the sequence RSGGDILQSGCUG (where U represents Secys),
which matches exactly the sequence of amino acids 486 to 498 at the
COOH terminus of the previously identified TrxR from rat liver
(18).
Cloning and Sequencing of TrxR2 cDNA--
TrxR2 cDNA was
amplified from a rat liver cDNA library by PCR with primers based
on the NH2- and COOH-terminal amino acid sequences of the
purified protein, as described under "Experimental Procedures." A
1.5-kilobase PCR product was obtained, the cloning and sequencing of
which revealed a 1463-base pair fragment that contained the precise
coding sequences in the same reading frame for the two internal tryptic
peptides derived from purified TrxR2 (Fig.
3). Additional 5' and 3' sequences were
obtained with the use of RACE-PCR, yielding a cumulative sequence of
1982 base pairs, excluding the poly(A) tail (Fig. 3). The translational
initiation site was assumed to be the methionine codon composed of
nucleotides 29 to 31, which was the first ATG triplet downstream of an
in-frame nonsense codon (TAA at nucleotides 14 to 16). Two
translational termination codons, TGA and TAA, occurred in-frame in the
sequence TGAGGTTAA (nucleotides 1601 to 1609).
As in other Secys-containing proteins, the TGA codon corresponds to the
penultimate Secys residue. Therefore, the TAA triplet was assumed to be
the termination codon. The open reading frame encodes a polypeptide of
526 amino acids, with a calculated molecular mass of 56,574.8 daltons.
The deduced protein sequence contained 36 residues upstream of the
experimentally determined NH2 terminus of purified TrxR2. This additional 36-residue sequence contains 6 arginine residues and no
acidic residues, and it is predicted to form an
The deduced TrxR2 sequence shows 54% identity (62% similarity) to
both rat (18) and human (20) TrxR1 sequences (Fig.
4). Rat TrxR proteins show a low sequence
homology to prokaryote and yeast TrxR enzymes (28 and 36% identity to
E. coli (16) and yeast (5) TrxR, respectively), and they are
distinguished from these enzymes by the presence of a COOH-terminal
extension containing the Secys residue. TrxR2 showed a relatively high
homology to human GR (38) (41% identity and 50% similarity) as well
as to putative GR sequences of Caenorhabditis elegans (39)
and Drosophila melanogaster (40) (48 to 50% identity, 57 to
58% similarity). Furthermore, unlike human and mouse GR proteins, the
C. elegans and D. melanogaster enzymes possess
long COOH-terminal regions that are similar to those of mammalian TrxR
proteins. The COOH termini of C. elegans and D. melanogaster GR proteins also end with the sequences GCCG and
SCCS, respectively, which resemble the GCUG motif at the COOH termini
of mammalian TrxR enzymes. As a result of the effort to sequence
C. elegans chromosome III (41), another putative GR gene
(GenBank accession number, U61947) has been identified on the basis of
its homology (36% identity and 45% similarity) to mammalian GR
enzymes. However, the gene product has not been shown to possess GR
activity, and, at the time the gene was characterized, mammalian TrxR
genes had not been identified. Comparison of the predicted amino acid
sequence of the protein encoded by the chromosome III GR gene with
those of TrxR1 and TrxR2 revealed higher homology (48 to 59% identity and 58 to 69% similarity) to these proteins than to mammalian GR
enzymes. Furthermore, as in mammalian TrxR enzymes, the codons for
Secys (TGA) and Gly (GGT) in the chromosome III gene are followed by
the stop codon TAA, indicating that the gene product could contain the
sequence GCUG at its COOH terminus. Therefore, we listed the product of
the chromosome III gene as a TrxR rather than as a GR in Fig. 4. TrxR2
showed low homology to other members of the flavoprotein disulfide
oxidoreductase family, such as human dihydrolipoamide reductase (42)
(28% identity) and Psuedomonas aeruginosa mercuric
reductase (43) (26% identity).
