Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

Hepoxilins are lipid signaling molecules derived from arachidonic acid through the 12-lipoxygenase pathway. These trans-epoxy hydroxy eicosanoids play a role in a variety of physiological processes, including inflammation, neurotransmission, and formation of skin barrier function. Mammalian hepoxilin hydrolase, partly purified from rat liver, has earlier been reported to degrade hepoxilins to trioxilins. Here, we report that hepoxilin hydrolysis in liver is mainly catalyzed by soluble epoxide hydrolase (sEH): i) purified mammalian sEH hydrolyses hepoxilin A₃ and B₃ with a V_max of 0.4–2.5 μmol/mg/min; ii) the highly selective sEH inhibitors N-adamantyl-N’-cyclohexyl urea and 12-(3-adamantan-1-yl-ureido) dodecanoic acid greatly reduced hepoxilin hydrolysis in mouse liver preparations; iii) hepoxilin hydrolase activity was abolished in liver preparations from sEH^−/− mice; and iv) liver homogenates of sEH^−/− mice show elevated basal levels of hepoxilins but lowered levels of trioxilins compared with wild-type animals. We conclude that sEH is identical to previously reported hepoxilin hydrolase. This is of particular physiological relevance because sEH is emerging as a novel drug target due to its major role in the hydrolysis of important lipid signaling molecules such as epoxyeicosatrienoic acids. sEH inhibitors might have undesired side effects on hepoxilin signaling.     

Immobilization of epoxide hydrolase purified from rat liver microsomes

Summary Purified epoxide hydrolase was immobilized by covalent binding to Sephadex G-150 activated with 1,1 carbonyl diimidazole under mild conditions Km^app values of free and immobilized epoxide hydrolase were 0.5 M and 2 M respectively towards benzo(a)pyrene-4,5-oxide, whereas Vmax^app was decreased from 300 nmol·min^–1·mg^–1 to 81 nmol·min^–1·mg^–1. Immobilization enhanced stability and allowed repeated use of the enzyme.     

Conformational study of purified epoxide hydrolase from rat liver

Hepatic epoxide hydrolase (EC 3.3.2.3) was purified from phenobarbital-treated rats by ion-exchange chromatography followed by hydrophobic chromatography. The enzyme had a specific activity of 300--400 nmol min-1 mg-1 protein with benzo[a]pyrene-4,5-oxide as the substrate. Circular dichroism (CD) spectra of the purified enzyme gave two negative bands, centered at 210 nm and 222 nm, respectively. The mean residue ellipticity at 222 nm was 12,9000 deg X cm(2) X dmol(-1), which indicated the presence of about 35% alpha-helical structures. Sodium dodecyl sulfate (SDS) greatly affected the shape of the CD spectra, which were gradually shifted to the blue. This suggested a decrease in the aggregation state of the protein. Electrostatic interactions were important in the organization of the enzyme structure since the conformation was stable between pH 7.4 and pH 10. At pH-values 5.0, 6.0 and 12.0, the CD bands underwent considerable changes in both amplitude and shape. Moreover there was a good correlation between the optimal pH range of the epoxide hydrolase activity and the organization state of the protein. After membrane reconstitution with liposomes, the conformation of the enzyme was not significantly modified by the presence of dimyristoyl L-alpha-phosphatidylcholine or other phospholipids. This constancy was obtained over a wide range of molar ratios of phospholipids to protein (0--500). However, phospholipids did increase the thermal stability of the enzyme. Fluorescence measurements of diphenylhexatriene (DPH) bound to dimyristoyl L-alpha-phosphatidylcholine indicated that addition of epoxide hydrolase modified the thermal transition of the lipid phase. On the other hand, electron paramagnetic resonance (EPR) signals of the nitroxide-labelled fatty acid, 2-(14-carboxy-tetradecyl)-2-ethyl-4,4-dimethyl-3,3,-oxazolidiny-oxyl, bound to the phospholipid, indicated that the presence of the protein decreased by about 53% the correlation time of the label, suggesting that its motion had increased. In conclusion, phospholipid-epoxide hydrolase interactions enhanced the fluidity of dimyristoyl L-alpha-phosphatidylcholine liposomes without changing the secondary structure of the enzyme. Electrostatic interactions also played an important role in the conformational stability of the protein.     

