Int. J. Biochem.

Vol. 22, No. 5, pp. 461470, Printed in Great Britain. All rights reserved

1990

0020-71 IX/90 $3.00 + 0.00 Copyright 0 1990 Pergamon Press plc

HUMAN AND MURINE CYTOSOLIC EPOXIDE HYDROLASE: PHYSICAL AND STRUCTURAL PROPERTIES ERIC C. DIETZE, JACQIJES MAGDALOU* and BRUCE D. HAMMOCKS Departments of Entomology and Environmental Toxicology, University of California, Davis, CA 95616, U.S.A. [Tel. (916) 752-75191 (Received 25 August 1989) Abstract-l. Human and murine liver cytosolic epoxide hydrolase (CEH) had an apparent M, of 59,000 by SDS-PAGE. 2. Peptide maps of CNBr, trypsin and Staphylococcus aureus V8 digests, as well as amino acid analysis, showed that human and murine CEH were similar. Uninduced and clofibrate induced murine CEH appeared qualitatively identical. 3. The CEHs shared antigenic determinants as determined by Western blotting. 4. Circular dichroism spectra indicate that human CEH had 39% a-helix. Uninduced and clofibrate induced murine CEH had 38 and 35% a-helix, respectively.

INTRODUCTION

Compounds

containing

the epoxide

functionality

are

common environmental contaminants. They are widely used as pesticides, steriliants and industrial synthetic precursors. Such compounds also occur as products, by-products of, or intermediates in, normal metabolism (Brash et al., 1988; Kadis, 1978; Meijer and DePierre, 1988; Ozawa et al., 1988; Puustinen et al., 1986) and as the result of spontaneous oxidation of membrane lipids (Sevanian et al., 1980). Epoxides are often very reactive and have been found to be cytotoxic, mutagenic and carcinogenic (El-Tantawy and Hammock, 1980; Hemminki and Vainio, 1984; Jones and Mackrodt, 1983; Meijer and DePierre, 1988; Ozawa et al., 1988; Sugiyama et al., 1987). Some arachadonic acid epoxides have potent physiological activity (Carroll et al., 1987; Cashman et al., 1987; Fitzpatrick et al., 1986) and squalene epoxide has been implicated in atherosclerotic plaque formation (Imai et al., 1980). Since man is constantly exposed to epoxide containing compounds from endogenous and exogenous sources, it is important to understand how this class of compounds is detoxified by the body and which compounds might inhibit this process. Epoxide hydrolases (EC 3.3.2.3) are a family of enzymes which hydrolyze the epoxide functionality to

*To whom all correspondence should be addressed. ICurrent address: Centre du Medicament, U.A. CNRS No. 597, 30 rue Lionnois, 54000 Nancy, France. Abbreoiutions: CEH, cytosolic epoxide hydrolase; MEH, microsomal epoxide hydrolase; TSO, trans-stilbene oxide; CSO, cis-stilbene oxide; HIV-I, human immunodeficiency virus-l; DPCC, diphenyl carbamyl chloride; TFA. trifluoroacetic acid: SDS-PAGE. sodium dodecvl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; and HPLC, high performance liquid chromatography.

diols. The resulting diol is more polar, usually less toxic, more easily excreted, and can be further metabolized. Thus, epoxide hydrolysis usually represents a detoxication process. Epoxide hydrolases have been found in all mammalian species tested and are found in most organs. They generally occur at the highest levels in liver and kidney (Gill and Hammock, 1980; Moody et al., 1986; Silva and Hammock, 1987; Wixtrom and Hammock, 1985). Intracellularly, epoxide hydrolases have been found in cytosol, nuclear membranes, mitochondria, peroxisomes and microsomes (Meijer et al., 1987b). The epoxide hydrolases include microsomal epoxide hydrolase (MEH), cholesterol epoxide hydrolase, leukotriene A4 hydrolase and cytosolic epoxide hydrolase (CEH) and can be distinguished from one another by such criteria as molecular weight, antigenic properties, pH optima, substrate specificity and differential induction (Haeggstrom et al., 1988; Meijer and DePierre, 1988; Wixtrom and Hammock, 1985). The relative importance and in vivo function of the various epoxide hydrolases has not been demonstrated. It is thought that MEH is involved in xenobiotic catabolism, cholesterol epoxide hydrolase in the breakdown of epoxidized sterols in cellular membranes, leukotriene A, hydrolase in the conversion of leukotriene A, to leukotriene B,, and CEH in the hydrolysis of epoxides generated during intermediary metabolism (Meijer and DePiere, 1988). Of the epoxide hydrolases, MEH is the best characterized in vitro. The primary amino acid sequence has been deduced from both cloned rat (Ruttus norvigicus) liver MEH cDNA (Porter et al., 1986) and purified rabbit (Oryctolugus cuniculus) liver MEH (Heinemann and Ozols, 1984). Histidine is crucial for catalytic activity (DuBois et al., 1978). MEH hydrolyzes cyclic, and mono-, 1,l di- and cis-1,2-disubstituted epoxides and is induced by a wide range of compounds including phenobarbital and Aroclor 1254 (Wixtrom and Hammock, 1985). Cholesterol

ERIC C. DIETZE et al.

