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B I O S Y N T H E S I S , E N Z Y M O L O G Y , A N D C H E M I C A L SYNTHESIS
[36]
the Km is lower (0.08/~M) but the Vm~xis markedly less (5 pmol x min -~ x mg-1). Acknowledgment This work was supported by Grants AI-22563, AR-38633 from the National Institutes of Health and in part by a grant-in-aid from the American Heart Association, Massachusetts Affiliate, Inc.
[36] Cytosolic L i v e r E n z y m e s C a t a l y z i n g H y d r o l y s i s of L e u k o t r i e n e A4 to L e u k o t r i e n e B4 and 5 , 6 - D i h y d r o x y e i c o s a t e t r a e n o i c Acid B y JESPER Z. HAEGGSTROM
The unstable allylic epoxide leukotriene (LT) A4 [5(S)-trans-5,6-oxido7,9-trans-1 I, 14-cis-eicosatetraenoic acid] is a key intermediate in the biosynthesis of the biologically active leukotrienes. Enzymatic hydrolysis of LTA4 may be catalyzed by two different enzymes with several functional and structural differences) Thus, LTA4 hydrolase (EC 3.3.2.6) converts the epoxide into LTB4, whereas cytosolic epoxide hydrolase (EC 3.3.2.3) generates 5(S),6(R)-dihydroxy-7,9-trans11,14-cis-eicosate-traenoic acid (5,6-DiHETE).2 Epoxide hydrolases, microsomal or cytosolic, are believed to be involved in detoxification of various harmful xenobiotic epoxides. Although this chapter is primarily concerned with LTA4 hydrolase and cytosolic epoxide hydrolase from guinea pig and mouse liver, respectively, the methods have a broader application since extended studies with synthetic LTA4 and xenobiotic epoxides have shown that both enzymatic activities have a widespread occurrence in mammalian tissues. 3-5 J. Haeggstr6m, J. Meijer, and O. RAdmark, J. Biol. Chem. 261, 6332 (1986). 2 j. Haeggstr6m, A. Wetterholm, M. Hamberg, J. Meijer, R. Zipkin, and O. R~tdmark, Biochim. Biophys. Acta 958, 469 (1988). 3 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 4 j. F. Medina, J. Haeggstr6m, M. Kumlin, and O. R~tdmark, Biochim. Biophys. Acta 961, 203 (1988). 5 S. S. Gill and B. D. Hammock, Biochem. Pharmacol. 29, 389 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A 4
325
Preparation of Liver Homogenates and Subcellular Fractions The liver of an anesthetized and heparinized guinea pig or mouse is perfused with cold 0.9% NaCl. After cautious removal of the gall bladder, the liver is homogenized in three parts (v/w) 50 mM potassium phosphate buffer, pH 7.4, utilizing a Potter-Elvehjem homogenizer. If the liver will be used for purification of LTA4 hydrolase, the homogenization should be carried out in l0 mM Tris/HC1, pH 8, 5 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. A simple subcellular fractionation is performed by sequential centrifugation at 20,000 g for 30 rain followed by 105,000 g for 60 min at 4°. The resulting pellets should be washed with buffer and recentrifuged 2-3 times prior to incubations. General Procedures for Incubations and Extractions Typically, 500-/zl aliquots of homogenates or subcellular fractions are incubated at 37° for 10 min with LTA4 lithium salt (10-20/~M) added as an ethanol solution. To retain an unimpaired activity of cytosolic epoxide hydrolase it is essential not to incubate with LTA4 dissolved in tetrahydrofuran, a solvent frequently used in saponification of LTA4 methyl ester. The formation of 5,6-DHETE from LTA4 is inhibited at low concentrations (1-2%) of tetrahydrofuran. 6 The reactions are terminated by the addition of 3 to 5 volumes of methanol, containing a defined amount of internal standard, e.g., prostaglandin (PG) B1. Precipitated proteins are removed by centrifugation or filtration. After acidification of the samples to an apparent pH ~ 3 with 0.1 M HCI, products may be conveniently extracted either with diethyl ether or on Sep-Pak C~8 cartridges (Waters Associates, Milford, MA) eluted with methyl formate after previous washings .7 In our hands, the former method gives more reproducible recoveries of LTA4 transformation products. The extract is evaporated under a stream of nitrogen and the residue reconstituted in an appropriate mobile phase for further analysis by HPLC. Characterization of Enzymatic Hydrolysis Products The basis for identification of LTB4 and 5,6-DHETE is reversed-phase HPLC. Free acids are separated by a Nucleosil Cls column (MaehereyNagel, Diiren, FRG) (250 x 4.5 mm) eluted with a mixture of methanol/ water/acetic acid (70:30:0.01, v/v/v) at 1 ml/min. The ultraviolet absorbance is monitored at 270 nm. A typical chromatogram is depicted in 6 j. Meijer and J. W. DePierre, Fur. J. Biochem. 150, 7 (1985). 7 W. S. Powell, Prostaglandins 20, 947 (1980).