Purification of an epoxide hydrolase from Rhodotorula glutinis

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 mol 1,2-epoxyhexane hydrolyzed min^–1 mg^–1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases.     

Purification and characterization of an epoxide hydrolase from the peroxisomal fraction of mouse liver

Epoxide hydrolase (EH) activity has been reported to occur in most subcellular fractions of mouse liver. The EHs in the microsomal and cytosolic fractions have been purified and characterized; however, the nature of the EH(s) in the peroxisomal fraction is not known. Therefore an EH, pEH, was purified from the solubilized 12,000g fraction, which contain peroxisomes. Previous studies have demonstrated that the EH activity in this crude solubilized 12,000g fraction resides mostly in the peroxisomes. Thus the crude 12,000g pellet from mouse liver, free from cytosolic contamination, was sonicated to obtain a 105,000g soluble fraction containing 80% of the original EH activity in this fraction. The pEH was purified, using trans-stilbene oxide (TSO) as substrate, by a combination of affinity and hydroxyapatite chromatography. The purified pEH had a native molecular weight of 57 kDa, a molecular weight of 59 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7. The purified pEH was observed to be immunologically similar to the cytosolic EH (cEH). The kinetics of hydrolysis of TSO, however, were slightly different. Lineweaver-Burk plots for the inhibition of pEH suggest a probable noncompetitive, mixed-type inhibition. The purified pEH thus appears to be very similar to the cEH. There are minor differences between the purified cEH and pEH, particularly in the kinetic parameters. However, these minor differences are insignificant. These results demonstrate that the cEH and pEH are substantially similar, if not identical.     

A new enzyme immunoassay of microsomal rat liver epoxide hydrolase

Antiserum against purified rat liver microsomal epoxide hydrolase was produced in the rabbit. We developed an enzyme-linked immunosorbent assay which is reliable with regard to its analytical criteria. The concentration of epoxide hydrolase was measured in liver microsomes of control rats and animals treated with F 1379 (250 mg/kg/day) for 5, 7, 14, and 21 days. This hypolipidemic drug was able to induce strong epoxide hydrolase activity and enhance protein concentration. The gradual increase in epoxide hydrolase concentration paralleled the increase of epoxide hydrolase activity, with stabilization occurring after the 14th until the 21st day of treatment.     

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.     

Nucleoside monophosphoramidate hydrolase from rat liver: Purification and characterization

• 1.1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol.
• 2.2. It consisted of two subunits of 14 kDa.
• 3.3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N^ω-phosphoarginine, 6-phospholysine and 3-phosphohistidine.
• 4.4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

     

Microsomal epoxide hydrolase of rat liver. Purification and characterization of enzyme fractions with different chromatographic characteristics.

Microsomal epoxide hydrolase was purified from rat liver, and different fractions of the purified enzyme, which varied in their contents of phospholipid, were obtained by ion-exchange chromatography. One fraction (A), which did not bind to CM-cellulose, had a high phospholipid content, and a second fraction (B), which was eluted from CM-cellulose at high ionic strength, had a low phospholipid content. Removal of most of the phospholipid from fraction A altered its chromatographic behaviour. When the delipidated material was re-applied to CM-cellulose, most of the enzyme bound to the cation-exchanger. The specific activities of all the fractions described (with styrene epoxide [(1,2-epoxyethyl)benzene] as substrate) were altered by adding the non-ionic detergent Lubrol PX or phospholipid. Lubrol PX inhibited enzyme activity, and phospholipid reversed this inhibition. The various enzyme fractions isolated appeared to be different forms of the same protein, as judged by their minimum Mr values and immunochemical properties. These results indicate that different fractions of epoxide hydrolase isolated by ion-exchange chromatography probably are not different isoenzyme forms.     

