1607328
©1992 S. Karger AG, Basel 0013-9432/92/0465-0221 $2 75/0
Enzyme 1992;46:221-228
Characterization of Multiple Forms of Carbonyl Reductase from Chicken Liver Tohru Nishinaka, Yumiko Kinoshita, Naomi Terada, Tomoyuki Terada, Tadashi Mizoguchi, Tsutomu Nishihara Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences. Osaka University, Osaka, Japan
Key Words. Carbonyl reductase, multiple forms • Liver, chicken • Estradiol-17 ß-dehydrogenase Abstract. Three enzyme forms (CRI, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR 1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17ß-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbo nyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.
Carbonyl reductase (secondary alcohol: NADP+ oxidoreductase, EC 1.1.1.184) is generally defined as a cytosolic monomeric enzyme that catalyzes the NADPH-dependent reduction of a great variety of carbonyl compounds such as quinones derived from polycyclic aromatic hydrocarbons, 9-ketoprostaglandins and the anthracycline antibi otic daunorubicin [1], This enzyme is widely distributed in various mammalian and non mammalian species [2, 3], Enzymes with similar properties, including aldehyde reduc
tase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21), have been described from a num ber of animal tissues and are generally re ferred to as the aldo-keto reductases [3]. We have been studying an aldo-keto reductase, and purified and characterized the aldehyde reductase from bovine liver cytosol [4], chicken liver cytosol [5] and mitochondria [6]. We also reported on the purification of carbonyl reductase from bovine liver [7], This time we investigated carbonyl reduc tase in chicken liver and found three multi ple forms which could be separated by chromatofocusing. The presence of multiple Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
Introduction
Nishinaka/Kinoshita/Terada/Terada/Mizoguchi/Nishihara
222
forms of carbonyl reductase has been re ported in many tissues, for example, chicken kidney [8], rat ovary [9], hamster liver [10], and human brain [1], The broad substrate specificity and the existence of multiple forms have led to confusion in understand ing the physiological function of carbonyl reductase. It is necessary to characterize each carbonyl reductase in detail in order to clarify the physiological significance of car bonyl reductase multiple forms. In this study, we demonstrated the purification of three multiple forms of carbonyl reductase in chicken liver and characterization of their properties.
bance at 340 nm. One unit was defined as 1 pmol product formation per min at 25 °C. In the purifica tion procedure, 4-benzoylpyridine was used as a typi cal substrate for carbonyl reductases [7, 9].
Purification of Multiple Forms of Carbonyl Reductase All the procedures were carried out below 4 ° C using the following buffers, to all of which 10 mmol/1 2-mercaptoethanol (2-ME) and 0.1 mmol/1 EDTA were added unless otherwise mentioned: buffer A, 3 mmol/1 Tris-HCl buffer (pH 7.4) containing 155 mmol/1 KCl, 1 mmol/1 EDTA and 0.5 mmol/1 phenylmethylsulfonyl fluoride; buffer B, 25 mmol/1 ethanolamine-acetate buffer (pH 8.9) without EDTA; buffer C, 2.5 mmol/1 sodium phosphate buffer (pH 7.0); buffer D, 2.5 mmol/1 sodium phosphate buffer (pH 7.0) without EDTA.
Extraction and Ammonium Sulfate Fractionation
Chicken liver was obtained from the local slaughter house. Ultrogel AcA 44 was from IBF, Villeneuve-la-Garenne, France. Polybuffer exchanger for chromatofocusing, PBE94, polybuffer 96, 2',5'-ADPSepharose and Superose 12 were purchased from Pharmacia LKB Biotechnology, Uppsala, Sweden. NADPH and NADH were from Oriental Yeast Co., Tokyo, Japan. Hydroxyapatite was from Wako Pure Chemical Industries, Osaka, Japan. Substrates and inhibitors were bought from Wako Pure Chemical Industries, Nacalai Tesque, Kyoto, Japan, and Sigma, St. Louis, Mo., USA. Polyvinylidene difluoride (PVDF) membrane was from Millipore, Bedford. Mass., USA. Wako POD-kit was supplied from Wako Pure Chemical Industries. Anti-chicken kidney carbo nyl reductase antibody was a kind gift from Dr. Hideo Sawada, Gifu Pharmaceutical University [8].
Enzyme Assay The carbonyl reductase activity assay system con sisted of substrate at the indicated concentration, 0.066 mmol/1 NADPH or NADH in 100 mmol/1 sodium phosphate buffer (pH 7.0). The activity was measured by monitoring the linear change in absor bance at 340 nm with respect to time. Dehydrogenase activity was also determined with 0.066 mmol/1 NADP+ or NAD+ monitoring the increase in absor
2',5'-ADP-Sepharose Affinity
Chromatography.
CR 1, CR2 and CR3 were dialyzed for 3 h against 2 times 2 liters of buffer C, and separately applied to
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Materials and Methods
Approximately 120 g of chicken liver was cut into small pieces and rinsed with ice-cold buffer A to remove excess blood. The liver was homogenized with 3 voi of buffer A in a Waring Blender at 10,000 rpm for 3 min. The homogenate was centrifuged at 10,000 g for 60 min, the pellet was discarded, and the supernatant was centrifuged at 105,000 £ for 60 min. The supernatant was fractionated by adding ammo nium sulfate with constant stirring and adjusting the pH to 7.0 with 3 N NH4OH; the protein, which pre cipitated between 40 and 70% saturation, was col lected by centrifugation at 10,000 g for 20 min and dissolved in the minimum volume of buffer B. The enzyme solution was dialyzed against 5 liters of buffer B for 3 h. Ultrogel AcA 44 Gel Filtration. After centrifuged at 10,000 g for 20 min, the dialysate was applied to Ultrogel AcA 44 column chromatography (5.0 X 150 cm) and eluted with buffer B. Fractions with the enzyme activity were pooled. Chromatofocusing. The pool from Ultrogel AcA 44 was subjected to chromatofocusing (1.6 X 35 cm) equilibrated with buffer B. The enzyme was eluted with 10-fold diluted polybuffer 96-acetate (pH 6.0). At this step, three active peaks were observed at pH 8.2, pH 7.8 and pH 7.6 (fig. 1). The three enzymes were separately collected and tentatively named CR1, CR2 and CR3, respectively.
