(Received for publication, November 29, 1994)
From the
Human thioredoxin reductase is a dimeric enzyme that catalyzes
reduction of the disulfide in oxidized thioredoxin by a mechanism
involving transfer of electrons from NADPH via FAD to a redox-active
disulfide. 1-Chloro-2,4-dinitrobenzene (DNCB) is an alkylating agent
used for depleting intracellular GSH and also showing distinct
immunomodulatory properties. We have discovered that low concentrations
of DNCB completely inactivated human or bovine thioredoxin reductase,
with a second order rate constant in excess of 200 M s
, which is almost
10,000-fold faster than alkylation of GSH. Total inactivation of 50
nM reduced thioredoxin reductase was obtained by 100
µM DNCB after 5 min of incubation at 20 °C also in the
presence of 1 mM GSH. The inhibition occurred with enzyme only
in the presence of NADPH and persisted after removal of DNCB,
suggesting alkylation of the active site nascent thiols as the
mechanism of inactivation. Thioredoxin reductase modified by DNCB
lacked reducing activity with oxidized thioredoxin,
5,5`-dithiobis-(2-nitrobenzoic acid), or sodium selenite. However, the
DNCB-modified enzyme oxidized NADPH at a rate of 4.7 nmol/min/nmol of
enzyme in the presence of atmospheric oxygen. This activity was not
dependent on the presence of DNCB in solution and constituted a 34-fold
increase of the inherent low NADPH oxidase activity of the native
enzyme. DNCB is a specific inhibitor of mammalian thioredoxin
reductase, which reacted 100-fold faster than glutathione reductase.
The inactivation of the disulfide reducing activity of thioredoxin
reductase and thioredoxin with a concomitant large increase of the
NADPH oxidase activity producing reactive oxygen intermediates may
mediate effects of DNCB on cells in vivo.
The thioredoxin (Trx) ()system (Trx and TR with
NADPH) serves as a hydrogen donor system for ribonucleotide reductase
and has general protein disulfide reductase
activity(1, 2, 3) . TR catalyzes the
reduction of oxidized Trx (Trx-S
) by NADPH, and reduced Trx
(Trx-(SH)
) is a powerful protein disulfide reductase ( and ).
TR from Escherichia coli is a dimeric enzyme with
subunits of 35 kDa that has been extensively characterized(1) .
The enzyme mechanism involves the transfer of reducing equivalents from
NADPH to a redox active disulfide (CATC) in the active site, via a FAD
prosthetic group. A crystal structure at 2-Å resolution has
demonstrated that the active site disulfide is located in a buried
position in the NADPH domain(4) . Results also strongly suggest
that upon generation of a dithiol in TR, the enzyme undergoes a large
conformational change to create a binding site for Trx-S and reduction by thiol-disulfide exchange(4) . TR from
mammalian cells has been purified to homogeneity and characterized from
calf liver and thymus(5) , rat liver(6) , and human
placenta(7) . All mammalian enzymes are dimeric but have a
higher native molecular weight (M
116,000)
compared with the E. coli enzyme (M
70,000). Unlike the E. coli TR, the mammalian enzymes
have a broad substrate specificity reducing thioredoxins also from
distant species as well as DTNB(5) ,
SeO
(8) ,
selenodiglutathione(9) , vitamin K(10) , and
alloxan(11) , indicating a more open active site structure.
Recent reports show that extracellular Trx plays several roles in
cell regulation. Thus, human Trx is identical to an adult T-cell
leukemia-derived factor from human T cell lymphotropic virus I-infected
cells, which up-regulates the receptor for interleukin-2(12) ,
and a factor promoting the growth of Epstein-Barr virus-immortalized
B-lymphocytes, which synergizes with several other
lymphokines(13) . Trx expression is induced in activated
lymphocytes, and Trx is secreted in a hormone-like manner in connection
with its immunostimulatory
effects(12, 14, 15, 16) . Trx also
up-regulates interleukin-6 in Epstein-Barr virus-infected B
cells(15) , affects lymphocyte rosetting as part of an early
pregnancy factor(16) , and regulates the activation of the
transcription factor NF-B intracellularly by redox control (17, 18, 19) . These effects of Trx are
related to the oxidation state and the activity of TR.
DNCB is an electrophilic compound alkylating SH groups used as a substrate in assays to determine glutathione S-transferase, which is involved in elimination of DNCB in vivo(20) . DNCB has also been used in cell culture experiments as a GSH-depleting agent. Furthermore, DNCB has an established use as an immunomodulatory agent to provoke delayed-type hypersensitivity(21) . Although proposed to function as a hapten, the mechanism of DNCB immunomodulation is not clear(22) . Recently, DNCB was also proposed as an immunostimulatory agent in AIDS treatment(23) .
Based on the reports described above in combination with the immunomodulatory and the chemical nature of DNCB, we determined to investigate possible interactions with the human Trx system.
DNCB was from Sigma, and stock solutions were made in 99.5%
ethanol. TR was purified from human placenta and calf thymus as
described(6) . The enzyme concentration was determined from A measurements or by determining the activity
with 5 mM DTNB, assuming that 1200 A
units/ml corresponded to 1 mg of protein/ml and an M
of 120,000 as described(6) . Human Trx
was a recombinant preparation(24) . Glutathione reductase was
prepared from rat liver(25) .