Secys Insertion Sequence Element in the 3'-Untranslated Region of
TrxR2 cDNA--
The encoding of Secys by TGA in eukaryotic
selenoproteins requires the presence of a Secys insertion sequence
(SECIS) element such as that located in the 3'-untranslated regions of
transcripts that encode thyroid hormone deiodinases, glutathione
peroxidases, and several types of selenoprotein P (44, 45). The spacing between the TGA codon and the SECIS element varies greatly. The SECIS
element has been defined on the basis of conserved sequence rather than
functional features. The conserved sequence includes the invariable
AUGA, three consecutive A residues that are separated by 9 to 12 residues from the AUGA motif, and the doublet GA that is separated by a
widely variable number of residues from the AAA triplet (Fig.
5A). Although the overall
sequence homology among SECIS elements is low, they exhibit conserved
stem-loop structures that can be divided into two types (46). Both type I and type II structures contain the conserved sequences AUGA and GA in
the 5' and 3' arms, respectively, of the stems. The two types differ in
that the unpaired AAA sequence is located in the apical loop in type I
structures but forms a bulge in the 5' arm of type II structures. The
AAA-containing bulge in type II structures is separated from the apical
loop by a predicted stem of 3 to 5 base pairs. SECIS elements
with a long sequence between the AAA triplet and GA dinucleotide
show a tendency to assume a type II structure (47, 48).
The 3'-untranslated region of TrxR2 cDNA contains a putative SECIS
element that conforms to the consensus sequence. A computer folding
program indicated that the TrxR2 SECIS element forms a type II
stem-loop structure that contains a AAA bulge and a stem of three GC
pairs below the apical loop of six nucleotides (Fig. 5B).
Improved TrxR Purification Procedure--
A rabbit antiserum to
TrxR2 was prepared by injection with a peptide (RSGLDPTVTGCCG) that is
identical to the COOH-terminal sequence of TrxR2 with the exception
that Secys was replaced by Cys. Immunoblot analysis with this
antipeptide serum and with rabbit antiserum to TrxR1 indicated that the
acidification of rat liver homogenate to pH 5 resulted in the
precipitation of TrxR2, whereas TrxR1 remained soluble (Fig.
6A). On the basis of this
result, the purification protocols for TrxR1 and TrxR2 were improved as
described under "Experimental Procedures." The modified approach,
which includes three successive column chromatographies after the acid
precipitation step, allowed us to obtain TrxR1 and TrxR2 with no
cross-contamination and with higher yields, because increased pooling
of column fractions was possible. Elution profiles for the three column
steps are shown for the purification of TrxR2 (Fig. 6, B-D).
The procedure yielded 6.5 mg of TrxR1 and 2.1 mg of TrxR2 with high
purity (>95%) from 1 kg of rat liver.
Homogeneity, Size, and Shape of TrxR Enzymes--
Like the TrxR
enzymes from prokaryotes and yeast, mammalian TrxR1 was shown to be a
dimer in its native state. Analysis by nondenaturing PAGE of TrxR1 and
TrxR2 purified by the improved procedure yielded a value for the
molecular mass of TrxR1 of 115 kDa, consistent with that expected for a
dimer. However, the mobility of TrxR2 was substantially less than that
of TrxR1. Electrophoresis performed overnight on an 8 to 16% gradient
gel yielded an estimated size of 300 kDa for TrxR2 (data not shown).
Furthermore, the molecular mass of TrxR2 determined from its mobility
was dependent on the time of electrophoresis. Because the isoelectric
point of mature TrxR2 (8.0) predicted from its amino acid sequence is
substantially higher than that of TrxR1 (5.9), it was possible that the
lower mobility of TrxR2 was due to its lower charge density under the conditions of electrophoresis rather than to a difference in
oligomerization state.
To verify this supposition, we subjected TrxR1 and TrxR2 to analytical
ultracentrifugation. A single, symmetrical sedimentation boundary was
observed for both TrxR1 and TrxR2 at pH 7.4 and 20 °C. Time
derivative analyses of four late concentration profiles are shown for
TrxR1 (Fig. 7A) and TrxR2
(Fig. 7B). The fit of g(s*) data for
TrxR1 and TrxR2 to a single Gaussian curve in each instance
demonstrated the homogeneity of the proteins. The
g(s*) function for TrxR2 (Fig. 7B)
appeared broader than that for TrxR1 (Fig. 7A) because TrxR2
was sedimented for a longer time and at a lower speed (40,000 rpm, 93 min) than was TrxR1 (48,000 rpm, 77 min) on the basis of the gel
electrophoresis data indicating that TrxR2 might be larger than
TrxR1.
