Aug 19, 1985 - ABSTRACT. When leukotriene (LT) A4 was incubated with subcellular fractions of sonicated rat basophilic leukemia. (RBL) cells in the ...
Proc. Nati. Acad. Sci. USA Vol. 82, pp. 8399-8403, December 1985
Biochemistry
Isolation and characterization of leukotriene C4 synthetase of rat basophilic leukemia cells (arachidonic acid)
TANIHIRO YOSHIMOTO, RoY J. SOBERMAN, ROBERT A. LEWIS, AND K. FRANK AUSTEN Department of Medicine, Harvard Medical School, and Department of Rheumatology and Immunology, Brigham and Women's Hospital, Boston, MA 02115
Contributed by K. Frank Austen, August 19, 1985
ABSTRACT When leukotriene (LT) A4 was incubated with subcellular fractions of sonicated rat basophilic leukemia (RBL) cells in the presence of glutathione, the enzyme producing LTC4, designated LTC4 synthetase, was found in the 105,000 x g pellet (microsomes) with a 3-fold enrichment in specific activity over that of the sonicate. The Identification of the reaction product as LTC4 was confirmed by its identical retention time on reverse-phase HPLC to that of synthetic LTC4, the incorporation of [3H]glutathione into the product, its reactivity in a radioimmunoassay, and its UV absorption spectrum. In contrast, glutathione S-transferase activity, measured spectrophotometrically with 1-chloro-2,4-dinitrobenzene, was detected predominantly in the 105,000 x g supernatant (89%) and also in the microsomes (7%). The microsomal glutathione S-transferase and LTC4 synthetase were solubilized with 0.4% Triton X-102 and separated by DEAE-Sephacel chromatography; the former appeared in the effluent and the latter in the eluate after the addition of 0.16 M NaCl to the equilibration buffer. Solubilized, microsomal glutathione S-transferase was inhibited by S-hexylglutathione with an IC50 of 36 ,IM and was stable at 40WC for 5 mi, whereas LTC4 synthetase was only slightly inhibited (IC50, 2.3 mM) by S-hexylglutathione and retained no activity after incubation at 40'C for 5 min. The partially purified LTC4 synthetase showed a specific activity of 1.34 ± 0.51 nmol of LTC4 per 10 min per mg of protein (mean ± SD, n = 9), representing a 10-fold purification from the sonicate and catalyzed the dose- and time-dependent production of LTC4 from LTA4 and glutathione. The apparent Km values for LTA4 and glutathione were estimated by Lineweaver-Burk plots to be 5-10 IAM and 3-6 mM, respectively. These results indicate that the conjugation of LTA4 with glutathione to form LTC4 is catalyzed by a unique microsomal enzyme.
homogenate of RBL cells. LTC4 synthetase has now been localized solely in the 105,000 x g pellet of RBL cells, solubilized, resolved chromatographically from detoxifying glutathione S-transferases by DEAE-anion-exchange chromatography, and characterized kinetically in this partially purified form. MATERIALS AND METHODS Materials. [14,15-3H]LTA4 methyl ester (50 Ci/mmol, 1 Ci = 37 GBq), [14,15-3H]LTC4 (35.7 Ci/mmol), [glycine-2-