462

epoxide hydrolase and leukotriene A4 hydrolase ap.5,6-oxide and pear selective for cholesterol leukotriene Aq. respectively, and have been less well studied. CEH has been purified and partially characterized from many mammalian species (Meijer and DePierre, 1988; Wixtrom and Hammock, 1985). The mouse offers the most ~nvenient system in which to study CEH due to a combination of ease of isolation and high stability of murine liver CEH. Murine CEH is a homodimer of IV, 120 kDa. The best substrates appear to be 1,2_disubstituted aliphatic epoxides (Hammock and Hasagawa, 1983; Wixtrom and Hammock, 1985). CEH is induced by clofibrate and other peroxisome proliferators (Meijer and DePierre, 1988). It is similar to epoxide hydrolases localized in the mitochondria and the peroxisomes (Gill and Hammock, 1980; Meijer and DePiere, 1988). The purpose of this study is to compare CEH from human (Homo sapiens) and murine (Mus musculus) liver and to test whether or not uninduced and clofibrate induced murine CEH are identical. (Induced murine CEH is routinely used to study CEH in vitro.) Murine CEH should be similar in structure and function to serve as a meaningful model for human CEH. In this study, affinity purified enzymes were compared by molecular weight, isoelectric point, peptide mapping, amino acid content, Western blotting and circular dichroism.

or female donors who died of traumatic injury or stroke. The livers were removed and held, perfused with urocolins, until they were not useful for transplantation. The livers were then transported, on ice, to SRI where they were frozen and stored in liquid nitrogen. The total time between removal of the livers and arrival at SRI varied between 1 and 13 hr. The histories of drug exposure were unknown. All livers used tested negative for hepatitis and HIV-l. En:yme preparation and assay

Murine Iiver CEH and human liver CEH were purified, dialyzed (Wixtrom et al., 1989). and assayed with [‘HITS0 as described (Wixtrom and Hammock, 1985). Cytosol was either used immediately or stored at -80°C. No loss of CEH activity occurred during storage (data not shown). Protein concentrations of cytosol and-nonbinding fractions were determined bv the Bradford method modified for use on a plate reader with computer readout (Hammock et al., 1986). The concentration of purified enzyme was determined using the Pierce RCA reagent. This method was modified for use in 96 well microtiter plates with a V,,,=p plate reader (Molecular Devices, Palo Alto, Calif.) and computer data processing with the associated SoftMaxTM software. The standard assay was carried out in quadrupli~ate and modified as follows. First 300 ~1 of BCA reagent was added to each well. Next 15~1 of protein solution (standard or unknown) was added and the plate floated in a 60°C water bath for 30min. The plate was removed from the water bath, wiped dry and read at 560 nm. The concentration of the added protein standards ranged from 5 to 80pg/ml. Correlation coefficients for standard curves were generally >0.98. Amino acid analysis

MATERIALS AND METHODS Chemicak

rrans-Stiibene oxide (TSO), CNBr, guanidine HCI and iodoacetamide were purchased from Aldrich (Milwaukee, Wis). Bovine serum albumin (fraction V), 2-mercaptoethanol, and DPCC treated trypsin were obtained from Sigma (St Louis, MO.). Endoproteinase Glu-C (V8 protease) was from Boehringer Mannheim (Indianapolis, Ind.). Bradford reagent, silver stain and SDS-PAGE electrophoresis reagents and standards were obtained from Bio-Rad (Richmond, Calif.). Ampholine PAG plates were obtained from LKB (Bromma, Sweden). BCA protein determination reagents were purchased from Pierce Chemical (Rockford, If.). Trifluoroacetic acid (TFA) was purchased from EM Industry (Cherry Hill, N.J.) and 88% formic acid was from Fisher Scientific (Pittsburgh, Pa.). HPLC grade methanol and acetonitrile were obtained from either Fisher, Aldrich. or J. T. Baker (Phillinsbure. N.J.). HPLC water was obtained from a M~lli~r~ (Bedford, ‘Mass.) Mini-Q system. Water for other uses was deionized and glass distilled. All other chemicals and solvents were of the best grade commercialy available. Tisslie samples

Male CS7B1/6 mice, 20-25, were purchased from Simonsen Laboratories (Gilroy, Calif.). They were housed in an environmentally controlled room (12 hr light cycle, 22.524.O”C, and constant humidity) and given water and Purina rodent chow ad libitum. The mice were held for one week after receipt and either killed by cervical dislocation or fed for two weeks on ground chow with 5% corn oil and 0.5% clofibrate (Ayerst Laboratories, N.Y.) by weight and killed. Livers were harvested, perfused with ice cold 1.15% KCI and immediately processed. Human liver samples were obtained from the SRI organ bank (Menlo Park, Calif.) and were stored at -80°C until needed. Total storage time of the livers was between 612 months. The livers used in this study were from either male

Amino acid analysis was perfotmed by the UC-Davis Protein Structure Laboratorv. Purified CEH (IO&200 tin) was hydrolyzed in 6 N HCl with or without prior performic acid oxidation. Amino acids were derivatized with ninhydrin and separated and quantitated on a Beckman 6300 amino acid analyzer. ~-Amino-~-guanidino propionic acid was used as an internal standard. Values reported are the average of two separate determinations. PAGE and Western blotting

SDS-PAGE was carried out according to the method of Laemmli (Laemmli, 1970). Samples (1 pg purified CEH or 1lo-120 ue cvtosol) were run on 12.5% eels with Bio-Rad (Richmond, Calif.) low molecular weight standards. The gels were stained with Coomassie Brilliant Blue R-250. Isoelectric focusing (IEF) gels were run on ampholine PAG plates at 4°C (Silva et al., 1988). After focusing was complete CEH was visualized using a Bio-Rad silver stain kit. ~-Lactoglobulins A [PI = 5.2_5land B IpI = 5.351(LKB, Bromma, Sweden) were used as standards to calculate pls for the human and murine CEH. Between 1 and 2pg of protein was loaded in each lane. Western blots were carried out according to standard procedure (Silva et a/., 1988). Blots were run with polyclonal antibodies, prepared as described (Hammock et al., 1986), against either purified human or murine liver CEH. Peptide preparation

