Purification and properties of two multiple forms of dihydrodiol dehydrogenase from guinea-pig testis

Biochimica et Biophysica Acta 912 (1987) 270 277 Elsevier

270

BBA 32813

P u r i f i c a t i o n and p r o p e r t i e s of two multiple f o r m s of dihydrodioi d e h y d r o g e n a s e f r o m guinea-pig testis Kazuya Matsuura, Akira Hara, Toshihiro Nakayarna, Makoto Nakagawa and Hideo Sawada Department of Biochemistry, Gifu Pharmaceutical University, Gifu (Japan) (Received 1 November 1986)

Key words: Dihydrodiol dehydrogenase multiple forms; trans-l,2-Dihydrobenzene-l,2-diol dehydrogenase; lmmunochemistry; Aldose reductase; Enzyme purification; (Guinea-pig testis)

NADP +-dependent dihydrodiol dehydrogenase (trans- 1,2-dihydrobenzene- 1,2-diol : NADP + oxidoreductase, EC 1.3.1.20) activity in the cytosol of guinea-pig testis was separated into two major and two minor peaks by Q-Sepharose chromatography; one minor form was immunologically cross-reacted with hepatic aldehyde reductase. The two major enzyme forms were purified to homogeneity. One form, which had the highest amount in the tissue, was a monomeric protein with a molecular weight of 32 000 and isoelectric point of 4.2, showed strict specificity for benzene dihydrodiol and NADP +, and reduced pyridine aldehydes, glyceraldehyde and diacetyl at low rates. Another form, with a molecular weight of 36 000 and isoelectric point of 5.0, oxidized n-butanol, glycerol and sorbitol as well as benzene dihydrodiol in the presence of NADP ÷ or NAD +, and exhibited much higher reductase activity towards various aldehydes, aldoses and diacetyl. The pl 5.0 form was more sensitive to inhibition by sorbinil and p-chloromercuriphenyl sulfonate than the pl 4.2 form and was activated by sulfate ion. The two enzymes did not catalyze the oxidation of hydroxysteroids and xenobiotic alicyclic alcohols and were immunologically different from hepatic 17fl-hydroxysteroid-dihydrodiol dehydrogenase. The results indicate that guinea-pig testis contains at least two dihydrodiol dehydrogenases distinct from the hepatic enzymes, one of which, the pl 5.0 enzyme form, may be identical to aldose reductase.

Introduction

A cytosolic NADP+-dependent dihydrodiol dehydrogenase ( trans-l,2-dihydrobenzene-l,2dioI:NADP + oxidoreductase, EC 1.3.1.20), that oxidizes benzene dihydrodiol (trans-l,2-dihydrobenzene-l,2-diol) to catechol [1,2], has been shown to be important for the metabolic inactivation of dihydrodiols or dihydrodiol epoxides derived from carcinogenic polycyclic hydrocarbons in that the Correspondence: H. Sawada, Department of Biochemistry, Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502, Japan.

enzyme decreases the rnutagenic activity of benzo[a]pyrene and benz[a]anthracene in the Ames test [3]. The enzyme from rat liver has been identified as 3a-hydroxysteroid dehydrogenase (EC 1.1.1.50) [4,5]. On the other hand, in mouse [6] and rabbit liver [5], multiple forms of cytosolic dihydrodiol dehydrogenase associated with 17/3hydroxysteroid dehydrogenase activity have been purified, and those of dihydrodiol dehydrogenase in guinea-pig liver cytosol have been identified as testosterone 17/3-hydroxysteroid dehydrogenase (NADP +) (EC 1.1.1.64) isozymes and aldehyde reductase (EC 1.1,1.2) [7,8]. The dihydrodiol dehydrogenase activity was de-

0167-4838/87/$03.50 © 1987 Elsevier Science Pubfishers B.V. (Biomedical Division)