The sedimentation properties of TrxR1 and TrxR2 are summarized in Table
I. The corrected sedimentation
coefficients for TrxR1 and TrxR2 were 6.08 and 6.29 S, and the
diffusion coefficients (±4%) were 4.85 × 10 Catalytic Activity--
The broad substrate specificity of
mammalian TrxR proteins has allowed the NADPH-dependent
reduction of 5,5'-dithiobis(2-nitrobenzoic acid) to be used as the
basis for an assay of TrxR activity (14, 15). In the present study, we
have used an assay of greater physiological relevance that is based on
the reduction of recombinant mammalian Trx after its oxidation by
H2O2. The specific activities of TrxR1 and
TrxR2 measured in the presence of saturating concentrations of oxidized
Trx and NADPH were 2.2 and 3.3 µmol/min/mg of protein, respectively
(Fig. 8A). The COOH-terminal
Secys of TrxR1 was recently shown to be essential for catalytic
activity (18, 35). To determine whether the Secys residue of TrxR2 was
similarly essential, freshly prepared TrxR2 was labeled with BIAM at pH
6.5 as described under "Experimental Procedures" and Fig. 2, with
the exception that the BIAM-labeled enzyme was not subsequently exposed
to iodoacetamide. Labeling with BIAM completely blocked the activity of
TrxR2 toward oxidized Trx (Fig. 8B). Labeling of TrxR2 as
described in Fig. 2 yielded only one major BIAM-labeled peptide, in
which Secys, but not the adjacent Cys, was modified. Like other
flavoprotein disulfide oxidoreductases, both TrxR1 and TrxR2 contain a
redox-active disulfide center comprising the sequence CVNVGC (residues
52 to 57 in mature TrxR2). However, the two cysteine residues in this sequence were not labeled by BIAM in the experiment shown in Fig. 2.
These results suggest that the inactivation of TrxR2 by BIAM resulted
from modification of the Secys residue, and thus that this residue is
essential for the catalytic activity of this protein. Because of the
high homology between TrxR2 and GR enzymes from human, C. elegans, and D. melanogaster, we also assayed TrxR2 for
GR activity with yeast GR as a control. TrxR2 did not exhibit detectable GR activity (Fig. 8C).
Selenium Content of TrxR Enzymes--
The selenium content of
TrxR1 purified from various sources was previously determined to be 0.6 to 0.93 mol of selenium per subunit (4, 18). TrxR appears to lose
selenium under conditions of increased oxidative stress, as indicated
by the observation that the selenium content of TrxR1 from HeLa cells
decreased by almost half when the oxygen level in the culture chamber
was increased (35). We measured the selenium content of freshly
purified TrxR1 and TrxR2 by atomic absorption spectrometry and
comparison with dilutions of a standard selenium stock solution. Five
independent measurements with TrxR enzymes in the concentration range
of 13.5 to 40.5 µg/ml yielded selenium contents of 0.75 ± 0.08 and 0.84 ± 0.20 mol of selenium per subunit (means ± S.E.)
for TrxR1 and TrxR2, respectively.
Tissue Distribution and Subcellular Localization of TrxR
Isoforms--
Total soluble fractions of sonicates prepared from
various rat tissues were subjected to immunoblot analysis with rabbit
antibodies specific for TrxR1 or TrxR2 (Fig.
9A). Comparison of the
intensities of the immunoreactive proteins in the various tissues with
those of purified TrxR proteins allowed us to estimate the amount of each isoform in micrograms of TrxR per milligram of total soluble protein. TrxR1 was abundant in all the tissues examined, varying in
amount from 0.6 to 1.6 µg/mg of soluble protein. However, TrxR2 was
relatively abundant (0.3 to 0.6 µg/mg of soluble protein) only in
liver, kidney, adrenal gland, and heart; in the other tissues, the
amount of TrxR2 was below the limit of detection (0.02 µg/mg).
The antibodies to TrxR2 detected two bands in the liver and kidney
(Fig. 9A). The lower band corresponded to TrxR2. Longer exposure of the immunoblot also revealed a faint upper band for the
other tissues. This upper band might correspond to a protein that shows
low cross-reactivity with the antibodies to TrxR2. However, the marked
intensity of the upper band in kidney is suggestive of the presence of
a third isoform of TrxR that is similar in size to TrxR1 but which
possesses a COOH-terminal sequence highly similar to that of TrxR2.
