Purification and initial characterization of microsomal epoxide hydrolase from the human adrenal gland

ELSEVIER Biochimica et Biophysica Acta 1206 (1994) 253-262

BB Biochi~ic~a et Biophysica A~ta

Purification and initial characterization of microsomal epoxide hydrolase from the human adrenal gland

Dimitris Papadopoulos a, Hans J6rnvall b, Jan Rydstr6m c, Joseph William DePierre a,, a Unit for Biochemical Toxicology, Department of Biochemistry, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden

b Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden c Department of Biochemistry, Chalmers Academy of Technology, S-412 58 Gothenberg, Sweden

Received 8 November 1993

Abstract

Microsomal epoxide hydrolase from the human adrenal gland was purified to a high degree of homogeneity in 10% overall yield using sequential chromatography on DE-52, FPLC Mono Q and FPLC Superose columns. The fact that the overall purification was only 7.3-fold indicates that approx. 14% of the total microsomal protein consisted of this enzyme, a uniquely high value. The human adrenal enzyme was found to resemble rat liver microsomal epoxide hydrolase closely in a number of respects, including molecular weight, N-terminal amino-acid sequence and response to low-molecular weight ligands. However, rabbit antibodies directed against human adrenal microsomal epoxide hydrolase crossreacted only weakly with the corresponding rat liver protein. The relatively high levels of microsomal epoxide hydrolase in the human adrenal gland suggest that this enzyme may be of particular importance in this tissue. However, very little cytochrome P-450-catalyzed metabolism of xenobiotics has been demonstrated in the human adrenal and our present results speak against the involvement of microsomal epoxide hydrolase in the steroid metabolism of this gland. Thus, the function of this enzyme in the human adrenal is enigmatic.

Key words: Microsomal epoxide hydrolase; Purification; Characterization; (Adrenal gland); (Human)

1. Introduction

A great deal is known about xenobiotic-metabolizing systems in various organs, especially liver, and in particular for rodents. Extensive efforts are now being made to determine the properties of these systems in human tissues. Our'studies on xenobiotic metabolism in the human adrenal gland [1-3] constitute one example.

Microsomal epoxide hydrolase is one of four known enzymes capable of hydrating epoxides of xenobiotics and even of certain endogenous substances (see [4] and [5] for reviews). Many xenobiotic epoxides arise as transient reac- tive intermediates during their metabolism to water-soluble conjugates for excretion. Such epoxides are thought to initiate many of the toxic and genotoxic effects of xenobiotics and microsomal epoxide hydrolase is thus considered to play a key role in our defenses against such deleterious effects.

One of our earlier findings was that the human adrenal cortex demonstrates relatively high levels of microsomal

* Corresponding author. Fax: +46 8 153024.

0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 4 8 3 8 ( 9 4 ) 0 0 0 3 1 - B

epoxide hydrolase activity, but little or no xenobiotic metabolism via the cytochrome P-450 monooxygenase system [1-3]. This finding may be relevant to the fact that the frequency of adrenal cancer in human beings is relatively low [6,7].

Microsomal and cytosolic epoxide hydrolases have been purified to homogeneity from a number of species and tissues, including human liver [4,5]. In the present investigation, we have purified the human adrenal enzyme to homogeneity, characterized its responses to inhibitors, possible role in steroid metabolism and the N-terminal amino-acid sequence. We have also prepared polyclonal rabbit antibodies toward the enzyme and demonstrated that these antibodies can be used for convenient quantitation of human microsomal epoxide hydrolase.

2. Materials and methods

2.1. Chemicals

[14C]Styrene-7,8-oxide (15 mC/mmol ) was purchased from the Radiochemical Centre (Amersham, UK). cis-

254 D. Papadopoulous et al. / Biochimica et Biophysica Acta 1206 (1994) 253-262

Stilbene oxide was synthesized by m-chloroperoxybenzoic acid oxidation of cis-stilbene (Merck, Darmstadt, Ger- many) and purifed by silica gel column chromatography with hexane/ethylacetate (95:5, v /v ) as the solvent system. cis-[3H]Stilbene oxide was synthesized at a specific radioactivity of approx. 2 Ci/mmol (74 GBq/mmol) and purified before use to > 99% by thin-layer chromatography on 0.2 mm silica gel plates (Merck, Darmstadt, Germany) with hexane/ethylacetate (95:5, v /v ) as eluent [8]. All other chemicals were of analytical grade.

