12
Biochimica ef Biophysics Acta, 1046 (1990) 12-18 Elsevier
BBALIP
53464
Purification and properties of 3whydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol Kei Kudo, Yoshiki Amuro,
Toshikazu
Hada and Kazuya Higashino
The Third Department of Internal Medicine, Hyogo College of Medicine, Nishinomi.va, Hyogo (Japan)
(Revised
Key words:
(Received manuscript
30 October 1989) received 1 February
Bile acid; Keto bile acid; Keto bile acid reduction;
1990)
3n-Hydroxysteroid
dehydrogenase;
(Human
liver)
The NADPH-dependent 3a-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5@holanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak l), 232-fold (peak 2) and 345fold (peak 3) and the recoveries of the enzymes were 0.4% (peak l), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34,7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7a,12a-dihydroxy-3-keto-5/3-cholanoic acid, 7a-hydroxy5/3-cholestan-3-one and 7u,l2a-dihydroxy-5@-cholestan-3-one to the corresponding 3a-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzymes required a sulfhydryl group for activity.
Introduction Cholic and chenodeoxycholic acids are synthesized from cholesterol in human and animal liver and deoxycholic and lithocholic acids are formed by bacterial ;rcY-dehydroxylation in the intestine from their respective precursor bile acids [l]. The bile acids are probably oxidized further to the corresponding keto bile acids by intestinal bacteria [2], as several keto bile acids have been found in the feces [3] and portal venous blood [4]. However, keto bile acids were not found in appreciable amounts in the bile, peripheral venous blood and urine, suggesting that keto bile acids could be reduced to the corresponding hydroxy bile acids during passage through the liver. Thus, a previous in vivo study with man [5] showed that radioactive 3,7,12_triketocholic acid could be reduced to 3a-hydroxy-7,12-diketo-S/3-cholanoic, 301,7a-
dihydroxy-12-keto-5P-cholanoic and 3a,7cq12a-trihydroxy-5/?-cholanoic (cholic) acids in the liver. These results indicate the presence of the enzymes catalyzing the reduction of keto-forms of cholanoic acid to the corresponding hydroxy-forms in human liver. Of the reducing enzymes, 3a-hydroxysteroid dehydrogenase would appear to be an enzyme catalyzing the reduction of 3-keto- to 3cY-hydroxy bile acids. The properties of the 3a-hydroxysteroid dehydrogenase as 3-keto bile acid reductase have, however, not been intensively studied, especially in human liver cytosol, although the enzyme from rat liver was found to be able to reduce the 3-keto group in a number of 3-keto steroids of the C19, C21, C24 and C27 series [6]. In this paper, we describe the purification and properties of the 3a-hydroxysteroid dehydrogenase, as 3-keto bile acid reductase, from human liver cytosol. Materials and Methods Chemicals
Correspondence: Medicine, Hyogo 0005-2760/90/$03,50
Y. Amuro, The Third Department of Internal College of Medicine, Nishinomiya, Hyogo, Japan. 0 1990 Elsevier Science Publishers
B.V. (Biomedical
The sources of the chemicals used in this study were as follows: trifluoroacetic acid anhydride was obtained Division)
13
park2
0.2 -T
0.1 ; J 2
100
Fraction number Fig. 1. CM-cellulose (CM-52) chromatography of the 4670% saturated ammonium were collected and analyzed for enzyme activity (0) and for protein concentration
