From the Institute of Environmental Medicine,
Division of Biochemical Toxicology and ¶ Division of
Experimental Asthma and Allergy Research, Karolinska Institutet, Box
210, SE-17177 Stockholm, Sweden
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ABSTRACT |
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Microsomal glutathione transferase-1 (MGST-1) is
an abundant protein that catalyzes the conjugation of electrophilic
compounds with glutathione, as well as the reduction of lipid
hydroperoxides. Here we report that leukotriene C4 is
a potent inhibitor of MGST-1. Leukotriene C4 was found to
be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for
the N-ethylmaleimide activated enzyme. This is the first
tight-binding inhibitor characterized for this enzyme. Leukotriene
C4 was competitive with respect to glutathione and
non-competitive toward the second substrate, CDNB. Analysis of
stoichiometry supports binding of one molecule of inhibitor per
homotrimer. Leukotrienes A4, D4, and
E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues,
which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In
particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078,
and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3.7, and 7.6 µM, respectively). In view of the partial
structural similarity between MGST-1, leukotriene C4
synthase, and 5-lipoxygenase activating protein (FLAP), it was of
interest that leukotriene C4 synthesis inhibitors (which
antagonize FLAP) also displayed significant inhibition
(e.g. IC50 for BAYx1005 was 58 µM). In contrast, selective 5-lipoxygenase inhibitors
such as zileuton only marginally inhibited activity at high
concentrations (500 µM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent
inhibitors of MGST-1 raises the possibility that MGST-1 influences the
cellular processing of leukotrienes. These findings may also have
implications for the effects and side-effects of drugs developed to
manipulate leukotrienes.
Glutathione transferases (see Refs. 1-4 for reviews) are a group
of enzymes involved in the detoxication of numerous carcinogenic, mutagenic, toxic, and pharmacologically active compounds (5). A
membrane-bound member of this family has been isolated (6) and named
microsomal glutathione transferase-1
(MGST-1).1 This homotrimeric
enzyme has a distinct amino acid sequence and immunological properties
that are different from its cytosolic counterparts (6, 7). Another
discriminating property is its ability to be activated (up to 15-fold)
by thiol reagents and proteolysis (8, 9). An important function of the
enzyme is thought to involve protection of intracellular membranes from oxidative modification as a result of oxidative stress (10), through
the reduction of phospholipid and fatty acid hydroperoxides (11). A
relationship between MGST-1 and the leukotriene pathway proteins
leukotriene (LT) C4-synthase and 5-lipoxygenase activating protein (FLAP) is indicated by similarities in size, hydropathy, and
primary structure (12, 13). Furthermore, MGST-1 can physically interact
with LTC4-synthase (14, 15). Interestingly, additional members of this putative protein superfamily that display common functional properties have recently been described (13, 16).
Leukotriene C4-synthase is a specialized membrane-bound
glutathione transferase involved in the production of cysteinyl
leukotrienes (LTC4, LTD4, and
LTE4). The cysteinyl leukotrienes are potent proinflammatory mediators that contribute to the pathophysiology of
asthma. Hence, drugs which block formation or receptors for cysteinyl-leukotrienes are currently introduced as new therapy in
asthma (17). LTC4-synthase is homologous to FLAP (18).
Microsomal GST-1 has been identified as a high capacity "low
affinity" binding protein for LTC4 and was found to bind
one molecule LTC4 per trimer (19, 20). The purified protein
did not display binding unless heat-inactivated microsomes had been added, implying the dependence on additional factors. The present study
was undertaken to determine whether LTC4 could in fact bind to MGST-1 under physiological conditions and whether such binding would
influence the activity of the enzyme. Having established that this was
indeed the case, we determined whether other leukotrienes and
clinically developed inhibitors of leukotriene formation or receptors
also were inhibitors of MGST-1. These studies indicate that
interactions involving MGST-1 could have implications for leukotriene
production and turn-over as well as for the effects or side-effects of
drugs which are used in the treatment of asthma.