Alternatively, the upper band might represent the TrxR2 preprotein with
the NH2-terminal 36 residues intact, which would suggest
that the translocation of the preprotein from the cytosol into
mitochondria is slower in kidney than in other tissues.
We next investigated the subcellular localization of TrxR1 and TrxR2 by
immunoblot analysis of cytosolic and mitochondrial fractions of rat
liver (Fig. 9B). Whereas TrxR1 was detected only in the
cytosolic fraction, TrxR2 was present predominantly in the
mitochondrial fraction.
While the present study was in progress, Rigobello et
al. (19) described the purification of TrxR from a rat liver
mitochondrial fraction. Like previously isolated mammalian TrxR enzymes
(15), the purified mitochondrial enzyme exhibited a broad substrate specificity. However, its chromatographic behavior differed from that
of the cytosolic enzyme and its size was smaller than that of the
cytosolic enzyme. It was not determined whether the purified mitochondrial protein was derived from the cytosolic enzyme or whether
it contained a Secys residue.
We have now established a relatively simple procedure for the
purification of TrxR1 and TrxR2 without cross-contamination, and we
have cloned a cDNA encoding rat TrxR2. Comparison of the amino acid
sequence deduced from the cDNA with the experimentally determined
sequence of the NH2-terminal region of purified TrxR2 indicated that TrxR2 is likely synthesized in the cytoplasm as a
pre-protein that is converted to the mature form in mitochondria by
removal of the 36 NH2-terminal residues. Like TrxR1, but
unlike TrxR proteins from prokaryotes and yeast, TrxR2 contains an
essential Secys residue in the COOH-terminal region. As in other
selenoproteins, the Secys residue of TrxR2 appears to be encoded by a
UGA codon under the influence of a stem-loop structure formed by a
SECIS element located in the 3'-untranslated sequence of TrxR2 mRNA.
Mammalian cells express two distinct forms of superoxide dismutase,
cytosolic CuZn-superoxide dismutase and mitochondrial Mn-superoxide
dismutase. We have previously shown that mammalian cells express a
family of peroxidases, termed the peroxiredoxin family, that reduce
H2O2 and lipid peroxides with the use of
electrons donated by Trx (26, 49). On reaction with hydroperoxides, the
redox-sensitve Cys residue of Prx is oxidized to Cys-SOH, which then
reacts with a neighboring Cys-SH of the other subunit to form an
intermolecular disulfide. This disulfide is specifically reduced by
Trx, but not by glutathione or glutaredoxin (5, 26). Whereas Prx I, II,
and IV isoforms are cytosolic proteins, Prx III is synthesized in the
cytosol and then transferred to mitochondria, where its 62 or 63 NH2-terminal residues are cleaved during maturation (26,
50, 51). Like TrxR2, Prx III is most abundant in adrenal gland, heart,
liver, and kidney.2 Recently,
a cDNA that encodes a second isoform of Trx (Trx2) with a
60-residue mitochondrial targeting sequence was cloned and its specific
expression in mitochondria confirmed (25).
Most of the reactive oxygen species generated in unstimulated mammalian
cells are generated as a result of the univalent reduction of
molecular oxygen to the superoxide anion (O Increased oxidative stress in mitochondria results in collapse of the
mitochondrial membrane potential, consequent impairment of oxidative
phosphorylation of ADP, and, ultimately, cell death (53, 54). Oxidative
stress in mitochondria also promotes the calcium-dependent,
nonspecific permeabilization of the inner membrane as a result of the
oxidation and cross-linking of thiol groups in membrane proteins (55,
56). Such increased nonspecific permeabilization has been suggested to
lead to the release of mitochondrial constituents, including cytochrome
c, into the cytosol, which in turn induces cell death by
apoptosis (57-61). Thus, the line of defense provided by PrxIII, Trx2,
and TrxR2 against H2O2 likely plays a critical
role in cell survival.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
70 °C.
70 °C.