from Tokyo Kasei (Tokyo, Japan); pyridine nucleotides, 3-keto-S/3-cholanoic acid, S/3-cholestan-3-one, S/3-pregnane-3,20-dione, SP-androstane-3,17-dione from Sigma Chemicals (St. Louis, MO); 7a,l2cr-dihydroxy-3-ketoS/3-cholanoic acid from Calbiochem-Behring (La Jolla, CA); 3,7-diketo-S/3-cholanoic acid, 7a-hydroxy-3-ketoSj&cholanoic acid from Steraloids (Wilton, NH); CMcellulose (CM-52), DEAE-cellulose (DE-52) from Whatman (U.K.); Affi-Gel blue from Bio-Rad (Richmond, CA); phenyl-Sepharose CL4B, Polybuffer 96, PBE 94 from Pharmacia Fine Chemicals (Sweden); Calibration proteins from Boehringer-Mannheim (F.R.G.) and Sigma. 7a-hydroxy-S/3-cholestan-3-one and 7a,l2a-dihydroxy-5fl-cholestan-3-one were kindly supplied by Dr. Kiyohisa Uchida (Shionogi Research Laboratory, Osaka, Japan). S/3-cholestane-3a,7a-diol and SP-cholestane3a,7a,12a-triol were synthesized as follows. The mixture in a total volume of 5 ml contained 2 units of 3ar-hydroxysteroid dehydrogenase (from Pseudomonas testosteroni, Sigma), 400 pg of 7a-hydroxy-SP-cholestan-3-one or 7a,l2cY-dihydroxy-5/I-cholestan-3-one in 100 ~1 of acetone, 0.1 M phosphate buffer (pH 7.4) and 2 mg of NADH was incubated at 37 o C for 20 min. The product formed was extracted with ethyl ether. All other chemicals were obtained from Wako Pure Chemicals (Osaka, Japan). Purification of 3a-hydroxysteroid dehydrogenase Normal human liver was obtained from a human cadaver several hours after death. The liver (200 g wet weight) was homogenized with 3 vol. of 0.25 M sucrose in a Teflon homogenizer. The homogenate was centrifuged at 10000 X g for 15 mm. The cytosol fraction was obtained by centrifuging the 10000 x g supernatant fluid at 105000 X g for 1 h. The precipitate obtained from 40-70% saturated ammonium sulfate fractionation of the cytosol was dissolved in 200 ml of 20 mM
sulfate fraction of the human liver cytosol. Fractions of 10 ml (0). The arrow indicates the start of a linear NaCl gradient.
sodium-potassium-phosphate buffer (pH 6.0) containing 20% (w/v) glycerol and 0.5 mM dithiothreitol (DTT) (buffer A) and dialyzed against the same buffer overnight. The fraction was centrifuged at 10000 X g for 10 min, and the supernatant was applied to a column of CM-cellulose (5.0 x 15.0 cm), previously equilibrated with buffer A. The column was washed with 300 ml of buffer A, and the protein was eluted with a linear gradient of concentration from 0 to 0.2 M NaCl in buffer A. Three peaks containing 3a-hydroxysteroid dehydrogenase activity were found. These three peaks were designated peaks 1, 2 and 3 (Fig. 1). The active fractions of peak 2 or 3 were applied to Affi-Gel blue column (3.2 x 6.0 cm), previously equilibrated with buffer A. The column was washed with the same buffer and the protein was eluted with a linear gradient of concentration from 0 to 2.0 M NaCl in buffer A. The fractions containing enzyme activity were dialyzed against 20 mM phosphate buffer (pH 7.4) containing 20% (w/v) glycerol, 0.5 mM DTT and 1.0 M ammonium sulfate (buffer B). The fractions were applied to phenyl-Sepharose CL-4B column (2.2 x 8.0 cm) equilibrated with buffer B. The column was washed with buffer B and the protein was eluted with a linear gradient of concentration from 1.0 to 0 M ammonium sulfate. The fractions containing enzyme activity were concentrated using Amicon Diaflo membrane and applied to Pharmacia Fast Protein Liquid Chromatography equipped with TSKgel G3000 SW column (7.5 mm X 60 cm), equilibrated with 20 mM phosphate buffer (pH 7.4) containing 20% glycerol and 0.5 mM DT-T. The active fractions of peak 1 from CM-cellulose column were dialyzed against 20 mM Tris-HCl buffer (pH 8.1) containing 20% (w/v) glycerol and 0.5 mM DTT (buffer C), and applied to a column of DEAE-cellulose (5.0 X 15.0 cm) equilibrated with buffer C. The