Chemicals and Enzyme--
Triton X-100,
N-ethylmaleimide (NEM), glutathione (GSH), arachidonic acid,
and fatty acid-free albumin were from Sigma. CDNB and CNBAM were from
Merck Co. (Darmstadt, Germany) and Alfred Bader Library of Rare
Chemicals, Division of Aldrich Chemical Company (Milwaukee, WI),
respectively. LTC4, LTD4, and LTE4
were from Cascade Biochemicals, Reading, U. K.. Zileuton A-64077 was from Abbott Pharmaceuticals, Chicago, IL. U 60,257 (piriprost) was from
Pharmacia Upjohn, Kalamazoo, MI. BAYx1005 and BAYu9773 were from Bayer
AG, Leverkusen, Germany. FPL 55712 was from Rhône Poulenc
(previously Fisons Pharmaceuticals), Loughborough, U. K.; ZD 230,487 and ICI 198,615 were from Zeneca Pharmaceuticals, Macclesfield, U. K.;
ONO-1078 (pranlukosl) was from ONO Pharmaceuticals, Osaka, Japan; SKF
104,353 was from Smith-Kline Beacham, Philadelphia, PA; and MK591,
MK886, MK-571, and MK-679 were from Merck Frosst, Montreal, Canada.
LTA4 was a kind gift from Dr. J. Haeggström, Department of Medical Biochemistry and Biophysics, Karolinska Institutet. All other chemicals were of the highest purity and were
purchased from common commercial sources.
Purification of MGST-1--
Microsomal GST-1 was purified from
male Sprague-Dawley rat liver as described (21). The protein
concentration was determined by the procedure of Peterson with bovine
serum albumin as the standard (22). The purity of MGST-1 was assessed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12.5%
gels (23). A single band of molecular weight 17,000 was obtained after
Coomassie Blue staining. Rat MGST-1 was used as a model for the human
enzyme upon verification that LTC4 is a strong inhibitor of
the human enzyme.
Measurement of MGST-1 Activity--
Microsomal GST-1 activity
was measured according to Morgenstern et al. (24), by
determination of the conjugation of glutathione with CDNB or CNBAM.
Unless stated differently, all enzyme activity determinations were
carried out in a standard buffer containing 100 mM
potassium phosphate and 0.1% Triton X-100, pH 6.5, at 30 °C., with
5 mM GSH and 0.5 mM second substrate. The
amount of enzyme used is indicated for each type of experiment. When
the effect of calcium was determined, phosphate was exchanged for PIPES
buffer to avoid precipitation.
Enzyme Activation--
Activation of the MGST-1 by NEM was
performed as described earlier (21, 24). In short, the enzyme was
incubated with 5 mM NEM on ice for up to 30 min; at maximal
activation, the reaction was stopped by adding 5 mM GSH
(adjusted to pH 7.0 by KOH).
Proteoliposomes--
Unilamellar liposomes were prepared by
sonication of 20 mg of phosphatidylcholine in 0.2 ml of 20% sodium
cholate under a stream of nitrogen, until a clear translucent solution
was obtained. The liposomes were diluted to 10 mg/ml with 10 mM potassium phosphate buffer, pH 7.0, containing 20%
glycerol, 0.1 mM EDTA, and 50 mM KCl. 2 mg of
purified MGST-1 (in 1.5-ml of purification buffer; Ref. 21) was then
added. The solution was dialyzed for 72 h against the dilution
buffer containing 1 mM GSH and 0.05% sodium cholate, and
for an additional 72 h omitting sodium cholate. The proteoliposome
solution obtained contained 0.58 mg of protein/ml. Enzyme activity of
MGST-1 in proteoliposomes was determined as described above,
except that Triton X-100 was omitted from the assay buffer. The effect
of LTC4 on the enzymatic activity of MGST-1 was determined
at a concentration of 116 nM MGST-1 trimer incorporated
into proteoliposomes.