326
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
BUFFER
[36]
CYTOSOL
25-
20-
% x c "glS-
E
"~1o-
.~_
C
lU
5-
o_r AUFS: 0.05
A U F S : 0.05
D
B
c
°l 0
E
1'0 2'0 3'0 4'0
o 1'o 2'o a'o io Minutes
FIG. 1. Reversed-phase HPLC analysis of products formed in an incubation of mouse liver cytosol (10 mg of protein/ml) with 20/~M PH]LTA4 (37°, 10 rain) as compared to a control incubation with 50 mM potassium phosphate buffer, pH 7.4. To each incubate (500 pd), 420 ng of PGB~ was added as internal standard. The column (Nucleosil C]s, 250 x 4.5 ram) was eluted with methanol/water/acetic acid (70 : 30 : 0.01, v/v/v) at I ml/min. Upper panels show the distribution of tritium in each collected fraction as determined by liquid scintillation counting. Lower panels show the continuous recordings of ultraviolet absorption at 270 nm. Peak A: A6-trans-LTB4; peak B: 12-epi-A6-trans-LTB4; peak C: LTB4; peak D: 5(S),6(R)-dihydroxy-7,9-trans-I 1,14-cis-eicosatetraenoic acid; peak E: Unidentified isomers of 5,6-DHETE.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A4
327
Fig. 1. In this chromatographic system, LTB4 and 5,6-DHETE elute approximately in 19 and 32 min, respectively. Both compounds may be further purified and examined by straightphase HPLC, UV spectroscopy, and gas chromatography coupled to mass spectrometry (GC-MS). The resolution of various isomers of 5,6-DHETE (methyl esters) is poor in straight-phase HPLC, which has limited our use of this technique. In ultraviolet spectroscopy, with methanol as the solvent, 5,6-DHETE appears as a conjugated triene with maximal absorbance at 272 nm and with shoulders at 263 and 284 nm. For 5,6-DHETE, we use an extinction coefficient of 4.0 x 104 M -I x cm -~ (at 272 nm) due to the structural similarity with LTA4. For GC-MS, the free acids of LTB4 and 5,6-DHETE are converted to methyl ester trimethylsilyl ethers and analyzed on a capillary column (SE-30, fused silica, 25 m x 0.25 mm) coupled directly to a mass spectrometer (VG 7070E). The gas chromatograph is operated isothermally (2400-250 °) with helium as the carrier gas. The analysis of 5,6-DHETE by GC-MS consistently results in two peaks with C values of 23.8 and 24.7 and with similar mass spectra (for details see Ref. 1). The latter peak most probably reflects an 11-cis to l l-trans isomerization of the parent compound during the analytical procedure.4 Subcellular Distribution of Epoxide Hydrolase and LTA4 Hydrolase in Liver Tissue The distribution of enzymatic activity in subcellular fractions of mouse liver homogenates differ between cytosolic epoxide hydrolase and LTA4 hydrolase. Typically, formation of LTB4 is almost exclusively detected in the high-speed supernatant whereas formation of 5,6-DHETE is seen in both the 105,000 g supernatant (cytosol) and the 20,000 g pellet. The activity in the pellet fraction most probably reflects the presence of mitochondrial/peroxisomal epoxide hydrolase, an enzymatic activity similar to cytosolic epoxide hydrolase with regard to molecular weight, substrate specificity, and antigenic properties, s Substrate Specificity Cytosolic (and microsomal) epoxide hydrolase has a broad substrate specificity and has frequently been characterized with stable aromatic and aliphatic xenobiotic epoxides. Besides LTA4, a number of other epoxides derived from arachidonic acid, e.g., 15(S)-trans-14,15-oxido-5,8-cis10,12-trans-eicosatetraenoic acid (14,15-LTA4), may serve as substrates 8 j. Meijer, G. Lundqvist, and J. W. DePierre, Eur. J. Biochem. 167, 269 (1987).