CNBr digests were carried out by dialyzing 0.1-1.0 mg of purified CEH against 2 1000 vol of 100 mM Tris (pH 8.3) 2 mM EDTA buffer for 2 hr at 4’C. The dialyzed CEH was reduced and denatured with 10mM dithiothreitol, 5M guanidine HCI, in the same buffer, for 2 hr under nitrogen at room temperature. The cysteine residues were then derivatized with 25 mM iodoacetamide for 1hr under nitrogen at room tem~rature, The derivatized CEH was precipitated by dialyzing overnight at 4°C against water containing 0.1% 2-mercaptoethanol. (Subsequent steps

Cytosolic epoxide hydrolase were performed in conical polypropylene tubes.) The precipitate was lyophilized and stored at -80°C until needed. The precipitate was suspended in 88% formic acid at a concentration of 10 mg/ml for CNBr digestion. The formic acid was then diluted to 70% with water. Solid CNBr was added to bring the solution to 100 mg/ml in CNBr, the tube flushed with N,, and incubated for 24 hr in the dark at 4°C. The digest was lyophilized after the addition of an equal volume of water. The digest was either used immediately or stored at -80°C. Protease digests were obtained by incubating 0.05505 mg of purified CEH with protease for 4 hr at 37°C with shaking. Additions of 1% protease (wtjw CEH) were made at 0 and 2 hr for trypsin and 0, 1.33 and 2.67 hr for V8 protease. Digestions were terminated by immersion in a dry ice/acetone bath and the frozen samples lyophilized. The samples were stored at -80°C until needed. All digestions were carried out in 76 mM Na/K phosphate (pH 7.4) 1 mM EDTA buffer. Liquid chromatography

Peptide digests were separated by reverse phase, ion pair HPLC. A Brownlee C-4 aquapore column (Brownlee Labs, Santa Clara, Calif.) was used with TFA as the counterion. A two solvent system was employed. The first (solvent A) was aqueous 5% (v/v) methanol, 0.1% (v/v) TFA and the second (solvent B) was aqueous 55% (v/v) acetonitrile, 0.07% (v/v) TFA. The gradient for separation of CNBr peptide fragments was 10% A/90% B to 100% B over 140min. The gradient used for the separation of the protease digests was 100% A to 100% B over 60min. In both types of separation 100% B was held for 10 min and the system was returned to 100% A over a period of 10 min. The gradients were linear. A Spectra Physics 8700 solvent delivery system (Spectra Physics, San Jose, Calif.) was used with a Kratos 757 u.v./Vis detector (Kratos Analytical Instruments, Ramsey, N.J.). Samples were injected after being dissolved in 70% aqueous formic acid (v/v). Between 10 and 100 pg of sample were loaded and peptides detected by their absorbance at 215 nm. Gel permeation chromatography was carried out with a Perkin Elmer 410 Bio solvent delivery system and LC-235 diode array detector (Perkin Elmer, Norwalk, Conn.). A Beckman TSK-3000 PW column (7.5 mm id. x 30cm, Beckman Instruments, Berkeley, Calif.) was used with a flow rate of 0.5 mlimin. The solvent was filtered 76 mM NaiK phosphate (pH 7.4), 1mM EDTA buffer. Protein was monitored at 220nm. The column was calibrated with Sigma native gel molecular weight standards. Circular dichroism

Circular dichroism (CD) spectra were obtained on a JASCO J-500C spectropolarimeter (JASCO, Tokyo, Japan) at room temperature. The instrument was calibrated with ammonium D-camphor-lo-sulfonate (0.5949 mg/ml in distilled water). CEH concentrations were 5.0 pg/ml for human and uninduced murine CEH and 7.5pg/ml for induced murine CEH. Measurements were taken using a 1 cm path length in a square quartz cell at room temperature. Each sample was scanned 16 times, from 260 to 199nm, at lOnm/min and the spectra were averaged. Buffer was scanned under identical conditions and background due to the buffer and cell substracted with a JASCO DP-5OlN data processing unit. A mean residue weight of 115 Da was used to calculate the molar ellipticity ([e]). RESULTS

PuriJication

All forms of CEH were pure as judged by SDS-PAGE and Coomassie Blue staining (Fig. 1). The results of purification are shown in Table 1.

463

Purified human liver CEH had a sp. act. of 360nmol.min-‘.mg-‘. Human liver CEH gave a yield of 17% and a 360-fold purification. Both uninduced and induced murine liver CEHs had a sp. act. of 1500 nmol.min-‘.mg-‘. Uninduced murine CEH showed a yield of 38% and a 212-fold purification. Induced murine CEH had a 49% yield and a 1IO-fold purification. The ca 2-fold induction of CEH in crude cytosol by clofibrate is in accord with previous reports (Loury et al., 1985; Meijer and dePierre, 1987; Moody et al., 1986; Pichare and Gill, 1985). Lower yields observed with uninduced murine and human liver CEH were probably due to decreased stability of CEH at low protein concentrations. In these preparations the concentration of purified CEH is significantly lower than in the preparations made using clofibrate induced mice. Significant loss of human CEH also occurred due to less efficient binding to the affinity column. Molecular

weight and isoelectric

point

Purified human CEH and both murine CEH preparations have an apparent IV, of 59 kD as determined by SDS-PAGE (Table 1). By HPLC gel permeation chromatography both human and the murine CEHs appeared to be monomeric. Their apparent M,, 63 kDa, was somewhat higher than the value obtained by gel electrophoresis. Reports in the literature indicate that native CEH is a homodimer (Gill, 1983; Meijer and DePierre, 1985a). However, under these conditions purified native CEH behaved as a monomer (Hammock et al., 1986; this study). Human CEH ran as a single, sharp band on both wide (pH 4.G9.0) and narrow (pH [email protected]) IEF gels. Both uninduced and induced murine CEH gave a tight doublet when focused on either wide or narrow range gels. Addition of 0.5 mM diisopropylfluorophosphate to the homogenization buffer abolished the minor band of this doublet. This band is probably the result of proteolytic cleavage during purification of murine CEH. Human CEH had an isoelectric point of 5.9 (Table 1). Reported isoelectric points for human CEH are 5.1-6.1 and 5.7 (Schladt et al., 1988; Wang et al., 1982). Both uninduced and clofibrate induced murine Table

I.