Determination of the Mechanism, Stoichiometry, and Ki
of Inhibition of Purified MGST-1 by LTC4--
The high
affinity of LTC4 for MGST-1 precluded analysis of the
kinetics of inhibition by examining double-reciprocal plots. To
determine the Ki of inhibition and stoichiometry for binding of LTC4 to MGST-1, we employed the graphical method
of Dixon (25). Inhibition by LTC4 of unactivated and
NEM-activated purified MGST-1 was determined at different
concentrations of LTC4. A graphical fit of empirical
velocity measurements at various concentrations of inhibitor and in the
absence of inhibitor (vo) was subsequently
generated. Points on the curve were selected at
vo/2, vo/3,
vo/4, vo/5, and
vo/6, and straight lines were generated from
vo through these points to the abscissa. The
distance values between the intersections (I2,
I3, I4,
I5, and I6) with the
abscissa were determined and averaged to obtain the
Kave at the particular concentration of
substrate. Kave was subsequently used to
determine I1 and I0. The
distance value between the origin and I0 on the abscissa reflects the concentration of binding sites present. A plot of
Kave versus substrate concentration,
with
To investigate whether LTC4 was acting as a slow,
tight-binding inhibitor, the difference in reaction velocities over
time were measured with the enzyme preincubated with LTC4
before starting the reaction with CDNB and GSH, or starting the
reactions by addition of enzyme (26).
To investigate the mode of inhibition of LTC4 with respect
to GSH and CDNB, competition experiments were performed. We employed a
method for tight-binding inhibitor analysis as described by Henderson
(27). The inhibitor concentration, divided by degree of inhibition
(i/(1 Leukotriene Synthesis Inhibitors and Cysteinyl Leukotriene
Receptor Antagonists--
IC50 values for the
5-lipoxygenase and FLAP inhibitors, as well as cysteinyl leukotriene
receptor antagonists were determined with 0.5 mM CDNB or
CNBAM as second substrates (24). CNBAM was used to measure the effect
of inhibitors only on that fraction of enzymatic activity that is not
due to activation of the enzyme (24). All experiments were performed at
90 nM MGST-1 trimer. After determining the uninhibited
rate, inhibitors were added in ethanol or dimethyl sulfoxide (maximally
1% (v/v) final concentration) while continuously monitoring the
decreased rate. The effects of the solvent alone (< 5%) were
corrected for. Plots of remaining activity versus inhibitor
concentration were constructed and the IC50 values
determined graphically.
Leukotriene C4 Is an Inhibitor of Enzyme
Activity--
Leukotriene C4 strongly inhibited the
enzymatic activity of MGST-1 incorporated into unilamellar
phosphatidylcholine-containing liposomes (Fig.
1). The inhibition was
concentration-dependent, and 50% inhibition was observed
at approximately 50 nM LTC4 at a protein
concentration of 116 nM trimer. Clearly, the tight-binding previously observed (19) results in inhibition.
Leukotriene C4 Is an Inhibitor of Purified MGST-1
Solubilized in Triton X-100-containing Buffer--
To address the
question of whether or not the presence of lipids is an absolute
requirement for the binding/inhibition, we also investigated the effect
of LTC4 on the enzymatic activity of purified MGST-1 in
Triton X-100-containing buffer. Also under these conditions, the
unactivated as well as the NEM-activated MGST-1 were strongly inhibited
by LTC4 in a concentration-dependent fashion
(Fig. 2, a and
b).
The Mechanism of Inhibition of Enzymatic Activity of MGST-1 by
LTC4--
Leukotriene C4 was found to be a
tight-binding inhibitor, i.e. stoichiometric amounts of
LTC4 bound to the MGST-1 trimer, thereby inhibiting
activity. The Ki of inhibition was found to be 5.4 nM for the unactivated enzyme, and 9.2 nM
for the activated enzyme (Fig. 2c and Table
I).
The purified enzyme was also inhibited by LTC4 if NAC was
used as a substrate instead of GSH. The Ki for the
unactivated enzyme was 4.4 nM (Table I) in this case. The
Ki for the activated enzyme could not be determined,
as only extrapolated data for the Km (NAC) of
activated MGST-1 exist. The stoichiometry of binding of
LTC4 to the MGST-1 trimer was in all cases close to one,
indicating that each trimer can bind one molecule of LTC4
(Table I).