3H]glutathione (892 mCi/mmol) (New England Nuclear), glutathione, S-hexylglutathione, 1-chloro-2,4-dinitrobenzene, diisopropyl fluorophosphate, Hepes, bovine serum albumin, nonionic detergents (Tritons X-100, X-102, X-114, X-165, X-305, Tween 20, and Tween 40), prostaglandin B2 (Sigma), DEAE-Sephacel (Pharmacia), HPLC-grade methanol and acetonitrile (Burdick and Jackson, Muskegon, MI) were purchased. LTA4 methyl ester, LTB4, LTC4, and LTD4 were synthesized (14-17) and provided by E. J. Corey of Harvard University. Hydrolysis of LTA4 Methyl Ester. Hydrolysis was carried out by a modification of the method described (18, 19) in which LTA4 methyl ester (0.3 mg) was dissolved in 3 ml of 60 mM lithium hydroxide in tetrahydrofuran/water (7:3, vol/vol) and allowed to react under argon in the dark at 240C for 15 hr. The solvent was removed by lyophilization for 6 hr, and the residue was dissolved in dry ethanol to give a concentration of 2 mM. The stock solution of LTA4 (lithium salt) was stored at -70'C under argon for at least 3 months without appreciable degradation. Preparation of Subcellular Fractions of RBL Cells. RBL cells were grown in RPMI 1640 supplemented with 10% (vol/vol) fetal calf serum, 100 units of penicillin/ml, 100 ,ug of streptomycin/ml, and 25 ,ug of gentamycin/ml, in suspension cultures to a cell density of 1-1.5 x 106 cells per ml at the Cell Culture Center at the Massachusetts Institute of Technology (Cambridge, MA). The cells were harvested, washed twice in 10 mM Tris HCl, pH 7.4/1 mM EDTA/0.25 M sucrose/2 mM diisopropylfluorophosphate, and suspended in the same buffer at a density of 108 cells per ml. The suspension of 1.3 x 109 cells was sonicated for 30 sec at 20 kHz with a Branson Sonifier model 350, and a portion of crude sonicate was retained. The remainder of the sonicate was centrifuged at 1000 x g for 10 min at 40C to remove unbroken cells and debris, and the supernatant was then centrifuged at 10,000 x g for 10 min at 40C. The resulting 10,000 x g precipitate was washed, recentrifuged, and then resuspended in 4.6 ml of the initial buffer. The initial 10,000
In the biosynthetic pathway of leukotrienes, arachidonic acid is first oxygenated by 5-lipoxygenase to form 5-hydroperoxy6,8,11,14-icosatetraenoic acid, which is then dehydrated to yield leukotriene (LT) A4 (1, 2). LTA4 can be enzymatically hydrolyzed to produce LTB4 by human leukocytes (3), rat basophilic leukemia (RBL) cells (4, 5), human blood plasma (6), and human erythrocytes (7). Alternatively, LTA4 can be conjugated with glutathione to form LTC4 by human leukocytes (8), RBL cells (9, 10), and rat liver (11). LTA4 hydrolase has been localized to the cytosol, purified to homogeneity, and analyzed kinetically (12); but the enzyme catalyzing the conjugation of LTA4 with glutathione, termed LTC4 synthetase, has not been well characterized or fully distinguished from glutathione S-transferases involved in the detoxification reaction of xenobiotics (13). LTC4 synthetase has been reported to be localized to the 10,000 x g pellet (9) or equally to the 25,000 x g and 105,000 x g pellets (10) of the
x g supernatant was centrifuged at 105,000 x g for 45 min at 4°C, and the supernatant, representing the cytosol, was separated from the pellet, which was enriched for micro-
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Abbreviations: LT, leukotriene; RBL, rat basophilic leukemia. 8399
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Proc. Natl. Acad. Sci. USA 82
Biochemistry: Yoshimoto et al.