328
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[36]
for cytosolic epoxide hydrolase. 9 The microsomal enzyme (from rat liver) has no detectable catalytic activity toward LTA4.1 In contrast, the substrate specificity of LTA4 hydrolase seems to be very narrow. I° Two isomers of LTA4, namely LTA3 and LTAs, may be enzymatically hydrolyzed by this enzyme. 11,12Thus, LTA5 is transformed into LTBs, although with lower efficiency as compared to hydrolysis of LTA4 into LTB4, whereas LTA3 is a very poor substrate. Purification of Cytosolic Epoxide Hydrolase from Mouse Liver Cytosol Cytosolic epoxide hydrolase can be purified to apparent homogeneity from mouse liver by a procedure that involves column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite, utilizing xenobiotic substrate in the assay of enzyme activity. 13 Purification of LTA4 Hydrolase from Guinea Pig Liver Cytosol
Step 1. Precipitate nucleic acids by adding streptomycin sulfate in water (10% w/v) to cytosol with continuous stirring on ice. After 30 min, remove the precipitate by centrifugation (10,000 g, 15 rain, 4°). Proceed with ammonium sulfate precipitation (on ice) and collect the 40-80% saturated fraction by centrifugation. Dissolve the pellet in 50 mM Tris/ HCI, pH 8.0, to give a protein concentration of 25 mg/ml. Step 2. The 40-80% ammonium sulfate fraction is further purified by molecular exclusion chromatography on a column packed with AcA 44 (LKB-produkter, Bromma, Sweden) or equivalent, equilibrated with 50 mM Tris/HC1, pH 8.0. For good resolution, the sample volume should be adjusted to less than 2% of the bed volume. Step 3. Pool the active fractions from step 2 and treat the sample with 2 rnM dithiothreitol for 30 min. Load this sample onto DEAE-cellulose (Whatman DE-52) preequilibrated with 10 mM Tris/HC1, pH 8.0. When nonadsorbing proteins are eluted, apply a linear gradient of KCI (50-250 raM) that increases with approx. 0.25 mM/ml. Active fractions will appear in the range of 90 to 130 mM KCI. In this chromatographic step, most of the cytosolic epoxide hydrolase activity will be separated from the LTA4 hy9 A. Wetterholm, J. Haeggstr6m, M. Hamberg, J. Meijer, and O, R~dmark, Eur. chem. 173, 531 (1988). io N. Ohishi, T. Izumi, M. Minami, S. Kitamura, Y. Seyama, S. Ohkawa, S. H. Yotsumoto, F. Takaku, and T. Shimizu, J. Biol. Chem. 262, 10200 (1987). 11 j. F. Evans, D. J. Nathaniel, R. J. Zamboni, and A. W. Ford-Hutchinson, J. Biol. 260, 10966 (1985). 12 D. J, Nathaniel, J. F. Evans, Y. Leblanc, C. L6veill6, B. J. Fitzsimmons, and Ford-Hutchinson, Biochem. Biophys. Res. Commun. 131, 827 (1985). t3 j. Meijer and J. W. DePierre, Eur. J. Biochem. 148, 421 (1985).
J. Bio-
Terao, Chem.
A. W.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A4
329
drolase activity. The former enzyme elutes in the flow-through peak and early fractions of the gradient. Step 4. Active fractions from step 3 are pooled and concentrated by ultrafiltration (Amicon PM10, Danvers, MA) in the presence of 2 mM dithiothreitol. The reducing agent should be added at this stage in order to prevent the appearance of multiple peaks of activity in the following chromatography. Anion-exchange separation with fast protein liquid chromatography (FPLC) is then performed on a column, Mono Q HR 5/5 (Pharmacia Fine Chemicals, Uppsala, Sweden), attached to the FPLC system and equilibrated at 10°-12° with 10 mM Tris/HC1, pH 8.0, supplemented with 0.1 mM dithiothreitol. The sample buffer is exchanged to the column equilibration buffer by molecular exclusion chromatography (PD-10, Pharmacia Fine Chemicals). Inject an aliquot of the enzyme pool, await the peak of nonadsorbing proteins, and then apply a gradient of KC1 (0-200 mM) in a total volume of 50 ml. Enzyme activity is recovered in fractions between 40-85 mM KCI. Step 5. Adsorption chromatography on hydroxyapatite is performed on a column (BioGel HPHT, Bio-Rad, Richmond, CA) attached to the FPLC system and equilibrated with 10 mM potassium phosphate buffer, pH 7.0, supplemented with 0.3 mM CaC12 and 0.1 mM dithiothreitol. Concentration and desalting of collected fractions from step 4 are conveniently carried out by repeated dilution and ultrafiltration of the sample