Leukotriene C4 was a competitive inhibitor with respect to
GSH. Slopes of the plot of inhibitor concentration divided by degree of
inhibition (i/(1
No difference in reaction velocities over time were seen whether the
enzyme was preincubated with LTC4 before starting the reaction with CDNB and GSH, or starting the reactions by addition of
enzyme (not shown), indicating that LTC4 does not inhibit
MGST-1 by slow, tight-binding inhibition.
It has been reported that the binding of LTC4 to MGST-1 was
dependent on divalent cations, such as calcium and magnesium (19). However, we found no such dependence nor any effect of either added
calcium or magnesium on inhibition (0.2 µM-20
mM, not shown).
Other Leukotrienes and Arachidonic Acid Are Weaker Inhibitors of
MGST-1 Activity--
Leukotrienes D4 and E4
were much less potent inhibitors of MGST-1 than LTC4 (at
least 3 orders of magnitude) with IC50 > 20 µM (higher concentrations were not tested). Leukotriene
A4 inhibited MGST-1 with an IC50 of 11 µM. The inhibitory potency of LTA4 was very
similar to that of arachidonic acid which displayed an IC50 of 9.7 µM (Table II).
Leukotriene Synthesis Pathway Inhibitors also Influence MGST-1
Activity--
Among the inhibitors of leukotriene biosynthesis, three
FLAP antagonists (MK-886, MK-591, and BAYx1005) and the compound
piriprost (U 60,257) were found to cause significant inhibition of
MGST-1 (Table III). The inhibitory
potencies against MGST-1 were similar for all four inhibitors although
piriprost and BAYx1005 appeared somewhat more potent. In contrast,
zileuton and ZD 230,487 displayed minimal inhibitory activity (Table
III).
With the exception of ICI 198,615, all tested antagonists of
cysteinyl-leukotrienes inhibited MGST-1 (Table III). The leukotriene analogues ONO-1078, BAYu9773, and in particular SKF 104,353, were the
strongest inhibitors of all tested compounds (Table III; Fig., 4). The
profile of inhibitory activity did not relate to whether CDNB or CNBAM
was the second substrate (Table III).
Published binding studies have indicated that MGST-1 contains a
binding site for LTC4 (19). However, radiolabeled
LTC4 was reported to bind to purified MGST-1 only if the
binding incubations were supplemented with a Triton X-100 extract of
dU937 cell membranes (that had been heat-inactivated to abolish
endogenous binding sites for LTC4). A lipid component was
therefore suggested to be required by purified MGST-1 for binding
LTC4 (19). Our studies document that a lipid component is
not required. Moreover, we discovered that LTC4 was an
extremely tight-binding inhibitor of MGST-1. The Ki
of LTC4 was 5.4 nM for the unactivated enzyme,
and 9.2 nM for the NEM-activated enzyme. This agrees well with the previously estimated dissociation constant (19), although in
our studies divalent cations are not required. In fact, the binding of
LTC4 was much stronger than that of GSH itself
(Kd = 18 µM) (28). The finding that
activated and unactivated MGST-1 display similar properties also agrees
with previous observations on GSH and glutathione sulfonate binding
(28). The activated enzyme did not exhibit an increased affinity for
substrates or inhibitory ligands.
It was suggested that GSH at physiological concentrations should be
able to displace LTC4 from MGST-1 (19). The present study
contradicts these findings. The inhibitory capacity of low (near
stoichiometric) levels of LTC4 was well evident at 5 mM GSH (c.f. Fig. 1). Thus, physiological levels
of GSH (1-10 mM; Ref. 29) will not effectively displace
LTC4 from MGST-1. Rather, the amount of intracellular
LTC4 might determine the fraction of MGST-1 available for
catalysis. Furthermore, there is no absolute requirement for a lipid
environment in order for LTC4 to inhibit MGST-1, as
demonstrated by the strong inhibition of purified MGST-1 dissolved in
Triton X-100. Differences between the direct binding studies (19) and
our kinetic data could perhaps be ascribed to some property of the
binding assay used.