somes. The microsomal fraction was suspended in the same buffer, and was centrifuged and resuspended in 5.4 ml of the same buffer using a Potter-Elvehjem homogenizer. Protein concentrations were determined by the method of Lowry et al. (20) in the presence of 0.8% NaDodSO4. Enzyme Assays. LTC4 synthetase was assayed in a standard reaction mixture containing 50 mM Hepes (pH 7.6), 20 AM LTA4 (lithium salt, 10 nmol of LTA4 in 5 ,1 of ethanol), 5 mM glutathione, and enzyme, in a final volume of 0.5 ml. The reaction was carried out at 240C for 10 min and was terminated by the addition of 0.4 ml of a solvent mixture of acetonitrile/methanol/acetic acid (50:50:1, vol/vol) containing 0.1 ug of prostaglandin B2 as an internal standard. The mixture was centrifuged at 26,000 x g for 10 min to precipitate the proteins, and 0.6 ml of the extract was injected into an Ultrasphere-ODS column (Beckman, 5-Am particle, 4.6 x 250 mm). The column was eluted with a solvent mixture of acetonitrile/methanol/0.1% acetic acid at pH 5.6 (50:50:100, vol/vol) at a flow rate of 1 ml/min. The absorbance at 280 nm was continuously monitored with a Beckman model 164 variable-wavelength detector equipped with a model C-1A integrator. The amount of LTC4 produced was calculated by taking a ratio of integrated absorbance values between LTC4 (retention time, 9.3 min) and prostaglandin B2 (14.8 min) and comparing this ratio to a standard curve. The recovery of 0.8 pM [3H]LTC4 incubated in the standard reaction mixture, resolved by reverse-phase HPLC, and quantitated in 1-ml fractions was 56.7 ± 1.8% (mean ± SD, n = 3). Glutathione S-transferase activity with 1 mM 1-chloro-2,4-dinitrophenol and 1 mM glutathione as substrate was measured spectrophotometrically by the method of Habig et al. (21). Radioimmunoassay of LTC4. LTA4 was incubated with the microsomes of RBL cells in the standard reaction mixture and chromatographed on reverse-phase HPLC in the standard solvent, and 1-ml fractions were collected. The solvent was removed under reduced pressure, and the residue was dissolved in 0.5 ml of 10 mM Tris HCl, pH 7.4/0.1% gelatin/0.9% NaCl/0.01% sodium azide. Samples (100 ILI) from each fraction were assessed for LTC4 by radioimmunoassay. The assay used a rabbit immune plasma in which the antibodies were elicited by a conjugate of LTD4 via its 5-hydroxy group with bovine serum albumin. In the assay with [3H]LTC4 used as the radioligand and the standard method of precipitating the immune complexes with a heterologous second (goat anti-rabbit IgG) antibody (22), the binding inhibition curve was linear between 0.033 and 1.0 ng of unlabeled LTC4, LTD4, or LTE4, and radioligand binding was inhibited by 50% with 0.20 ng of LTC4, 0.40 ng of LTD4, and 0.58 ng of LTE4. Glutathione, LTB4, monohydroxyicosatetraenoic acids, and prostaglandins were not recognized at quantities up to 100 ng in this assay.
RESULTS Subcellular Localization of LTC4 Synthetase. The sonicate of RBL cells (2.8 mg of protein, equivalent to 1.6 x 107 cells) was incubated with 20 ,M LTA4 and 5 mM glutathione at 24°C for 10 min, and the extract of the reaction mixture was analyzed by reverse-phase HPLC with monitoring of absorbance at 280 nm to detect a conjugated triene. Four maor peaks were resolved in addition to that of the internal standard, prostaglandin B2 (retention time, 14.8 min). The generated peaks corresponded to LTC4 (9.3 min), LTB4 (27.8 min), and the nonenzymatic conversion products of LTA4, diastereoisomeric 6-trans-LTB4 (21.6 min and 23.4 min) (Fig. LA). When [3H]LTA4 was incubated and the radioactivity of the 1-ml fractions of reverse-phase HPLC was determined by scintillation counting, the compounds that chromatographed together with LTC4 and LTB4 accounted for 2.8% and 5.2%,
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FIG. 1. Transformation of LTA4 by subcellular fractions of RBL cells. The reactions were carried out in the standard reaction mixture containing 2.8 mg (equivalent to 1.6 x 10' cells) of sonicate (A), 0.75 mg of the 10,000 x g pellet (B), 2.9 mg of cytosol (C), or 1.9 mg of microsomes (D) and the products were analyzed by reverse-phase HPLC. Arrows indicate the retention times of authentic compounds. PGB2, prostaglandin B2.