with the column equilibration buffer. The sample is applied to the column at a flow rate of 0.25 ml/min and after elution of nonadsorbing proteins, the flow is increased to 0.5 ml/min and a linear gradient of phosphate in two steps (10-160 mM in 50 ml followed by 160-310 mM in 5 ml) is developed by mixing with 500 mM potassium phosphate buffer, pH 7.0, supplemented with 6/~M CaCI2 and 0.1 mM dithiothreitol. Enzymatic activity is collected between 60-85 mM potassium phosphate. Step 6. The final purification is achieved by chromatofocusing on a Mono P column (HR 5/20, Pharmacia Fine Chemicals) equilibrated with 25 mM triethanolamine, the pH adjusted to 8.3 with iminodiacetic acid, and supplemented with 0.1 mM dithiothreitol. The sample buffer from step 5 is exchanged to the alkaline starting buffer by repeated concentration and dilution as described above. To achieve maximal resolution in the chromatography, the column should be washed with 1-2 ml of 2 M sodium iminodiacetate and reequilibrated prior to each sample injection. Proteins are eluted with a pH gradient (8-5) by changing the buffer to a mixture of Polybuffer 74 and 96 (70 : 30 by volume, Pharmacia), the pH adjusted to 5.0 with iminodiacetic acid, and supplemented with 0.1 mM dithiothreitol, at a flow rate of 0.5 ml/min. Immediately after each chromatographic run, the pH should be measured in collected fractions and restored to an alkaline pH with 1 M Tris/HC1, pH 8.0, to prevent inactivation of the enzyme. In
330
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[36]
TABLE I PURIFICATION OF ETA4 HYDROLASE FROM GUINEA PIG LIVER
Fraction Cytosol Precipitations AcA 44b DEALcellulose Mono QC Hydroxyapatite Mono pd
Volume (ml) 102 40 265 80 21 13.5 1.1
Total protein (rag) 1835 975 500 62 18 2.6 0.15
Totala activity (U)
Specific activity (U/rag)
Yield (%)
Purification (-fold)
0.73 1.42 1.45 0.86
0.0004 0.0015 0.0029 0.014
-100 102 61
-1 2 9
0.80 0.44 0.27
0.044 0.17 1.8
56 31 19
29 114 1200
a Aliquots of the enzyme from each step of purification were incubated with LTA4 (100/zM) at 37° for 1 min. Analysis and quantitation of LTB4 were performed with reversed-phase HPLC as described in the text. Enzymatic activity was expressed as micromoles of LTB4 formed per minute. In crude cytosol, lower amounts of activity were detected as compared to following steps most probably due to the presence of competing enzyme activities. Therefore, the activity in the ammonium sulfate precipitate was set to 100%. b Molecular exclusion chromatography. c FPLC, anion-exchange separation. d FPLC, chromatofocusing. this system, guinea pig liver LTA4 hydrolase is consistently r e c o v e r e d at a p H equal to 19/of 6.2. By this p r o c e d u r e the protein is purified m o r e than 1000-fold to near h o m o g e n e i t y with a yield of about 20% (Table I). The total activity found in the 4 0 - 8 0 % a m m o n i u m sulfate precipitate is usually higher than in crude cytosol which contains o t h e r e n z y m e s , e.g., cytosolic epoxide hydrolase and glutathione transferases, that could c o m p e t e with LTA4 hydrolase for the substrate. In general, results of e n z y m e activity determinations v a r y considerably depending on the incubation conditions, especially t e m p e r a ture and substrate solvent. Thus, the activity seems to be s o m e w h a t reduced w h e n LTA4 is added in tetrahydrofuran and incubations on ice m a y give m o r e p r o d u c t than incubations at 37 °, p r o b a b l y due to increased substrate stability. It should be noted that the purified e n z y m e is inactivated upon freezing. E n z y m e Kinetics Performing e n z y m e kinetic e x p e r i m e n t s with LTA4 confronts the investigator with the p r o b l e m o f substrate instability. At 25 ° in a buffer o f p H 7.4 without a n y organic solvent, the half-life o f LTA4 is
B I O S Y N T H E S I S , E N Z Y M O L O G Y , A N D C H E M I C A L SYNTHESIS
[36]
the Km is lower (0.08/~M) but the Vm~xis markedly less (5 pmol x min -~ x mg-1). Acknowledgment This work was supported by Grants AI-22563, AR-38633 from the National Institutes of Health and in part by a grant-in-aid from the American Heart Association, Massachusetts Affiliate, Inc.