271 tected in several extrahepatic tissues of rats [9], and 3a-hydroxysteroid dehydrogenase, with properties similar to the hepatic enzyme, also presents in the tissues [10], which suggests that 3a-hydroxysteroid dehydrogenase acts as dihydrodiol dehydrogenase in rat tissues. In contrast, in guinea pigs aldehyde reductase is immunologically detected in almost all extrahepatic organs, but testosterone 17fl-dehydrogenase immunologically identical to the hepatic enzyme exists only in kidney [8]. The distribution of dihydrodiol dehydrogenase activity in guinea pigs (liver >> kidney > testis > spleen > heart > lung > brain) [8] is not consistent with that of aldehyde reductase activity (liver > kidney >> lung > testis = brain > heart > spleen) [11]. The data suggest that dihydrodiol dehydrogenase(s) different from hepatic testosterone 17fl-dehydrogenase presents in some tissues of guinea pigs. Therefore, we have examined guinea-pig testis for benzene dihydrodiol dehydrogenase activity and have found that the enzyme exists in four multiple forms, of which one minor form was immunologically identified as aldehyde reductase. This paper describes the purification and properties of the two testicular enzyme forms which were distinct from aldehyde reductase. Materials and Methods

Materials. Liver and testis were excised from adult male guinea pigs of the Hartley strain. Aldehyde reductase and a major isozyme (pI 7.8) of testosterone 17/3-dehydrogenase were purified from the livers by the procedures described previously [8]. Pyridine nucleotides and p I markers were from Oriental Yeast Co. (Tokyo, Japan); steroids, soybean trypsin inhibitor, pepstatin A, indomethacin and succinic semialdehyde from Sigma Chemical Co.; 1-acenaphthenol and glyceraldehydes from Aldrich Chemical Co.; and dienstrol from Tokyo Chemical Industry (Japan). Sorbinil was a kind gift from Dr. Y. Ohta (Fujita-Gakuen Health University, Aichi, Japan). Benzene dihydrodiol was synthesized as described by Platt and Oesch [12]. Sephadex G-100, Q-Sepharose and standard proteins for molecular weight determination were obtained from Pharmacia Fine Chemicals; HA-Ultrogel from LKB Product AB; and Matrex Red A from Amicon Corporation.

Enzyme purification. All operations during the purification of dihydrodiol dehydrogenase from guinea-pig testes were performed at 0 - 4 ° C. Testes (100 g) were homogenized in a Waring Blendor with 3 vol. of 50 mM Tris-HC1 buffer (pH 8.5)/ 0.15 M KC1/5 mM EDTA. The homogenate was centrifuged for 10 min at 10000 x g. The supernate obtained was further centrifuged for 60 min at 105 000 x g. The microsome-free supernate was fractionated by the addition of solid (NH4)2SO 4. The 0.40 to 0.80 (NH4)2SO 4 precipitate was collected by centrifugation for 15 min at 12000 X g, suspended in 20 ml of 10 mM Tris-HC1 buffer (pH 8.5)/0.25 M sucrose/0.5 mM E D T A / 5 mM 2-mercaptoethanol (buffer A), and then dialyzed overnight against buffer A. The dialyzed enzyme solution was passed through a Sephadex G-100 column (4.8 x 100 cm) in buffer A. The fractions with the enzyme activity were collected and directly applied to a Q-Sepharose column that had been equilibrated with buffer A. After the column had been washed with this buffer, the enzyme was eluted with two steps of NaC1 linear gradient; the first gradient was performed from 0 to 0.05 M and the second from 0.05 M to 0.5 M (Fig. 1). This resulted in the resolution of four distinct peaks of dihydrodiol dehydrogenase activity which were designated DD1, DD2, DD3 and DD4. The fractions of DD1, DD2 and DD4 were separately concentrated by ultrafiltration through an Amicon YM-10 membrane. The concentrates of DD1 and DD2 were stored at 4°C. DD3 and DD4 were further purified by chromatography on Matrex Red A and HA-Ultrogel. DD3 was directly applied to a Matrex Red A column (1.6 x 15 cm) equilibrated with buffer A, whereas DD4 was applied t o the same size of Matrex Red A column after dialysis against buffer A. The columns were washed with buffer A containing 0.1 M NaCI, and the enzymes were eluted with the buffer containing 0.5 M NaC1. These enzyme fractions were separately concentrated by ultrafiltration, dialyzed overnight against 50 vol. of 10 mM Tris-HC1 buffer ( p H 7.5)/0.25 M sucrose/5 mM 2mercaptoethanol (buffer B), and then applied to HA-Ultrogel columns (each 1.6 x 15 cm) equilibrated with buffer B. The two enzymes were eluted with a linear 0-50 mM potassium phosphate gradient in buffer B. These enzyme active