respectively, of added [3H]LTA4 (data not shown). A parallel sonicate was subjected to subcellular fractionation to define the localization of the LTC4 generating activity. A small UV-absorbing peak cochromatographed with authentic LTC4 when the 10,000 x g pellet was incubated with LTA4 (Fig. 1B). With the cytosol fraction (Fig. 1C), a peak coelutdng with authentic LTB4 was apparent, whereas only a small peak cochromatographing with LTC4 was observed. The microsomal fraction yielded a prominent UV-absorbing peak corresponding to authentic LTC4 (Fig. 1D). The product formed by the incubation of LTA4 and glutathione with 1.7 mg of RBL cell microsomes under the standard reaction conditions was isolated by reverse-phase HPLC (Fig. 2A) and analyzed by scanning spectroscopy from 240 to 320 nm. It showed a characteristic UV spectrum of authentic LTC4 (23) with a peak at 280 nm and shoulders at 270 nm and 291 nm. When 1-ml fractions were collected and analyzed by radioimmunoassay, a single peak was identified, which corresponded to the retention time of authentic LTC4 (Fig. 2B). As determined by radioimmunoassay, this peak contained 0.92 ,g of LTC4, similar to the value of 0.73 ,g obtained by integrated absorbance. In the parallel reaction mixture, in which [3H]glutathione was included as a substrate, 0.6% of the added radioactivity cochromatographed with the product exhibiting a retention time identical to that of authentic LTC4 (Fig. 2C). Based on these criteria, the product formed by the incubation of LTA4 with the microsomal fraction of RBL cells in the presence of glutathione was identified as LTC4. Table 1 summarizes the subcellular localization of both LTC4 synthetase and glutathione S-transferase with LTA4 and 1-chloro-2,4-dinitrobenzene, respectively, used as substrates. The microsomal fraction represented 70% of recovered LTC4 synthetase activity and shqwed 3.3-fold enrichment of the enzyme. Approximately 8% of the LTC4 synthetase was associated with the 10,000 x g pellet and 22% was associated with the cytosol. The specific activity of the microsomal LTC4 synthetase in five experiments was found to be 0.55 ± 0.10 nmol of LTC4 per 10 min per mg of protein (mean ± SD). In contrast, 89% of the recovered glutathione S-transferase activity was found in the cytosol, with a 50% increase in specific activity, whereas only 7% of the recov-
Biochemistry: Yoshimoto et al.
Proc. Natl. Acad. Sci. USA 82 (1985)
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detergent at a concentration of 0.5% (wt/vol). The mixture was stirred for 30 min at 4°C and then centrifuged at 105,000
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Retention time, min FIG. 2. Identification of the product formed from LTA4 and glutathione by microsomes of RBL cells. Three parallel reaction mixtures containing 1.7 mg of microsomal protein, 20 ALM LTA4, and 5 mM glutathione (A and B) or supplemented [3H]glutathione (C) were resolved by reverse-phase HPLC and assessed for LTC4 by integrated absorbance (A), immunoreactivity (B), and radioactivity (C). Arrows indicate the retention times of authentic LTC4 and prostaglandin B2 (PGB2).
ered activity was associated with microsomes and had a decreased specific activity. Solubilization, Partial Purification, and Kinetic Characterization of LTC4 Synthetase. The nonionic detergents used to solubilize LTC4 synthetase of RBL microsomes that were examined are as follows: Tritons X-100 (hydrophilic lipophilic balance value, 13.5), X-102 (14.6), X-114 (12.4), X-165 (15.8), and X-305 (17.3), Tween 20 (16.7), and Tween 40 (15.6). A portion of the microsomal fraction, at a concentration of 5.6 mg of protein/ml, was mixed with each
x g for 45 min. The LTC4 synthetase activity in the supernatant was determined with LTA4 used as a substrate in the standard assay system. In a comparative experiment, Tritons X-100, X-102, and X-165 were found to effectively solubilize 61, 58, and 54% ofthe microsomal enzyme activity, respectively. The preparation of LTC4 synthetase solubilized with Triton X-102 gave higher specific activity than those with Tritons X-100 and X-165. In a concentration-dependent analysis, 74 ± 12% (mean ± SD, n = 4) of the microsomal LTC4 synthetase activity was solubilized with 0.4% Triton X-102, and the solubilized enzyme showed a specific activity of 0.70 ± 0.19 nmol of LTC4 per 