Purification, molecular weight, and isoelectric human and murine CEHs*

Cytosol Total protein (mg) Total activity (nmol’min) Flow through Total protein (mg) Total activity (nmol’min) CEH Total protein (mg) Total activity (nmol ‘min) Sp. act. (nmol~min~mg) Fold purification Yield (%) K @Da) SDS-PAGE gel permeation nl *Activity measured SDS-PAGE.

point

Human liver

Uninduced murine liver

Induced murine liver

833 866

383 2710

533 7410

116 168

327 280

441 163

0.42 150 360 343 17

0.68 1020 1500 212 38

58.8 * 3.9 57.9 + 6.2 5.9

59.1 + 3.4 60.7 + 5.5 51

at pH 7.4 with TSO. Molecular

of

2.4 3610 1500 110 49 59.2 + 3.7 62.6 i 6.2 5.7 weight

from

464

ERIC C. DIETZE et al. Table 2. Amino acid composition murine CEHs Human liver Ala Arg Asx CYS Glx GlY His Ile Lell LYS Met Phe Pro Ser Thr TOP TY~ Val Total

Uninduced murine liver

40.0 26.5 44.4 13.2 64.2 39. I 9.04 21.7 58.4 34.2 21.1 23.6 34.7 29.8 29.8 ND I I.4 33.9

of human and Induced murine liver 39.3 23.6 44.5 12.7 60.8 32.6 8.24 29.4 47.2 33.0 26.4 30.2 37.4 27.4 30. I ND 11.7 31.4

41.5 25.5 38.7 12.2 56.0 45.2 10.3 32.6 50.1 38.0 21.6 28.5 40.7 31.5 29. I ND 12.2 37.3

535

1977; Metzger et al., 1968). By either method the most closely related enzymes pairs were rabbit MEH/rat MEH, human CEH/induced murine CEH, and uninduced murine CEH/induced murine CEH (Table 3). Human and murine CEH do not appear to be related to rabbit CEH or to any of the other enzymes considered.

551

Peptide mapping

526

CEH had an isoelectric point of 5.7. The reported value for the pI of murine CEH is 5.5 (Hammock et al., 1986; Meijer and DePierre, 1985a). Amino ncid analysis

The amino acid composition of human and murine CEH’s was found to be very similar (Table 2). Human CEH contained significantly more leucine residues than either uninduced or induced murine CEH. However, human CEH also had fewer isoleucine and phenylalanine residues. The most striking difference in composition between uninduced and induced murine CEH was the increased number of glycine residues found in uninduced murine CEH. The values for amino acid composition of induced and uninduced murine CEH are in close agreement with reported values (Meijer and DePierre, 1985a; Hammock et al., 1986). Two exceptions are the high glycine content of uninduced murine CEH in this report and the low value for methionine reported by Hammock et al. (Hammock er al., 1986). Relatedness of human, induced murine and uninduced murine CEH to each other and to rabbit CEH (Waechter et al., 1982) rabbit MEH (Heinemann and Ozols, 1984) rat MEH (Porter et al., 1986) guinea pig (Cacia porcellus) leukotriene A, hydrolase (Haeggstrom et al., 1988) and human leukotriene A, hydrolase (Funk et al., 1987) was assessed by comparing their amino acid compositions (Cornish-Bowden, Table 3. Difference HCEH HCEH M( -)CEH M(+)CEH RBCEH RBMEH RTMEH GPLTA4 HLTA4

M(-)CEH 210/220

5.8 5.0 7.3 14 15 9.5 9.1

5.2 7.9 II II I? I2

analysis

M( + )CEH 1601220 1601220 a.4 14 I3 I3 I3

Human liver CEH peptide maps were not identical to those of murine liver CEH (Figs 2-4). All three types of cleavage generated a few peptides which were simlar, or identical, based on retention time. However, the overall peptide maps were substantially different. The CNBr peptide maps were the most closely related. In all CNBr digests there was a good correlation between the number of peaks observed and the number of methionines found in the amino acid analyses. This indicates that most of the cleavage occurred at methionine residues. A similar correlation cannot be made with either of the proteolytic digests since CEH was not denatured prior to protease digestion. Both uninduced and induced murine liver CEH were qualitatively identical by peptide mappings (Figs 2-4). CNBr, trypsin or V8 protease cleavage each generated a unique set of identical peptide maps. Thus, fragments generated by cleavage at methionine, lysine/arginine or aspartate/glutamate could not be differentiated. These results confirm and extend earlier findings by Meijer et al. (1987a). Western blotting

