Purification of human liver cytosolic epoxide hydrolase and comparison to the microsomal enzyme

Epoxide hydrolase (EC 3.3.2.3) was purified to electrophoretic homogeneity from human liver cytosol by using hydrolytic activity toward trans-8-ethylstyrene 7,8-oxide (TESO) as an assay. The overall purification was 400-fold. The purified enzyme has an apparent monomeric molecular weight of 58 000, significantly greater than the 50 000 found for human (or rat) liver microsomal epoxide hydrolase or for another TESO-hydrolyzing enzyme also isolated from human liver cytosol. Purified cytosolic TESO hydrolase catalyzes the hydrolysis of cis-8-ethylstyrene 7,8-oxide 10 times more rapidly than does the microsomal enzyme, catalyzes the hydrolysis of TESO and trans-stilbene oxide as rapidly as the microsomal enzyme, but catalyzes the hydrolysis of styrene 7,8-oxide, p-nitrostyrene 7,8-oxide, and naphthalene 1,2-oxide much less effectively than does the microsomal enzyme. Purified cytosolic TESO hydrolase does not hydrolyze benzo[a]pyrene 4,5-oxide, a substrate for the microsomal enzyme. The activities of the purified enzymes can explain the specific activities observed with subcellular fractions. Anti-human liver microsomal epoxide hydrolase did not recognize cytosolic TESO hydrolase in purified form or in cytosol, as judged by double-diffusion immunoprecipitin analysis, precipitation of enzymatic activity, and immunoelectrophoretic techniques. Cytosolic TESO hydrolase and microsomal epoxide hydrolase were also distinguished by peptide mapping. The results provide evidence that physically different forms of epoxide hydrolase exist in different subcellular fractions and can have markedly different substrate specificities.     

Human liver cytosolic epoxide hydrolases

Human liver epoxide hydrolases were characterized by several criteria and a cytosolic cis-stilbene oxide hydrolase (cEHCSO) was purified to apparent homogeneity. Styrene oxide and five phenylmethyloxiranes were tested as substrates for human liver epoxide hydrolases. With microsomes activity was highest with trans-2-methylstyrene oxide, followed by styrene 7,8-oxide, cis-2-methylstyrene oxide, cis-1,2-dimethylstyrene oxide, trans-1,2-dimethylstyrene oxide and 2,2-dimethylstyrene oxide. With cytosol the same order was obtained for the first three substrates, whereas activity with 2,2-dimethylstyrene oxide was higher than with cis-1,2-dimethylstyrene oxide and no hydrolysis occurred with trans-1,2-dimethylstyrene oxide. Generally, activities were lower with cytosol than with microsomes. The isoelectric point for both microsomal styrene 7,8-oxide and cis-stilbene oxide hydrolyzing activity was 7.0, whereas cEHCSO had an isoelectric point of 9.2 and cytosolic trans-stilbene oxide hydrolase (cEHTSO) of 5.7. The cytosolic epoxide hydrolases could be separated by anion-exchange chromatography and gel filtration. The latter technique revealed a higher molecular mass for cEHCSO than for cEHTSO. Both cytosolic epoxide hydrolases showed higher activities at pH 7.4 than at pH 9.0, whereas the opposite was true for microsomal epoxide hydrolase. The effects of ethanol, methanol, tetrahydrofuran, acetonitrile, acetone and dimethylsulfoxide on microsomal epoxide hydrolase depended on the substrate tested, whereas both cytosolic enzymes were not at all, or only slightly, affected by these solvents. Effects of different enzyme modulators on microsomal epoxide hydrolase also depended on the substrates used. Trichloropropene oxide and styrene 7,8-oxide strongly inhibited cEHCSO whereas cEHTSO was moderately affected by these compounds. Immunochemical investigations revealed a close relationship between cEHCSO and rat liver microsomal, but not cytosolic, epoxide hydrolase. Interestingly, cEHTSO has no immunological relationship to rat microsomal, nor to rat cytosolic epoxide hydrolase. cEHTSO from human liver differed also from its counterpart in the rat in that it was only moderately affected by tetrahydrofuran, acetonitrile and trichloropropene oxide. Five steps were necessary to purify cEHCSO. The enzyme has a molecular mass (49 kDa) identical to that of rat liver microsomal epoxide hydrolase.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Expression of microsomal and cytosolic epoxide hydrolases in cultured rat hepatocytes and hepatoma cell lines