272 fractions were separately concentrated by ultrafiltration and stored at 4 ° C. Enzyme assay. Dihydrodiol dehydrogenase activity was assayed fluorometrically by recording NADPH formation as described [7]. The standard reaction mixture consisted of 100 mM glycineNaOH buffer (pH 10.0), 1.8 mM benzene dihydrodiol, 0.25 mM NADP + and enzyme in a total volume of 2.0 ml. Dehydrogenase activity for other alcohols was determined similarly in the same reaction mixture, except that 1-acenaphthenol dehydrogenase activity was assayed spectrophotometrically by monitoring NADPH absorbance at 340 nm. Inhibitors were added to the reaction mixture before the reaction was initiated by the addition of the enzyme. The reverse reaction was assayed spectrophotometrically in a 2.0 ml system containing 80 mM potassium phosphate buffer (pH 6.0), 50/~M NADPH, and various concentrations of carbonyl compound and enzyme. One unit of enzyme is defined as the amount that catalyses reduction or oxidation of 1 #tool NADPH per rain at 25°C. For study of pH dependence of the dehydrogenase or reductase activity of the enzyme, 0.1 M buffers composed of sodium phosphate (pH 5.8-8.0 and 11.0-11.8), Tris-HC1 (pH 8.3-9.0) and glycine-NaOH (pH 9.0-11.0) were used. Protein determination. Protein concentration was determined by the method of Lowry et al. [13] using bovine serum albumin as a standard. Immunochemical experiments. Antibodies against guinea-pig liver testosterone 17fl-dehydrogenase and aldehyde reductase were raised in young female rabbits as described [8]. Immunological comparison between the enzymes from testis and liver was carried out by the double-diffusion technique of Ouchterlony [14], using 1.2% agarose gel in potassium phosphate-buffered saline (pH 7.5), and by immunoprecipitation as described [15]. Electrophoresis. Polyacrylamide disc gel electrophoresis was performed by the method of Davis [16]. Electrophoresis in the presence of sodium dodecyl sulfate (SDS) was carried out according to the method of Laemmli [17] on a 10% polyacrylamide slab gel. Dehydrogenase and reductase activities in the disc gels were stained with 1.3 mM benzene dihydrodiol and 1 mM pyridine-3-al-

dehyde as substrate, respectively, as described [18] and protein in the gel was stained with Coomassie brilliant blue R-250. Molecular weight determination. The molecular weights of the native enzymes were estimated by gel filtration on a Sephadex G-100 column (1.9 × 97 cm) in buffer A, and those of the denatured enzymes by SDS-polyacrylamide slab gel electrophoresis using the standard proteins. Isoelectric focusing. Isoelectric focusing on 5% polyacrylamide disc gel containing 2% (w/v) Ampholine (pH 3.5-10.0) was performed at 4 ° C as described [19]. Dihydrodiol dehydrogenase activity in the gel was stained as described above. The pH gradient in the gel was assayed by focusing p I markers simultaneously. Results

Purification and purity The results of purification of dihydrodiol dehydrogenases from guinea pig liver cytosol are summarized in Table I. The enzyme activity resolved into four peaks (DD1, DD2, DD3 and DD4) by Q-Sepharose chromatography (Fig. 1). When the four enzyme fractions were analyzed by immunodiffusion test with the antisera against hepatic aldehyde reductase and testosterone 17fl-dehydrogenase, the anti-aldehyde reductase serum formed a precipitin line only against DD2, and the pre-

0.5

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lo

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o ~

o.2 O.l

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ool

~2 ~oo3i

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IS ik , i

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1 O0

200

r

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Fraction number

Fig. 1. Resolution of four forms of guinea-pig testis dihydrodiol dehydrogenaseon a Q-Sepharose column. The fractions (10 ml each) were analyzed for protein (. . . . . . ) and activity (0). , NaCI concentration.