Crude and purified CEH from human, uninduced murine, and induced murine liver were run on 12.5% SDS-polyacrylamide, transferred to nitrocellulose and blotted with either rabbit anti-murine liver CEH (Fig. 5a) or rabbit anti-human liver CEH (Fig. 5b). Human and murine liver CEH were shown to be antigenically related by this technique. Rabbit anti-human CEH displayed a much stronger crossreaction with murine liver CEH than the anti-murine CEH with human liver. Since the sera were polyclonal, it is possible that human CEH shares most of its antigenic determinants with murine CEH while murine CEH has antigenic determinants that human CEH lacks. It is also possible that differences in the degree of crossreactivity were due to variation between the two polyclonal sera (serum samples from different animals were not pooled and standardized).

of CEH amino acid compositions* RBCEH

RBMEH

RTMEH

GPLTA4

HLTA4

280/2lO 29012 IO 36012 IO

6601186 500/186 640/186 980/189

830/186 450/186 6lO/l86 I ooo/ I90 136/191

4181225 6371231 7351221 696/218 55Ojl9l 478/191

3851225 650123 I 1010/221 819/218 426/191 1770/191 48 I/256

I3 I6 I3 I3

5.5 I4 I2

I2 I7

6.9

‘Upper right corner: difference index of Cornish-Bowden (1977)iindex value for 95% confidence interval. Lower corner: difference index of Metzger er al. (1968). Abbreviations: Human CEH (HCEH), uninduced murine CEH [M(-)CEH]), induced murine CEH [M(+)CEH], rabbit CEH (RBCEH), rabbit MEH (RBMEH), rat MEH (RTMEH), guineapig leukotriene A, hydrolase (GPLTA4) and human leukotriene A, hydrolase (HLTA4).

97.4

66.2

42.7

31.0

21.5-

14.4-

MW

(kDax10-3)

1

. 2

3

4

5

wfvl

6

(kDax10e3)

Fig. 1. SDS -PAGE of human and murine CEHs and cytosol. Lanes are: (I) affinity purified, uninduced murine liver cytosol; (3) affinity purified, induced murine liver CEH; murine liver CEH; (2) uninduced (4) induced murine liver cytosol; (5) affinity purified human liver CEH; and (6) human liver cytosol.

465

(a) Blot with

rabbit

anti-murine

liver CEH antibody

97.4 66.2

66.2

42.7

,42.7

31 .o

31.0

21.5

21.5

14.4 Mw

14. 1

2

3

4

5

6

(kDa x 10e3)

(b) Blot with rabbit

anti-human

liver CEH antibody

66.2

66.2

42.7

42.7

31 .o

31.0

21.5

21.5

14.4

14.4

Mw (kDa x 10m31

4

MW (kDa ~10~~)

MW (kDa x 10s3)

Fig. 5. Western blot of human and murine liver CEHs and cytosol using: (a) rabbit anti-murine liver CEH antibody and (b) rabbit anti-human liver CEH antibody. Lanes are: (I) affinity purified, uninduced murine liver CEH; (2) uninduced murine liver cytosol; (3) affinity purified, induced murine liver CEH; (4) induced murine liver cytosol; (5) affinity purified human liver CEH; and (6) human liver cytosol.

466

Cytosolic epoxide hydrolase

467

same degree to either anti-murine or anti-human CEH antibodies (Fig. 2). Induction of murine CEH was clearly seen when the respective crude preparations were compared. Circular dichroism loo-

b

m E % f?i 8

Time (mln)

Fig. 2. HPLC separation of CNBr digests of affinity purified CEH: (a) uninduced murine liver CEH; (b) induced murine liver CEH; and (c) human liver CEH. A linear gradient of 10% B to 100% B over 140 min was used. The conditions were: column-Brownlee C4; flow-l ml/min; 0.1 absorbance units full scale. spectrophotometer-A,,,;

There are conflicting reports in the literature on the antigenic relatedness of primate and rodent CEH (Meijer et al., 1987b; Schladt et al., 1988). This variation may also be due to the use of uncharacterized polyclonal antibodies. Uninduced and induced murine CEH appeared to be antigenically identical. Both responded to the

100

1

The CD spectra of human, uninduced and induced liver CEH (Fig. 6) showed that human CEH, uninduced murine CEH, and induced murine CEH all differed slightly from one another. The spectra were typical of proteins with a moderate content of both cc-helix and p-sheet. Estimates of the a-helix content were made using the method of Chen et al. (1972). The values were: human CEH - [0]222 = 9400 mdeg.cm*.dmol-’ and 39% a-helix, uninduced murine CEH - [0]222= 9200 mdeg.cm2.dmoll’ murine CEH and 38% cc-helix, and induced - [6]222= 8300 mdeg.cm*.dmoland 35% a-helix. The maximum value of [e] was seen at 220 nm for human, uninduced murine and induced murine CEH.

DISCUSSION

The major goal of this study was to test the physical similarity of human and murine liver CEH. A variety of methods were chosen which would be likely to find differences, should they exist, and provide a broad comparison to assess the relative degree of similarity. These methods range from simple molecular weight determination to amino acid composition to antigenic relatedness. The secondary goal of this work was to establish the degree of similarity of uninduced and clofibrate induced murine CEH.

a

OY 1001

n

O-0

0

SO Time (mln)

Fig. 3. HPLC separation of trypsin digests of affinity purified CEH: (a) uninduced murine liver CEH; (b) induced murine liver CEH; and (c) human liver CEH. A linear gradient of 0% B to 100% B over 60min was used. The conditions were: column-Brownlee C4; flow-l ml/min; 0.1 absorbance units full scale. spectrophotometer-A,,,;

Time (mln)

Fig. 4. HPLC separation of V8 protease digests of affinity purified CEH: (a) uninduced murine liver CEH; (b) murine liver CEH; and (c) human liver CEH. A linear gradient of 0% to 100% B over 60min was used. The conditions were: column-Brownlee C4; flow-l ml/min; spectrophotometer-A,,, ,. 0.05 absorbance units full scale.