Microsomal epoxide hydrolase activity, determined using benzpyrene 4,5-oxide and styrene 7,8-oxide, increased in cultured hepatocytes compared to freshly isolated cells. In contrast, cytosolic epoxide hydrolase activity, assayed using trans-stilbene oxide, had decreased 80% by 24 hr and was barely detectable after 96 hr in culture. There was no difference in enzyme activity between freshly isolated hepatocytes and the two rat hepatoma cell lines McA-RH 7777 and H4-II-E, when styrene 7,8-oxide was used as substrate. However, benzpyrene 4,5-oxide hydrolase activity of the McA-RH 7777 and H4-II-E cell lines were 55 and 10%, respectively, of freshly isolated hepatocytes. These results show that hepatoma cell lines provide a suitable system for studying the regulation of both the microsomal and cytosolic epoxide hydrolase enzymes.     

Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species

Sex and species differences in hepatic epoxide hydrolase activities towards cis- and trans-stilbene oxide were examined in common laboratory animals, as well as in monkey and man. In general trans-stilbene oxide was found to be a good substrate for epoxide hydrolase activity in cytosolic fractions, whereas the cis isomer was selectively hydrated by the microsomal fraction (with the exception of man, where the cytosol also hydrated this isomer efficiently). The specific cytosolic epoxide hydrolase activity was highest in mouse, followed by hamster and rabbit. Epoxide hydrolase activity in the crude 'mitochondrial' fraction towards trans-stilbene oxide was also highest in mouse and low in all other species examined. Microsomal epoxide hydrolase activity was highest in monkey, followed by guinea pig, human and rabbit, which all had similar activities. Sex differences were generally small, but where significant, male animals had higher catalytic activities than females of the same species in most cases. Antibodies raised against microsomal epoxide hydrolase purified from rat liver reacted with microsomes from all species investigated, indicating structural conservation of this protein. Antibodies directed towards cytosolic epoxide hydrolase purified from mouse liver reacted only with liver cytosol from mouse and hamster and with the 'mitochondrial' fraction from mouse in immunodiffusion experiments. Immunoblotting also revealed reaction with rat liver cytosol. The cytosolic and 'mitochondrial' epoxide hydrolases in all three mouse strains and in both sexes for each strain were immunochemically identical. The anomalies in human liver epoxide hydrolase activities observed here indicate that no single common laboratory animal is a good model for man with regard to these activities.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunochemical characterization of human lung epoxide hydrolases

Immunochemical techniques were used to investigate the biochemical properties of human lung epoxide hydrolases. Two epoxide hydrolases with different immunoreactive properties were identified. These two epoxide hydrolases were found in both cytosolic and microsomal cell fractions. Immunotitration of enzyme activity showed that enzymes that catalyze the hydration of benzo(a)pyrene 4,5-oxide react with antiserum to rat microsomal epoxide hydrolase; those that hydrate trans-stilbene oxide do not. Immunotitration and Western blot experiments showed that microsomal and cytosolic benzo(a)pyrene 4,5-oxide hydrolases have significant structural homology. Immunohistochemical staining of human lung benzo(a)pyrene 4,5-oxide hydrolase showed that the enzyme is localized primarily in the bronchial epithelium. No cell type-specific localization was observed. An enzyme-linked immunosorbent assay was developed which allows direct quantitation of benzo(a)pyrene 4,5-oxide hydrolase protein. Levels of enzyme protein detected by this assay correlated well with enzyme levels determined by substrate conversion assays.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Characterization of optimal assay conditions, substrate specificity and effects of modulators on the catalytic activity