14 column was washed with 300 ml of buffer C and the protein was eluted with a linear gradient of concentration from 0 to 0.2 M NaCl in buffer C. The fractions of enzyme activity from DEAE-cellulose column were purified further with Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography described above and chromatofocusing. Chromatofocusing The enzyme preparations were dialyzed against 0.025 M monoethanolamine-acetate buffer (pH 9.4) and was applied to a chromatofocusing column (1.0 X 20.0 cm) packed with Polybuffer exchanger 94 equilibrated with the same buffer. The protein was eluted with 160 ml of a 1 : 10 dilution of Polybuffer 96-acetate (pH 6.0) at a flow rate 30 ml/h. Amino acid analysis The enzymes purified (70-200 pg/rnl) were dialyzed two times overnight against 2000 ml of distilled water and then hydrolyzed in 6 M HCl at 110 ’ C for 24 and 72 h. Amino acid analysis was performed using a Hitachi L-8500 amino acid analyzer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to
Laemmh [7] using a 15% acrylamide gel. The proteins were located using a commercially available silver stain kit (Wako Pure Chemicals, Osaka, Japan). Determination of protein concentration Protein concentration was determined by the method of Lowry et al [8] or from the absorbance at 280 nm with bovine serum albumin as standard. Incubation procedure and analysis of the product formed Activity of 3ar-hydroxysteroid dehydrogenase catalyzing the reduction of 3-keto-5P-cholanoic acid to lithocholic acid was assayed by the method as described previously [9]. Briefly, the standard incubation mixture in a total volume of 3.0 ml contained 178 PM 3-ketoSfi-cholanoic acid in 100 ~1 of ethanol, an appropriate amount of enzyme preparation, 0.1 M phosphate buffer (pH 7.4), and 0.8 mM NADPH. The mixture was incubated at 37” C for 10 min and the reaction was stopped by adding 1.0 ml of 4 M hydrochloric acid. Then 100 pg of 5P-cholanoic acid was added as an internal standard. The bile acids extracted with diethyl ether from the incubation mixture were converted to the hexafluoroisopropyl-trifluoroacetyl derivatives [lo] and were applied to gas chromatography (Shimadzu GC9A) on a glass column (3.0 mm x 1.6 m) of 3% Silicon DC QF-1 at 220’ C. The amount of lithocholic acid formed
TABLE I Purification of 3a-hydroxysteroid
dehydrogenase Protein (mg)
Peak 1 cytosol (N&)$0., 4&70% CM-52 DE-52 Affi-Gel blue phenyl-Sepharose TSK-3000SW Chromatofocusing Peak 2 cytosol (NH&Q 40-704; CM-52 Affi-Gel blue phenyl-Sepharose TSK-3000SW Peak 3 cytosol (f’+)& 40-7046 CM-52 Affi-gel blue phenyl-Sepharose TSK-3000SW
Total enzyme activity (nmol/min)
3936
3818
1866 266 89.7 25.5 7.8 0.3 0.05
2221 2012 1947 1626 997 57.3 15.3
3936
12534
1866 150 25.9 5.5 1.2
8417 6318 5 286 2197 892.5
3936
6303
1866 156.7 23.8 7.4 0.42
4232 3 183 1815 630.6 231.7
Spec. act. (nmol/min per mg)
Yield (%)
Purification (-fold)
1.0
100.0
1.0
1.2 7.6 21.7 63.8 127.3 184.8 306.0
58.2 52.7 51.0 42.6 26.1 1.5 0.4
1.2 7.8 22.4 65.8 131.1 190.5 315.5
3.2
100.0
1.0
4.5 42.1 203.8 402.1 743.8
67.1 50.4 42.1 17.5 7.1
1.4 13.2 64.1 126.4 232.4
1.6
100.0
1.0
2.3 20.3 76.4 85.8 551.7
67.1 50.5 28.8 10.0 3.7
1.4 12.7 47.8 53.7 344.8
15
2.0
0.4
1.0
0.2
0.5
0
0 ^
0
0.2
'T‘ :
1.0
j &
E, %E.c
L 2.0
50
35
0.1
u
I.