2.2. Human adrenal tissue

The material was obtained from adult kidney transplant donors or autopsies at the Urological Clinics of the Upp- sala Academic Hospital and Huddinge Hospital. The tissue samples were frozen immediately and maintained at - 18°C until use. This study was approved by the Ethical Commit- tee at Karolinska Hospital.

2.3. Subcellular fractionation

After thawing the adrenals to 4°C, the cortex and medulla were collected by dissection. All subsequent steps were performed at 4°C. The tissue was weighed and homogenized as a 20% (w/v) suspension in 0.25 M sucrose using 4 up-and-down strokes of a Potter-Elvehjem homogenizer at 440 rpm. Subfractionation of this ho- mogenate was performed by differential centrifugation as described [9].

2.4. Purification of microsomal epoxide hydrolase

Adrenal microsomes were suspended in a buffer containing 10 mM sodium Tricine (pH 8.0), 0.2 mM dithiothreitol and 2 mM EDTA. After addition of a 10% solution of Triton X-100 to give a final detergent concentration of 2%, the suspension was solubilized by homogenization for 20 min with cooling in an ice bath. After centrifugation at 30000 X gay for 20 rain, the resulting supernatant (containing almost all of the epoxide hydrolase activity) was dialyzed, first for 4 h against 2 1 20 mM sodium Tricine (pH 8.0), containing 0.2 mM dithiothreitol, 0.1% Triton X-100 and 2 mM EDTA and, subsequently, overnight against 4 1 of the same solution.

The dialysate was passed through an anion exchange column (DE-52 cellulose) previously equilibrated with the same solution.

After washing (100 ml of this solution), the column was eluted with a 400 ml linear gradient of KCI (0-0.5 M) in the same buffer; flow rate 9.33 ml/min and fraction collection time 20 min. The fractions containing the highest activities of epoxide hydrolase were pooled, dialyzed overnight against 4 1 of the FPLC buffer (20 mM sodium Tricine (pH 8.0), 0.2 mM dithiothreitol and 0.1% Triton

X-100) and lyophilized. The dried sample was redissolved in FPLC buffer (1 /5 the volume of the original ho- mogenate) and dialyzed twice against 2 1 of this buffer, 3 h each time.

FPLC on Mono Q HR 10/10 (1 ml) was performed after pre-equilibration with ultrafiltered and degassed FPLC buffer. Using the Motor Valve MV7 injector with a loop of 500/xl, the filtered and degassed dialysate from the DE-52 cellulose column was applied to the FPLC column with a flow rate of 1 ml/min. Epoxide hydrolase was then eluted with a 0-0.5 M linear KC1 gradient in FPLC buffer (gradient slope 6.25 mM per ml). 0.2 ml fractions were collected and the fractions containing most epoxide hydrolase (as determined by SDS-polyacrylamide gel electrophoresis) pooled and dialyzed against 4 1 FPLC buffer twice, for 5 h each time.

After concentration by lyophilization (to 1/5 the orginal volume), filtration and degassing, the dialysate (containing approx. 8 /zg protein) was subjected to gel filtration on Superose 6 prepacked HR 10/30 (pre-equilibrated with 0.5 M KCI in FPLC buffer) using a 500 /zl loop. Elution was performed with the same solution at a flow of 0.5 ml /min and the fractions containing the highest epoxide hydrolase activity were pooled.

2.5. Assay of epoxide hydrolase activity

Epoxide hydrolase activity was assayed with t4C-labeled styrene oxide essentially according to Seideg~rd et al. [10]. Incubation mixtures contained 1.6 mM substrate (unless otherwise stated), 50 /~g protein and 125 mM Tris-C1 (pH 7.5), in a total volume of 100 /zl and the assay was performed at 37°C for 5 min. These conditions give optimal epoxide hydrolase activity with human adrenal microsomes, as determined earlier ([2]; cf. the results in Section 3).