We have characterized certain catalytic properties of cytosolic epoxide hydrolases purified from untreated and clofibrate-treated mouse liver. The enzyme activity was found to be sensitive to oxygen, but nitrogen-saturated buffers containing dithiothreitol maintained high activity for at least 12 h at 0 degrees C. Linearity of the hydration of trans-stilbene oxide with time and protein was established, the pH optimum was broad (6.5 to 7.4) and the temperature optimum was close to 50 degrees C for both forms. The activity was independent of ionic strength, with the exception of the control form in the absence of dithiothreitol, where a lower activity was observed at low ionic strength. The activity decreased when ethanol was replaced by acetone or acetonitrile as solvent for the substrate. Tetrahydrofuran was found to be highly inhibitory, while dimethylsulfoxide had less pronounced effects. The apparent Km values were 4.9 microM, 73 microM and 1980 microM for the control form with trans-stilbene oxide, cis-stilbene oxide and styrene oxide as substrates, respectively. The Km values for the enzyme from clofibrate-treated mice were in the same range, although the V values were higher for all three substrates with this form. The highest turnover was found for trans-beta-propylstyrene oxide as substrate, followed by trans-beta-ethylstyrene oxide. Little or no activity was observed with benzo[a]pyrene 4,5-oxide or cholesterol 5,6 alpha-oxide. The enzymes were found to be sensitive to 5,5'-dithiobis(2-nitrobenzoic acid) and a phenylmercuric salt. alpha-Naphthoflavone, beta-naphthoflavone and chalcone derivatives also inhibited the activity, while none of the compounds known to activate microsomal epoxide hydrolase activated the cytosolic forms.     

Selective inhibition of cytosolic epoxide hydrolase activity in vitro by compounds that inhibit catalase

The ability of a number of known inhibitors of catalase activity to affect cytosolic and microsomal epoxide hydrolase activities in vitro, measured as enzymatic trans-stilbene oxide hydrolysis and styrene oxide hydrolysis, respectively, was investigated. Catalase and cytosolic epoxide hydrolase activities are inhibited by hydroxylated metabolites of 2-amino-4,5-diphenylthiazole (DPT). The metabolite hydroxylated on the 4-phenyl ring (4OH-DPT) and the metabolite hydroxylated on both phenyl rings (4,5-DIOH-DPT) are potent inhibitors of both enzymes; the metabolite hydroxylated on the 5-phenyl ring (5OH-DPT) is less potent. Unmetabolized DPT has no effect on either enzyme. 4OH-DPT inhibits, but 5OH-DPT enhances, microsomal epoxide hydrolase activity. 4,5-DIOH-DPT and DPT have no effect on this enzyme. Other compounds that inhibit both catalase and cytosolic epoxide hydrolase activities, but do not inhibit microsomal epoxide hydrolase activity, are nordihydroguaiaretic acid and 2-aminothiazole. Microsomal epoxide hydrolase activity is enhanced by 2-aminothiazole and levamisole in vitro. Thus these inhibitors of catalase are selective epoxide hydrolase inhibitors in that they inhibit cytosolic epoxide hydrolase activity in vitro, but have either no effect on, or increase the activity of, microsomal epoxide hydrolase in vitro. Conversely, the selective cytosolic epoxide hydrolase inhibitors 4-phenylchalcone oxide and 4'-phenylchalcone oxide do not inhibit catalase activity, nor does trichloropropene oxide, a selective microsomal epoxide hydrolase inhibitor.     

Inhibition of epoxide hydrolases and glutathione S-transferases by 2-, 3-, and 4-substituted derivatives of 4'-phenylchalcone and its oxide

4'-Phenylchalcones, chalcone oxides, and related compounds were synthesized and tested as inhibitors of cytosolic epoxide hydrolase, microsomal epoxide hydrolase, and glutathione S-transferases from mouse and rat liver. Several compounds were more potent inhibitors of the cytosolic epoxide hydrolase than the parent 4'-phenylchalcone oxide while large substituents in the 4- and especially the 2-position caused a reduction in inhibition. The chalcone oxides showed selectivity as inhibitors of the cytosolic epoxide hydrolase acting on trans-stilbene oxide, while chalcones were inhibitors of cytosolic glutathione S-transferase acting on cis-stilbene oxide. Data are consistent with the hypothesis that much of the inhibition of the glutathione S-transferase is caused by the glutathione conjugate of the chalcone.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.     