N-acetyl-L-cysteine is a useful analytical
alternative to GSH because it is specifically utilized by MGST-1 and
not by cytosolic glutathione transferases (30). Here we demonstrate
that LTC4 inhibits MGST-1 just as well when this
alternative thiol donor is used to assay the enzyme. Therefore, NAC, in
conjunction with LTC4 inhibition should provide a helpful
tool to determine the amount and activity of MGST-1 in complex
biological systems.
The binding of LTC4 to MGST-1 was reversible, as
demonstrated by the reversibility of inhibition by fatty acid-free
albumin. From the competition experiments, it appears likely that
LTC4 and GSH bind to the same binding site on MGST-1 and
that this site forms part of the active site of the enzyme. However,
LTC4 does not compete for the binding of CDNB, indicating
that a fatty acid-binding site may be present in the MGST-1 trimer
which is different from the hydrophobic substrate-binding site. Another likely explanation is the possibility that CDNB does not form a
kinetically significant complex to MGST-1.
Previous studies have determined that there is one binding site for GSH
per MGST-1 trimer (28). Tight-binding inhibitors can also be used as
tools for the evaluation of stoichiometry. LTC4 inhibition
measured in the present study indicates that there is one
LTC4 binding site per trimer in agreement with direct
binding studies with radiolabeled LTC4 (19). The observed
stoichiometry raises important issues regarding the enzymatic mechanism
of MGST-1. Spatially equivalent overlapping mutually exclusive sites or
non-overlapping sites can be envisioned in the homo-trimeric protein
(31). If non-overlapping sites are present, conformational information upon binding of GSH or LTC4 has to be transmitted, possibly
resulting in alternating site catalysis. Structural studies presently
performed are hoped to yield sufficient resolution to address these questions.
The high affinity binding of LTC4 to MGST-1 stimulated us
to explore whether or not compounds related to LTC4, or
drugs which inhibit the leukotriene pathway at different points in the
cascade, interfered with MGST-1 activity. Many of the compounds indeed inhibited MGST-1.
The antagonists of the receptors for cysteinyl-leukotrienes were potent
inhibitors of MGST-1. In particular, the compounds which may be
classified as analogues of cysteinyl-leukotrienes (BAY u9773, SKF
104,353, and ONO-1078) were the most potent (Fig. 4). The potency of SKF 104,353 was
striking, causing 50% inhibition in the submicromolar concentration
range, which in fact suggests greater or equal affinity of this
compound for MGST-1 in comparison with the CysLT1 receptor
(pA2 values for SKF 104,353 against LTD4 in
guinea-pig and human airways being 8.6 and 8.0; Ref. 32). For BAY u9773
and ONO-1078, the activity against MGST-1 was also evident in the same
concentrations as those required for antagonism of CysLT1
receptors. However, there were also discrepancies between the compounds
documented relative potency as pharmacological antagonists of the
functional responses to cysteinyl-leukotrienes and the potency we
observed as inhibitors of MGST-1. For example, the early prototype of
CysLT1-antagonists, FPL 55712, was as potent an inhibitor
of MGST-1 as the considerably more potent leukotriene antagonist
MK-571. Likewise, MK-679, which is the active enantiomer of the racemic
compound MK-571, was 5-fold less active than MK-571. Moreover, ICI
198,615, which is the most potent antagonist of CysLT1-receptors among the tested compounds
(pA2 values in different systems ranging between 10.1-9.3;
Ref. 33), failed to cause significant inhibition of MGST-1.
INTRODUCTION
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Abstract
Introduction
References
MATERIALS AND METHODS
Km intersecting the abscissa, gives the
Ki of LTC4 as the intercept with the
ordinate (for a competitive inhibitor; see below). These experiments
were performed at 90 nM trimer MGST-1. A typical
representative of three-five independent experiments is shown. For
some experiments, GSH was replaced with
N-acetyl-L-cysteine (NAC) as indicated.
vi/vo)), was plotted
against velocity without inhibitor divided by velocity with inhibitor
(vo/vi). The mechanism of
inhibition was determined from the replot of the variation of the slope
at different GSH (2, 5, 10, and 20 mM) or CDNB
concentrations (50, 200, and 500 µM). These experiments
were performed at 90 nM trimer MGST-1. Because the plots
deviated from linearity at low inhibition, this method was used to
evaluate the type of inhibition in a qualitative manner, whereas the
Dixon method (which does not rely on values at very low inhibition) was
used for the quantitative estimations. Nevertheless, the
Ki value derived by the Henderson method (17 nM) agrees reasonably to that obtained with the Dixon method.