Activity was also measured using cis-stilbene oxide as substrate. In this case, a radiometric procedure involving separation of the product from remaining substrate by extraction with n-dodecane after termination of the reaction [11] was employed, in 0.1 M glycine (pH 9.2). Optimal conditions for this assay are described below (Section 3).

All effectors added to the assay medium were dissolved in ethanol in volumes such that the final ethanol concentration was 4.75%. Control incubations contained the same amount of ethanol to compensate for effects on enzyme activity.

2.6. Protein

Protein was quantitated according to Lowry et al. [12] or Peterson [13] using bovine serum albumin as standard. The different assay procedures resulted in very similar values.

D. Papadopoulous et al. / Biochimica et Biophysica Acta 1206 (1994) 253-262 255

2.7. SDS-PAGE 2.10. Investigation of possible effects on steroid metabolism

SDS-PAGE was performed using 7.5% polyacrylamide and bisacrylamide [14]. Visualization was accomplished by Coomassie brilliant blue staining [15] or by a silver staining procedure [16].

2.8. Antibody preparation and immunoquantitation

The band from the SDS-polyacrylamide gel containing microsomal epoxide hydrolase was eluted electrophoreti- cally [17] and polyclonal antibodies against this protein were raised in rabbits. Subsequent immunochemical quan- tification was performed by electrophoretic transfer of the protein to nitrocellulose sheets [18] and immunoblotting [19].

2.9. Structural analysis

N-Terminal sequence analysis was performed with the dimethylaminoazobenzene isothiocyanate procedure [20], using by-products to assist the identifications [21].

Human adrenal microsomes, prepared as described above, were incubated with 50 /zM [14C]testosterone or [14C]-progesterone (New England Nuclear, Boston, MA) (1.6 × 105 cpm per incubation), an NADPH-generating system (1 mM NADPH, 10 mM isocitrate (Sigma, St. Louis, MO.) and 1.5 U isocitrate dehydrogenase (Sigma)), 4 mM MgCI 2 and 100 mM Tris-chloride (pH 7.5), in a total volume of 0.5 ml for 60 min at 37°C. The reaction was terminated by extracting the products and remaining substrate into 1 ml water-saturated ethyl acetate. The or- ganic solvent phase was dried under a stream of nitrogen and the residue redissolved in 0.5 ml acetone. Products and remaining substrate were separated by thin-layer chromatography on silica gel plates with a concentration zone (Merck, Darmstadt, Germany) using elution for 70 min with chloroform:ethyl acetate, 4:1 (v/v), followed by elution in the same direction with cyclohexane:ethyl acetate, 1:1 (v/v) twice for 50 min each time and thereafter visualization by autoradiography using Kodak X-ray film.

A O O o o

~ C e,, O O

.= O

gate or Contaminant 200. KD (Myosin)

116.

92.

KD (8-Galactosidase)

KD (Phosphorylase)

66. KD (BSA)

( Human Adrenal ) "H ~Epoxide Hydrolase

45, KD (Ovalbumin)

Fig. 1. SDS-PAGE of the microsomal epoxide hydrolase isolated from human adrenal. Microsomal epoxide hydrolase was isolated from human adrenal microsomes as described in Section 2 and documented in Table 1. The purified protein was submitted to SDS-PAGE according to Laemmli [14].


Table 1 Purification of microsomal epoxide hydrolase from human adrenal microsomes

Preparation Total activity

Microsomes Microsomes solubillized and centrifuged Pooled and dialyzed fractions from the DE-52 column Pooled and dialyzed fractions from the Mono Q column Pooled and dialyzed fractions from the Superose 6 column

(nmol styrene glycol produced/min)

2420 3110 2950 325 248

Specific activity Purification Yield (nmol styrene glycol factor (%) produced/mg per min)

18.2 1.0 100 23.9 1.3 129 75.8 4.2 122 91.4 5.0 13.4

133 7.3 10.2

The effects of additions were examined-including 8 /xg purified human adrenal microsomal epoxide hydrolase per ml; 20 /~1 rabbit antiserum towards this enzyme; 0.8 mM 1,1,1-trichloropropene oxide; 0.8 mM unlabeled styrene oxide or 0.2% Triton X-100.

