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INTRODUCTION |
Several proteins from bacteria and animals contain selenocysteine
in their primary structure (1). Each of the cDNA clones of these
selenoproteins contains one TGA codon, which corresponds to UGA in
mRNA, in the open reading frame (1, 2). This UGA, known formerly
only as a stop codon, encodes and directly incorporates selenocysteine
into these selenoproteins (1, 3).
The first identified mammalian selenoprotein was glutathione peroxidase
(GPx1; EC 1.11.1.9). GPx
catalyzes the reduction of hydrogen peroxide and organic hydroperoxide
by glutathione (GSH) and functions in the protection of cells against
oxidative damage. At least four forms of GPx are reported to exist and
differ in many properties, including their localization, subunit
structure, primary structure, and enzymatic nature. The classical
cellular GPx (cGPx), found in the cytosol of various tissues and blood
cells, reduces hydrogen peroxide and organic hydroperoxides, but cannot
reduce phospholipid hydroperoxides. A second GPx, phospholipid
hydroperoxide GPx (PH-GPx), is believed to be mostly cytosolic and
partly membrane-bound (4, 5). This enzyme is able to reduce
phospholipid hydroperoxide in addition to hydrogen peroxide (4, 5, 6).
A third GPx in plasma, now called extracellular GPx (eGPx), is reported
to reduce both hydrogen peroxide and phospholipid hydroperoxides (7,
8). A fourth GPx, originally called GPx-related selenoprotein (9), is
now called gastrointestinal GPx (GI-GPx) and has been found to be
highly expressed in the gastrointestinal tract mucosal epithelium (10).
Furthermore, type I, type II, and type III iodothyronine deiodinase
(11-13) and thioredoxin reductase (14) were identified as
selenoproteins. In all mammalian selenoenzymes belonging to the class
of oxidoreductase characterized to date, the selenocysteine residue is
a catalytically active site.
Further mammalian selenoproteins with as-yet-unidentified functions
have been reported, such as selenoprotein P (SeP) from plasma (15, 16)
and selenoprotein W from rat muscle (17). SeP is a selenium-rich
extracellular protein and is major selenoprotein in rat and human
plasma (16). SeP has been purified recently from human and rat (18, 19)
including the immunoaffinity chromatography step, and characterized as
containing 7-8 selenium atoms per molecule as selenocysteine. The
sequence of the cloned DNA predicts that SeP contains 10 selenocysteines encoded by UGA stop codons in the open reading frame of
its mRNA (20, 21). The deduced amino acid sequence of SeP is
reported to have no similarities with sequences of other known
selenoproteins (20).
A key issue that remains unresolved is the catalytic function of SeP.
Recent in vivo studies suggest that SeP plays an important role in antioxidative defense (22, 23). It is quite possible that SeP
lost its catalytic activity during the isolation step, perhaps in
acidic elution, of immunoaffinity chromatography (18, 19). Thus, we
first describe the newly developed procedure for isolating SeP from
human plasma without using immunoaffinity chromatography. Second, we
report that SeP exhibits PH-GPx-like activity and characterize the
enzymatic nature of SeP.
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EXPERIMENTAL PROCEDURES |
Chemicals--
Heparin-Sepharose CL-6B and Q-Sepharose Fast Flow
were obtained from Amersham Pharmacia Biotech, Uppsala, Sweden;
nickel-nitrilotriacetic acid (Ni-NTA)-agarose was from Qiagen Inc.,
Chatsworth, CA.; diisopropyl fluorophosphate was from Kishida Chemical
Co., Osaka, Japan; polyethylene glycol (number 4,000), tertiary butyl
hydroperoxide, cumene hydroperoxide, and hydrogen peroxide were from
Nacalai, Kyoto, Japan; 1-palmitoyl-2-linoleoyl-3-phosphatidylcholine (PLPC), GSH, and GSH reductase were from Sigma; and soybean lipoxidase was from Biozyme Laboratories Ltd, Blaenavon, Great Britain. For high
performance liquid chromatography (HPLC), distilled water was further
purified by Organo Puric model-S water purifier. Acetonitrile (HPLC
grade) was purchased from Wako Pure Chemical Co. (Osaka, Japan).
Methanol and 2-propanol were distilled before use. Human out-dated
frozen plasma was kindly donated from Hokkaido Red Cross Blood Center.
Other chemicals were of the highest quality commercially available.