10 min per mg of protein (mean ± SD, n = 4). Higher concentrations of Triton X-102 were less effective, e.g., 54% (mean, n = 2) ofthe microsomal enzyme activity was solubilized with 1% Triton X-102. Glutathione S-transferase activity in the microsomes was also solubilized by 0.4% Triton X-102 with a recovery of 46 ± 4% (mean + SD, n = 4). A 6-ml portion of the solubilized microsomal enzymes at a concentration of 5 mg of protein/ml was applied to a DEAE-Sephacel column (1.5 x 6 cm) that had been equilibrated with 20 mM sodium phosphate (pH 7.6), 1 mM EDTA, and 0.1% Triton X-102. The column was washed with equilibration buffer and eluted with the same buffer containing 0.16 M NaCi. Fractions of 2.4 ml were collected and examined for the presence of both LTC4 synthetase and glutathione S-transferase activities. Glutathione S-transferase activity was detected in the effluent with a recovery of 74%, and LTC4 synthetase was found in the eluate with a recovery of 66% (Fig. 3). The partially purified enzyme showed a specific activity of 1.34 ± 0.51 nmol of LTC4 per 10 min per mg of protein (mean ± SD, n = 9). To further distinguish LTC4 synthetase from glutathione S-transferase, their stability at various temperatures and their inhibition by S-hexylglutathione, which is a known inhibitor of glutathione S-transferase of rat liver (24), were examined after separation by DEAE-Sephacel chromatography. When each enzyme was incubated at 40°C for 5 min, 90%o of glutathione S-transferase activity remained, whereas all of the LTC4 synthetase activity was lost. When increasing concentrations of S-hexylglutathione were added to 0.36 mg of protein containing LTC4 synthetase and to 0.094 mg of protein containing glutathione S-transferase, this compound inhibited the glutathione S-transferase in a dose-dependent manner with an IC50 of 36 ,uM. In contrast, the LTC4 syn-
Table 1. Subcellular localization of LTC4 synthetase and glutathione S-transferase Activity LTC4 synthetase Glutathione
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Total nmol of LTC4 per 10 min 34.9 1.96 17.2
Specific S-transferase nmol of Total Specific LTC4 nmol of nmol of per 10 product* product min per per mn per mg per mg min
Protein, Fraction mg Sonicate 276 0.13 6195 22.4 Pellet 13.8 0.14 173 12.5 Microsomes 40.5 0.43 338 8.3 116 Cytosol 5.52 0.05 4049 34.9 LTC4 synthetase and glutathione S-transferase were assayed under standard conditions with LTA4 or 1-chloro-2,4-dinitrobenzene, respectively, as substrate. The pellet was from a 10,000 x g centrifugation. *Product refers to the conjugate of reduced glutathione and 1-chloro2,4-dinitrobenzene.
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Fraction FIG. 3. Resolution of LTC4 synthetase and glutathione S-transferase of RBL cell microsomes by DEAE-Sephacel chromatography. Enzyme activity of LTC4 synthetase (e) and glutathione S-transferase (o) was determined by taking 300-,u1 and 200-,lI samples from the 2.4-ml fractions, respectively, under the standard assay conditions. The arrow indicates the increase in the salt concentration of the buffer to 0.16 M NaCl. The dotted line indicates protein concentration.
Biochemistry: Yoshimoto et al.
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Proc. Natl. Acad. Sci. USA 82
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FIG. 4. Protein dependence and time course of LTC4 production from LTA4 and glutathione with the partially purified enzyme. (A) Reactions were carried out under the standard conditions with increasing amounts ofpartially purified enzyme (o) and with partially purified enzyme that had been boiled for 5 miin (e). (B) Time course of LTC4 generation by partially purified LTC4 synthetase, 0.69 rug, under the standard reaction conditions. Each point is an average of two experiments.
thetase was not affected by concentrations of S-hexylglutathione less than 100 ,uM, and an IC50 of 2.3 mM was obtained. The protein dependence and the time course of the reaction of the partially purified LTC4 synthetase with 20 pM LTA4 and 5 mM glutathione were examined. The reaction velocity increased in a linear fashion over the range of 0-0.8 mg of protein, and heat-inactivated enzyme was totally inactive (Fig. 4A). The transformation of LTA4 to LTC4 approached a maximum at about 15 min (Fig. 4B). The addition of bovine serum albumin (18) (