[36] Cytosolic L i v e r E n z y m e s C a t a l y z i n g H y d r o l y s i s of L e u k o t r i e n e A4 to L e u k o t r i e n e B4 and 5 , 6 - D i h y d r o x y e i c o s a t e t r a e n o i c Acid B y JESPER Z. HAEGGSTROM
The unstable allylic epoxide leukotriene (LT) A4 [5(S)-trans-5,6-oxido7,9-trans-1 I, 14-cis-eicosatetraenoic acid] is a key intermediate in the biosynthesis of the biologically active leukotrienes. Enzymatic hydrolysis of LTA4 may be catalyzed by two different enzymes with several functional and structural differences) Thus, LTA4 hydrolase (EC 3.3.2.6) converts the epoxide into LTB4, whereas cytosolic epoxide hydrolase (EC 3.3.2.3) generates 5(S),6(R)-dihydroxy-7,9-trans11,14-cis-eicosate-traenoic acid (5,6-DiHETE).2 Epoxide hydrolases, microsomal or cytosolic, are believed to be involved in detoxification of various harmful xenobiotic epoxides. Although this chapter is primarily concerned with LTA4 hydrolase and cytosolic epoxide hydrolase from guinea pig and mouse liver, respectively, the methods have a broader application since extended studies with synthetic LTA4 and xenobiotic epoxides have shown that both enzymatic activities have a widespread occurrence in mammalian tissues. 3-5 J. Haeggstr6m, J. Meijer, and O. RAdmark, J. Biol. Chem. 261, 6332 (1986). 2 j. Haeggstr6m, A. Wetterholm, M. Hamberg, J. Meijer, R. Zipkin, and O. R~tdmark, Biochim. Biophys. Acta 958, 469 (1988). 3 T. Izumi, T. Shimizu, Y. Seyama, N. Ohishi, and F. Takaku, Biochem. Biophys. Res. Commun. 135, 139 (1986). 4 j. F. Medina, J. Haeggstr6m, M. Kumlin, and O. R~tdmark, Biochim. Biophys. Acta 961, 203 (1988). 5 S. S. Gill and B. D. Hammock, Biochem. Pharmacol. 29, 389 (1980).
METHODS IN ENZYMOLOGY, VOL. 187
Copyright © 1990by Academic Press, Inc. All fights of reproduction in any form reserved.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A 4
325
Preparation of Liver Homogenates and Subcellular Fractions The liver of an anesthetized and heparinized guinea pig or mouse is perfused with cold 0.9% NaCl. After cautious removal of the gall bladder, the liver is homogenized in three parts (v/w) 50 mM potassium phosphate buffer, pH 7.4, utilizing a Potter-Elvehjem homogenizer. If the liver will be used for purification of LTA4 hydrolase, the homogenization should be carried out in l0 mM Tris/HC1, pH 8, 5 mM phenylmethylsulfonyl fluoride, and 2 mM EDTA. A simple subcellular fractionation is performed by sequential centrifugation at 20,000 g for 30 rain followed by 105,000 g for 60 min at 4°. The resulting pellets should be washed with buffer and recentrifuged 2-3 times prior to incubations. General Procedures for Incubations and Extractions Typically, 500-/zl aliquots of homogenates or subcellular fractions are incubated at 37° for 10 min with LTA4 lithium salt (10-20/~M) added as an ethanol solution. To retain an unimpaired activity of cytosolic epoxide hydrolase it is essential not to incubate with LTA4 dissolved in tetrahydrofuran, a solvent frequently used in saponification of LTA4 methyl ester. The formation of 5,6-DHETE from LTA4 is inhibited at low concentrations (1-2%) of tetrahydrofuran. 6 The reactions are terminated by the addition of 3 to 5 volumes of methanol, containing a defined amount of internal standard, e.g., prostaglandin (PG) B1. Precipitated proteins are removed by centrifugation or filtration. After acidification of the samples to an apparent pH ~ 3 with 0.1 M HCI, products may be conveniently extracted either with diethyl ether or on Sep-Pak C~8 cartridges (Waters Associates, Milford, MA) eluted with methyl formate after previous washings .7 In our hands, the former method gives more reproducible recoveries of LTA4 transformation products. The extract is evaporated under a stream of nitrogen and the residue reconstituted in an appropriate mobile phase for further analysis by HPLC. Characterization of Enzymatic Hydrolysis Products The basis for identification of LTB4 and 5,6-DHETE is reversed-phase HPLC. Free acids are separated by a Nucleosil Cls column (MaehereyNagel, Diiren, FRG) (250 x 4.5 mm) eluted with a mixture of methanol/ water/acetic acid (70:30:0.01, v/v/v) at 1 ml/min. The ultraviolet absorbance is monitored at 270 nm. A typical chromatogram is depicted in 6 j. Meijer and J. W. DePierre, Fur. J. Biochem. 150, 7 (1985). 7 W. S. Powell, Prostaglandins 20, 947 (1980).