3. R e s u l t s

3.1. Purification of microsomal epoxide hydrolase from human adrenal gland to homogeneity

The protein was purified to homogeneity according to the procedure in Table 1, as described in detail under section 2. Solubilization from the adrenal microsomes with 1% Triton X-100 resulted in an increase in epoxide hydrolase activity, observed for the rat liver enzyme as well [4]. The most effective purification steps were chromatography on DE-52 cellulose and FPLC Superose. Chromatography on an FPLC mono Q column led to extensive loss of the microsomal epoxide hydrolase without much purification. However, this step was necessary for removal of a contam- inating protein observed on SDS-polyacrylamide gel elec- trophoretograms. After the three column chromatographic steps, resulting in an overall purification of 7.3-fold, the protein was obtained in 10% yield and highly homoge- neous form (Table 1).

The isolated protein gave a single band upon SDS-polyacrylamide gel electrophoresis (Fig. 1) at a position corresponding to a subunit molecular weight of 51 000 Da, as is the case for other mammalian microsomal epoxide hydrolases. The N-terminus of human adrenal microsomal epoxide hydrolase was not blocked and conventional methods of sequence analysis could be employed. Manual sequenc- ing of the intact protein for a few steps revealed a single N-terminal amino-acid sequence highly similar or even identical to that of the human liver form (Table 2).

The purification procedure has been tested 3 times with identical overall results.

3.2. Characterization of the assay procedure

We have demonstrated earlier that with microsomes, the epoxide hydrolase activity toward styrene oxide is satu-

rated at a substrate concentration of 1.6 raM, at which the activity is linear for about 5 rain and up to about 100 /xg protein [2]. Using our standard assay procedure, the activity of purified microsomal epoxide hydrolase toward this same substrate is linear up to approx. 1.5 /xg protein. In addition, 1.6 mM styrene oxide is saturating in this case as well (not shown).

With cis-stilbene oxide as substrate, epoxide hydrolase in situ in human adrenal microsomes is linear for much longer (at least 30 min), but at much lower protein concentrations (maximally 10 /xg per assay) [2]. The activity of the purified enzyme toward this substrate was saturated at a concentration of 60 /xM and was linear up to 1 /xg protein (Fig. 2).

3.3. Production of antibodies in rabbits and immunoblotting

Upon injection of human adrenal microsomal epoxide hydrolase into rabbits, polyclonal antibodies were readily obtained. As shown in Fig. 3, this antibody preparation was suitable for quantitation of human adrenal microsomal epoxide hydrolase by immunoblotting. Indeed, we have utilized this procedure to measure the levels of the enzyme in benign and malignant tumors of the human adrenal gland [3]. The immunoblotting pattern in Fig. 3 reconfirms the homogeneity of our purified protein, since only a single band was obtained using both the purified prepara-

Table 2 N-Terminal segment of the microsomal epoxide hydrolase isolated from human adrenal and, for comparison, corresponding parts of other mammalian forms of this enzyme reported

Tissue Sequence

Human adrenal (Met) (XXX) Leu Glu Ile Leu Leu Human liver [22] Met Trp Leu Glu lie Leu Leu Rat liver [23,24] Met Trp Leu Glu Leu Val Leu Rabbit liver [25] Met Leu Leu Glu Leu Leu Leu

The N-terminus of microsomal epoxide hydrolase isolated from human adrenal microsomes was unblocked and submitted directly to a few steps of manual sequence degradation with the dimethylaminoazobenzene isothiocyanate double coupling method [20]. Residues at the early positions within parentheses were difficult to identify, because of multiple assignments probably derived from the presence of free amino-acid contami- nants. However, subsequent cycles gave single assignments, supporting protein purity and identifying subunit start position.