Figure 1: Effects of DNCB on the NADPH-dependent reduction of insulin disulfide by thioredoxin reductase and thioredoxin. Human TR (0.05 µM) was preincubated with 200 µM NADPH and 1 µM Trx at room temperature for 10 min, whereafter 50 µM DNCB was added and the preincubation was continued for 15 min. Then 50 µl of insulin (10 mg/ml) was added, and the absorbance at 340 nm was followed. After 6.5 min, fresh TR (0.05 µM) was added, as indicated.
Incubation of 25 nM TR
with NADPH and 100 µM DNCB for 10 min at 20 °C also
destroyed all the activity of TR (>98%) with either 5 mM DTNB or 20 µM selenite. These small molecules are
reduced by TR (5, 6, 9) via the active site
dithiol. Selenite (SeO) was used in
further experiments since it lacks thiol groups after reduction.
Preincubation of 50 nM TR with DNCB for 15 min in the
absence of NADPH followed by addition of 200 µM NADPH and
20 µM SeO resulted in no
inhibition. This demonstrated that only the dithiol form of TR was
susceptible to inactivation by DNCB.
The inhibition of TR activity by DNCB was dependent both on the concentration of the inhibitor and the preincubation time. Removal of DNCB from the enzyme using Sephadex G-25 chromatography showed that the enzyme had lost activity (Fig. 2) and strongly suggested alkylation of one or both of the active site thiols as the mechanism of inactivation.
Figure 2:
Reduction of selenite by thioredoxin
reductase after incubation with DNCB followed by removal of the
inhibitor by gel filtration. Calf thymus TR (0.36 µM) was
preincubated with either 500 µM DNCB () or with only
ethanol as control (
) at 20 °C for 15 min in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mg/ml bovine serum
albumin, and 250 µM NADPH. The enzyme in the preincubation
mixtures was then isolated using Sephadex G-25 columns. Then 250
µM NADPH and 20 µM SeO
was added to the enzyme
solutions, and NADPH consumption was followed by the decrease in
absorbance at 340 nm, in each case against a blank with only
NADPH.
Figure 3:
Inhibition of the activity of TR by
varying concentrations of DNCB in the presence of 1 mM GSH.
Human TR (0.05 µM) was incubated with 200 µM NADPH and different concentrations of DNCB in the presence of 1
mM GSH in both reference and sample cuvettes for 5 min at 20
°C. Then 20 µM SeO was
added to the sample cuvette, and TR activity was measured as the
initial decrease in absorbance at 340 nm.
Figure 4: NADPH oxidation activity of thioredoxin reductase under anaerobic and aerobic conditions after inactivation by DNCB. Human TR (0.3 µM), 200 µM NADPH, and 25 µM DNCB (substituted by ethanol in the reference cuvette) was incubated under anaerobic conditions for 60 min, and the absorbance was followed at 340 nm. Then air was admitted with mixing in both the reference and sample cuvettes, and the absorbance at 340 nm was recorded.
In this paper we have shown that DNCB is an efficient and
specific irreversible inhibitor of TR from human placenta or calf
thymus. Inhibition only occurred after reduction of the active site
disulfide in the enzyme by NADPH, strongly suggesting alkylation of one
or both nascent thiols in the enzyme. The inhibition was a fast
process; compared with alkylation of GSH it was almost 10 times faster, and glutathione reductase was 100-fold less
sensitive than TR. These results clearly suggest that a prime target
for DNCB in vivo will be TR. The concentration range of DNCB
in this study was about 100-fold lower than that used in clinical
applications of the agent. Furthermore, since previous work has shown
that TR is localized at the plasma membrane in many cells (27, 28, 29, 30) the enzyme can be
envisioned to be easily accessible to the membrane-permeable DNCB upon
topical application.
Inactivation of TR by DNCB completely inhibited
the reduction of Trx but increased the NADPH oxidase activity,
independent of free DNCB in solution. Thus, the disulfide reducing
activity of Trx-(SH) required for deoxyribonucleotide and
DNA synthesis and its general disulfide reduction in the cell will be
lost. In addition, Trx has several newly discovered roles in cell
regulation of receptors or transcription factors by redox reactions
that may be affected. The NADPH oxidase activity of the
DNCB-inactivated enzyme will produce reactive oxygen intermediates,
which either are causing oxidative stress or operate as mediators of
growth (18) in cell signaling. Since the DNCB-inactivated
enzyme showed the NADPH oxidase activity independent of the presence of
DNCB in solution, the production of reactive oxygen intermediates will
be effective and long lasting even after clearance of DNCB from the
cell by GSH and glutathione S-transferase.
In cell culture experiments, DNCB is often used as a GSH-depleting agent in order to study processes dependent on intracellular GSH levels (31) . Obviously inactivation of TR with loss of the disulfide reducing activity of Trx is a previously unknown effect of DNCB. This may be of much greater importance than the effect on GSH levels, which obviously must be dependent on the highly variable presence of GSH S-transferase activity since we have shown that a direct reaction between GSH and DNCB is negligible. As an example the induction of leukotriene synthesis in lymphoblastoid B-cells by DNCB was not correlated to lowering of GSH(32) . A possible hypothesis is production of reactive oxygen intermediates by DNCB-modified TR since peroxides will activate lipoxygenases(33) . Further studies on the effects of DNCB on TR will hopefully help to explain the immunomodulatory effects of DNCB.