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Molecular Weight Analysis The native molecular weights of the three carbo nyl reductases were analyzed by gel filtration with Superose 12 (0.9 X 30 cm) equilibrated with 50 mmol/1 sodium phosphate buffer (pH 7.0) containing 300 mmol/1 NaCl at a flow rate of 0.5 ml/min. The molecular weights of the three enzymes were calcu lated on the basis of the retention times of the enzymes and molecular weight standards. The sub unit size was determined by SDS-PAGE. Purified enzymes were run on a 12.5% SDS-polyacrylamide gel and stained with silver. The Rm values of the three enzymes were measured, and the subunit weight was calculated. Bovine serum albumin, ovalbumin, a-chymotrypsinogen A and cytochrome c were used as molecular weight standards in both procedures.
Western Blotting Western blot analysis was performed according to the method of Towbin et al. [11], Antigens (1 gg) were subjected to 12.5 % SDS-PAGE. After the electropho resis, proteins were electrically transferred to the PVDF membrane at 200 mA for 60 min. The mem brane was incubated with anti-chicken kidney carbo nyl reductase antiserum and then reacted with a 1,500-fold dilution of peroxidase-labeled goat anti rabbit IgG antibody. The blotted proteins were visulalized by Wako POD-kit at 25 °C for 30 min.
Results and Discussion Three enzyme forms (CRI, CR2 and CR3) of carbonyl reductase were found in chicken liver cytosol. They were separated
o.io
».0
E 8.0
0.0S
sB. 7.0
< 0.00
0
10
20
30
40
6.0 50
Fraction number
Fig. 1. Separation of three carbonyl reductase multiple forms in chicken liver by chromatofocusing. The condition is described in the text. Fractions (2.5 ml) were analyzed for pH ( ) and for enzyme activity (•), which was assayed at pH 7.0 with 4-benzoylpyridine.
by chromatofocusing. That is, three active peaks were observed at pH 8.2 (CR1), pH 7.8 (CR2) and pH 7.6 (CR3), respectively (fig. 1). The three enzymes were further puri fied as described in Materials and Methods. The final preparations exhibited specific ac tivity toward 4-benzoylpyridine of 1.42 units/mg for CR1, 0.355 units/mg for CR2 and 3.05 units/mg for CR3. The molecular weights of CRI, CR2 and CR3 were esti mated to be 25, 32 and 32 kD, respectively, by SDS-PAGE, and to be 48, 32 and 32 kD, respectively, by gel filtration with Superose 12 (fig. 2). These results indicate that the most basic enzyme, CR1, is composed of two identical subunits and the other two en zymes, CR2 and CR3, are monomeric en zymes. Since most carbonyl reductases are monomeric enzymes [1], CR1 is a very cu rious enzyme having two subunits. The substrate specificity of the three en zymes toward carbonyl compounds is shown in table 1. CR1 had a broad substrate speciDownloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
2',5'-ADP-Sepharose (1.5 X 1.0 cm) equilibrated with buffer C. Each enzyme was eluted with a linear gradient (40 ml of buffer C and 40 ml of the same buffer with 600 mmol/1 KC1) after washing the col umn with 50 ml of buffer C. These enzymes were dia lyzed for 3 h against 2 times 2 liters of buffer D. Hydroxyapatite The three enzymes were sepa rately applied to hydroxyapatite (0.8 X 2.5 cm) equil ibrated with buffer D, and eluted with a linear gra dient (30 ml of buffer D and 30 ml of 100 mmol/1 sodium phosphate, pH 7,0, containing 10 mmol/1 2-ME) after washing the column with 50 ml of buffer D.
Nishinaka/Kinoshita/Terada/Terada/Mizoguchi/Nishihara
224
4.8
4.8 JS BG
4.4
E
BG
BG
-j 4.0 0.0
0.5 Rm
1.0
4.0 12
14
16
Elution volume (ml)
ficity and could utilize both NADPH and NADH as a cofactor. On the other hand, CR2 and CR3 showed the activity only to ward 4-benzoylpyridine, menadione and 9,10-phenanthrenequinone so far tested, while CR1 exhibited the activity also toward cyclohexanone, 4-nitrobenzaldehyde and pyridine-3-aldehyde. All the three enzymes exhibited little activity toward Z)-glucuronic acid which is a typical substrate for aldehyde reductase [4-6]. NADH could not be a cofac tor for CR2 and CR3 in contrast to the case of CR1. Since some carbonyl reductases from rat [12], guinea pig [13] and rabbit [14] have been reported to have oxidoreductase activity toward steroid compounds, we also examined the steroid oxidoreduction by chicken liver carbonyl reductases. CR1 could catalyze the oxidoreduction of 17ß-hydroxysteroids such as esterone and 17ß-estradiol as well as 3-ketosteroid reduc tion toward 5a-dihydrotestosterone. CR2 and CR3, however, had little activity toward such steroids. Table 2 shows the Km values of the three carbonyl reductases toward their typical substrates. CR1 had low Km values toward all the substrates, especially toward
steroids. CR2 and CR3 had similar Km val ues toward 4-benzoylpyridine, menadione and 9,10-phenanthrenequinone. Table 3 shows the inhibitor sensitivity of the three enzymes. Quercitrin was a com mon inhibitor for them. Among the three enzymes, only CR1 was sensitive for barbi tal. The sulfhydryl modifiers, 5,5'-dithiobis(2-nitrobenzoic acid) and CuCl2 strongly inhibited both CR2 and CR3 though these reagents did not affect the CR 1 activity, sug gesting that both CR2 and CR3 might have essential cysteine residue(s) in their active sites. CR2 and CR3 were also sensitive to the nonsteroidal anti-inflammatory drug, indomethacin, the same as the case for the hu man [1], rat [15], guinea pig [13] and bovine enzyme [7], These results suggested that CR1 was quite different from CR2 and CR3, and CR2 and CR3 were similar to each other in the enzymatic properties of substrate speci ficity and inhibitor sensitivity. The proper ties of CR2 and CR3 are also similar to those of kidney enzymes reported by Hara et al. [8],
The immunological properties of the three enzymes were analyzed by Western Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
4.4
E
Fig. 2. Molecular weight analy sis of chicken liver carbonyl reduc tase by SDS-PAGE and Superose 12 gel filtration. The conditions of SDS-PAGE and gel filtration are described in the text. Molecular standards are: 1 = bovine serum al bumin (Mr 66.000); 2 = ovalbumin (Mr 45,000); 3 = a-chymotrypsinogen A (Mr 24,500), and 4 = cyto chrome c (Mr 12,400).