1 5 0

A

• ~: 1 0 0

E 5 0

I I

0 1 2 3

~tg p r o t e i n

Fig. 2. Enzymatic activity of isolated human adrenal microsomal epoxide hydrolase towards cis-stilbene oxide as a function of protein amount. Incubation with radioactive substrate was performed for 5 rain at 37°C, after which it was terminated by extraction with n-dodecane, which removes remaining substrate while leaving almost all of the product in the aqueous phase. Subsequently, radioactivity in the product and remaining substrate was determined by scintillation counting.

tion and microsomes. Rabbit antisera toward the human

adrenal microsomal epoxide hydrolase crossreacted only weakly with the corresponding rat liver protein (Fig. 4; cf. Section 4).

3.4. Effects o f low-molecular weight ligands

The activity of rat liver microsomal epoxide hydrolase toward styrene oxide can be stimulated or inhibited by a number of low-molecular weight ligands [4]. In order to characterize further the human adrenal enzyme and com- pare it to the rat protein, the effects of ligands on the

human enzyme were investigated (Table 3). Benzil and clotrimazole are strong activators of the human microso-

mal epoxide hydrolase activity toward styrene oxide, just

as they are for the rat liver enzyme. Metyrapone and

chalcone epoxide are moderate activators and cyclohexene oxide and 1,1,1-trichloropropene oxide are strong inhibitors of both the human and rat activities, trans-Stilbene oxide, which is a selective substrate for cytosolic epoxide hydrolase [5], gave a moderate stimulation, as was also the case with trans-stilbene.

A confusing property of rat liver microsomal epoxide hydrolase is that certain low-molecular weight effectors

influence the activities toward different substrates differently [4]. In order to determine whether this is also the case with the human adrenal enzyme, the experiment with

styrene oxide was repeated using cis-stilbene oxide as substrate (Table 3). There are, indeed, considerable differ- ences between the effects with these two substrates. With

cis-stilbene oxide as substrate, benzil and clotrimazole actually inhibited the activity, whereas chalcone epoxide and metyrapone were without effect. On the other hand,

cyclohexene oxide and 1,1,1-trichloropropene oxide were potent inhibitors of the activities of the human enzyme toward both these substrates, as is also the case with the rat enzyme [4].

Very similar effects were obtained using 0.8 mM effector, i.e., half the concentration employed in the experiments shown in Table 3.

Fig. 5 illustrates that ethanol also affects the activities of human adrenal microsomal epoxide hydrolase toward styrene oxide and cis-stilbene oxide differently.

3.5. Studies on possible incoh,ement in steroid metabolism

Since the human adrenal gland demonstrates relatively

little cytochrome P-450-catalyzed monooxygenase activ-

Table 3 Influence of different effectors on the activity of human adrenal microsomal epoxide hydrolase toward styrene oxide and cis-stilbene oxide

Effector Specific activity Specific activity

(1.6 mM generally) (nmol styrene glycol/ (%) (nmol stilbeoe glycol/ (%) min per mg protein) min per mg protein)

None 34.6 + 3.8 22.1 + 1.4 Ethanol 4.75% 62.9 + 2.8 100 19.5 + 1.3 100 Benzil 104.7 + 7.0 166 12.5 + 1.6 64 Betamethasone 72.4 + 5.7 115 22.8 -I- 1.1 117 Chalcone epoxide 75.2 ___ 3.7 119 18.4 + 0.9 94 Clotrimazole 183.0 + 8.0 291 7.2 + 0.7 37 Cholestane-3fl,5a,6fl-triol a 49.7 + 2.3 79 25.7 + 3.4 132 Cholestane-5a,6a-epoxy-3fl-ol 65.7 + 7.4 104 18.8 5:1.3 96 Cholesterol-5a,6a-epoxide 63.1 + 5.4 100 23.2 5:0.8 119 Cyclohexene oxide 11.1 ::1:1.1 18 3.7 + 0.3 19 Dexamethasone 96.4 + 2.0 153 21.6 5:1.8 111 Metyrapone 73.6 + 3.1 117 19.9 ___ 2.9 102 1,1,1-Trichloropropene oxide 0 5:0 0 0 5:0 0 5-Pregnen-16a,17a-epoxy-3fl-ol-20-one 69.8 + 4.5 111 21.4 + 1.9 110 trans-Stilbene h 62.0 + 3.5 99 18.2 5:1.4 93 trans-Stilbene oxide b 58.8 5:0.1 93 12.7 5:1.2 65