SE ga
-
_ Y
0.5
z
0
0
O
0 0.2
20
2.0
10
0
10
20
30
40
Fraction
50
60
70
0
number
%
2
z
10
20
30
Fraction
40
50
60
70
is v v
0 1.0
0.1
0.5
0 Lt
0
;i 5
number
Fig. 2. Affi-Gel blue chromatography of peak 1 (A) from DEAE column and of peak 2(B) and peak 3(C) from CM column. Fractions of 10 ml were collected and analyzed for enzyme activity (0) and for protein concentration (0). The arrow indicates the start of a linear NaCl gradient.
Fig. 3. Phenyl-Sepharose chromatography of peak 1 (A), peak 2 (B) and peak 3 (C) from Affi-Gel blue column. Fractions of 6.0 ml were collected and analyzed for enzyme activity (0) and for protein concentration (0). The arrow indicates the start of a linear ammonium sulfate gradient.
was determined by measuring the peak areas of lithocholic acid and the internal standard.
Size exclusion chromatography
Results Enzyme purification The overall purifications
When the enzymes from phenyl-Sepharose chromatography were applied to high performance size exclusion chromatography on a column of TSKgel G3000 SW, the peaks of the three enzymes appeared in the
of 3a-hydroxysteroid dehydrogenases are summarized in Table I. The peaks 1, 2 and 3 (Fig. 1) from CM-cellulose chromatography of 40-708 saturated ammonium sulfate fractionation were purified further about 316-, 232- and 345fold as compared to the original cytosol with yields of about 0.4, 7.1 and 3.78, respectively. The specific activities of the purified enzymes were 306 (peak l), 744 (peak 2) and 552 (peak 3) nmol lithocholic acid formed/mm per mg protein. Affi-Gel blue chromatography
Fig. 2 shows the elution profiles of the enzymes on Affi-Gel blue chromatography. Peak 1 eluted with 0.88 M NaCl (Fig. 2A), peak 2 with 0.96 M NaCl (Fig. 2B) and peak 3 with 1.04 M NaCl (Fig. 2C). Phenyl-Sepharose
chromatography
Fig. 3 shows the elution profiles of the enzymes on phenyl-Sepharose Chromatography. Peak 1 eluted with 0.4 M ammonium sulfate (Fig. 3A), peak 2 with 0.51 ammonium sulfate (Fig. 3B) and peak 3 with 0.48 M ammonium sulfate (Fig. 3C). Chromatofocusing
Fig. 4 shows the elution profiles of peaks 1, 2 and 3 on chromatofocusing. Peak 1 eluted at pH 7.34, peak 2 at pH 7.46 and peak 3 at pH 7.88.
Fraction number
Fig. 4. Chromatofocusing of the enzymes. A, 40-70X saturated ammonium sulfate fraction of the cytosol(lO0 mg of protein); B, peak 1 (50 pg of protein) from TSKgel G3000 SW; C, peak 2 (150 cg of protein) from phenyl-Sepharose; D, peak 3 (150 cg of protein) from phenyl-Sepharose. Fractions of 3.0 ml were collected and analyzed for enzyme activity (0).
16 TABLE
II
TABLE
Amino acid composition of 3a-hydroxysteroid
dehydrogenase
Substrate specificity
Values represent the average of values after 24 and 72 h hydrolysis time, except for four amino acids indicated in the footnotes. Amino acid
Aspartic acid Threonine a Setine a Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine b Leucine Tyrosine a Phenylalanine Histidine Lysine Arginine
(mol amino acid/m01
protein)
peak 1
peak 2
peak 3
22.3 16.7 27.9 30.4 16.2 31.2 25.5 19.1 3.6 12.5 28.3 3.8 11.2 6.9 11.9 13.1
21.8 10.3 26.5 24.3 0.0 27.8 19.1 17.4 7.2 10.0 22.6 4.6 7.7 6.7 11.5 8.3
22.2 12.4 24.7 25.4 0.0 29.6 20.4 15.9 6.4 11.6 21.7 5.6 8.5 7.7 12.8 8.5
a Values obtained by extrapolation to zero hydrolysis b Values after 72 h hydrolysis time.
time.
same region of molecular weight of about 32000. These results were similar to those from size exclusion chromatography in the absence of DTT and glycerol. Sodium dodecyl sulfate-polyactylamide gel electrophoresis The electrophoretogram of SDS-PAGE of the purified enzymes showed single bands in the molecular weight range of about 32000 for each (Fig. 5). Amino acid composition The amino acid compositions of the enzymes are shown in Table II. Peaks 2 and 3 had closely similar amino acid composition except for tryptophan and cysteine, which were not determined in this study. Peak 1 had a significant amount of proline, which was not contained in peaks 2 and 3.