Chicken Liver Carbonyl Reductase
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Table 1. Substrate specificity of chicken liver carbonyl reductases toward various carbonyl compounds and steroids Substrate
Concentration mmol/1
Relative acitvity, % --------------------------CR 1
CR2
CR3
NADPH
NADH
NADPH
NADH
NADPH
NADH
100
53 33 16
100
ND ND ND
100
ND ND ND
Reductase activity 4-Benzoylpyridine Menadione 9,10-Phenanthrenequinone 4-N itroacetophenone Cyclohexanone 4-N itrobenzaldehyde Pyridine-3-aldehyde D-glucuronic acid Androsterone Testosterone Estrone 5a-Androstane-3,17-dione 5ß-Androstane-3,l 7-dione 5a-Diyhdrotestosterone 5ß-Dihydrotestosterone
1.0 0.1 0.05 0.1 3.3 0.1 1.0 3.3 0.05 0.1 0.025 0.1 0.1 0.1 0.1
63 196 ND 104 81 53 ND ND ND 32 76 76 54 45
Dehydrogenase activity Androsterone Testosterone 17ß-Estradiol 5a-Dihydrotestosterone 5ß-Dihydrotestosterone
0.05 0.1 0.025 0.1 0.1
ND 11 51 ND ND
21 61 11
ND 25 ND ND ND
ND ND
76 436 9 ND 7 11 4 ND ND ND ND ND ND
83 303 ND ND ND 10 13 ND ND ND 13 ND ND ND
ND ND ND ND ND
ND ND ND ND ND
20
Relative activity is expressed as percentage of the reduction of 4-benzoylpyridine, determined under the conditions described in the text. The 100% values are 1.42 units/mg for CR1,0.355 units/mg for CR2 and 3.05 units/mg for CR3. ND = Not detectable; - = not determined.
tibody (data not shown). These results sug gested that CR2 and CR3 were immunologically similar to each other, and also similar to kidney enzymes [8], CR1 is a unique enzyme. Several oligo meric enzymes have been reported in the dog [14], rabbit [16], hamster liver [10] and guinea pig lung [17], Since these enzymes Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
blotting with anti-chicken kidney carbonyl reductase antibody [8] (fig. 3). CR2 and CR3 are strongly cross-reacted with this antibody though CR1 did not, suggesting that CR1 was different from CR2 and CR3 in their immunological properties. Additionally, all three enzymes did not cross-react with antibovine liver high-Km aldehyde reductase an
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226
Table 2. Kinetic properties of chicken liver carbonyl reductases CR2
CR1
Substrate
4-Benzoylpyridine Menadione 9,10-Phenanthrenequinone 4-N itrobenzaldehyde Pyridine-3-aldehyde Estrone 5a-Androstane-3,17-dione 17ß-Estradiol
Km
V v max
Km
32.5 2.7 0.7 4.1 486 1.1 1.7 5.7
77 54 112 51 63 51 55 48
2,890 274 0.7
CR3 V v max
44 22 56
Km 2,010 246 0.9
V v max
294 305 412
Kinetic constants were calculated from double reciprocal plots of substrate concentration versus enzyme activity in the presence of 0.066 mmol/1 NADPH as a cofactor. The Km values are expressed as the concentration (pmol/1). The V max values are expressed as the specific activity (mol product/min/mol enzyme). - = Not determined.
CR1
CR2
CR3
Fig. 3. Western blot analysis of chicken liver car bonyl reductase. Experimental procedure is described in the text. Anti-chicken kidney carbonyl reductase antibody was used as a first antibody.
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32K
were not active toward steroids, CR1 is dif ferent from such enzymes. CR1 is consid ered to be the same as estradiol-17ß dehy drogenase reported by Renwick et al. [18] on the basis of its molecular weight, subunit composition and substrate specificity. But its activity toward endogenous carbonyl compounds was as strong as toward steroids, suggesting that CR1 might participate in both the detoxication of xenobiotics and me tabolism of both estrogen and androgen, such as estradiol and testosterone. On the other hand, CR2 and CR3 may belong to the same enzyme group as the kidney enzymes [8]. The chicken kidney carbonyl reductase as well as the enzymes in human brain [1] and rat liver [ 19] were reported to exhibit the 9-keto-reductase activity of prostaglandin E2. CR2 and CR3 also showed as much activity as kidney enzymes toward prosta glandin E? (data not shown), suggesting that they may participate in the metabolism of
Chicken Liver Carbonyl Reductase
227
Inhibitor
Barbital Coumarin Quercitrin Indomethacin Medroxyprogesterone DTNB CuCl2 Pyrazole
Concentration mmol/1 0.1 1.0 0.1 0.1 0.05
0.01 0.01 10.0
Inhibition, % ---------------------CRI CR2 CR3 67 56 85 3 21 0 0 15
3 13 90 24 0 55 80 14
14 51 100 28 16 44 39 19
Percent inhibition of 4-benzoylpyridine-reducing activity is expressed. NADPH was used as a cofactor. DTNB = 5,5'-Dithiobis(2-nitrobenzoic acid).