ERICC. DIETZEet al.

468

As compared by molecular weight, human, murine and induced murine liver CEH were identical. All forms had an apparent M,, of 59 or 63 kDa depending on whether SDS-PAGE or gel permeation chromatography was used to determine molecular weight. This is consistent with reported values which range from 5840 kDa (Gill, 1983; Meijer and DePierre, 1985a; Prestwich and Hammock, 1985). However, IEF showed that human and murine CEH were not identical. Human CEH had a p1 of 5.9 while both uninduced and induced murine CEH had a p1 of 5.7 when run on the same focusing gel. These values were in the range seen for typical CEHs and well below the p1 of ca 7.0 seen for human and rat MEHs (Schladt et al., 1988). All purified CEHs were found to be homogeneous by both SDS-PAGE and IEF which indicated the presence of one form of affinity purified CEH in both species. By amino acid analysis and peptide mapping, human and murine liver CEH were not identical. The similarity of the amino acid analyses indicated that CEH from human and murine liver are probably closely related. The CNBr digests were the most nearly identical while the trypsin and V8 digests were substantially different. Less variability would be expected in the number and position of methionine residues than either lysine/arginine or aspartate/glutamate residues. The identity of the uninduced and induced murine CEH peptide maps and near identity of amino acid composition confirmed reports in the literature (Meijer et af., 1987). It is clear from the digestion patterns and amino acid composition that murine CEH purified from uninduced and induced animals was, at worst, only slightly different. Without the amino acid sequence for uninduced and induced murine CEH it is impossible to determine whether or not the differences seen are real or due to experimental variability.

_‘I a

0

1x1o.4

7

b

gt .

0.

NE i = d

l!!??L 1x10’4 C 0

1x10-4k

200

230

260

Wavelength (nm)

Fig. 6. CD of affinity purified CEH: (a) uninduced murine liver; (b) induced murine liver; and (c) human liver.

Western blots showed that human and murine CEH are antigenically related. The anti-murine CEH crossreacted only weakly with purified human CEH and no band could be seen with human liver cytosol. However, when anti-human liver CEH was used there was a strong crossreaction with murine CEH. This is clear indication that human and murine CEH share common antigenic determinants. Monoclonal antibodies could be used to characterize the antigenic determinants on both human and murine CEH. Structural similarity of the crossreacting determinants could be ascertained. Uninduced and induced murine CEH were indistinguishable by Western blotting. Both stained with the same intensity with either anti-human or anti-murine CEH antibody. The differences in the CD spectra were minor as reflected by the estimated cc-helical content of 39% for human CEH, 38% for uninduced murine CEH and 35% for induced murine CEH. The differences

are not significant and subtraction of the spectra from one another showed only minor differences (data not shown). Overall the spectra of all three enzymes have similar shape and amplitude which indicates similar secondary structure content. The content of a-helix also highlighted structural similarity between CEH and MEH. MEH was found to have 35% cc-helical structure (Magdalou et al., 1982). Clearly, uninduced and clofibrate induced murine liver CEH were essentially identical proteins. To the extent that murine CEH is a valid in vitro model for

human CEH, it appears that either uninduced or induced murine CEH may be utilized. Both enzyme preparations were homogeneous and had identical molecular weight, isoelectric point and specific activity with TSO. The CNBr, tryptic and V8 protease digests appeared qualitatively identical. The uninduced and induced murine CEH were indistinguishable by either Western blotting or CD. Both the uninduced and induced CEH stain to the same intensity with either anti-murine or anti-human antibodies. Within experimental error the percent cc-helix was the same. Amino acid analysis indicated possible microheterogeneity in the composition of uninduced and induced murine CEH. This microheterogeneity, while reproducible, may have been the result of differences in minor contaminants of the purified preparations or, as has been proposed, differences in CEH processing between uninduced and induced animals (Meijer and DePierre, 1988). Differences have also been found in the physical and kinetic properties of uninduced and induced murine CEH (Meijer and DePierre, 1985b). A definitive answer will require comparison of the primary sequences and/or isolation of multiple genes, or mRNAs, for murine CEH. From the data presented it is clear that human and murine liver CEH were closely related. Human and murine CEH have the same molecular weight by both SDS-PAGE and gel permeation chromatography. The pIs are similar and clearly different from the pI typically seen for MEHs. By the difference index of Cornish-Bowden, the more quantitative of the two difference indexes used, there was a significant relationship between human and murine CEH. Differences did occur in the amino acid compositions