Selenoprotein P Purification--
All purification procedures
were conducted at 4 °C, and diisopropyl fluorophosphate (2 mM) was added to each pooled fraction to avoid the
proteolytic cleavage of SeP. Human plasma (1,000 ml) was mixed with
50 g of polyethylene glycol under stirring. After 1 h, the
precipitate was removed by centrifugation at 8,000 rpm for 10 min. The
supernatant was applied to heparin-Sepharose CL-6B column (50 ml)
equilibrated with 20 mM phosphate buffer, pH 7.4, containing 0.15 M NaCl and 0.5 mM EDTA, at a
flow rate of 100 ml/h. After washing with the equilibrated buffer, the
bound proteins were eluted by linear gradient increase of NaCl
concentration with each 500 ml of 0.15 and 0.6 M NaCl in
the buffer. Among three selenium-containing proteins in human plasma,
only SeP reportedly bound to heparin-Sepharose. So, we detected SeP by
measuring selenium contents after the heparin-Sepharose fractionation.
The fractions containing SeP were pooled and diluted with 6 volumes of
20 mM Tris-HCl, pH 8.0, and directly applied to a column
(20 ml) of Q-Sepharose Fast Flow equilibrated with 20 mM
Tris-HCl, pH 8.0, containing 0.06 M NaCl, at a flow rate of
30 ml/h. The bound proteins were eluted by linear gradient increase of
NaCl concentration with each 150 ml of 0.06 and 0.3 M NaCl
in the buffer. The fractions containing SeP were pooled, and imidazole
and NaCl were added to the pooled fractions at a final concentration of
20 mM and 1 M, respectively. The fractions were
applied to a column (1 ml) of Ni-NTA-agarose equilibrated with 20 mM Tris-HCl, pH 8.0, containing 20 mM imidazole
and 1 M NaCl, at a flow rate of 30 ml/h. After washing with
the equilibrated buffer, the bound proteins were eluted with 20 mM Tris-HCl, pH 8.0, containing 250 mM
imidazole and 1 M NaCl. For further study, imidazole and
NaCl were removed by passing through PD-10 gel filtration column
equilibrated with the desired buffer.
Selenium Assay--
Levels of selenium in plasma and in column
fractions was determined according to the fluorometric method of
Bayfield and Romalis (24).
Protein Assay--
Protein concentrations of samples at each
chromatographic step were determined measuring absorbance at 280 nm.
Protein content of purified preparation was determined using a protein
assay kit (Bio-Rad) with bovine immunoglobulin G as a standard. To
determine the selenocysteine content per mole of SeP, quantitative
amino acid analysis was applied.
Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis--
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis was performed according to the method of Laemmli (25)
in slab gels (10% gel) under reducing or nonreducing conditions. After electrophoresis, proteins in a gel were stained with 0.1% Coomassie Brilliant Blue R-250 in 10% acetic acid and 30% methanol for 30 min
and then the gel was destained in 10% acetic acid and 30% methanol.
Amino Acid Analysis--
The protein was dialyzed against
distilled water and hydrolyzed in 6 M HCl for 24, 48, or
72 h or in 3 M mercaptoethanesulfonic acid for 24 h. After removal of solvent in vacuo, amino acid analysis was performed on a Pico-Tag system (Water, Millipore Corp.).
Amino Acid Sequence Analysis--
After electrophoresis,
proteins in the gel were transferred to a Pro Blot membrane (Applied
Biosystems) in 20 mM Tris containing 150 mM
glycine and 20% methanol electrically. The proteins on the membrane
were stained with 0.1% Coomassie Brilliant Blue R-250 in 1% acetic
acid and 40% methanol for 1 min, and then the membrane was destained
with 50% methanol. The protein bands were cut off and their
NH2-terminal amino acid sequences were analyzed with a 473A
Protein Sequencer (Applied Biosystems).
Preparation of Monoclonal Antibody and
Immunoprecipitation--
A method to prepare hybridomas producing rat
monoclonal antibodies was used (26, 27). The enlarged medial iliac
lymph nodes from rats injected via hind footpads with an emulsion of purified SeP and Freund's complete adjuvant were used for cell fusion,
followed by hybridization, cloning, and establishment of hybridomas.
Eleven hybridomas producing specific monoclonal antibodies against
human SeP were obtained. Details will be described elsewhere. One
monoclonal antibody (BD1, IgG2a,
) was used for immunoprecipitation
study. The BD1 antibody was coupled to agarose using cyanogen bromide
(28). After the addition of increasing amounts of BD1-agarose to the
purified SeP preparation, the enzyme activities of the supernatant were
measured as described below.