prostaglandins as well as detoxication of ex ogenous compounds. Chicken liver has three multiple forms of carbonyl reductase. Such a phenomenon is also observed in chicken kidney [8], rat ovary [9], hamster liver [10], human brain [1], Though the physiological significance re mains unclear, these enzymes may coopera tively function in the metabolism of both endogenous compounds such as steroids and prostaglandins and exogenous compounds such as metabolites of polycyclic aromatic hydrocarbons because their substrate speci ficities and inhibitor sensitivities are vari able and different from each other.
Acknowledgement The authors thank Dr. Hideo Sawada. Gifu Phar maceutical University, for providing them with anti chicken kidney carbonyl reductase antibody.
References 1 Wermuth B: Purification and properties of an NADPH-dependent carbonyl reductase from hu man brain: Relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase. J Biol Chem 1981;256:1206-1213. 2 Nakayama T, Hara A, Yashiro K, Sawada H: Reductase for carbonyl compounds in human liv er. Biochem Pharmacol 1985;34:107-117. 3 Wermuth B: Aldo-keto reductases. Prog Clin Biol Res 1985;174:209-230. 4 Terada T, Kohno T, Samejima T, Hosomi S, Mizoguchi T, Uehara K: Purification and proper ties of beef liver aldehyde reductase catalyzing the reduction of D-erythrose 4-phosphate. J Biochem (Tokyo) 1985;97:79-87. 5 Kohno T, Yasuda M, Murai K, Hosomi S, Mizo guchi T: Separation of the molecular forms of chicken liver cytosolic aldehyde reductase and comparison with mitochondrial aldehyde reduc tase. Chem Pharm Bull 1986;34:5034-5039. 6 Kohno T, Hosomi S, Mizoguchi T: Partial purifi cation and some properties of mitochondrial alde hyde reductases from chicken liver. Chem Pharm Bull 1984;32:31 18-3127. 7 Terada T, Niwase K, Shinagawa K, Koyama I, Hosomi S, Mizoguchi T: Bovine liver cytosolic aldehyde reductase and carbonyl reductase. Puri fication and characterization. Prog Clin Biol Res 1989;290:293-305. 8 Hara A, Deyashiki Y, Nakagawa M, Nakayama T, Sawada H: Isolation of proteins with carbonyl reductase activity and prostaglandin 9-ketoreductase activity from chicken kidney. J Biochem (To kyo) 1982;92:1753-1762. 9 Iwata N, Inazu N, Satoh T: The purification and characterization of NADPH-dependent carbonyl reductase from rat ovary. Prog Clin Biol Res 1989;290:307-321. 10 Hara A, Seiriki K, Nakayama T, Sawada H: Dis crimination of multiple forms of diacetyl reduc tase in hamster liver. Prog Clin Biol Res 1985; 174:291-304. 11 Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some appli cations. Proc Natl Acad Sci USA 1979;76:4350— 4354. 12 Sawada H, Hara A, Hayashibara M, Nakayama T: Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
Table 3. Inhibitor sensitivity of chicken liver car bonyl reductases
13
14
15
16
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Guinea pig liver aromatic aldehyde-ketone reduc tases identical with 17ß-hydroxysteroid dehydro genase isozymes. J Biochem (Tokyo) 1979;86: 883-892. Hasebe K, Hara A, Nakayama T, Hayashibara M, Inoue Y, Sawada H: Inhibition of hepatic 17ßand 3a-hydroxysteroid dehydrogenase by anti inflammatory drugs and nonsteroidal estrogens. Enzyme 1987;37:109-114. Hara A, Nakayama T, Deyashiki Y, Kariya K, Sawada H: Carbonyl reductase of dog liver: Puri fication properties and kinetic mechanism. Arch Biochem Biophys 1986;244:238-247. Penning TM, Mukharji I, Barrows S, Talalay P: Purification and properties of a 3a-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs. Biochem J 1984;222:601-611. Sawada H, Hara A, Nakayama T, Kato F: Reduc tases for aromatic and ketones from rabbit liver: Purification and characterization. J Biochem (To kyo) 1980;87:1153-1165.
17 Nakayama T, Hara A, Sawada H: Purification and characterization of a novel pyrazole-sensitive car bonyl reductase in guinea pig lung. Arch Biochem Biophys 1982;217:564-573. 18 Renwick AGC, Soon CY, Chambers SM, Brown CR: Estradiol-17ß dehydrogenase from chicken liver. J Biol Chem 1981;256:1881-1887. 19 Penning TM, Sharp RB: Prostaglandin dehydro genase activity of purified rat liver 3a-hydroxysteroid dehydrogenase. Biochem Biophys Res Com mun 1987;148:646-652.