The values presented are the means :1: S.D. of 3 to 4 trials of 3 samples per trial from 2 different individuals. a The concentrations of cholestane-3/$,5a,6/3-triol were 0.2 and 0.4 mM. b The concentration of trans-stilbene and trans-stilbene oxide was 60 ~M.


- r ) , m 2: +

k . . . . . . . •

References

0.81xg

1.7~tg

3.4gg

(n

-o )>

o m

References

e -

O.8~g 0 .

0 C ) 1.71.9 " 0

.4:

3.4gg

t = r

> m -l-

Fig. 3. Immunochemical titration of human adrenal microsomal epoxide hydrolase by Western blotting employing polyclonal antibodies raised in rabbits. After SDS/polyacrylamide eleetrophoresis, human adrenal microsomal epoxide hydrolase was transferred etectrophoretically to nitrocellulose paper, exposed in turn to polyclonal rabbit antibodies toward the human enzyme, to 131I-protein A and finally to autoradiography. The left half of the figure illustrates the SDS-polyacrytamide gel electrophoretic pattern corresponding to the immunochemical titration. Quantitites given indicate the amounts of protein applied.

ity, the importance of the high levels of microsomal epoxide hydrolase in this tissue for xenobiotic metabolism can be questioned. Therefore, it seemed desirable to examine other possible functions of this enzyme. Many enzymes metabolizing xenobiotics are also active with endogenous compounds and the adrenal gland is specialized for steroid production. Thus, the hypothesis raised here is that human

P u r i f i e d H u m a n - r

Adrelal Epoxlde Ratt Adrenal Ratt Adrenal Hydrolase (HAEH) Mlcroaomes MItochondrla

Fig. 4. Dot-blot of human adrenal epoxide hydrolase and rat adrenal microsomes and mitochondria employing rabbit polyclonal antibodies raised against the human enzyme. Purified human adrenal epoxide hydrolase and rat adrenal microsomes and mitoehondria were dotted onto nitrocellulose paper and then immunoquantitated using polyclonal rabbit antibodies towards the human enzyme as described in the legend to Fig. 3. Quantities indicate the amounts of protein applied.

adrenal microsomal epoxide hydrolase is involved in steroid metabolism. This same hypothesis has previously been presented by other investigators, primarily Oesch and

8 0 A -+i

._o .- 4 0 E

~t~ m

I

10 2 0

% E t O H Fig. 5. Effects of ethanol on the activities of isolated human adrenal microsomal epoxide hydrolase toward styrene oxide (SO) and c/s-stilbene oxide (SbO). Incubation was performed with 100 and 1 /xg enzyme and radioactive substrate for 5 and 10 rain, respectively, at 37"C in the presence of various concentrations of ethanol, after which product and remaining substrate were separated by extraction and subjected to scintillation counting.


coworkers, on the basis of totally different findings, including the observation that microsomal epoxide hydrolase can hydrate certain steroid epoxides (see [4] for a review). It is worth noting in this respect that an epoxide hydrolase relatively specific for cholesterol oxide and distinct from the xenobiotic-metabolizing microsomal epoxide hydrolase has been identified (see [5] for a review).

Two approaches were employed to test this hypothesis. In one, various steroids and steroid epoxides-- including betamethasone, cholestane-3fl ,5 ,6f l- t r iol , cholestane- 5a,6a-oxide, cholesterol oxide, dexamethasone and 5- pregnen-16a,17a-epoxy-3fl -ol-20-one--were tested as po- tential competitive inhibitors of the activities of human adrenal microsomal epoxide hydrolase toward both styrene oxide and c is -s t i lbene oxide (Table 3). In no case was any inhibition observed. Indeed, the various steroids and steroid epoxides stimulated the activity toward styrene oxide.