Preparation and Purification of
1-Palmitoyl-2-(13-hydroperoxy-cis-9,trans-11-octadecadienoyl)
Phosphatidylcholine (PLPC-OOH)--
PLPC-OOH was prepared from PLPC by
oxidation with lipoxidase (29, 30). The reaction mixture, containing 3 mM sodium deoxycholate and 0.13 mM PLPC in 500 ml of 0.2 M Tris-HCl buffer (pH 8.8) was incubated with 50 mg soybean lipoxidase at room temperature for 4 h, under
continuous stirring. PLPC-OOH was extracted from the reaction mixture
with ethyl acetate. The ethyl acetate extract after evaporation under
reduced pressure was dissolved in methanol and subjected to preparative
HPLC on an ODS column with a mobile phase of
acetonitrile/methanol/water (75:21:4) to obtain PLPC-OOH. The PLPC-OOH
was dissolved in methanol and stored at
15 °C. The concentrations
of hydroperoxides were determined by microiodometric methods (31, 32),
using cumene hydroperoxide as standard for calibration. PLPC-OOH was
confirmed by fast atom bombardment mass spectrometry (FAB-MS). The
deprotonated molecular ion of PLPC-OOH was observed at
m/z 788 (negative FAB-MS). PLPC-OOH was reduced to PLPC-OH by the addition of sodium borohydride. PLPC-OH was the only
product of the reduction and was isolated from the reduction product by
preparative HPLC on an ODS column with a mobile phase of methanol/water
(91:9). The deprotonated molecular ion of PLPC-OH was observed at
m/z 772 (negative FAB-MS).
Enzyme Assay--
GPx activities were examined by following the
oxidation of NADPH in the presence of GSH reductase, which catalyzes
the reduction of oxidized GSH formed by GPx (33) with a slight
modification. Both samples and reference cuvettes contained 0.1 M Tris-HCl, pH 8.0, 0.2 mM NADPH, 0.5 mM EDTA, 2 mM GSH, and 1 unit of GSH reductase
in a total volume of 1 ml. An aliquot of enzyme was added to the sample
cuvette only. The reaction mixture was preincubated at 37 °C for 2 min, after which the reaction was started by the addition of peroxide
in both cuvettes. In the case of phospholipid hydroperoxide, Triton
X-100 and deoxycholate were added to the reaction mixture at an
appropriate concentration. The oxidation of NADPH was followed at 340 nm at 37 °C. To confirm the reduction of PLPC-OOH, HPLC assay was
also conducted. After incubating PLPC-OOH under appropriate conditions,
a 9-fold volume of ice-cold 2-propanol was added to the reaction
mixture. An aliquot of the reaction mixture was directly injected into
the HPLC system. The HPLC conditions were basically those of Bao
et al. (34). An ODS column (5 µm, 4.6
× 250 mm, TSK
ODS-80Ts, Tosoh Co., Tokyo, Japan) was used for analysis and
determination. The mobile phase was acetonitrile/methanol/water (75:21:4; v/v/v, containing 10 mM choline chloride), and
the flow rate was 1.5 ml/min. The UV peaks of phospholipids were
monitored at 235 nm. The temperature of the column was maintained at
40 °C in a column oven.
Kinetic Analysis--
Kinetic analysis was carried out following
the time progress curves of substrate consumption (NADPH oxidation) in
the coupled test with GPx as described previously (35). The substrate
concentration and the reaction rate, at each time interval (typically
from 1 to 3 min) thus obtained were then fitted to the Dalziel
equation, indicating a ping-pong mechanism as in the case of cGPx,
eGPx, and PH-GPx.
The rate constants k+1 and
k'+2 were calculated from the Dalziel
coefficients, as described previously (35, 36). The first step is the
oxidation of selenol anion (E-Se
) by PLPC-OOH to yield a
selenenic acid derivative (E-SeOH) and PLPC-OH. The
selenenic acid derivative is then reduced by two molecules of GSH to
yield a selenol anion.
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RESULTS |
Purification of SeP from Human Plasma--
SeP was purified
13,000-fold from human plasma, with an overall yield of 16% (Table
I). The final preparation gave a single stained band on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis with mobility corresponding to 69 kDa under nonreducing
conditions and 66 kDa under reducing conditions (Fig.