Received: May 1, 1992 Accepted after revision: July 2, 1992 Tohru Nishinaka Laboratory of Biochemistry Faculty of Pharmaceutical Sciences Osaka University 1-6 Yamada-oka, Suita Osaka 565 (Japan)
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©1992 S. Karger AG, Basel 0013-9432/92/0465-0221 $2 75/0
Enzyme 1992;46:221-228
Characterization of Multiple Forms of Carbonyl Reductase from Chicken Liver Tohru Nishinaka, Yumiko Kinoshita, Naomi Terada, Tomoyuki Terada, Tadashi Mizoguchi, Tsutomu Nishihara Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences. Osaka University, Osaka, Japan
Key Words. Carbonyl reductase, multiple forms • Liver, chicken • Estradiol-17 ß-dehydrogenase Abstract. Three enzyme forms (CRI, CR2 and CR3) of carbonyl reductase were purified from chicken liver with using 4-benzoylpyridine as a substrate. CR 1 was a dimeric enzyme composed of two identical 25-kD subunits. CR2 and CR3 were monomeric enzymes whose molecular weights were both 32 kD. CR1 exhibited 17ß-hydroxysteroid dehydrogenase activity as well as carbonyl reductase activity in the presence of both NADP(H) and NAD(H). CR2 and CR3 had similar properties with regard to substrate specificity and inhibitor sensitivity. They could exhibit the activity only with NADPH and had no hydroxysteroid dehydrogenase activity. CR2 and CR3 cross-reacted with anti-chicken kidney carbo nyl reductase antibody, though CR1 did not. The results suggest that CR1 is a hydroxysteroid dehydrogenase, and CR2 and CR3 are similar to each other and to the kidney enzymes.
Carbonyl reductase (secondary alcohol: NADP+ oxidoreductase, EC 1.1.1.184) is generally defined as a cytosolic monomeric enzyme that catalyzes the NADPH-dependent reduction of a great variety of carbonyl compounds such as quinones derived from polycyclic aromatic hydrocarbons, 9-ketoprostaglandins and the anthracycline antibi otic daunorubicin [1], This enzyme is widely distributed in various mammalian and non mammalian species [2, 3], Enzymes with similar properties, including aldehyde reduc
tase (EC 1.1.1.2) and aldose reductase (EC 1.1.1.21), have been described from a num ber of animal tissues and are generally re ferred to as the aldo-keto reductases [3]. We have been studying an aldo-keto reductase, and purified and characterized the aldehyde reductase from bovine liver cytosol [4], chicken liver cytosol [5] and mitochondria [6]. We also reported on the purification of carbonyl reductase from bovine liver [7], This time we investigated carbonyl reduc tase in chicken liver and found three multi ple forms which could be separated by chromatofocusing. The presence of multiple Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
Introduction
Nishinaka/Kinoshita/Terada/Terada/Mizoguchi/Nishihara
222
forms of carbonyl reductase has been re ported in many tissues, for example, chicken kidney [8], rat ovary [9], hamster liver [10], and human brain [1], The broad substrate specificity and the existence of multiple forms have led to confusion in understand ing the physiological function of carbonyl reductase. It is necessary to characterize each carbonyl reductase in detail in order to clarify the physiological significance of car bonyl reductase multiple forms. In this study, we demonstrated the purification of three multiple forms of carbonyl reductase in chicken liver and characterization of their properties.
bance at 340 nm. One unit was defined as 1 pmol product formation per min at 25 °C. In the purifica tion procedure, 4-benzoylpyridine was used as a typi cal substrate for carbonyl reductases [7, 9].
Purification of Multiple Forms of Carbonyl Reductase All the procedures were carried out below 4 ° C using the following buffers, to all of which 10 mmol/1 2-mercaptoethanol (2-ME) and 0.1 mmol/1 EDTA were added unless otherwise mentioned: buffer A, 3 mmol/1 Tris-HCl buffer (pH 7.4) containing 155 mmol/1 KCl, 1 mmol/1 EDTA and 0.5 mmol/1 phenylmethylsulfonyl fluoride; buffer B, 25 mmol/1 ethanolamine-acetate buffer (pH 8.9) without EDTA; buffer C, 2.5 mmol/1 sodium phosphate buffer (pH 7.0); buffer D, 2.5 mmol/1 sodium phosphate buffer (pH 7.0) without EDTA.
Extraction and Ammonium Sulfate Fractionation
Chicken liver was obtained from the local slaughter house. Ultrogel AcA 44 was from IBF, Villeneuve-la-Garenne, France. Polybuffer exchanger for chromatofocusing, PBE94, polybuffer 96, 2',5'-ADPSepharose and Superose 12 were purchased from Pharmacia LKB Biotechnology, Uppsala, Sweden. NADPH and NADH were from Oriental Yeast Co., Tokyo, Japan. Hydroxyapatite was from Wako Pure Chemical Industries, Osaka, Japan. Substrates and inhibitors were bought from Wako Pure Chemical Industries, Nacalai Tesque, Kyoto, Japan, and Sigma, St. Louis, Mo., USA. Polyvinylidene difluoride (PVDF) membrane was from Millipore, Bedford. Mass., USA. Wako POD-kit was supplied from Wako Pure Chemical Industries. Anti-chicken kidney carbo nyl reductase antibody was a kind gift from Dr. Hideo Sawada, Gifu Pharmaceutical University [8].
Enzyme Assay The carbonyl reductase activity assay system con sisted of substrate at the indicated concentration, 0.066 mmol/1 NADPH or NADH in 100 mmol/1 sodium phosphate buffer (pH 7.0). The activity was measured by monitoring the linear change in absor bance at 340 nm with respect to time. Dehydrogenase activity was also determined with 0.066 mmol/1 NADP+ or NAD+ monitoring the increase in absor
2',5'-ADP-Sepharose Affinity
Chromatography.