273

TABLE I P U R I F I C A T I O N OF D I H Y D R O D I O L D E H Y D R O G E N A S E S F R O M G U I N E A - P I G TESTIS Step

Total protein (mg)

Total activity (munits)

105 000 x g supernate 0.4-0.8 (NH4)2SO 4 Sephadex G-100 Q-Sepharose DD1 DD2 DD3 DD4 Matrex Red A DD3 DD4 HA-Ultrogel DD3 DD4

9 630 1 360 320

4160 3 260 2 830

2.42 0.90 26.9 3.41 2.60 0.41 1.57 0.19

57.6 19.3 245 1590 109 1230 69.2 593

cipitin line fused with that formed between the antiserum and hepatic aldehyde reductase. The enzyme activity of DD2 was also completely immunoprecipitated by the anti-aldehyde reductase serum. Not all the enzyme fractions reacted with the antiserum against testosterone 17fl-dehydrogenase. Since the amounts of DD1 and DD2 were small and DD1 was unstable under the conditions (about half of the activity was lost within 1 week during the storage at 4 o C), the two enzymes could not be further purified and the nature of DD1 has not been determined. In contrast to DD1, DD3 and DD4 were relatively stable and were further purified to homogeneity as evidenced by the observation of single coincident bands of protein stain and enzyme activity with benzene dihydrodiol as a substrate on polyacrylamide disc gel electrophoresis (Fig. 2). It should be noted that such multiple forms were also observed by Q-Sepharose chromatography of the sample obtained from testicular cytosol by ammonium sulfate fractionation and subsequent gel filtration in the presence of 10 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 5 m g / l i t e r pepstatin A and 1 m g / l i t e r soybean trypsin inhibitor.

Molecular weight and isoelectric point DD2, DD3 and DD4 showed similar molecular weights of about 30 000 on Sephadex G-100 filtra-

Specific activity (munits/mg)

Recovery (%)

0.4 2.4 8.9

Purification (-fold)

100 78 68

1 6 22

23.8 21.4 9.1 466

1.4 0.5 6.0 56

60 54 23 1160

41.9 3 000

2.6 30

105 7 500

44.1 3120

1.7 14

110 7 800

tion, and the molecular weights of the denatured DD3 and DD4 estimated by SDS gel electrophoresis were 36000 and 32000, respectively. When the enzyme forms were analyzed on gel focusing, the dihydrodiol dehydrogenase activity DD4

DD3

®

a

b

c

o

D

c

Fig. 2. Polyacrylamide disc gel electrophoresis of testicular dihydrodiol dehydrogenases. Approx. 10 ~g of DD3 and D D 4 was run on a 7.5% polyacrylamide gel at 4 ° C. The gels were stained for protein (a), benzene dihydrodiol dehydrogenase activity (b) and pyridine-3-aldehyde reductase activity (c). The direction of migration was from top to bottom.

274 TABLE II SUBSTRATE SPECIFICITY OF TESTICULAR D I H Y D R O D I O L D E H Y D R O G E N A S E S Relative steroids steroids with 1.8

velocities (V) with indicated concentrations of the substrates to that with benzene dihydrodiol are expressed. 3a-Hydroxyare 5a- and 5fl-androstan-3a-ol-17-ones, 3/3-hydroxysteroids are 5a- and 5fl-androstan-3/~-ol-17-one, and 17/3-hydroxyare 5a- and 5fl-androstan-17/~-ol-3-ones, testosterone and 17/3-estradiol. The activities towards cofactors were determined mM benzene dihydrodiol as a substrate and the velocity with N A D + is relative to that with N A D P ÷. n.d., not determined.