) of the dialysate buffer (10 mM sodium
phosphate-1.8 mM potassium phosphate (pH 7.4), 137 mM NaCl, 2.7 mM KCl, 1 mM EDTA, and
1 mM 2-mercaptoethanol (substituted for 1 mM
DTT in centrifugation studies)) was determined to be 1.00546 g/ml at
20.00 ± 0.01 °C with a Paar DMA 58 densitometer, and the
relative viscosity was determined to be 1.020 (28). The partial
specific volumes (
) of TrxR1 and TrxR2 were
calculated to be 0.720 and 0.722 ml/g (29), respectively, from the
amino acid sequences. The protein sample (0.34 ml) and dialysate buffer (0.35 ml) were loaded into the right and left sides, respectively, of a
4° Kel-F coated double-sector centerpiece in 12-mm cells that were
equipped with plane ultraviolet-quartz windows. Sedimentation velocity
experiments were performed at 48,000 and 40,000 rpm for TrxR1 and
TrxR2, respectively, while scanning in a continuous mode (0.003-cm
steps) with triple averaging at 280 nm and 4-min intervals (after
equilibration and radial calibration at 3000 rpm, at which speed radial
and wavelength (9 to 11 averages at 1-nm resolution) scans were
collected). The TRACKER program of A. P. Minton
() was used to monitor
the progress of runs. Observed sedimentation coefficients
(sobs) were corrected to the density and
viscosity of water at 20.0 °C, yielding
s20,w values of 1.0393 sobs and 1.0395 sobs for
TrxR1 and TrxR2, respectively. The time derivative method of Stafford
(30, 31) was used to estimate the molecular weights of TrxR1 and TrxR2;
the diffusion coefficient (D) and sedimentation coefficient
(s) were obtained from the half-width and maximum,
respectively, of the Gaussian fit to the g(s*)
distribution pattern from four late scans (Origin Windows
g(s*) Velocity Program of Beckman Instruments),
and the solute molecular weight (M) was calculated from the
Svedberg equation, M = RTs/[D(1
)]. The
relation of D to the half-width or standard deviation of the
Gaussian fit is given by D = (
rm
2t)2/2t, where
t is the sedimentation time in seconds,
2 is
the angular velocity of the rotor, and rm is the radial position of the meniscus (30, 31); t,
2t, and rm can be read
from the output file from DC_DT in the Beckman
g(s*) program.
RESULTS
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Fig. 1.
SDS-PAGE (A), immunoblot
(B), and spectral (C) analysis of
TrxR1 and TrxR2. A, 1 µg each of purified TrxR1
(lane 1) and TrxR2 (lane 2) was subjected to
SDS-PAGE on a 10% gel and stained with Coomassie Brilliant Blue. The
positions of molecular size standards are indicated in kilodaltons.
B, TrxR1 and TrxR2 (0.2 µg each) were subjected to
SDS-PAGE as in A, transferred to a nitrocellulose membrane,
and analyzed by immunoblotting with rabbit antiserum to TrxR1 (1:4000
dilution). Immune complexes were detected with horseradish
peroxidase-conjugated antibodies to rabbit immunoglobulin G and ECL
reagents. C, ultraviolet-visible spectrum of TrxR2 (1.42 mg/ml) in a solution containing 10 mM sodium phosphate, 1.8 mM potassium phosphate (pH 7.4), 137 mM NaCl, 1 mM EDTA, and 1 mM 2-mercaptoethanol.
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Fig. 2.
Purification of tryptic peptides derived from
BIAM-labeled TrxR2. A, TrxR2 was labeled with BIAM and
digested with trypsin, and the resulting peptides were analyzed on a
C18 column, with elution monitored on the basis of
A215. The positions of BIAM-labeled peptide
a and of two other peptides, b and c,
that were subjected to sequence determination are indicated.
B, each fraction from the C18 column in
A was assayed for BIAM-labeled peptides with the use of
horseradish peroxidase-conjugated streptavidin and the peroxidase
substrate 3,3',5,5'-tetramethyl benzidine, the oxidation of which was
monitored spectrophotometrically at 405 nm. C, the tryptic
digest of BIAM-labeled TrxR2 was subjected to affinity purification
with Neutravidin beads, and the resulting purified peptides were
analyzed on a C18 column as described in A. The
position of BIAM-labeled peptide a is indicated.
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Fig. 3.