1). The results of amino acid analysis
and selenium analysis are shown in Table
II. The determined values of amino acid
residues in purified SeP were in good agreement with the predicted
values for the full-length SeP except for the number of selenocysteine
residues, indicating the presence of 6.3 selenocysteines for a
polypeptide mass of 41 kDa (as estimated from the sums of amino acid
compositions). The NH2-terminal amino acid sequence
(E-S-Q-D-Q-S-S-L-C-K-Q-P-P-A-W-S) obtained for purified SeP confirmed
the sequence predicted from SeP cDNA (21).

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Fig. 1.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of purified selenoprotein P. 3 µg of purified
selenoprotein P was run on a 10% gel under nonreducing conditions
(A) and reducing conditions (B). The gel was
stained with Coomassie Brilliant Blue R-250. Molecular mass standard
locations are shown on the left.
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Reduction of PLPC-OOH by SeP--
We measured the ability of SeP
to catalyze the GSH-dependent reduction of various
hydroperoxides by coupled enzymatic assay with GSH reductase. We
detected little activity (less than 0.02 µmol/min/mg of protein)
using hydrogen peroxide or t-butyl hydroxyperoxide. This
value is about 3 orders of magnitude lower than that of cGPx (37).
Next, we examined the reducing activity using PLPC-OOH as a substrate.
As shown in Fig. 2A, SeP
caused an immediate and rapid decrease of A340,
suggesting that PLPC-OOH is reduced by SeP. There was good agreement
between PLPC-OOH values (60 nmol) calculated from these measurements
and the absolute values (60 nmol) of total PLPC-OOH determined
independently by the iodometric assay. Reducibility of PLPC-OOH was
also studied by HPLC analysis of reaction products. Fig. 2B
shows elution profiles of untreated PLPC-OOH, hydroxy derivative from
PLPC-OOH (PLPC-OH), and PLPC-OOH treated with SeP for 5 min,
respectively. The retention times of PLPC-OOH and PLPC-OH elution were
10.6 and 11.4 min, respectively. The identity of the two peaks was
confirmed by FAB-MS, as described under "Experimental Procedures."
The time course of the formation of PLPC-OH and the loss of PLPC-OOH by
SeP in the presence of GSH is shown in Fig. 2C. The loss of
substrate was equal to the formation of product. Furthermore, the rate
(2.03 µmol/min/mg of protein) of conversion of PLPC-OOH to PLPC-OH by
SeP was equal to that determined by coupling assay (2.23 µmol/min/per
mg of protein).

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Fig. 2.
Reduction of PLPC-OOH by selenoprotein
P. A, PLPC-OOH (60 µM) was reacted with
SeP (5 µg) in a coupled enzymatic assay system. B, HPLC
separation of PLPC-OOH and PLPC-OH. PLPC-OOH (100 pmol) (panel
a) and PLPC-OH (100 pmol) (panel b) were eluted at 10.6 and 11.4 min, respectively. Panel c, PLPC-OOH (200 pmol)
treated for 5 min with SeP in the coupled assay system was applied to a
HPLC column. C, time course of total PLPC-OOH decay ( )
and the formation of PLPC-OH ( ) determined by HPLC assay. At the
indicated times, samples from reaction mixture were removed for
determination of PLPC-OOH and PLPC-OH. Points with error bars are
means ± deviation of values from duplicate determinations.
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To confirm that SeP is responsible for the enzyme activity, an
immunoprecipitation assay was conducted. After addition of various
amounts of BD1 (rat monoclonal antibody against human SeP)-agarose (1.2 mg/ml agarose) to the purified SeP preparation (25 µg per ml), the
enzyme activities of the supernatant were measured. When 10% (v/v) of
BD1-agarose was added, less than 5% activity was observed in the supernatant.
Enzymatic Characterization of SeP--
To determine the optimal
conditions for SeP enzymatic activity, the effect of detergent
concentration was studied. In the absence of detergent, no reducing
activity of SeP was observed toward PLPC-OOH in dispersed form. The
addition of Triton X-100 stimulated the enzyme activity of SeP (Fig.
3A). Maximum activity was
found at 0.025% Triton X-100. The further addition of 0.3 mM deoxycholate to the assay mixture produced an increase
in activity of 50%. As shown in Fig. 3B, the addition of
deoxycholate alone little enhanced SeP activity. In the presence of
0.025% Triton X-100, a maximum activity was observed from 0.3 to 1.0 mM deoxycholate. Thus, the enzyme activity of SeP in the
following experiments was assayed in the presence of 0.025% Triton
X-100 and 0.3 mM deoxycholate. Under this standard
condition, the specific activity of SeP against PLPC-OOH is 2.03 µmol/min/mg of protein.