Cytosolic epoxide hydrolase

Epoxide hydrolase activity is recovered in the high-speed supernatant fraction from the liver of all mammals so far examined, including man. For some as yet unexplained reason, the rat has a very low level of this activity, so that cytosolic epoxide hydrolase is generally studied in mice. This enzyme selectively hydrolyzes trans epoxides, thereby complementing the activity of microsomal epoxide hydrolase, for which cis epoxides are better substrates. Cytosolic epoxide hydrolase has been purified to homogeneity from the livers of mice, rabbits and humans. Certain of the physicochemical and enzymatic properties of the mouse enzyme have been thoroughly characterized. Neither the primary amino acid, cDNA nor gene sequences for this protein are yet known, but such characterization is presently in progress. Unlike microsomal epoxide hydrolase and most other enzymes involved in xenobiotic metabolism, cytosolic epoxide hydrolase is not induced by treatment of rodents with substances such as phenobarbital, 2-acetylaminofluorene, trans-stilbene oxide, or butylated hydroxyanisole. The only xenobiotics presently known to induce cytosolic epoxide hydrolase are substances which also cause peroxisome proliferation, e.g., clofibrate, nafenopin and phthalate esters. These and other observations indicate that this enzyme may actually be localized in peroxisomes in vivo and is recovered in the high-speed supernatant because of fragmentation of these fragile organelles during homogenization, i.e., recovery of this enzyme in the cytosolic fraction is an artefact. The functional significance of cytosolic epoxide hydrolase is still largely unknown. In addition to deactivating xenobiotic epoxides to which the organism is exposed directly or which are produced during xenobiotic metabolism, primarily by the cytochrome P-450 system, this enzyme may be involved in cellular defenses against oxidative stress.     

Continuous spectrophotometric assays for cytosolic epoxide hydrolase

Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.     

Subcellular distribution, catalytic properties and partial purification of epoxide hydrolase in the human adrenal gland

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Purification of microsomal epoxide hydrolase from liver of rhesus monkey: partial separation of cis- and trans-stilbene oxide hydrolase

Solubilized rhesus monkey liver microsomes were used as the starting material for the purification of epoxide (cis-stilbene oxide) hydrolase. Successive chromatography over DEAE-Sephacel followed by CM-cellulose resulted in two peaks of activity, CM A and CM B. Passage of these two eluates over separate hydroxyapatite columns resulted in two peaks of activity from CM A, HA A1, and HA A2, and one peak from CM B and HA B, with respective recoveries of 1, 7, and 0.2% of cis-stilbene oxide hydrolase activities. A similar recovery was found for benzo[a]pyrene-4,5-oxide hydrolase, while trans-stilbene oxide hydrolase activity coeluted only in HA A2. Fraction HA A1 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblots of the three eluates and solubilized microsomes incubated with anti-HA A1 demonstrated a single band at 49 kDa in each fraction. The three eluates were differentially affected by the inhibitors of epoxide hydrolase, trichloropropene oxide and 4-phenylchalcone oxide, and addition of Lubrol PX and phospholipid. Immunoprecipitation of HA A2 resulted in coprecipitation of cis- and trans-stilbene oxide hydrolase activity. Upon immunoprecipitation of solubilized microsomes, all the cis-stilbene oxide and benzo[a]pyrene-4,5-oxide, but only 50-60% of trans-stilbene oxide hydrolase activity was precipitated. These studies support findings with other species that (i) an immunochemically distinct cytosolic-like epoxide hydrolase exists in microsomes, and (ii) microsomal epoxide hydrolase activity can be separated during ion-exchange chromatography giving proteins with similar molecular weights and immunochemical cross-reactivity. The precipitation of cis- and trans-stilbene oxide hydrolase activity in eluate HA A2 provides convincing evidence that these isozymes are not structurally identical.     

Enzymatic hydration of leukotriene A4. Purification and characterization of a novel epoxide hydrolase from human erythrocytes