RESULTS
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Fig. 1.
Inhibition of MGST-1 incorporated in
phosphatidylcholine-containing liposomes by LTC4.
Assayed with CDNB as the second substrate, as described under
"Materials and Methods."
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Fig. 2.
Inhibition by LTC4 of unactivated
(a) and N-ethyl maleimide activated
(b) purified MGST-1. The curved line represents a
manual fit of empirical velocity measurements (open squares)
at various concentrations of inhibitor and in the absence of inhibitor
(vo). Points on the curve were
selected at vo/2, vo/3,
vo/4, vo/5, and
vo/6 and straight lines were
generated from vo through these points to the
abscissa. The distance values between the intersections of the straight
lines (I2, I3,
I4, I5, and
I6) with the abscissa were determined and
averaged to obtain the Kave at the particular
concentration of substrate. Kave was
subsequently used to determine I1 and
I0. The distance value between the origin and
I0 on the abscissa estimates the concentration
of binding sites present. c, graphical determination of the
LTC4 inhibition constant (Ki) for the
unactivated and N-ethylmaleimide-activated purified MGST-1.
Values of Kave are from panels a and
b and Km values (abscissa) are taken from
Ref. 30.
Inhibition of MGST-1 by LTC4; dependence on state of
activation and substrate
vi/vo)) versus
velocity without inhibitor divided by velocity with inhibitor
(vo/vi) changed linearly
with changes in GSH (Fig. 3a).
Because inhibition was competitive, it must be reversible. As expected,
the inhibition by LTC4 could be partially reversed also by
addition of fatty acid-free albumin (maximally 30%, not shown). In
contrast, LTC4 was a non-competitive inhibitor with respect
to the second substrate CDNB (Fig. 3b).
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Fig. 3.
Determination of the type of inhibition
exerted by LTC4. Shown is a replot of the slope of
i/(1 vi/vo) versus
vo/vi against
concentration of GSH (a) or CDNB (b).
Inset shows plot of i/(1
vo/vi) versus
vo/vi at different
concentrations of GSH and CDNB, respectively.
Leukotrienes and arachidonic acid as inhibitors of MGST-1 activity,
assayed in vitro in 0.1 M potassium phosphate-buffer
containing 0.1% Triton X-100, pH 6.5, 30 °C
Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor
antagonists as inhibitors of MGST-1 activity, assayed in vitro in
0.1 M potassium phosphate buffer containing 0.1%
Triton X-100, pH 6.5, 30 °C
DISCUSSION
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Fig. 4.
Graphical display of IC50 values
of 5-lipoxygenase inhibitors (open squares), FLAP
inhibitors (open circles), and cysteinyl leukotriene
antagonists (closed circles). The IC50
values were obtained from inhibition studies of MGST-1 with CDNB as a
second substrate.
Among the inhibitors of leukotriene formation, two relatively selective inhibitors of the 5-lipoxygenase (the iron-chelator zileuton and the active site inhibitor ZD 230,487) failed to inhibit MGST-1, whereas three different FLAP antagonists displayed significant potency as inhibitors (Fig. 4). The prostacyclin analogue piriprost inhibits leukotriene formation by mechanisms that have not been completely elucidated. Interestingly enough, interference with GST activity (34) has been discussed as one possible mechanism of its action, and piriprost was found to inhibit MGST-1 in this study.