Nucleotide sequence of rat liver TrxR2
cDNA and the deduced amino acid sequence of the encoded
protein. The ATG codon encoding the initiating methionine, the TGA
triplet encoding Secys (U), and the termination codon TAA are indicated
by bold type. The experimentally determined
NH2-terminal sequence of purified rat liver TrxR2, as well
as the sequences of two internal tryptic peptides and of the
BIAM-labeled peptide, are underlined. Nucleotide sequences
used for RACE experiments are also underlined. Double
underlines indicate the putative mitochondrial targeting sequence
in the predicted protein and the nucleotide sequences of the three
segments conserved in the SECIS element. Nucleotide residue numbers are
indicated on the left. The nucleotide sequence of rat TrxR2
has been deposited in GenBank under the accession number
AF072865.
-helical structure
(Plotstructure program of the University of Wisconsin Genetics Computer
Group). The predicted high isoelectric point and
-helical structure
are hallmarks of most mitochondrial leader peptides (36). Furthermore,
like many mitochondrial precursor proteins, the predicted TrxR2 protein
contains an arginine residue at position
10 (relative to the
NH2-terminal residue of the mature protein) (37).
Therefore, the mature TrxR2 comprises 490 amino acids, with a
calculated molecular mass of 53,036 daltons; for comparison, the
calculated molecular mass of the 498-residue rat TrxR1 is 54,491 daltons (18). The minor NH2-terminal sequence GQQNFD
obtained from purified TrxR2 was likely derived from products of
cleavage by aminopeptidases.
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Fig. 4.
Alignment of the amino acid sequences of TrxR
and GR proteins from various species. Dashes within
sequences represent gaps introduced to optimize alignment. Residue
numbers are shown on the right. The last two amino acids
(underlined) of C. elegans TrxR were translated
as Secys and Gly on the basis of the published nucleotide sequence
(GenBank accession number, U61947).
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Fig. 5.
Comparison of the nucleotide sequence of the
rat TrxR2 SECIS element with those of SECIS elements of other
selenoproteins (A) and the predicted secondary
structure of the rat TrxR2 SECIS element (B).
A, the rat TrxR2 SECIS element was compared with those of
rat and human TrxR1 (18); human and murine 15-kDa selenoprotein
(15K SELP) (63); rat and human deiodinase (43); rat, human,
murine, and bovine glutathione peroxidase (GPX) (43); and
rat and human selenoproteins 1 and 2 (SELP1 and
SELP2) (43) with the Pileup program (University of Wisconsin
Genetics Computer Group). The position of the first base in each
sequence from rat, or in human 15K SELP, is indicated in
parentheses. B, the stem-loop structure of the
rat TrxR2 SECIS element was generated with the Foldrna program
(University of Wisconsin Genetics Computer Group).
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Fig. 6.
Separation of TrxR2 from TrxR1 by acid
precipitation (A) and purification of TrxR2 from the
acid-precipitated proteins (B-D). A,
immunoblot analysis with antisera to TrxR1 ( TrxR1) and to
TrxR2 (
TrxR2) of rat liver homogenate (crude extract) as
well as of the supernatant and precipitate obtained after acidification
of the homogenate to pH 5.0. B-D, purification of TrxR2 by
sequential chromatography of the acid-precipitated proteins on columns
of DEAE-Sephacel (B), 2', 5'-ADP-agarose (C), and
Phenyl-5PW (D). Column fractions were subjected to
immunoblot analysis with antibodies to TrxR2 (insets). See
"Experimental Procedures" for further details.
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Fig. 7.
Sedimentation velocity analysis at 20.0 °C
and time derivative analysis of four late concentration profiles at 280 nm for TrxR1 (A) and TrxR2 (B), with
harmonic average sedimentation times of 76.6 and 92.8 min at 48,000 and
40,000 rpm, respectively. The concentrations of TrxR1 and TrxR2
were 0.92 and 1.05 mg/ml, respectively. Unsmoothed data from
triple-averaging in 0.003-cm steps (open circles) in each
panel show the apparent distribution function,
g(s*), versus the sedimentation
coefficient (s*) in Svedberg units
(S). The solid line in each panel represents a
single Gaussian fit for either the TrxR1 or TrxR2 data set. The
observed sedimentation and diffusion coefficients of the solute are
given by the s* values at the maximum and the
half-width, respectively, of the g(s*) curve
(28, 29).
