Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers.

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 microM, 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 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). 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.

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 C 4 is a potent inhibitor of MGST-1. Leukotriene C 4 was found to be a tight-binding inhibitor, with a K i 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 C 4 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 A 4 , D 4 , and E 4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C 4 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 (IC 50 values: 0.13, 3.7, and 7.6 M, respectively). In view of the partial structural similarity between MGST-1, leukotriene C 4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C 4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC 50 for BAYx1005 was 58 M). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 M). Our discovery that leukotriene C 4 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) C 4 -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 LTC 4 -synthase (14,15). Interestingly, additional members of this putative protein superfamily that display common functional properties have recently been described (13,16).
Leukotriene C 4 -synthase is a specialized membrane-bound glutathione transferase involved in the production of cysteinyl leukotrienes (LTC 4 , LTD 4 , and LTE 4 ). 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). LTC 4 -synthase is homologous to FLAP (18).
Microsomal GST-1 has been identified as a high capacity "low affinity'' binding protein for LTC 4 and was found to bind one molecule LTC 4 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 LTC 4 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.

MATERIALS AND METHODS
Chemicals and Enzyme-Triton X-100, N-ethylmaleimide (NEM), glutathione (GSH), arachidonic acid, and fatty acid-free albumin were * 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. This 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.
ʈ 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 LTC 4 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 LTC 4 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 K i of Inhibition of Purified MGST-1 by LTC 4 -The high affinity of LTC 4 for MGST-1 precluded analysis of the kinetics of inhibition by examining doublereciprocal plots. To determine the K i of inhibition and stoichiometry for binding of LTC 4 to MGST-1, we employed the graphical method of Dixon (25). Inhibition by LTC 4 of unactivated and NEM-activated purified MGST-1 was determined at different concentrations of LTC 4 . A graphical fit of empirical velocity measurements at various concentrations of inhibitor and in the absence of inhibitor (v o ) was subsequently generated. Points on the curve were selected at v o /2, v o /3, v o /4, v o /5, and v o /6, and straight lines were generated from v o through these points to the abscissa. The distance values between the intersections (I 2 , I 3 , I 4 , I 5 , and I 6 ) with the abscissa were determined and averaged to obtain the K ave at the particular concentration of substrate. K ave was subsequently used to determine I 1 and I 0 . The distance value between the origin and I 0 on the abscissa reflects the concentration of binding sites present. A plot of K ave versus substrate concentration, with ϪK m intersecting the abscissa, gives the K i of LTC 4 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.
To investigate whether LTC 4 was acting as a slow, tight-binding inhibitor, the difference in reaction velocities over time were measured with the enzyme preincubated with LTC 4 before starting the reaction with CDNB and GSH, or starting the reactions by addition of enzyme (26).
To investigate the mode of inhibition of LTC 4 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 Ϫ v i /v o )), was plotted against velocity without inhibitor divided by velocity with inhibitor (v o /v i ). 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 M). 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 K i value derived by the Henderson method (17 nM) agrees reasonably to that obtained with the Dixon method.
Leukotriene Synthesis Inhibitors and Cysteinyl Leukotriene Receptor Antagonists-IC 50 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 (Ͻ 5%) were corrected for. Plots of remaining activity versus inhibitor concentration were constructed and the IC 50 values determined graphically.

RESULTS
Leukotriene C 4 Is an Inhibitor of Enzyme Activity-Leukotriene C 4 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 LTC 4 at a protein concentration of 116 nM trimer. Clearly, the tight-binding previously observed (19)