The substrate for leukotriene formation, arachidonic acid, and the immediate precursor of LTC4, LTA4, both inhibited MGST-1, but their effects were only observed at relatively high concentrations. Successive reductions of the glutathionyl side chain in LTC4 yields LTD4 and LTE4. Neither product was particularly potent as inhibitor of MGST-1, which is interesting because LTD4 and LTE4 are presumably formed extracellularly and not in the same compartments as MGST-1 and LTC4.
Considered together, the new finding that not only LTC4 but also certain antagonists and inhibitors of leukotrienes inhibited MGST-1 raises a number of questions relating to the function of MGST-1 and related intracellular proteins. There are, as discussed, observations suggesting physical proximity and interactions between FLAP, LTC4 synthase and MGST-1. Our results, in addition, suggest previously unknown functional interactions between MGST-1 on the one hand and enzymes, substrates, products, and cofactors in the leukotriene pathway on the other hand. For example, binding of LTC4 to MGST-1 may represent a means for transient storage of preformed LTC4. Furthermore, binding of LTC4 to MGST-1 may reduce product inhibition at the level of LTC4 synthase, which may be desirable at times when there is a strong drive for leukotriene generation. Inhibition of enzymatic activity of MGST-1 by LTC4 is also expected to interfere with the reduction of phospholipid and fatty acid hydroperoxides, which are important cofactors for the synthesis of leukotrienes and several other eicosanoids.
All in all, our findings generate the hypothesis that compounds which have some relation to FLAP or LTC4 synthase also may interact with MGST-1. Tight binding of LTC4 by another, cytosolic, GST has also been observed (35). In this context, it should be noted that synthesis of LTC4 will take place at the perinuclear membrane (36, 37), which means that intracellular proteins are exposed to LTC4. In addition, transport mechanisms in the cell membrane which share affinity for LTC4 and inhibitors of the leukotriene pathway have been described (38). Therefore, we suggest that our observations represent an indication that many different but related proteins must be considered in future equations and schemes attempting to comprehend how leukotrienes are formed and handled in the cell as well as released to the exterior with the aid of transport mechanisms. The observed interactions between LTC4 and MGST-1, and in particular MGST-1 and several leukotriene antagonists, may in addition have bearing on the effects and side-effects of inhibitors of leukotrienes that currently are introduced as new treatment of asthma and other inflammatory diseases. For example, some drugs have caused liver function test abnormalities, and the effects we have observed in this study could well contribute to effects such as peroxisome proliferation that in fact has been documented for some of the drug candidates. The most potent inhibitors of MGST-1 we found could perhaps also be used to probe the role of the enzyme in cellular and organismal detoxication/toxication reactions. For instance, the suggested role for MGST-1 in the protection against oxidative stress (10) and as the first step in the biotransformation of many nephrotoxic poly-halogenated hydrocarbons (39) can be further studied.
In conclusion, our findings have shown that LTC4 is a
tight-binding inhibitor of MGST-1. This finding suggests an important role of MGST-1 in the intracellular management of LTC4.
Future studies will therefore address the role of MGST-1 in the
biosynthesis and intracellular transport of LTC4.
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ACKNOWLEDGEMENTS |
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Lilian Larsson and Eli Zahou are gratefully acknowledged for practical assistance.
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FOOTNOTES |
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* This work was supported in part by the Swedish Cancer Society, the Medical Research Council (project 71X-9071), the Heart Lung Foundation, the Foundation for Health Care Sciences and Allergy Research (Vårdal), the Association Against Asthma and Allergy and Funds from the Karolinska Institutet.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.
§ Tenure was generously supported by the Wenner Grenska Samfundet and the Karolinska Institutet.
Supported by a European Community Erasmus Fellowship.
** To whom correspondence should be addressed. Tel:. 46-8-7287574; Fax: 46-8-334467; E-mail: ralf.morgenstern{at}IMM.K1.SE.
The abbreviations used are: MGST-1, microsomal glutathione transferase-1; CDNB, 1-chloro-2,4-dinitrobenzene; CNBAM, 4-chloro-3-nitrobenzamide; FLAP, 5-lipoxygenase activating protein; LT, leukotriene; NEM, N-ethylmaleimide; NAC, N-acetyl-L-cysteine; PIPES, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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