Leukotriene C synthetase, a special glutathione S-transferase: Properties of the enzyme and inhibitor studies with special reference to the mode of action of U-60,257, a selective inhibitor of leukotriene synthesis

Leukotriene C synthetase, a special glutathione S-transferase: Properties of the enzyme and inhibitor studies with special reference to the mode of action of U-60,257, a selective inhibitor of leukotriene synthesis Michael K. Bach, Ph.D., John R. Brashler, B.S., Rebecca E. Peck, B.S., and Douglas R. Morton, Jr., Ph.D. Kalamazoo, Mich.

The cytosolic glutathione S-transferases qf rat liver have been fractionated by chromatofocusing into IO distinct fractions based on their reactivity with 2,4-dinitrochlorobenzene. All these fractions were capable of generating leukotriene C, (LTC,) from leukotriene A, (LTA,) to some extent. An inhibitor of leukotriene synthesis, U-60,257, inhibited the activity of these enzymes. The cytosolic glutathione S-transferases of rat basophil leukemia (RBL) cells have been similarly fractionated. U-60,257 inhibited the activity of some of these fractions but not that of others. None of the fractions of the enzyme from RBL cells formed LTC, from LTA,. The microsomal glutathione S-transferase from rat liver also produced LTC, from LTA,. It difers from the microsomal LTC synthetase of RBL cells in at least two respects: (1) The enzyme from RBL cells did not react with chromophoric substrates like dinitrochlorobenzene while the enzyme from liver did react. (2) Triton X-100 potentiated the activity of the enzyme from basophil leukemia cells and solubilized it, while it inhibited the activity of the leukotriene-synthesizing enzyme in the rat liver preparation. These results, along with a distinctly different inhibitor projile, indicate that LTC synthetase is a new and distinct glutathione S-transferase. (.I ALLERGY CLIN IMMUN~L 74:353, 1984.)

Our interest in regulation of the biosynthesisof the sulfidopeptideleukotrienesled to the discovery of 6,9deepoxy-6,9-(phenylimino)-A6z8prostaglandinI, (or U-60,257), which is a selective inhibitor of leukotriene biosynthesis.’ Initial mode-of-action studies in human PMNs indicated that low concentrationsof this inhibitor, in the presence of O.lmM arachidonate, caused a stimulation of the formation of both 5,12dihydroxy-eicosatetsaenoic acids and 5-hydroxy-eicosatetraenoic acid, while higher concentrations ( 104M) causedan inhibition. This suggestedto us that U-60,257 may inhibit leukotriene biosynthesis by inhibiting the terminal step in the biosynthetic pathway, the coupling of glutathione with LTA,. Indeed, we found that this compound was a potent inhibitor of

From the Departmentsof Hypersensitivity DiseasesResearchand Experimental Sciences I, The Upjohn Company, Kalamazoo, Mich. Reprint requests: M. K. Bach, Ph.D., Hypersensitivity Diseases Research,The Upjohn Company, Kalamazoo, MI 49001.

Abbreviations used PMNs: Polymorphonuclear leukocytes

LTA,: DCNB: RBL LTC,: pI:

LeukotrieneA, 1,2-Dichloro-4-nitrobenzene

Rat basophilleukemia Leukotriene C, Isoelectric point, based on elution in chro-

matofocusing DNCB: 2,4-Dinitrochlorobenzene ENPP: 1,2-Epoxy-3-(p-nitrophenoxy)-propane EC,,:

50% Inhibitory dose (micromolar)

the crude, mixed glutathione S-transferasesof rat liver when the activity of these enzymeswas assayedwith a typical chromogenic substrate, DCNB.’ We therefore embarkedon a more detailed examination of the glutathione S-transferasesand particularly on a study of their ability to synthesize the sulfidopeptide leukotrienes and their susceptibility to inhibition by U60,257. These studies form the basis of this presentation. 353

354

J ALLERGY

Bach et al

ii/a t-Er,r,o,-34”Balr I tslro”e-3-h”ftate /*In01 , “3 1 lOeM I +--+-+---VI” p4 FIG. 1. Effect of various inhibitors on cytosolicglutathione S-transferase activity from rat liver and RBL cells and on the formation of sulfidopeptide leukotrienes by crude homogenates of RBL cells with arachidonate used as substrate. 0 = Results with rat liver enzymes with ENPP as substrate; ~2 -- results with rat liver enzymes with DCNB as substrate; A = results with enzyme from RBL cells with DCNB as substrate; A := sulfidopeptide leukotriene production (as LTD,) with an RBL cell homogenate and O.tmM arachidonate assubstrate. (Reproduced from Bach MK, Brashler JR, Morton DR, Steel LK, Kaliner MA, Hugli TE: Formation of leukotrienes C and D pharmacologic madulation of their synthesis. Adv Prostaglandin Thromboxane Leukotriene Res 9:103, 1982.1