CR 1, CR2 and CR3 were dialyzed for 3 h against 2 times 2 liters of buffer C, and separately applied to
Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
Materials and Methods
Approximately 120 g of chicken liver was cut into small pieces and rinsed with ice-cold buffer A to remove excess blood. The liver was homogenized with 3 voi of buffer A in a Waring Blender at 10,000 rpm for 3 min. The homogenate was centrifuged at 10,000 g for 60 min, the pellet was discarded, and the supernatant was centrifuged at 105,000 £ for 60 min. The supernatant was fractionated by adding ammo nium sulfate with constant stirring and adjusting the pH to 7.0 with 3 N NH4OH; the protein, which pre cipitated between 40 and 70% saturation, was col lected by centrifugation at 10,000 g for 20 min and dissolved in the minimum volume of buffer B. The enzyme solution was dialyzed against 5 liters of buffer B for 3 h. Ultrogel AcA 44 Gel Filtration. After centrifuged at 10,000 g for 20 min, the dialysate was applied to Ultrogel AcA 44 column chromatography (5.0 X 150 cm) and eluted with buffer B. Fractions with the enzyme activity were pooled. Chromatofocusing. The pool from Ultrogel AcA 44 was subjected to chromatofocusing (1.6 X 35 cm) equilibrated with buffer B. The enzyme was eluted with 10-fold diluted polybuffer 96-acetate (pH 6.0). At this step, three active peaks were observed at pH 8.2, pH 7.8 and pH 7.6 (fig. 1). The three enzymes were separately collected and tentatively named CR1, CR2 and CR3, respectively.
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Chicken Liver Carbonyl Reductase
Molecular Weight Analysis The native molecular weights of the three carbo nyl reductases were analyzed by gel filtration with Superose 12 (0.9 X 30 cm) equilibrated with 50 mmol/1 sodium phosphate buffer (pH 7.0) containing 300 mmol/1 NaCl at a flow rate of 0.5 ml/min. The molecular weights of the three enzymes were calcu lated on the basis of the retention times of the enzymes and molecular weight standards. The sub unit size was determined by SDS-PAGE. Purified enzymes were run on a 12.5% SDS-polyacrylamide gel and stained with silver. The Rm values of the three enzymes were measured, and the subunit weight was calculated. Bovine serum albumin, ovalbumin, a-chymotrypsinogen A and cytochrome c were used as molecular weight standards in both procedures.
Western Blotting Western blot analysis was performed according to the method of Towbin et al. [11], Antigens (1 gg) were subjected to 12.5 % SDS-PAGE. After the electropho resis, proteins were electrically transferred to the PVDF membrane at 200 mA for 60 min. The mem brane was incubated with anti-chicken kidney carbo nyl reductase antiserum and then reacted with a 1,500-fold dilution of peroxidase-labeled goat anti rabbit IgG antibody. The blotted proteins were visulalized by Wako POD-kit at 25 °C for 30 min.
Results and Discussion Three enzyme forms (CRI, CR2 and CR3) of carbonyl reductase were found in chicken liver cytosol. They were separated
o.io
».0
E 8.0
0.0S
sB. 7.0
< 0.00
0
10
20
30
40
6.0 50
Fraction number
Fig. 1. Separation of three carbonyl reductase multiple forms in chicken liver by chromatofocusing. The condition is described in the text. Fractions (2.5 ml) were analyzed for pH ( ) and for enzyme activity (•), which was assayed at pH 7.0 with 4-benzoylpyridine.
by chromatofocusing. That is, three active peaks were observed at pH 8.2 (CR1), pH 7.8 (CR2) and pH 7.6 (CR3), respectively (fig. 1). The three enzymes were further puri fied as described in Materials and Methods. The final preparations exhibited specific ac tivity toward 4-benzoylpyridine of 1.42 units/mg for CR1, 0.355 units/mg for CR2 and 3.05 units/mg for CR3. The molecular weights of CRI, CR2 and CR3 were esti mated to be 25, 32 and 32 kD, respectively, by SDS-PAGE, and to be 48, 32 and 32 kD, respectively, by gel filtration with Superose 12 (fig. 2). These results indicate that the most basic enzyme, CR1, is composed of two identical subunits and the other two en zymes, CR2 and CR3, are monomeric en zymes. Since most carbonyl reductases are monomeric enzymes [1], CR1 is a very cu rious enzyme having two subunits. The substrate specificity of the three en zymes toward carbonyl compounds is shown in table 1. CR1 had a broad substrate speciDownloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
2',5'-ADP-Sepharose (1.5 X 1.0 cm) equilibrated with buffer C. Each enzyme was eluted with a linear gradient (40 ml of buffer C and 40 ml of the same buffer with 600 mmol/1 KC1) after washing the col umn with 50 ml of buffer C. These enzymes were dia lyzed for 3 h against 2 times 2 liters of buffer D. Hydroxyapatite The three enzymes were sepa rately applied to hydroxyapatite (0.8 X 2.5 cm) equil ibrated with buffer D, and eluted with a linear gra dient (30 ml of buffer D and 30 ml of 100 mmol/1 sodium phosphate, pH 7,0, containing 10 mmol/1 2-ME) after washing the column with 50 ml of buffer D.