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.     

Purification and characterization of rat-liver cytosolic epoxide hydrolase

Rat liver cytosolic epoxide hydrolase has been purified and characterized. The enzyme was purified from tiadenol-induced rat liver 540-fold with respect to trans-stilbene oxide as a substrate. Similar purification was obtained with the substrates trans-beta-ethyl styrene oxide and styrene 7,8-oxide, the specific activities decreasing in the order trans-beta-ethyl styrene oxide greater than styrene 7,8-oxide greater than trans-stilbene oxide. The enzyme exerts highest activity at pH 7.4 Km and Vmax of the pure enzyme for trans-stilbene oxide were 1.7 microM and 205 nmol x min-1 x mg protein-1 respectively. With trans-stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol less than methanol less than acetone less than isopropanol = N,N-dimethyl formamide less than acetonitrile less than tetrahydrofuran. The native enzyme, with a molecular mass of 120 kDa, consists of two 61-kDa subunits. Digestion of rat liver cytosolic and microsomal epoxide hydrolase by three proteases resulted in markedly different peptide maps. Western-blot analysis with antiserum against rat liver cytosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofibrate-induced rats. No cross-reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes. A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig, Syrian hamster and New Zealand white rabbit but not with that of green monkey.     

Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes

This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.     

Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A4 epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A4 (LTA4) to leukotriene B4 (LTB4) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 microM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA4 to LTB4. This method was used to produce 700 mg of LTB4 from LTA4 methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca+2, Mn+2, Fe+2, Zn+2 and Cu+2 were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 microM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 microM. From these inhibition studies, it can be theorized that the epoxide hydrolase has at least one hydrophobic and one hydrophilic binding site.     

Purification and characterization of a recombinantCaulobacter crescentus epoxide hydrolase

ACaulobacter crescentus epoxide hydrolase (CCEH) from a recombinantEscherichia coli was purified to homogeneity using a three-step procedure. The CCEH protein was purified 7.3-fold with a 22.9% yield in overall activity. The optimal reaction temperature and pH were determined to be 37°C and pH 8.0, respectively. The addition of 10% (v/v) dimethylsulfoxide as a cosolvent improved the enantioselectivity of CCEH for a batch kinetic resolution of racemic indene oxide.     

Purification and characterization of a long-chain acyl-CoA hydrolase from rat liver microsomes.

A long-chain acyl-CoA hydrolase from rat liver microsomes has been purified by solvent extraction and gel chromatography to homogeneity as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The enzyme was a monomer of molecular weight 59 000. In a sucrose gradient it sedimented at 4.3 S. The isoelectric point, pI was 6.9, and the Stokes radius was approx. 31 A. The enzyme hydrolyzed long-chain fatty acyl-CoA (C7--C18) with maximum activity for palmitoyl-CoA. Bovine serum albumin activation of the enzyme was related to the ratio acyl-CoA/bovine serum albumin, and at high ratios, acyl-CoA inhibited the enzyme activity. Disregarding the substrate inhibition, an apparent Km of 65 nmol/mg protein or 1-10(-7) M and a V of 750 nmol/mg protein per min were calculated. The enzyme was inhibited by p-hydroxymercuribenzoate and N-ethylmaleimide. Reactivation by means of dithiothreitol was not complete.     

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.     

Perinatal development of styrene monooxygenase and epoxide hydrolase in rat liver microsomes and nuclei

Nuclear enzymes were found to develop earlier than the corresponding microsomal activities. In fact styrene monooxygenase enzymatic activity at 18 days gestational age reached about half the values of adult animals, whereas fetal microsomal activity was only about $\text{1}\text{20}$ the adult level at the same age. In microsomes and nuclei the ontogenic development of epoxide hydrolase is slightly slower than styrene monooxygenase. This suggests that fetuses and newborn animals are exposed to higher risk of accumulation of styrene-7,8-oxide, a toxic and possibly teratogenic product of styrene monooxygenase.