326
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
BUFFER
[36]
CYTOSOL
25-
20-
% x c "glS-
E
"~1o-
.~_
C
lU
5-
o_r AUFS: 0.05
A U F S : 0.05
D
B
c
°l 0
E
1'0 2'0 3'0 4'0
o 1'o 2'o a'o io Minutes
FIG. 1. Reversed-phase HPLC analysis of products formed in an incubation of mouse liver cytosol (10 mg of protein/ml) with 20/~M PH]LTA4 (37°, 10 rain) as compared to a control incubation with 50 mM potassium phosphate buffer, pH 7.4. To each incubate (500 pd), 420 ng of PGB~ was added as internal standard. The column (Nucleosil C]s, 250 x 4.5 ram) was eluted with methanol/water/acetic acid (70 : 30 : 0.01, v/v/v) at I ml/min. Upper panels show the distribution of tritium in each collected fraction as determined by liquid scintillation counting. Lower panels show the continuous recordings of ultraviolet absorption at 270 nm. Peak A: A6-trans-LTB4; peak B: 12-epi-A6-trans-LTB4; peak C: LTB4; peak D: 5(S),6(R)-dihydroxy-7,9-trans-I 1,14-cis-eicosatetraenoic acid; peak E: Unidentified isomers of 5,6-DHETE.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A4
327
Fig. 1. In this chromatographic system, LTB4 and 5,6-DHETE elute approximately in 19 and 32 min, respectively. Both compounds may be further purified and examined by straightphase HPLC, UV spectroscopy, and gas chromatography coupled to mass spectrometry (GC-MS). The resolution of various isomers of 5,6-DHETE (methyl esters) is poor in straight-phase HPLC, which has limited our use of this technique. In ultraviolet spectroscopy, with methanol as the solvent, 5,6-DHETE appears as a conjugated triene with maximal absorbance at 272 nm and with shoulders at 263 and 284 nm. For 5,6-DHETE, we use an extinction coefficient of 4.0 x 104 M -I x cm -~ (at 272 nm) due to the structural similarity with LTA4. For GC-MS, the free acids of LTB4 and 5,6-DHETE are converted to methyl ester trimethylsilyl ethers and analyzed on a capillary column (SE-30, fused silica, 25 m x 0.25 mm) coupled directly to a mass spectrometer (VG 7070E). The gas chromatograph is operated isothermally (2400-250 °) with helium as the carrier gas. The analysis of 5,6-DHETE by GC-MS consistently results in two peaks with C values of 23.8 and 24.7 and with similar mass spectra (for details see Ref. 1). The latter peak most probably reflects an 11-cis to l l-trans isomerization of the parent compound during the analytical procedure.4 Subcellular Distribution of Epoxide Hydrolase and LTA4 Hydrolase in Liver Tissue The distribution of enzymatic activity in subcellular fractions of mouse liver homogenates differ between cytosolic epoxide hydrolase and LTA4 hydrolase. Typically, formation of LTB4 is almost exclusively detected in the high-speed supernatant whereas formation of 5,6-DHETE is seen in both the 105,000 g supernatant (cytosol) and the 20,000 g pellet. The activity in the pellet fraction most probably reflects the presence of mitochondrial/peroxisomal epoxide hydrolase, an enzymatic activity similar to cytosolic epoxide hydrolase with regard to molecular weight, substrate specificity, and antigenic properties, s Substrate Specificity Cytosolic (and microsomal) epoxide hydrolase has a broad substrate specificity and has frequently been characterized with stable aromatic and aliphatic xenobiotic epoxides. Besides LTA4, a number of other epoxides derived from arachidonic acid, e.g., 15(S)-trans-14,15-oxido-5,8-cis10,12-trans-eicosatetraenoic acid (14,15-LTA4), may serve as substrates 8 j. Meijer, G. Lundqvist, and J. W. DePierre, Eur. J. Biochem. 167, 269 (1987).