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4.8
4.8 JS BG
4.4
E
BG
BG
-j 4.0 0.0
0.5 Rm
1.0
4.0 12
14
16
Elution volume (ml)
ficity and could utilize both NADPH and NADH as a cofactor. On the other hand, CR2 and CR3 showed the activity only to ward 4-benzoylpyridine, menadione and 9,10-phenanthrenequinone so far tested, while CR1 exhibited the activity also toward cyclohexanone, 4-nitrobenzaldehyde and pyridine-3-aldehyde. All the three enzymes exhibited little activity toward Z)-glucuronic acid which is a typical substrate for aldehyde reductase [4-6]. NADH could not be a cofac tor for CR2 and CR3 in contrast to the case of CR1. Since some carbonyl reductases from rat [12], guinea pig [13] and rabbit [14] have been reported to have oxidoreductase activity toward steroid compounds, we also examined the steroid oxidoreduction by chicken liver carbonyl reductases. CR1 could catalyze the oxidoreduction of 17ß-hydroxysteroids such as esterone and 17ß-estradiol as well as 3-ketosteroid reduc tion toward 5a-dihydrotestosterone. CR2 and CR3, however, had little activity toward such steroids. Table 2 shows the Km values of the three carbonyl reductases toward their typical substrates. CR1 had low Km values toward all the substrates, especially toward
steroids. CR2 and CR3 had similar Km val ues toward 4-benzoylpyridine, menadione and 9,10-phenanthrenequinone. Table 3 shows the inhibitor sensitivity of the three enzymes. Quercitrin was a com mon inhibitor for them. Among the three enzymes, only CR1 was sensitive for barbi tal. The sulfhydryl modifiers, 5,5'-dithiobis(2-nitrobenzoic acid) and CuCl2 strongly inhibited both CR2 and CR3 though these reagents did not affect the CR 1 activity, sug gesting that both CR2 and CR3 might have essential cysteine residue(s) in their active sites. CR2 and CR3 were also sensitive to the nonsteroidal anti-inflammatory drug, indomethacin, the same as the case for the hu man [1], rat [15], guinea pig [13] and bovine enzyme [7], These results suggested that CR1 was quite different from CR2 and CR3, and CR2 and CR3 were similar to each other in the enzymatic properties of substrate speci ficity and inhibitor sensitivity. The proper ties of CR2 and CR3 are also similar to those of kidney enzymes reported by Hara et al. [8],
The immunological properties of the three enzymes were analyzed by Western Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
4.4
E
Fig. 2. Molecular weight analy sis of chicken liver carbonyl reduc tase by SDS-PAGE and Superose 12 gel filtration. The conditions of SDS-PAGE and gel filtration are described in the text. Molecular standards are: 1 = bovine serum al bumin (Mr 66.000); 2 = ovalbumin (Mr 45,000); 3 = a-chymotrypsinogen A (Mr 24,500), and 4 = cyto chrome c (Mr 12,400).
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Table 1. Substrate specificity of chicken liver carbonyl reductases toward various carbonyl compounds and steroids Substrate
Concentration mmol/1
Relative acitvity, % --------------------------CR 1
CR2
CR3
NADPH
NADH
NADPH
NADH
NADPH
NADH
100
53 33 16
100
ND ND ND
100
ND ND ND
Reductase activity 4-Benzoylpyridine Menadione 9,10-Phenanthrenequinone 4-N itroacetophenone Cyclohexanone 4-N itrobenzaldehyde Pyridine-3-aldehyde D-glucuronic acid Androsterone Testosterone Estrone 5a-Androstane-3,17-dione 5ß-Androstane-3,l 7-dione 5a-Diyhdrotestosterone 5ß-Dihydrotestosterone
1.0 0.1 0.05 0.1 3.3 0.1 1.0 3.3 0.05 0.1 0.025 0.1 0.1 0.1 0.1
63 196 ND 104 81 53 ND ND ND 32 76 76 54 45
Dehydrogenase activity Androsterone Testosterone 17ß-Estradiol 5a-Dihydrotestosterone 5ß-Dihydrotestosterone
0.05 0.1 0.025 0.1 0.1
ND 11 51 ND ND
21 61 11
ND 25 ND ND ND
ND ND
76 436 9 ND 7 11 4 ND ND ND ND ND ND
83 303 ND ND ND 10 13 ND ND ND 13 ND ND ND
ND ND ND ND ND
ND ND ND ND ND
20
Relative activity is expressed as percentage of the reduction of 4-benzoylpyridine, determined under the conditions described in the text. The 100% values are 1.42 units/mg for CR1,0.355 units/mg for CR2 and 3.05 units/mg for CR3. ND = Not detectable; - = not determined.
tibody (data not shown). These results sug gested that CR2 and CR3 were immunologically similar to each other, and also similar to kidney enzymes [8], CR1 is a unique enzyme. Several oligo meric enzymes have been reported in the dog [14], rabbit [16], hamster liver [10] and guinea pig lung [17], Since these enzymes Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
blotting with anti-chicken kidney carbonyl reductase antibody [8] (fig. 3). CR2 and CR3 are strongly cross-reacted with this antibody though CR1 did not, suggesting that CR1 was different from CR2 and CR3 in their immunological properties. Additionally, all three enzymes did not cross-react with antibovine liver high-Km aldehyde reductase an
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Table 2. Kinetic properties of chicken liver carbonyl reductases CR2
CR1
Substrate
4-Benzoylpyridine Menadione 9,10-Phenanthrenequinone 4-N itrobenzaldehyde Pyridine-3-aldehyde Estrone 5a-Androstane-3,17-dione 17ß-Estradiol
Km
V v max
Km
32.5 2.7 0.7 4.1 486 1.1 1.7 5.7
77 54 112 51 63 51 55 48
2,890 274 0.7
CR3 V v max
44 22 56
Km 2,010 246 0.9
V v max
294 305 412
Kinetic constants were calculated from double reciprocal plots of substrate concentration versus enzyme activity in the presence of 0.066 mmol/1 NADPH as a cofactor. The Km values are expressed as the concentration (pmol/1). The V max values are expressed as the specific activity (mol product/min/mol enzyme). - = Not determined.
CR1
CR2
CR3
Fig. 3. Western blot analysis of chicken liver car bonyl reductase. Experimental procedure is described in the text. Anti-chicken kidney carbonyl reductase antibody was used as a first antibody.