Human erythrocytes contained a soluble cytosolic epoxide hydrolase for stereospecific enzymatic hydration of leukotriene A4 into leukotriene B4. The enzyme was purified 1100-fold, to apparent electrophoretic homogeneity, by conventional DEAE-Sephacel fractionation followed by high performance anion exchange and chromatofocusing procedures. Its characteristics include a molecular weight of 54,000 +/- 1,000, an isoelectric point 4.9 +/- 0.2, a Km apparent from 7 to 36 microM for enzymatic hydration of leukotriene A4, and a pH optimum ranging from 7 to 8. The enzyme was partially inactivated by its initial exposure to leukotriene A4. There was slow but detectable enzymatic hydration (pmol/min/mg) of certain arachidonic acid epoxides including (+/-)-14,15-oxido-5,8-11-eicosatrienoic acid and (+/-)-11,12-oxido-5,8,14-eicosatrienoic acid, but not others, including 5,6-oxido-8,11,14-eicosatrienoic acid. Human erythrocyte epoxide hydrolase did not hydrate either styrene oxide or trans-stilbene oxide. In terms of its physical properties and substrate preference for leukotriene A4, the erythrocyte enzyme differs from previously described versions of epoxide hydrolase. Human erythrocytes represent a novel source for an extrahepatic, cytosolic epoxide hydrolase with a potential physiological role.     

Hepatic levels of cytosolic, microsomal and 'mitochondrial' epoxide hydrolases and other drug-metabolizing enzymes after treatment of mice with various xenobiotics and endogenous compounds

This study was performed in order to study the response of epoxide hydrolases in different subcellular compartments of mouse liver to treatment with various compounds. Male C57BL/6 mice were treated with 31 different compounds--including traditional inducers of xenobiotic-metabolizing systems, liver carcinogens, stilbene derivatives, endogenous compounds and various other drugs and xenobiotics. The effects on liver somatic index; protein contents in 'mitochondria', microsomes and cytosol prepared from the liver; epoxide hydrolase activity towards trans- or cis-stilbene oxide in these three fractions; microsomal cytochrome P-450 content; cytosolic and 'mitochondrial' glutathione transferase activity and cytosolic DT-diaphorase activity were then determined. Cytosolic epoxide hydrolase activity was induced by chlorinated paraffins, di(2-ethylhexyl)phthalate and clofibrate and depressed by alpha-naphthylisothiocyanate, 3-methylcholanthrene, benzil and quercitin. Radial immunodiffusion revealed similar changes in the amount of enzyme protein present, except for two cases, where the increase in amount was larger; and the enzyme seems to be inhibited by benzil. Microsomal epoxide hydrolase activity was induced by these same compounds and several others as well, including dibenzoylmethane, butylated hydroxyanisole and polychlorinated biphenyls. 'Mitochondrial' epoxide hydrolase activity towards trans-stilbene oxide was not affected by those compounds which induced the cytosolic enzyme, but increased about two-fold after treatment with 2-acetylaminofluorene, DL-ethionine, aflatoxin B1 and phenobarbital. There does not seem to be any co-regulation of different forms of epoxide hydrolase in mouse liver. In general small effects were observed on liver weight and protein contents in the different subcellular fractions. Polychlorinated biphenyls were the most potent of the 8 compounds which induced cytochrome P-450, while butylated hydroxyanisole induced cytosolic glutathione transferase activity to the highest extent. 'Mitochondrial' glutathione transferase activity was most induced by certain of the stilbene derivatives. The most potent inducers of DT-diaphorase activity were 3-methylcholanthrene, polychlorinated biphenyls and dinitrotoluene.     

Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. Purification procedure and physiochemical characterization of the pure enzymes

Cytosolic epoxide hydrolase was purified from the liver of untreated and clofibrate-treated male C57Bl/6 mice. The purification procedure involves chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxyapatite, takes two days to perform and results in a 120-fold purification and approximately 35% yield of the enzyme from untreated mice. The purified enzyme is a dimer with a molecular mass of 120 kDa, a Stokes' radius of 4.2 nm, a frictional ratio of 1.0 and an isoelectric point of 5.5. The subunits behave identically upon isoelectric focusing in 8 M urea and only one band with a molecular mass of 60 kDa is seen after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The form purified from clofibrate-treated mice had very similar properties and was apparently identical to the control form as judged by amino acid analysis and peptide mapping as well. These analyses also demonstrated that the cytosolic enzyme is clearly different from microsomal epoxide hydrolase isolated from rat liver. Furthermore, Ouchterlony immunodiffusion using antibodies raised in rabbits towards the control form of cytosolic epoxide hydrolase revealed identity between the two forms of cytosolic epoxide hydrolase, but no reaction with the microsomal epoxide hydrolase was observed. These findings indicate large structural differences between the cytosolic and microsomal forms of epoxide hydrolase in the liver.