In the other approach, human adrenal microsomes were allowed to metabolize radioactive testosterone or progesterone and the products obtained were characterized using thin-layer chromatography. The effects of adding purified human adrenal microsomal epoxide hydrolase, antibodies towards this enzyme, the inhibitor 1,1,1-trichloropropeno- xide or styrene oxide (as a competitive inhibitor) to this system were then examined. In no case was steroid metabolism altered by such additions. One experiment representative of the 10 different experiments of this type which we performed is shown in Fig. 6.

4. D i s c u s s i o n

Microsomal epoxide hydrolase from human adrenal gland can be purified to a high degree of homogeneity and

(A) ~ro~u~t

- 4

(o ~ ::3

o

Irenal

!1

1 xide o

~pene

Fig. 6. Effects of pretreatments on progesterone metabolism by human adrenal microsomes. Human adrenal microsomes (90 /.tg protein) were incubated with [J4C]progesterone (1.6.105 cpm) for 60 min at 37°C. The mixture also contained an NADPH-regenerating system. The reaction was terminated by extraction and products identified by thin-layer chromatography and subsequent autoradiography. (A) Effects of boiling, 1% dimethylsulfoxide, 0.8 mM 1,1,1-trichloropropene oxide and rabbit antibodies. (B) Effects of boiling, rabbit preimmune serum, rab bit antibodies, 2 /~g purified human adrenal mi crosomai epoxide hydrolase (HAEH) and 0.2 % Triton X-100 (C). Effects of boiling, 0.6% ethanol, 60/~M cis-stilbene oxide in ethanol, 1% acetonitrile, 1.6 mM styrene oxide in acetonitrile and rabbit antibodies


in satisfactory yield using simple, conventional procedures. The fact that the overall apparent purification was only 7.3-fold suggests that approx. 14% of the total microsomal protein may consist of this enzyme, a uniquely high value. The corresponding value for the rat liver enzyme is 'only' 2% [4]. However, it is particularly difficult to quantitate levels of microsomal epoxide hydrolase protein on the basis of activity, since this enzyme demonstrates different specific activities upon solubilization, in the presence of different detergents or certain other small ligands, in com- plex with different phospholiplids and/or detergents, etc. Therefore, reliable determination of the amount of human adrenal microsomal protein which is accounted for by microsomal epoxide hydrolase requires futher studies, e.g., utilizing immunoquantitation.

The human adrenal enzyme was found to resemble rat liver microsomal epoxide hydrolase closely in a number of respects, including molecular weight, N-terminal amino- acid sequence and response to various low-molecular- weight ligands. It is also known that the coding regions of

the genes for human and rat microsomal epoxide hydrolase demonstrate 83% homology [26].

In light of the structural and functional similarities between the rat and human enzymes, it is surprizing that polyclonal rabbit antibodies raised toward human adrenal microsomal epoxide hydrolase crossreact only weakly with the corresponding protein isolated from rat liver. This difference would seem to suggest that those regions of microsomal epoxide hydrolase that constitute the strongest immunological epitopes are neither strongly involved in the enzymatic catalysis nor evolutionarily conserved.

The conservation of the properties of microsomal epoxide hydrolase indicates that this enzyme has an important function, while the relatively high levels of this protein in the human adrenal gland suggest that it may be of particular importance in this tissue. However, very little cytochrome P-450-catalyzed metabolism of xenobiotics has been demonstrated in the human adrenal [2] and our present results speak against the involvement of microsomal epoxide hydrolase in the steroid metabolism of this

(B) Product

Fig. 6 (,continued.).

"O B ~b

¢1

B

Boiled human adrenal mtcrosomes

Rabbit pre-immune serum

Rabbit potyclonal antibodies against HAEH

None

Purified Human Adrenal Epoxide Hydrolase

0.2% Triton X-100


(c) Products

.= r~

~ o ~- ~.