Cytosolic epoxide hydrolase

of human and murine CEH. These may be the result of conservative changes in amino acids. Human CEH contained 58 leucine residues but induced murine CEH contained only 47 leucine residues. However, the sum of isoleucine and phenylalanine residues, both have bulky, hydrophobic side chains, is 46 for human CEH and 59 for induced murine CEH. The lack of leucine residues in induced murine CEH may be compensated for by the increased number of isoleucine and phenylalanine residues. The apparent relationship between human CEH and induced murine CEH was as good as that between rabbit and rat MEH. Since rabbit and rat MEH have 81% sequence homology (Porter et al., 1986), it is probable that human and murine CEH are also highly related. Peptide mapping showed clearly that there were differences between human and murine CEH. Although there appeared to be some peptides which might have been similar or identical, all three digests of human and murine CEH were qualitatively different. Western blotting and CD both demonstrated that human and murine CEH had extensive similarity in their secondary and tertiary structures. Both CEHs shared antigenic determinants and both had an identical content of ~-helix. Until both proteins are sequenced and X-ray crystal structures are available, the exact degree of structural homology will remain uncertain. Despite the clear physical and structural similarity of the human and murine CEH, it is evident from reports in the literature that they may be functionally distinct and can be differentiated by substrate specificity (Gill et al., 1983; Meijer and DePierre, 1988; Meijer et al., 1987b; Schladt et al., 1988). The diagnostic preference for TSO vs CSO for murine CEH at pH 7.4 is reversed for human CEH (Gill et al., 1983). This may be explained, in part, by the recent discovery of two distinct epoxide hydrolase activities in human cytosol (Schladt ef al., 1988). One of the enzymes was similar to MEH in both its physical properties and substrate preference. The second enzyme closely resembled murine CEH in its properties. As a result further comparison of human and murine CEH must be undertaken in order to validate the use of murine CEH as a model for the function of one, or both, human CEHs. These studies should include a continued search for multiple forms of CEH as well as extensive comparison of substrate specificity, primary and tertiary structure determination, and comparison of catalytically important amino acids. However it appears likely that the mouse and murine CEH will be a good model system in which to study the catalytic mechanism and in uiuo function of CEH. Acknowledgements-We would like to thank DC Dieter W. Gruenwedel for assistance with the CD measurements and

Ayerst Pharmaceutical for the gift of clofibrate. The SRI organ bank is supported by Grant ES-55109 from NIEHS and Grant NOl-DK-8-2235 from NIDDK. This work was sutmorted by NIEHS ESO27IO. Eric Dietze was supported: *in part,- by NIEHS predoctoral training Grant ES07059 and a Jastro-Shields Graduate Research Fellowship from the University of California, Davis. Jacques Magdalou was partially supported by a Grant from CNRS. Bruce Hammock is a Burroughs-Wellcome Scholar

in Toxicology.

469 REFERENCES

Brash A. R., Baertschi S. W., Ingram C. D. and Harris T. M. (1988) Isolation and characterization of natural allene oxides: unstable intermediates in the metabolism of lipid hydroperoxides. Proc. nnrn. Acad. Sci. U.S.A. 85, 3382-3386. Carroll M. A., Schwartzman M., Capdevila J., Flack J. R. and McGiff J. C. (1987) Vasoactivity of arachidonic acid epoxides. Eur. J. Phurmuc. 138, 281-283. Cashman J. R.. Hanks D. and Weiner R. I. (1987) Epoxy derivatives of arachadonic acid are potent stimulators of prolactin secretion. Neuroendocrinology 46, 246-25 1. Chen Y. H., Yang J. T. and Martinez H. M. (1972) Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 11, 4120-4131. Cornish-Bowden A. (1977) Assessment of protein sequence identity from amino acid composition data. J. theor. Biol. 65, 735-742.

DuBois G. C., Appella E., Levin W., Lu A. Y. H. and Jerina D. M. (1978) Hepatic microsomal euoxide hydrolase: involvement of a histidine at the active site suggests a nucleophilic mechanism. J. &al. Chem. 253, 2932-2939.

El-Tantawy M. A. and Hammock B. D. (1980) The effect of hepatic microsomal and cytosolic subfractions on the mutagenic activity of epoxide-containing compounds in the Salmonella assay. Mural. Res. 79, 59-71. Fitzpatrick F. A., En& M. D., Baze M. E., Wynalda M. A., McGee J. E. and Liezett W. F. (1986) Inhibition of cyclooxygenase activiyi and platelet aggregation by epoxyeicosattrienoic acids. J. biol. Chem. 261, 1533415338. Funk C. D., Radmark O., Fu J. Y., Matsumoto T., Jomvall H., Shimizu T. and Samuelsson B. (1987) Molecular cloning and amino acid sequence of leukotriene A4 hydrolase. Proc. natn. Acad. Sci. U.S.A. 84, 66776681. Gili S. S. (1983) Purification of mouse liver cytosolic enoxide hvdrolase. Biochem. bionhvs. ^ , Rex Commu~. 112. 7k3-769.

-

Gill S. S. and Hammock B. D. (1980) Distribution and properties of a mammalian soluble epoxide hydrolase. Biochem. Pharmac. 29, 389-395. Gill S. S., Ota K., Ruebner B. and Hammock B. D. (1983) Microsomal and cytosolic epoxide hydrolase in rhesus monkey liver and in normal and neoplastic human liver. Life Sri. 32, 2693-2100. Haeggstrom J., Bergman T., Jornvall H. and Radmark 0. (1988) Guinea pig leukotriene A4 hydrolase. Eur. J. Biochem. 174, 717-724. Hammock B. D. and Hasagawa L. S. (1983) Differential substrate selectivity of murine hepatic cytosolic and microsomal epoxide hydrolases. Bioehem. Pharmac. 32, 1155-1164. Hammock B. D., Prestwich G. D., Loury D. N., Cheung P. Y. K.. Ene W. S.. Park S. K.. Moodv D. E.. Silva M. H. and Wixtiom R.‘N. (1986) domparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice. Archs Biochem. Biophys. 244, 292-309. Heinemann F. S. and 0~01s J. (1984) The covalent structure of hepatic microsomal epoxide hydrolase. J. biol. Chem. 259, 797-804. Hemminki K. and Vainio H. (1984) Genotoxicity of epoxides and epoxy compounds. In Industrial Hazards of Plastics and Synthetic Elastomers (Edited by Jarvisalo J., Pfaffli P. and Vainio H.), pp. 373-384. Liss, New York. Imai H., Werthessen N. T., Subramanyam V., Lequesne P. W., Soloway A. H. and Kanisawa M. (1980) Angiotoxicity of oxygenated sterols and possible precursors. Science 207, 651-653.