We first carried out a comparison of the inhibition profiles of the crude cytosolic glutathione S-transferasesof rat liver and of RBL cells on the one hand. :tnd the capacity of a whole RBL cell homogenateto generateleukotrienes from arachidonateon the other., using a variety of well-known inhibitors of the glu.. tathione S-transfemses.’As shown in Fig. 1, inhibition of all thesereactionscould be achievedbut there were markeddifferencesin the susceptibility of the different reactions to inhibition: the bukotriene-generating reactions were, on the whole, the least sensitiveto these inhibitors. Thesefindings suggestedthat the synthesisof LTC, may be atypical as far asthe glutathione S-transferases are concerned. The assay for LTA,: glutathione Stransferases,or LTC synthetase, was therefore opti-

i!~.-.,-ad..A, 0.2 0.4 mg

.Li 0.6 0.8

PROTEIN

LO

CCIN. IMMUNOL. SEPTEMBER 1984

I 0

10

20

-/.-

30

MINUTES

FIG. 2. Optimizaton of the LTC synthetase reaction with Triton X-lOO-solubilized particulate enzyme from RBL cetls. Except as noted, incubations contained 0.45 mg protein in a substrate mixture (0.5 ml) that was 1mM glutathione, 25pM LTA, (lithium salt), 0.3% Triton X-100, and 25mM HEPEs, at pH 7.0, and that contained 0.5 mg bovine serum albumin. Incubations were at 37” C for 10 minutes. (Data from Bach MK, Brashler JR, Morton DR: Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel particulate glutathione S-transferase. Arch Biochem Biophys 230: 455, 1984.)

mized by useof enzyme?from rat liver and RBL cells ( Fig. 21, Briefly, it was necessary to stabilize the substrateLTA, by incorporating bovine serumalbumin into the incubations.’ The glutathione concentration was optimal in the 3mM to 5mM range and it was not possible to saturatethe reaction with LTA, within the concentrations of this precious substratethat we could afford to use (up to 50pM). The reaction was linear for at least 10 minutes and the reaction product was characterized as LTC, by cochromatographyon high-pressureliquid chromatographywith a synthetic standard. It is well known that rat liver cytosol contains a farge assortmentof isozymes of glutathione S-transferasethat differ somewhatin pl and in their substrate specificity.’ Before any meaningful analysis of the substrateor inhibition profiles of this mixture could be made, it was necessaryto resolve the mixture and to examine the isolated, purified subtypesof the glu-

VOLUME NUMBER

74 3, PART 2

Leukotriene

C synthetase

355

Bilirubin Estradiol-3sullate-17 slucuronide U-60257@ Bromosulfo phthalein E&one-3sulfate Estriol-3sulfate EstrW; A5 Androstene3,17-dione Probenecid p-Aminohippuric acid I 1

I 10

I 100 (PM) E&o

I 1,000

I 10,000

FIG. 3. Effect of inhibitors of glutathione S-transferases on mixed cytosolic glutathione S-transferases of RBL cells (solid bars/ and on the solubilized microsomal LTC synthetase (hatched bars. *No inhibition at the highest inhibitor concentration tested. **Stimulation of enzyme activity.

tathione S-transferase.This proved to be a massive task becauseof the unexpectedcomplexity of the mixture of enzymespresent.A preliminary separationthat used chromatofocusing resulted in 10 regions of activity when DNCB was used as the substrate,rather than the sevenisotypes that had been describedin the literature.4 Someof theseregions showedfurther heterogeneity when other chromophoric substrateswere employed in place of DNCB, but despitethis, activity was pooled basedon the results with DNCB and the pooled fractions were then comparedfor their activity with DNCB, DCNB, ENPP, and LTA, as substrates. The results indicated that all the fractions were capable of generatingLTC, from LTA4, and with the exception of a very small fraction with a very low specific activity for this enzyme (p1 10.3 based on chromatofocusing and total activity representing only 0.03% of that of the total homogenate), the molar ratio of the LTC-generatingactivity to the activity with DNCB as substraterangedbetween 1.Oand 8.0 X 10e5.The highest ratio was found in a fraction, which appeared to be homogenous(pl around 8.1), which accounted for 10% of the total LTC-generating activity of the homogenate and which had relative activities with DCNB and ENPP as substratessimilar to those of most of the other fractions. Clearly, the capacity to generateLTC, from LTA, is not limited to a unique