8. 8" 8. - q ' ~ = a ¢D a

J,

"0 a

8. a

,L

Boiled human adrenal microsomes

None

Ethanol

cis-Stilbene oxide

Acetonitrile

Styrene oxide (30 min before the incubation start)

Styrene oxide

Rabbit polyclonal antibodies against HAEH

Fig. 6 (continued).

gland. Thus, the function of this enzyme in the human adrenal remains a mystery.

Acknowledgments

This study was supported by grants from the Swedish Cancer Society.

References

[1] Papadopoulos, D., Seideg~rd, J. and Rydstr/~m, J. (1984) Cancer Lett. 22, 23-30.

[2] Papadopoulus, D., Seideg~rd, J., Georgellis, A. and RydstriSm, J. (1985) Chem. Biol. Interact. 55, 249-260.

[3] Papadopoulus, D., Gr6ndal, S., Rydstr6m, J. and DePierre, J.W. (1992) Cancer Biochem. Biophys. 12, 283-291.

[4] Seideg~d, J.-E. and DePierre, J.W. (1983) Biochim. Biophys. Acta 695, 251-270.

[5] Meijer, J. and DePierre, J.W. (1988) Chem. Biol. Interact. 64, 207-249.

[6] Cancer Incidence in Sweden 1979 (1982) National Board of Health and Welfare, The Cancer Registry, Stockholm, Sweden.

[7] Cutler, S.J. and Young, J.L. (Eds.) (1975) Third National Cancer Survey: Incidence Data, Natl. Cancer Inst. Monogr. Vol. 41, pp. 1-454, National Cancer Institute, Stockholm.

[8] Meijer, J. and DePierre, J.W. (1987) Chem. Biol. Interact. 62, 249-269.

[9] Ernster, L., Siekevitz, P. and Palade, G.E. (1962) J. Cell Biol. 6, 541-562.

[10] Seideg[lrd, J., DePierre, J.W., Moron, M.S., Johannesen, K.A.M. and Ernster, L. (1977) Cancer Res. 37, 1075-1082.

[11] Gill, S.S., Ota, K. and Hammock, B.D. (1983) Anal. Biochem. 131, 273-282.

[12] Lowry, O.H., Roseborough, N.J., Farr, A.L. and Randall, G. (1951) J. Biol. Chem. 193, 265-275.

[13] Peterson, G.L. (1977) Anal. Biochem. 83, 346-356. [14] Laemmli, U.K. (1970) Nature (London) 227, 680-685. [15] Wilson, C.M. (1979) Anal. Biochem. 96, 263-278. [16] Goldman, P., Sedman, S.A. and Ebert, M.H. (1981) Science 211,

1437-1438. [17] Mendel-Harvig, I. (1982) Anal. Biochem. 121, 309-334.


[18] Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354.

[19] Bumette, W.N. (1981)Anal. Biochem. 112, 195-203. [20] Chang, J.Y., Brauer, D. and Wittmann-Liebold, B. (1978) FEBS

Lett. 93, 205-214. [21] von Bahr-LindstriSm, H., Hempel, J. and JSrnvall, H. (1982) J.

Protein Chem. 1, 257-261. [22] DuBois, G.C., Appella, E., Ryan, D.E., Jerina, D.M. and Levin, W.

(1982) J. Biol. Chem. 257, 2708-2712.

[23] DuBois, G.C., Appella, E., Armstrong, R., Levin, W., Lu, A.Y.H. and Jerina, D.M. (1979) J. Biol. Chem. 254, 6240-6243.

[24] Porter, T.D., Beck, T.W. and Kasper, C. (1986) Arch. Biochem. Biophys. 248, 121-129.

[25] Heinemann, F. and Ozols, J. (1984) J. Biol. Chem. 259, 797-804. [26] Wilson, N.M. and Omiecinski, C.J. (1989) Biochim. Biophys. Acta

1008, 357-358.

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Purification and initial characterization of microsomal epoxide hydrolase from the human adrenal gland