470

ERIC C. DIETZEet al.

Jones R. B. and Mackrodt W. C. (1983) Structuregenotoxicity relationship for aliphatic epoxides. Biochem. Pharmac. 32, 2359-2362. Kadis B. (1978) Steroid epoxides in biological systems: a review. J. steroid Biochem. 9, 75-81. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 221, 680-685. Loury D. N., Moody D. E., Kim B. W. and Hammock B. D. (1985) Effect of dietary clofibrate on epoxide hydrolase activity in tissues of mice. Biochem. Pharmac. 34, 1827-1833. Magdalou J., Kiffel L., Balland M., Thirion C., Le-Meste M. and Siest G. (1982) Conformational study of purified epoxide hydrolase from rat liver. Chem.-biol. Interact. 39, 245-256. Meijer J. and DePierre J. W. (1985a) Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. I. Purification procedure and physicochemical characterization of the pure enzymes. Eur. J. Biochem. 148, 421430. Meijer J. and DePierre J. W. (1985b) Properties of cytosolic epoxide hydrolase purified from the liver of untreated and clofibrate-treated mice. II. Characterization of optimal assay conditions, substrate specificity and effects of modulators on the catalytic activity. Eur. J. Biochem. 150, 7-16. Meijer J. and DePierre J. (1987) Hepatic levels of cytosolic, microsomal and “mitochondrial” epoxide hydrolases after treatment of mice with various xenobiotics and endogenous compounds. Chem.-biol. Interact. 62, 249-269. Meijer J. and DePierre J. W. (1988) Cytosolic epoxide hydrolase. Chem.-biol. Inreracl. 64, 207-249. Meijer J., DePierre J. W. and Jornvall H. (1987a) Cytosolic epoxide hydrolase from liver of control and clofibratetreated mice. Structural comparison by HPLC peptide mapping. Biosci. Rep. 7, 891-896. Meijer J., Lundqvist G. and DePierre J. W. (1987b) Comparison of the sex and subcellular distributions, catalytic and immunochemical reactivities of hepatic epoxide hydrolases in seven mammalian species. Eur. J. Biochem. 167, 269-279. Metzger H., Shapiro M. B., Mosimann J. E. and Vinton J. E. (1968) Assessment of compositional relatedness between proteins. Nature 219, 11661168. Moody D. E., Silva M. H. and Hammock B. D. (1986) Epoxide hydrolysis in the cytosol of rat liver, kidney and testis. Biochem. Pharmac. 35, 2073-2080. Ozawa T., Sugiyama S., Hayakawa M., Taki F. and Hanaki Y. (1988) Neutrophil microsomes biosynthesize linoleate epoxide (9, IO-epoxy- 12-octadecenoate), a biologically

active substance. Biochem. biophys. Res. Commun. 152, 13lCk1318. Pichare M. M. and Gill S. S. (1985) The regulation of cytosolic epoxide hydrolase in mice. Biochem. biophys. Res. Commun. 133, 233-238. Porter T. D., Beck T. W. and Kasper C. B. (1986) Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase. Archs Biochem. Biophys. 248, 121Ll29. Prestwich G. D. and Hammock B. D. (1985) Rapid purification of cytosolic epoxide hydrolase from normal and induced animals by affinity chromatography. Proc. natn. Acad. Sci. U.S.A. 82, 1663-1667. Puustinen T., Webber S. E., Nicolaou K. C., Haeggstrom J., Serhan C. N. and Samuelsson B. (1986) Evidence for a S(6)-epoxytetraene intermediate in the biosynthesis of lipoxins in human leukocytes. FEES Left. 207, 127-132. Schladt L., Thomas T., Hartmann R. and Oesch F. (1988) Human liver cytosolic epoxide hydrolase. Eur. J. Biochem. 176, 715-723. Sevanian A., Mead J. F. and Stein R. F. (1980) Lipid epoxidation in the lung. In Molecular Basis of Environmental Toxicology (Edited by Bhatnager R. S.), pp. 213-228. Ann Arbor Science, Michigan. Silva M. H. and Hammock B. D. (1987) Affinity purification of cytosolic epoxide hydrolase from human, rhesus monkey, baboon, rabbit, rat, and mouse liver. Camp. Biochem. Physiol. 87B, 95-102. Silva M. H., Wixtrom R. N. and Hammock B. D. (1988) Epoxide-metabolizing enzymes in mammary gland and liver from Balb/c mice and effects of inducers on enzyme activity. Cancer Res. 48, 139&1397. Sugiyama S., Hayakawa M., Nagai S., Ajioka M. and Ozawa T. (1987) Leukotoxin, 9, IO-epoxy- 12-octadecanoate, causes cardiac failure in dogs. Life Sci. 40, 225-23 1. Waechter F., Merdes M., Bieri F., Staubel W. and Bentley P. (1982) Purification and characterization of a soluble epoxide hydrolase from rabbit liver. Eur. J. Biochem. 125, 457461. Wang P., Meijer J. and Guengerich F. P. (1982) Purification of human liver cytosolic epoxide hydrolase and comparison with the microsomal enzyme. Biochemistry 21, 5769-5776. Wixtrom R. N. and Hammock B. D. (1985) Membranebound and soluble fraction epoxide hydrolases. In Biochemical Pharmacology and Toxicology, Vol. 1 (Edited by Zakim D. and Vessey D. A.), pp. l-93. Wiley, New York. Wixtrom R. N., Silva M. H. and Hammock B. D. (1989) Affinity purification of cytosolic epoxide hydrolase using derivatized epoxy-activated Sepharose gels Analyt. Biochem. 169, 71-80.