Substrate

Benzene dihydrodiol Glycerol Benzyl alcohol 1-Acenaphthenol n-Butanol D-Sorbitol 1-Indanol 3a-Hydroxysteroids 3fl-Hydroxysteroids 17/3-Hydroxysteroids NADP ÷ NAD +

Concentration

DD2

DD3

DD4

(mM)

V (%)

Km (mM)

V (%)

Km (mM)

V (%)

Km (mM)

1.8 100 10 0.5 100 100 1.0 0.05 0.05 0.05 0.5 0.5

100 92 43 2 0 0 0 0 0 0 100 9

4.5 192 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.020 n.d.

100 305 305 4 141 122 0 0 0 0 100 70

3.5 166 1.4 n.d. 9.4 n.d. n.d. n.d. n.d. n.d. < 0.001 0.010

100 0 0 2 0 0 0 0 0 0 100 0

1.5 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.015 n.d.

TABLE III COMPARISON OF REDUCTASE ACTIVITY FOR CARBONYL COMPOUNDS A M O N G TESTICULAR D I H Y D R O D I O L DEHYDROGENASES The activity was assayed at pH 6.0 and the velocities (V) with indicated concentrations of the substrates are relative to the benzene dihydrodiol dehydrogenase activity determined under the standard reaction mixture. D-Fructose, 17-ketosteroids such as 5a- and 5fl-androstan-3a-ol-17-ones and androstane-3,17-diones and aromatic ketones such as 4-nitroacetophenone and 4-benzoylpyridine were not reduced by the enzymes. The activities towards cofactors were assayed with 1.0 mM pyridine-3-aldehyde as the substrate, and the velocity with N A D H is relative to that with NADPH. n.d., not determined. Substrate

4-Nitrobenzaldehyde Pyridine-3-aldehyde 4-Carboxybenzaldehyde Pyridine-4-aldehyde D-Glucuronate D-Glucuronolactone Succinic semialdehyde D-Glyceraldehyde Diacetyl L-Glyceraldehyde D-Xylose D-Glucose D-Galactose NADPH NADH

Concen-

DD2

tration (mM)

V (%)

Km (raM)

DD3 V (%)

Km (raM)

DD4 V (%)

Km (raM)

1.0 1.0 1.0 1.0 10 10 22 1.0 1.0 1.3 100 100 100 0.07 0.07

5 700 4800 4650 4100 2 860 1750 1700 990 380 300 290 0 0 100 1

0.26 0.83 0.053 0.77 8.1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 0.005 n.d.

6 0130 4480 1350 3 950 1000 3 330 1720 5 550 2 550 1340 2 440 1350 836 100 12

0.016 0.025 n.d. 0.023 n.d. 2.7 22 0.50 0.68 0.50 26 121 n.d. 0.002 0.26

0 71 0 23 0 0 46 20 71 20 0 0 0 100 0

n.d. 0.55 n.d. 1.1 n.d. n.d. 39 13 0.72 7.1 n.d. n.d. n.d. 0.003 n.d.

275 bands of DD2, D D 3 and D D 4 were observed at about p H 5.4, 5.0 and 4.2, respectively.

Catalytic properties The three testicular dihydrodiol dehydrogenases showed different substrate specificity for the various alcohols other than benzene dihydrodiol (Table II). D D 2 oxidized glycerol, and DD3 butanol, glycerol and sorbitol, whereas DD4 did not oxidize tile alsohols. DD3 exhibited dihydrodiol dehydrogenase activity with N A D ÷ as a cofactor, but the other enzymes were essentially NADP+-dependent. The K m value of D D 4 for benzene dihydrodiol was lower than those of D D 2 and DD3. The NADP+-dependent activities of DD3 and D D 4 gave p H optima around 10.9 and 10.5, respectively, and the p H optimum of NAD+-dependent activity by DD3 was 9.2, while D D 2 showed a broad p H optimum from 8.6 to 9.7. When the reverse reactions by the three enzymes were examined with N A D P H as a cofactor, the enzymes reduced pyridine-3-aldehyde, showing similar p H optima at 6.0. The reductase activities of DD3 and D D 4 stained at the same mobilities as those of protein and dihydrodiol dehydrogenase activity on polyacrylamide gel electrophoresis (Fig. 2), which indicates that the reductase activity resides in the same enzyme proteins of DD3 and DD4. The substrate specificity of the two enzymes in the reverse reaction was compared to that of D D 2 (Table III). The high reductase activity of D D 2 towards aldehydes and D-glucuronate is similar to that of hepatic aldehyde reductase [7,8]. DD3 also highly reduced aldoses as well as the carbonyl substrates for DD2 and its K m values for the aldehydes except some aldoses were lower than that for benzene dihydrodiol, whereas D D 4 reduced some aldehydes and diacetyl at low rates. Even in the reverse reaction, only DD3 exhibited dual cofactor specificity for N A D P H and N A D H .