isozyme among the soluble glutathione S-transferases in rat liver. But rat liver is not generally considered to be a major source of leukotrienes in the body. We were therefore interested in studying the samereactions in a tissue that is capable of generating large amounts of leukotrienes, and we chose RBL cells for this purpose. In agreementwith the published results of Jakschik et al. ,5we found that LTC, synthesistook place in the microsomal fraction of RBL cell homogenates. By contrast, the cytosolic enzymes from these cells were devoid of LTC synthetaseactivity even though there was a respectableamount of glutathione S-transferase activity present in these preparations. The microsomal fraction was devoid of the more usual glutathione S-transferaseactivity. The LTC synthetase activity of the microsomal fraction was solubilized by use of Triton X-100 and there was a modest potentiation of enzyme activity in the presence of Triton X-100. We used solubilized enzyme preparation to optimize the conditions for the assayof the LTC synthetase. We had tested some 25 different detergents, including NP-40, which is structurally very similar to Triton X-100, but only Triton was effective in solubilizing the LTC synthetase. These observations were reminiscent of a recent description of the solubilization and purification of the

356

9.7

2.5

9.6

1.3

8.9

3.6

8.2

6.3

T.3

J. ALLERGY

Bach et al

* l

15.0~

6.0

21.0

5.3

42.0 ----.I---0.1

1.0

ECsc Concentration

-A.-----. 10

(~frrl)

FIG. 4. Effect of Inhibitors of glutathione S-transferases i>n subfractions of the glutathione S-transferases from RBL cells. The pl values and the relative abundance of the respective fractions are shown beneath each group of bars. All results are based on the use of DNCB as substrate. *Lack of inhibition at the highest dose tested. Solid bars = bilirubin; hatched bars = bromosulfophthalein; open bars = U-60,257; crosshatched bars = estrone-3. sulfate: stippled bars = estradiol-3-sulfate,

rnicrosomal glutathione S-transferase of rat liver,” and before we could conclude that the LTC synthetase ol RBL cells was indeed a new enzyme, we needed to compare the enzyme from RBL cells with the particulate enzyme from liver. We found that the micro5omal enzyme from liver was capable of generating LTC.,. We confirmed the reported uc’fivation of this enzyme by brief incubation with N-ethylmaleimide when DNCB was used as the substrate. However, we found no such activation when LTA, was used as a substrate. Neither was the enzyme from RBL cells activated by this treatment. More strikingly, we found that, whereas Triton X-100 had been reported to sulubilize the glutathione S-transferase in liver microsomes, it inhibited the LTC-generating activity of these preparations in the same concentration range in which it was both solubilizing and potentiating the activity of the enzyme from RBL cells. These observations Ied us to believe that these two enzymes are distinct.

CLIN. IMMUNOL. SEPTEMBER 1984

#Zsalready mentioned, the known inhibitors of glutathione S-transferase seemed much less active in inhibiting leukotriene synthesis in intact RBL cells than they were in inhibiting the DCNB-dependent glutathione S-transferase activity of the same cells. Examination of the activity of several of these inhibitors on the LTC synthetase of these cells ( Fig. 3) revealed that, on the whole, these compounds did not inhibit this reaction. Most noteworthy is bilirubin, which, it may be recahed (Fig. l), was a potent inhibitor of leukotriene synthesis in the intact cells and which was inactive up to the highest concentration that could be achieved when it was studied with the solubilized enzyme. U-60,257, the inhibitor of leukotriene synthesis that started us on this long road, also failed to inhibit LTC synthetase of RBL cells. It should be parenthetically mentioned that further work by Frank Sun and Jim McGuire’ at our laboratories has demonstrated that when the exogenous arachidonic acid concentration was reduced, U-60,257 inhibited the 5lipoxygenase of PMNs with an EC,, of about 1pM. As far as we know, this is the primary mode of action of’ U-60,257 in these cells. But U-60,257 had yet another trick in store for us. Examination of the activity of this compound on crude cytosolic glutathione S-transferase of RBL cells disclosed that it was relatively easy to achieve a 75% inhibition. But even a hundredfold increase in inhibitor concentration failed to cause any further inhibition of the reaction with DNCB. This suggested that the isozymes of glutathione S-transferase that are present in RBL cells may differ markedly in their susceptibility to inhibition by this drug. To examine this. we fractionated the cytosol of RBL cells by chromatofocusing after we had first isolated the mixed glutathione S-transferases by affinity chromatography on glutathione-sepharose. Again, we were able to make 10 pools of more or less defined regions of activity with DNCB as substrate. These pools differed from those obtained from rat liver in that by far most of the enzyme activity was distributed in the fractions that had a pl of less than 7.0, while the liver enzymes were predominantly in fractions having pl greater than 9.6. An examination of the susceptibility of these pools to inhibition by t-J-60,257 revealed that the pool with a pl of X.2 was completely resistant to this compound while the EC,, values for the other pools spanned a tenfold range in concentrations (Fig. 4). In conclusion, therefore. we believe that the LTC synthetase of RBL ceils is a unique enzyme. We have also presented evidence that both RBL cell and rat liver cytosolic glutathione S-transferases may be a more complicated mixture than had been hitherto suspected. The substrate specificity of the enzymes from