results in inhibition.
Leukotriene C 4 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 LTC 4 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 LTC 4 in a concentration-dependent fashion (Fig. 2, a and b).
The Mechanism of Inhibition of Enzymatic Activity of MGST-1 by LTC 4 -Leukotriene C 4 was found to be a tightbinding inhibitor, i.e. stoichiometric amounts of LTC 4 bound to the MGST-1 trimer, thereby inhibiting activity. The K i of inhi- bition 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 LTC 4 if NAC was used as a substrate instead of GSH. The K i for the unactivated enzyme was 4.4 nM (Table I) in this case. The K i for the activated enzyme could not be determined, as only extrapolated data for the K m (NAC) of activated MGST-1 exist. The stoichiometry of binding of LTC 4 to the MGST-1 trimer was in all cases close to one, indicating that each trimer can bind one molecule of LTC 4 (Table I).
Leukotriene C 4 was a competitive inhibitor with respect to GSH. Slopes of the plot of inhibitor concentration divided by degree of inhibition (i/(1 Ϫ v i /v o )) versus velocity without inhibitor divided by velocity with inhibitor (v o /v i ) changed linearly with changes in GSH (Fig. 3a). Because inhibition was competitive, it must be reversible. As expected, the inhibition by LTC 4 could be partially reversed also by addition of fatty acid-free albumin (maximally 30%, not shown). In contrast, LTC 4 was a non-competitive inhibitor with respect to the second substrate CDNB (Fig. 3b).
No difference in reaction velocities over time were seen whether the enzyme was preincubated with LTC 4 before starting the reaction with CDNB and GSH, or starting the reactions by addition of enzyme (not shown), indicating that LTC 4 does not inhibit MGST-1 by slow, tight-binding inhibition.
It has been reported that the binding of LTC 4 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 M-20 mM, not shown).
Other Leukotrienes and Arachidonic Acid Are Weaker Inhibitors of MGST-1 Activity-Leukotrienes D 4 and E 4 were much less potent inhibitors of MGST-1 than LTC 4 (at least 3 orders of magnitude) with IC 50 Ͼ 20 M (higher concentrations were not tested). Leukotriene A 4 inhibited MGST-1 with an IC 50 of 11 M. The inhibitory potency of LTA 4 was very similar to that of arachidonic acid which displayed an IC 50 of 9.7 M (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). DISCUSSION Published binding studies have indicated that MGST-1 contains a binding site for LTC 4 (19). However, radiolabeled LTC 4 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 abol-   (19). Our studies document that a lipid component is not required. Moreover, we discovered that LTC 4 was an extremely tight-binding inhibitor of MGST-1. The K i of LTC 4 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 LTC 4 was much stronger than that of GSH itself (K d ϭ 18 M) (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 LTC 4 from MGST-1 (19). The present study contradicts these findings. The inhibitory capacity of low (near stoichiometric) levels of LTC 4 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 LTC 4 from MGST-1. Rather, the amount of intracellular LTC 4 might determine the fraction of MGST-1 available for catalysis. Furthermore, there is no absolute requirement for a lipid environment in order for LTC 4 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 LTC 4 inhibits MGST-1 just as well when this alternative thiol donor is used to assay the enzyme. Therefore, NAC, in conjunction with LTC 4 inhibition should provide a helpful tool to determine the amount and activity of MGST-1 in complex biological systems.
The binding of LTC 4 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 LTC 4 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, LTC 4 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. LTC 4 inhibition measured in the present study indicates that there is one LTC 4 binding site per trimer in agreement with direct binding studies with radiolabeled LTC 4 (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 LTC 4 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 LTC 4 to MGST-1 stimulated us to explore whether or not compounds related to LTC 4 , 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 CysLT 1 receptor (pA 2 values for SKF 104,353 against LTD 4 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 CysLT 1 receptors. However, there were also discrepancies between the compounds documented relative potency as pharmacological antagonists of the functional responses to cysteinylleukotrienes and the potency we observed as inhibitors of MGST-1. For example, the early prototype of CysLT 1 -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 CysLT 1 -receptors among the tested compounds (pA 2 values in different systems ranging between 10.1-9.3; Ref. 33), failed to cause significant inhibition of MGST-1.
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 LTC 4 , LTA 4 , both inhibited MGST-1, but their effects were only observed at relatively high concentrations. Successive reductions of the glutathionyl side chain in LTC 4 yields LTD 4 and LTE 4 . Neither product was particularly potent as inhibitor of MGST-1, which is interesting because LTD 4 and LTE 4 are presumably formed extracellularly and not in the same compartments as MGST-1 and LTC 4 .
Considered together, the new finding that not only LTC 4 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, LTC 4 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 LTC 4 to MGST-1 may represent a means for transient storage of preformed LTC 4 . Furthermore, binding of LTC 4 to MGST-1 may reduce product inhibition at the level of LTC 4 synthase, which may be desirable at times when there is a strong drive for leukotriene generation. Inhibition of enzymatic activity of MGST-1 by LTC 4 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 LTC 4 synthase also may interact with MGST-1. Tight binding of LTC 4 by another, cytosolic, GST has also been observed (35). In this context, it should be noted that synthesis of LTC 4 will take place at the perinuclear membrane (36,37), which means that intracellular proteins are exposed to LTC 4 . In addition, transport mechanisms in the cell membrane which share affinity for LTC 4 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 LTC 4 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 LTC 4 is a tightbinding inhibitor of MGST-1. This finding suggests an important role of MGST-1 in the intracellular management of LTC 4 . Future studies will therefore address the role of MGST-1 in the biosynthesis and intracellular transport of LTC 4 .