7 and
5.57 × 10
7 cm2/s, respectively, as
calculated from Gaussian fits of the g(s*)
distributions shown in Fig. 7. These values and the partial specific
volumes calculated from the amino acid compositions (29) yielded
molecular weight values within 4% of those expected for dimers of
TrxR1 and TrxR2. The determined frictional coefficients (f)
were slightly higher than those (f0) calculated
for spherical dimer particles, indicating that shape or volume effects
reduce sedimentation rates.
Sedimentation properties of TrxR1 and TrxR2
)/Ns) and from
the frictional coefficient of a sphere having a volume equal to that of
an ellipsoid: f0 = 6
(3M
/4
N)1/3 (62).
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Fig. 8.
Measurement of TrxR and GR activities of
TrxR2. A, the Trx-reducing activities of 1 µg of
TrxR1 ( ) and TrxR2 (
) were measured by coupling the reduction of
oxidized Trx to NADPH oxidation and monitoring the decrease in
A340. An assay mixture lacking enzyme served as
a control (
). B, the Trx-reducing activities of 1 µg of
BIAM-labeled (
) or unlabeled (
) TrxR2 were measured as in
A. An assay mixture lacking TrxR2 served as a control (
).
TrxR2 was labeled with BIAM as described under "Experimental
Procedures," with the exception that subsequent incubation with
iodoacetamide was omitted. C, the GR activities of 2 µg of
yeast GR (
), rat TrxR1 (
), and rat TrxR2 (
) were measured by
coupling the reduction of GSSG to NADPH oxidation and monitoring the
decrease in A340. An assay mixture lacking
enzyme served as a control (
).
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Fig. 9.
Tissue distribution (A) and
subcellular localization (B) of TrxR1 and TrxR2.
A, protein samples (23 µg of the total soluble fraction of
various rat tissues and the indicated amounts of purified enzymes) were
fractionated by SDS-PAGE on a 10% gel, transferred to a nitrocellulose
membrane, and subjected to immunoblot analysis with antibodies specific
for TrxR1 or TrxR2. Immune complexes were detected with the use of
horseradish peroxidase-conjugated secondary antibodies and ECL
reagents. B, immunblot analysis of rat liver total extract
(20 µg), cytosolic proteins (20 µg), and mitochondrial proteins (20 µg) with antibodies to TrxR2, TrxR1, and cytochrome c
oxidase VIc subunit (cyt. ox.). Purified TrxR1 (50 ng) and
TrxR2 (10 ng) were also analyzed as controls.
DISCUSSION
2) by
electrons that leak from the mitochondrial respiratory chain; the
O
2 then undergoes spontaneous or enzyme-mediated dismutation
to H2O2. Adrenal cortical mitochondria contain
several cytochrome P-450 enzymes that participate in the hydroxylation
of cholesterol during the synthesis of adrenal steroid hormones (52).
The cytochrome P-450 electron carrier system also leaks electrons and
generates O
2 and H2O2. Thus, our
discovery of TrxR2, which presumably functions together with
Mn-superoxide oxidase, PrxIII, and Trx2, highlights a
mitochondria-specific defense system against O
2 and
H2O2.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. T. C. Stadtman for encouragement, M. Nauman for peptide sequencing, A. K. Aumock for assistance, and Dr. C. Y. Choi for discussion.
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FOOTNOTES |
---|
* 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF072865.
§ Present address: Dept. of Biochemistry, College of Medicine, Yeungnam University, Taegu 705-035, Korea.
¶ Present address: Korea Research Institute of Bioscience and Biotechnology, Taejon 305-60, Korea.
** To whom correspondence should be addressed: Bldg. 3, Rm. 122, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-9646; Fax: 301-480-0357; E-mail: sgrhee{at}nih.gov.
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ABBREVIATIONS |
---|
The abbreviations used are: Trx, thioredoxin; TrxR, Trx reductase; GR, glutathione reductase; Secys, selenocysteine; BIAM, N-(biotinoyl)-N'-(iodoacetyl)ethylenediamine; DTT, dithiothreitol; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; PAGE, polyacrylamide gel electrophoresis; HPLC, high-performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; SECIS, Secys insertion sequence; Prx, peroxiredoxin; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol.
2 S. W. Kang and S. G. Rhee, unpublished observation.
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REFERENCES |
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