328
BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[36]
for cytosolic epoxide hydrolase. 9 The microsomal enzyme (from rat liver) has no detectable catalytic activity toward LTA4.1 In contrast, the substrate specificity of LTA4 hydrolase seems to be very narrow. I° Two isomers of LTA4, namely LTA3 and LTAs, may be enzymatically hydrolyzed by this enzyme. 11,12Thus, LTA5 is transformed into LTBs, although with lower efficiency as compared to hydrolysis of LTA4 into LTB4, whereas LTA3 is a very poor substrate. Purification of Cytosolic Epoxide Hydrolase from Mouse Liver Cytosol Cytosolic epoxide hydrolase can be purified to apparent homogeneity from mouse liver by a procedure that involves column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxyapatite, utilizing xenobiotic substrate in the assay of enzyme activity. 13 Purification of LTA4 Hydrolase from Guinea Pig Liver Cytosol
Step 1. Precipitate nucleic acids by adding streptomycin sulfate in water (10% w/v) to cytosol with continuous stirring on ice. After 30 min, remove the precipitate by centrifugation (10,000 g, 15 rain, 4°). Proceed with ammonium sulfate precipitation (on ice) and collect the 40-80% saturated fraction by centrifugation. Dissolve the pellet in 50 mM Tris/ HCI, pH 8.0, to give a protein concentration of 25 mg/ml. Step 2. The 40-80% ammonium sulfate fraction is further purified by molecular exclusion chromatography on a column packed with AcA 44 (LKB-produkter, Bromma, Sweden) or equivalent, equilibrated with 50 mM Tris/HC1, pH 8.0. For good resolution, the sample volume should be adjusted to less than 2% of the bed volume. Step 3. Pool the active fractions from step 2 and treat the sample with 2 rnM dithiothreitol for 30 min. Load this sample onto DEAE-cellulose (Whatman DE-52) preequilibrated with 10 mM Tris/HC1, pH 8.0. When nonadsorbing proteins are eluted, apply a linear gradient of KCI (50-250 raM) that increases with approx. 0.25 mM/ml. Active fractions will appear in the range of 90 to 130 mM KCI. In this chromatographic step, most of the cytosolic epoxide hydrolase activity will be separated from the LTA4 hy9 A. Wetterholm, J. Haeggstr6m, M. Hamberg, J. Meijer, and O, R~dmark, Eur. chem. 173, 531 (1988). io N. Ohishi, T. Izumi, M. Minami, S. Kitamura, Y. Seyama, S. Ohkawa, S. H. Yotsumoto, F. Takaku, and T. Shimizu, J. Biol. Chem. 262, 10200 (1987). 11 j. F. Evans, D. J. Nathaniel, R. J. Zamboni, and A. W. Ford-Hutchinson, J. Biol. 260, 10966 (1985). 12 D. J, Nathaniel, J. F. Evans, Y. Leblanc, C. L6veill6, B. J. Fitzsimmons, and Ford-Hutchinson, Biochem. Biophys. Res. Commun. 131, 827 (1985). t3 j. Meijer and J. W. DePierre, Eur. J. Biochem. 148, 421 (1985).
J. Bio-
Terao, Chem.