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Fig. 3.
Effect of Triton X-100 and deoxycholate on
the activity of selenoprotein P. The activity of selenoprotein P
toward PLPC-OOH was measured as described under "Experimental
Procedures" in the presence of various concentrations of Triton X-100
and 0.3 mM DOC (A) or DOC and 0.025% Triton
X-100 (B). The amount of enzyme used was 5 µg.
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To investigate the enzymatic nature of SeP, the kinetics of SeP for
PLPC-OOH were studied following the time progress curves of substrate
consumption (NADPH oxidation) in the coupled assay with GSH reductase
(35, 36). Linear Lineweaver-Burk plots were obtained from reactions
rate and substrate concentration values measured from progress curves
(Fig. 4). Different GSH concentrations led to parallel lines, indicating a ping-pong mechanism as in the case
of cGPx (38), eGPx (7), and PH-GPx (35). This allowed the distinct
measurement of k+1 and
k'+2 for the oxidative reaction and the sum of
the two reductive steps of the peroxidatic reaction, respectively. The
k+1 and k'+2 values
calculated are 2,360 mM
1 min
1
and 29.3, respectively.

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Fig. 4.
Lineweaver-Burk plot of the activity of
selenoprotein P on PLPC-OOH. The plots were traced through
analysis of single progression curves. The GSH concentration was 1 mM ( ), 1.5 mM ( ), or 2 mM
( ).
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As PH-GPx is reported to reduce peroxides using thiols besides GSH as a
reductant (4, 34), the thiol specificity of SeP was assayed by
measuring the reduction of PLPC-OOH by HPLC assay. As shown in Table
III, dithiothreitol, mercaptoethanol,
cysteine, and homocysteine were also effective as a reducing
substance.
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DISCUSSION |
Burk and his groups reported the purification of SeP from human
and rat plasma (18, 19), using immunoaffinity chromatography. Although
Eberle and Haas purified human SeP without immunoaffinity chromatography, their final preparation reportedly contained some contaminants (39). Until now, no one has succeeded in purifying SeP to
homogeneity using conventional means. To investigate the structure and
function of this distinctive selenoprotein, we first established the
procedure for isolation of SeP. Prior to the column chromatography,
polyethylene glycol was added to the plasma to remove some coagulation
factors and macromolecular weight proteins. SeP was separated from the
two other selenium-containing proteins, eGPx and albumin, by
heparin-Sepharose chromatography. After Q-Sepharose chromatography, we
selected immobilized metal chelate affinity chromatography. This column
was generally used for the purification of recombinant proteins, which
contain six consecutive histidine residues at their amino terminus. SeP
has two histidine-rich domains; one of which includes a run of four
histidines followed by a lysine, histidine, lysine for a total of seven
consecutive basic amino acid residues. As expected, SeP binds strongly
to the resin and is eluted by competition with imidazole, a histidine
analogue. During the purification, diisopropylfluorophosphate, one of
the most potent serine-protease inhibitors, was added to each fraction. If diisopropyl fluorophosphate was not added, the recovery of SeP was
very low, suggesting that a proteolysis of SeP occurs in the course of
purification without diisopropyl fluorophosphate.
The purified SeP yielded a single band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis. The amino acid composition and amino-terminal amino acid sequence of the final preparation is
similar to those postulated for the deduced polypeptide (21), confirming the homogeneity of our final preparation. As shown in Table
I, 800 µg of SeP was isolated from 1 liter of plasma. Multiple forms
of rat SeP are reportedly present in plasma (40). As we only measured
selenium contents to detect SeP, it is probable that only the major
isoform of SeP was detected, and other isoforms of SeP were missed.
Only 6.3 mol of selenium was found in the selenium and quantitative
amino acid analysis of purified SeP, even though 10 mol was predicted.
Under the same analysis conditions, 1.1 mol of selenium was detected in
the purified eGPx preparation, as
expected.2 This discrepancy
was also observed in a full-length form of SeP purified from rat plasma
(41). Further structure analysis of purified SeP will be necessary to
understand this difference.