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32K
were not active toward steroids, CR1 is dif ferent from such enzymes. CR1 is consid ered to be the same as estradiol-17ß dehy drogenase reported by Renwick et al. [18] on the basis of its molecular weight, subunit composition and substrate specificity. But its activity toward endogenous carbonyl compounds was as strong as toward steroids, suggesting that CR1 might participate in both the detoxication of xenobiotics and me tabolism of both estrogen and androgen, such as estradiol and testosterone. On the other hand, CR2 and CR3 may belong to the same enzyme group as the kidney enzymes [8]. The chicken kidney carbonyl reductase as well as the enzymes in human brain [1] and rat liver [ 19] were reported to exhibit the 9-keto-reductase activity of prostaglandin E2. CR2 and CR3 also showed as much activity as kidney enzymes toward prosta glandin E? (data not shown), suggesting that they may participate in the metabolism of
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Inhibitor
Barbital Coumarin Quercitrin Indomethacin Medroxyprogesterone DTNB CuCl2 Pyrazole
Concentration mmol/1 0.1 1.0 0.1 0.1 0.05
0.01 0.01 10.0
Inhibition, % ---------------------CRI CR2 CR3 67 56 85 3 21 0 0 15
3 13 90 24 0 55 80 14
14 51 100 28 16 44 39 19
Percent inhibition of 4-benzoylpyridine-reducing activity is expressed. NADPH was used as a cofactor. DTNB = 5,5'-Dithiobis(2-nitrobenzoic acid).
prostaglandins as well as detoxication of ex ogenous compounds. Chicken liver has three multiple forms of carbonyl reductase. Such a phenomenon is also observed in chicken kidney [8], rat ovary [9], hamster liver [10], human brain [1], Though the physiological significance re mains unclear, these enzymes may coopera tively function in the metabolism of both endogenous compounds such as steroids and prostaglandins and exogenous compounds such as metabolites of polycyclic aromatic hydrocarbons because their substrate speci ficities and inhibitor sensitivities are vari able and different from each other.
Acknowledgement The authors thank Dr. Hideo Sawada. Gifu Phar maceutical University, for providing them with anti chicken kidney carbonyl reductase antibody.
References 1 Wermuth B: Purification and properties of an NADPH-dependent carbonyl reductase from hu man brain: Relationship to prostaglandin 9-ketoreductase and xenobiotic ketone reductase. J Biol Chem 1981;256:1206-1213. 2 Nakayama T, Hara A, Yashiro K, Sawada H: Reductase for carbonyl compounds in human liv er. Biochem Pharmacol 1985;34:107-117. 3 Wermuth B: Aldo-keto reductases. Prog Clin Biol Res 1985;174:209-230. 4 Terada T, Kohno T, Samejima T, Hosomi S, Mizoguchi T, Uehara K: Purification and proper ties of beef liver aldehyde reductase catalyzing the reduction of D-erythrose 4-phosphate. J Biochem (Tokyo) 1985;97:79-87. 5 Kohno T, Yasuda M, Murai K, Hosomi S, Mizo guchi T: Separation of the molecular forms of chicken liver cytosolic aldehyde reductase and comparison with mitochondrial aldehyde reduc tase. Chem Pharm Bull 1986;34:5034-5039. 6 Kohno T, Hosomi S, Mizoguchi T: Partial purifi cation and some properties of mitochondrial alde hyde reductases from chicken liver. Chem Pharm Bull 1984;32:31 18-3127. 7 Terada T, Niwase K, Shinagawa K, Koyama I, Hosomi S, Mizoguchi T: Bovine liver cytosolic aldehyde reductase and carbonyl reductase. Puri fication and characterization. Prog Clin Biol Res 1989;290:293-305. 8 Hara A, Deyashiki Y, Nakagawa M, Nakayama T, Sawada H: Isolation of proteins with carbonyl reductase activity and prostaglandin 9-ketoreductase activity from chicken kidney. J Biochem (To kyo) 1982;92:1753-1762. 9 Iwata N, Inazu N, Satoh T: The purification and characterization of NADPH-dependent carbonyl reductase from rat ovary. Prog Clin Biol Res 1989;290:307-321. 10 Hara A, Seiriki K, Nakayama T, Sawada H: Dis crimination of multiple forms of diacetyl reduc tase in hamster liver. Prog Clin Biol Res 1985; 174:291-304. 11 Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some appli cations. Proc Natl Acad Sci USA 1979;76:4350— 4354. 12 Sawada H, Hara A, Hayashibara M, Nakayama T: Downloaded by: University of Exeter 144.173.6.94 - 1/26/2020 7:01:52 PM
Table 3. Inhibitor sensitivity of chicken liver car bonyl reductases
13
14
15
16
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Guinea pig liver aromatic aldehyde-ketone reduc tases identical with 17ß-hydroxysteroid dehydro genase isozymes. J Biochem (Tokyo) 1979;86: 883-892. Hasebe K, Hara A, Nakayama T, Hayashibara M, Inoue Y, Sawada H: Inhibition of hepatic 17ßand 3a-hydroxysteroid dehydrogenase by anti inflammatory drugs and nonsteroidal estrogens. Enzyme 1987;37:109-114. Hara A, Nakayama T, Deyashiki Y, Kariya K, Sawada H: Carbonyl reductase of dog liver: Puri fication properties and kinetic mechanism. Arch Biochem Biophys 1986;244:238-247. Penning TM, Mukharji I, Barrows S, Talalay P: Purification and properties of a 3a-hydroxysteroid dehydrogenase of rat liver cytosol and its inhibition by anti-inflammatory drugs. Biochem J 1984;222:601-611. Sawada H, Hara A, Nakayama T, Kato F: Reduc tases for aromatic and ketones from rabbit liver: Purification and characterization. J Biochem (To kyo) 1980;87:1153-1165.
17 Nakayama T, Hara A, Sawada H: Purification and characterization of a novel pyrazole-sensitive car bonyl reductase in guinea pig lung. Arch Biochem Biophys 1982;217:564-573. 18 Renwick AGC, Soon CY, Chambers SM, Brown CR: Estradiol-17ß dehydrogenase from chicken liver. J Biol Chem 1981;256:1881-1887. 19 Penning TM, Sharp RB: Prostaglandin dehydro genase activity of purified rat liver 3a-hydroxysteroid dehydrogenase. Biochem Biophys Res Com mun 1987;148:646-652.
Received: May 1, 1992 Accepted after revision: July 2, 1992 Tohru Nishinaka Laboratory of Biochemistry Faculty of Pharmaceutical Sciences Osaka University 1-6 Yamada-oka, Suita Osaka 565 (Japan)
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