Inhibitors and activators Since DD3 and D D 4 displayed aldehyde reductase activity, we examined effects of inhibitors for hepatic dihydrodiol dehydrogenase, aldehyde reductase and aldose reductase (EC 1.1.1.21) on DD2, DD3 and D D 4 (Table IV). The dihydrodiol

TABLE IV EFFECTS OF VARIOUS COMPOUNDS ON TESTICULAR DIHYDRODIOL DEHYDROGENASES PCMPS, p-chloromercuriphenylsulfonate. Compound

Concen- Relativeactivity (%) tration DD2 DD3 DD4 (mM)

None Stilbestrol Hexestrol Sorbinil Quercitrin PCMPS Indornethacin Barbital Valproate Potassium phosphate Na2SO4 Li2SO4 (NH4)2SO4 NaCI Potassium phosphate (360 mM) + Na2SOa (400 mM)

0.01 0.01 0.01 0.1 0.1 0.1 1.0 1.0 360 400 400 400 400

100 48 62 56 23 99 63 28 103 176 151 125 135 88

100 46 65 37 36 38 92 100 100 193 196 170 163 120

100 57 75 I00 41 100 62 88 100 125 126 130 25 92

-

202

377

109

dehydrogenase activities of the enzymes were similarly inhibited by the synthetic estrogens, which have been reported as potent inhibitors of guineapig liver testosterone 17fl-dehydrogenase [20], and by quercitrin, a common inhibitor for aldehyde and aldose reductases [21], but the sensitivities of the enzymes to Sorbinil, p-chloromercuriphenyl sulfonate, indomethacin and barbital were different. On the other hand, the activities of the enzymes were enhanced by phosphate and sulfate ions at their high concentrations of more than 200 mM, but not by neutral salts such as NaC1 and KC1. Sulfate ion was more effective with DD3 than with the other enzymes and the enhancement of DD3 by phosphate and sulfate ions were additive. The sulfates also activated about 1.5-times the N A D P H - d e p e n d e n t pyridine-3-aldehyde reductase activity of DD3, but not those of DD2 and DD4. Discussion We have demonstrated that guinea-pig testis contains four soluble multiple forms of dihy-