““LVME NUMBER

Leukotriene C synthetase

74 3, PART 2

liver, but not that of those from RBL cells, is sufficiently broad that LTA, can be recognized and converted to LTC,. It may be possible to capitalize on the specificity of the LTC synthetase for developing selective inhibitors of sulfidopeptide leukotriene synthesis. REFERENCES

I. Bach MK, Brashler JR, Smith HW, Fitzpatrick FA, Sun FF, McGuire JC: 6,9-Deepoxy-6,9-(phenylimino)-~6~*-prostaglandin I,, (U-60,257), a new inhibitor of leukotriene C and D synthesis:In vitro studies. Prostaglandins23:759, 1982 2. Bach MK, Brashler JR, Morton DR. Steel LK, Kaliner MA, Hugli TE: Formationof leukotrienesC and D andpharmacologic modulation of their synthesis.Adv ProstaglandinThromboxane Leukotriene Res 9:103, 1982 3. Fitzpatrick FA, Morton DP, Wynalda MA: Albumin stabilizes leukotriene A,. J Biol Chem 257:4680, 1982 4. Jakoby WB, Ketley JN, Habig WG: Rat glutathione S-transferases:Binding and physical properties.In Arias IW, Jakoby WB, editors: Glutathione: Metabolism and function. New York, 1976, Raven Press, p 213 5. Jakschik BA, Kuo CG: Subcellular localization of leukotriene forming enzymes.Adv ProstaglandinThromboxaneLeukotriene Res 11:141, 1983 6. Morgenstem R, GuthenbergC, DePierre JW: Microsomal glutathione S-transferase.Purification, initial characterization,and demonstrationthat it is not identical to the cytosolic glutathione S-transferasesA, B and C. Eur J Biochem 128:243, 1982 7. Sun FF, McGuire JC: Inhibition of human neutrophil arachidonate S-lipoxygenaseby 6,9-deepoxy-6,9-(phenylimino)-A6,*prostaglandin I, (U-60257). Prostaglandins26:211, 1983

357

DISCUSSION Anthony Ford-Hutchinson: Do you have any results of clinical trials with inhibitor U-60,257? Michael Bach: Unfortunately, we arenot yet in a position to discuss the clinical results. Aaron Marcus: Does U-60,257 inhibit 12-lipoxygenase activity? If it does not, then its action would be more specific.

M. Bach: The U-60,257 has no effect on 12-hydroxyeicosatetraenoicacid production in human platelets up to 104M, which was the highest dose tested. Edward Goetzl: It appearsthat more than 75% of your LTC, synthetaseactivity was found as pH levels of 7.0 or lower, by chromatofocusing,and theseactivities had similar profiles of inhibition by severalantagonists.To what extent are the quantitatively more minor activities at other pH levels, which show different inhibitor profiles, sufficiently characterizedto be consideredsubclassesof the enzyme? M. Bach: The previously described isozymes differ mainly by p1.Thus chromatofocusingseemedan ideal method to separatethesesubclasses.Indeed, we find many more forms than previously described.Those in RBL cells are on the whole acidic, while those in liver are largely basic. Robert Murphy: Have you looked at the sterochemistry of LTC, produced from LTA, by meansof the soluble enzyme isolated from rat liver? M. Bach: All we know is that stereochemically pure LTA, lithium is converted to biologic activity. We do not even know whether LTC, or LTD, is formed.