Selenoproteins with known enzymatic activity are redox enzymes and
contain selenocysteine in their active sites. The function of SeP is
not known. Its appearance in plasma correlates with protection against
free radical injury of the liver by diquat, suggesting that SeP plays
an important role in antioxidative defense (22, 23). Previous reports
show that SeP does not reduce hydrogen peroxide in the presence of GSH
(42). As we could prepare a highly purified SeP without denaturing
conditions, it seems worth reexamining the GPx-like activity of SeP. We
confirmed that SeP lacks the enzymatic activity of cGPx. Next, we
investigated the PH-GPx activity of SeP. Using two different assays,
coupled assay and HPLC assay, it is shown that SeP reduces the same
amounts of PLPC-OOH in the presence of GSH. The conversion rate (2.03 µmol/min/mg of protein) of PLPC-OOH to PLPC-OH by SeP, directly determined by HPLC, was equal to that indirectly determined by coupling
assay, in which PH-GPx activity is coupled to the oxidation of NADPH by
GSH reductase. Immunoprecipitation experiments with monoclonal antibody
against SeP rules out the possibility of contamination by another
enzyme in our final preparation. As it is impossible to measure PH-GPx
activity of plasma, it is unclear whether or not SeP alone is
responsible for PH-GPx activity of plasma. Further studies to confirm
this hypothesis are currently in progress.
In the presence of 0.025% Triton X-100 and 0.3 mM
deoxycholate, the highest PH-GPx activity of SeP was observed. The
addition of Triton X-100 to the assay mixture resulted in the
transformation of the lipid bilayer into an optically clear solution of
mixed micelles. This suggested that SeP catalyzes a reaction at the lipid-water interface, as is the case of PH-GPx (35). Deoxycholate reportedly can stimulate PH-GPx activity in the presence of Triton X-100 (30). The same stimulatory effect by deoxycholate was observed in
our experiments using SeP.
A difference in peroxide specificity in the GPx family was reported.
cGPx reduces hydrogen peroxide and t-butyl hydroperoxide, but does not
reduce phospholipid hydroperoxide. PH-GPx reduces phospholipid
hydroperoxide and is also reactive against hydrogen peroxide (4, 5).
eGPx reduces hydrogen peroxide and t-butyl hydroperoxide and
shows some reactivity against phospholipid hydroperoxides (7, 8). We
found that SeP reduces phospholipid hydroperoxide and has no activity
against hydrogen peroxide and t-butyl hydroperoxide. The
peroxide specificity of SeP was more similar to that of PH-GPx than to
that of cGPx or eGPx. Approximately 100-fold lower
k+1 and k'+2 values were
observed in human SeP as compared with pig PH-GPx (35), suggesting that
SeP is less reactive to PLPC-OOH, an artificial substrate, than is
PH-GPx. Further studies to understand the reactivities of SeP against
various hydroperoxides in physiological conditions must be done.
Previous reports indicated that PH-GPx, unlike cGPx, did not exhibit a
strict requirement for glutathione as reducing agent. Using SeP as
enzyme, we observed that 1,4-dithiothreitol has a stronger activity
than glutathione. Other thiols, including 2-mercaptoethanol, cysteine,
and homocysteine, also serve as reductant, as is already known in the
case of PH-GPx (4, 34).
In conclusion, we isolated SeP from human plasma and characterized the
enzymatic nature of the purified preparation. Immunoprecipitation with
monoclonal antibodies against SeP results in the almost total loss of
PH-GPx-like activity in the supernatant of the purified preparation.
This rules out the possibility of contamination by another enzyme in
our final preparation. Although SeP can reduce PLPC-OOH, it is
approximately 100 times slower than the cellular counterpart, PH-GPx.
It is currently unknown whether SeP is able to react with certain
phospholipid hydroperoxides under physiological conditions. Using
selenium-deficient rats supplemented with selenium, it was demonstrated
that SeP appearance correlated with disappearance of diquat-induced
lipid peroxidation (22). Furthermore, SeP was reportedly associated
with protection against oxidant injury from GSH depletion in the
selenium-deficient rat (23). These in vivo studies and our
in vitro results described above strongly suggest that SeP
serves to protect the plasma membrane from oxidative damage in the
presence of GSH. Even though the GSH concentration in plasma is low, it
is released continuously from the cells (43), and it may represent a
significant source of reductant for SeP enzymatic activity.
SeP and eGPx are located in the extracellular fluids, and cGPx, PH-GPx,
and GPx-GI are found in the cytosol. These enzymes exhibit different
substrate specificities and collaborate to protect the biological
molecules from oxidative stress inside and outside the cells, respectively.