A. W.
[36]
ENZYMATIC HYDROLYSIS OF LEUKOTRIENE A4
329
drolase activity. The former enzyme elutes in the flow-through peak and early fractions of the gradient. Step 4. Active fractions from step 3 are pooled and concentrated by ultrafiltration (Amicon PM10, Danvers, MA) in the presence of 2 mM dithiothreitol. The reducing agent should be added at this stage in order to prevent the appearance of multiple peaks of activity in the following chromatography. Anion-exchange separation with fast protein liquid chromatography (FPLC) is then performed on a column, Mono Q HR 5/5 (Pharmacia Fine Chemicals, Uppsala, Sweden), attached to the FPLC system and equilibrated at 10°-12° with 10 mM Tris/HC1, pH 8.0, supplemented with 0.1 mM dithiothreitol. The sample buffer is exchanged to the column equilibration buffer by molecular exclusion chromatography (PD-10, Pharmacia Fine Chemicals). Inject an aliquot of the enzyme pool, await the peak of nonadsorbing proteins, and then apply a gradient of KC1 (0-200 mM) in a total volume of 50 ml. Enzyme activity is recovered in fractions between 40-85 mM KCI. Step 5. Adsorption chromatography on hydroxyapatite is performed on a column (BioGel HPHT, Bio-Rad, Richmond, CA) attached to the FPLC system and equilibrated with 10 mM potassium phosphate buffer, pH 7.0, supplemented with 0.3 mM CaC12 and 0.1 mM dithiothreitol. Concentration and desalting of collected fractions from step 4 are conveniently carried out by repeated dilution and ultrafiltration of the sample with the column equilibration buffer. The sample is applied to the column at a flow rate of 0.25 ml/min and after elution of nonadsorbing proteins, the flow is increased to 0.5 ml/min and a linear gradient of phosphate in two steps (10-160 mM in 50 ml followed by 160-310 mM in 5 ml) is developed by mixing with 500 mM potassium phosphate buffer, pH 7.0, supplemented with 6/~M CaCI2 and 0.1 mM dithiothreitol. Enzymatic activity is collected between 60-85 mM potassium phosphate. Step 6. The final purification is achieved by chromatofocusing on a Mono P column (HR 5/20, Pharmacia Fine Chemicals) equilibrated with 25 mM triethanolamine, the pH adjusted to 8.3 with iminodiacetic acid, and supplemented with 0.1 mM dithiothreitol. The sample buffer from step 5 is exchanged to the alkaline starting buffer by repeated concentration and dilution as described above. To achieve maximal resolution in the chromatography, the column should be washed with 1-2 ml of 2 M sodium iminodiacetate and reequilibrated prior to each sample injection. Proteins are eluted with a pH gradient (8-5) by changing the buffer to a mixture of Polybuffer 74 and 96 (70 : 30 by volume, Pharmacia), the pH adjusted to 5.0 with iminodiacetic acid, and supplemented with 0.1 mM dithiothreitol, at a flow rate of 0.5 ml/min. Immediately after each chromatographic run, the pH should be measured in collected fractions and restored to an alkaline pH with 1 M Tris/HC1, pH 8.0, to prevent inactivation of the enzyme. In
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BIOSYNTHESIS, ENZYMOLOGY, AND CHEMICAL SYNTHESIS
[36]
TABLE I PURIFICATION OF ETA4 HYDROLASE FROM GUINEA PIG LIVER
Fraction Cytosol Precipitations AcA 44b DEALcellulose Mono QC Hydroxyapatite Mono pd
Volume (ml) 102 40 265 80 21 13.5 1.1
Total protein (rag) 1835 975 500 62 18 2.6 0.15
Totala activity (U)
Specific activity (U/rag)
Yield (%)
Purification (-fold)
0.73 1.42 1.45 0.86
0.0004 0.0015 0.0029 0.014
-100 102 61
-1 2 9
0.80 0.44 0.27
0.044 0.17 1.8
56 31 19
29 114 1200
a Aliquots of the enzyme from each step of purification were incubated with LTA4 (100/zM) at 37° for 1 min. Analysis and quantitation of LTB4 were performed with reversed-phase HPLC as described in the text. Enzymatic activity was expressed as micromoles of LTB4 formed per minute. In crude cytosol, lower amounts of activity were detected as compared to following steps most probably due to the presence of competing enzyme activities. Therefore, the activity in the ammonium sulfate precipitate was set to 100%. b Molecular exclusion chromatography. c FPLC, anion-exchange separation. d FPLC, chromatofocusing. this system, guinea pig liver LTA4 hydrolase is consistently r e c o v e r e d at a p H equal to 19/of 6.2. By this p r o c e d u r e the protein is purified m o r e than 1000-fold to near h o m o g e n e i t y with a yield of about 20% (Table I). The total activity found in the 4 0 - 8 0 % a m m o n i u m sulfate precipitate is usually higher than in crude cytosol which contains o t h e r e n z y m e s , e.g., cytosolic epoxide hydrolase and glutathione transferases, that could c o m p e t e with LTA4 hydrolase for the substrate. In general, results of e n z y m e activity determinations v a r y considerably depending on the incubation conditions, especially t e m p e r a ture and substrate solvent. Thus, the activity seems to be s o m e w h a t reduced w h e n LTA4 is added in tetrahydrofuran and incubations on ice m a y give m o r e p r o d u c t than incubations at 37 °, p r o b a b l y due to increased substrate stability. It should be noted that the purified e n z y m e is inactivated upon freezing. E n z y m e Kinetics Performing e n z y m e kinetic e x p e r i m e n t s with LTA4 confronts the investigator with the p r o b l e m o f substrate instability. At 25 ° in a buffer o f p H 7.4 without a n y organic solvent, the half-life o f LTA4 is