276 drodiol dehydrogenase. Of the multiple forms of the enzyme, one minor form, DD2, cross-reacted with hepatic aldehyde reductase and exhibited substrate specificity in both oxidation and reduction reactions similar to that of hepatic aldehyde reductase [7,8], which indicates that the enzyme form is aldehyde reductase. Since the other three enzyme forms did not react with the antisera against hepatic aldehyde reductase and testosterone 17fl-dehydrogenase, which have been demonstrated to act as dihydrodiol dehydrogenases in guinea-pig liver [7,8], the enzymes in testis are clearly distinct from the two hepatic enzymes. Of the three enzyme forms, DD3 and DD4 were obtained in a pure form, and their physical and catalytic properties were fairly distinctive. Since the multiple forms of dihydrodiol dehydrogenase were observed in the sample prepared in the presence of the proteinase inhibitors, the multiplicity of the enzyme may not be artifactual formation during purification. DD3, with the second highest amount of enzyme activity in testis, was an acidic monomeric protein with a molecular weight of 36 000, oxidized glycerol and n-butanol, and highly reduced aromatic aldehydes, aldoses, diacetyl and succinic semialdehyde. These properties of DD3 resemble those of aldose reductases from other mammalian tissues [21-27]. The specific activity of the enzyme form in the reduction of glyceraldehyde or pyridine-3-aldehyde can be calculated to be 2.44 or 1.97 units/mg, which values are compatible with those of homogeneous aldose reductases purified from other tissues [23-25]. In addition, the other properties of DD3, including dual cofactor specificity, relatively high sensitivity to Sorbinil and activation by sulfate ion, further support the idea that this enzyme form is identical to aldose reductase. Aldose reductase has been identified in testis and seminal vesicle of other animal species [28,29] and is thought to be involved in fructose formation in the tissues. The role of the enzyme in the detoxication of polycyclic hydrocarbons may be unlikely because of its low ability to oxidize benzene dihydrodiol compared with its reductase activity. However, the present finding suggests that in addition to aldehyde reductase as previously reported [7], aldose reductase might be partly responsible for the multiplicity of dihydrodiol de-

hydrogenase in tissues which contain aldose reductase. DD4 was a monomeric protein with a molecular weight of 32 000, similar to hepatic dihydrodiol dehydrogenases [7,8], but its high acidity and inability to oxidize alicyclic alcohols and hydroxysteroids represent marked differences from the hepatic enzymes. In these respects, the testicular enzyme differs from dihydrodiol dehydrogenases in rat [2,5,9], mouse [6] and rabbit liver [30], which oxidize the alcohols highly. Hepatic dihydrodiol dehydrogenases from guinea pig [8], rat [2,5,9] and rabbit [30] reduce various carbonyl compounds, including aldehydes, aromatic ketones, and ketosteroids, whereas DD4 exhibited low reductase activity only towards glyceraldehydes, pyridine aldehydes, diacetyl and succinic semialdehyde of the various carbonyl compounds tested. Since DD4 has the highest amount of enzyme activity in testis and showed the lowest K m value for benzene dihydrodiol, this enzyme form is the predominant dihydrodiol dehydrogenase in the tissue. Recently, Nakayama et al. [31] have reported that carbonyl reductase (EC 1.1.1.184), which specifically distributes in guinea pig and mouse lung, also exerts benzene dihydrodiol dehydrogenase activity. The present identification of the unique dihydrodiol dehydrogenase in testis indicates that different enzymes act as dihydrodiol dehydrogenase, depending on the tissue, at least in guinea pigs, although further studies of the occurrence and physiological function of this testicular enzyme are required. These data contrast with the data showing ubiquitous distribution of 3a-hydroxysteroid dehydrogenase with properties similar to hepatic enzyme in several rat tissues, including lung and testis, which may dehydrogenate benzene dihydrodiol [10]. The presence of a microsomal mixed-function oxidase system [32,33] and epoxide hydrolase [33,34] in testis has been shown and suggests that polycyclic hydrocarbons are metabolized to ultimate metabolites, dihydrodiol epoxides, via dihydrodiol intermediates in the tissue. Of the multiple forms of testicular dihydrodiol dehydrogenase, DD4 was the predominant enzyme form with affinity for benzene dihydrodiol similar to that of rat liver 3a-hydroxysteroid-dihydrodiol dehydrogenase, which oxidizes dihydrodiols of poly-

277 cyclic a r o m a t i c h y d r o c a r b o n s as well as b e n z e n e d i h y d r o d i o l [35]. T h e i d e n t i f i c a t i o n of the d i h y d r o d i o l d e h y d r o g e n a s e i n testis suggests a n alternative pathway of cathechol formation from a r o m a t i c h y d r o c a r b o n s via d i h y d r o d i o l s i n the tissue as o b s e r v e d i n liver [36], a l t h o u g h the d e h y d r o g e n a s e activity i n testicular cytosol is a b o u t o n e - f i f t h of t h a t i n liver c y t o s o l of g u i n e a pig.

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