6
III
The substrates, 178 pM, were incubated with the enzymes purified in the presence of NADPH under standard incubation conditions described in text. In the experiment in which 3-ketosteroids were used as substrate, the steroids extracted were directly analyzed by gas chromatography (3% QF-1) at 210 o C. Substrates
(Product
(178 PM)
peak 1
peak 2
peak 3
3-Keto-Sb-cholanoic acid 7a-Hydroxy-3-ketoSD-cholanoic acid 3.7-Diketo-SB-cholanoic acid 7a,l2a-Dihydroxy-3keto-5/&cholanoic acid 5/3-Cholestan-3-one 7a-Hydroxy-SFcholestan-3-one 7a,l2a-DihydroxySP-cholestan-3-one 5/?-Pregnane-3,20dione SP-Androstane-3,17dione
374.5
771.5
551.8
135.4
268.8
176.8
126.3
259.0
146.4
0.0
0.0
0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0
0.0
0.0
0.0
0.0
0.0
96.3
171.5
117.9
formed
nmol/min
per mg)
Substrate specificity The substrate specificity for the purified enzymes is shown in Table III. The enzymes could reduce 7a-hydroxy-3-keto-5/%cholanoic and 3,7-diketo-SP-cholanoic acids to the corresponding 3cY-hydroxy bile acids. However, the enzymes could not reduce 7cql2cY_dihydroxy3-keto-S/3-cholanoic acid, Sfi-cholestan-3-one, 7a-hydroxy-5/3-cholestan-3-one and 7ql2a-dihydroxy-58cholestan-3-one to the corresponding 3a-hydroxysteroids. Effect of pH and reversibility of reaction As shown in Fig. 6, the reaction of the enzymes was found to be reversible. The optimum pH of the enzymes
A
LO-
C
B
X 2 .P f j 2
Mr 45,000 Ire 2.0.
Mr 29,000 26,000 s
-
I
Mr 12,500 -W
s I
1.0 ’ 0.5 Fig. 5. Sodium dodecyl
-
sulfate-polyacrylamide
1.0
1.5
Relative mobility gel electrophoresis of the purified enzymes. Migration B, peak 2; lane C, peak 3 (3 pg of each).
was from top to bottom.
Lane A, peak 1; lane
17 TABLE
IV
Effect of pCMB on reduction of 3-keio-.5b-cholanoic acid to liihocholic acid In
this experiment, 5.6 pg of the enzymes was used.
pCMB added
peak 3
peak 2
peak 1
inhibition
enzyme activity (nmol/min)
(I)
enzyme activity (nmol/min)
(I)
enzyme activity (nmol/min)
None 0.05 pM 0.10 pM 0.10 pM
1.70 0.76 0.00
0.0 55.3 100.0
4.32 0.59 0.00
0.0 86.5 100.0
3.09 1.20 0.00
100.0
+D’l’TlmM
1.79
0.0
4.47
0.0
3.12
0.0
inhibition
for the reduction was about 7.4, whereas that for the oxidation was about 8.4. Effects of concentration of pyridine nucleotides and substrate The apparent K, values for pyridine nucleotides
and 3-keto-S/%cholanoic acid of the enzymes were calculated by nonlinear least squares method using Canon Small Computer CX-1 (Canon, Tokyo, Japan). The values of peaks 1, 2 and 3 for NADPH were 116 PM, 102 PM and 120 PM, respectively. Those for 3-keto-5fi-cholanoic acid of 76 PM, 80 PM and 82 PM, respectively. Effect of pCMB
Table IV shows the effect of sulfhydryl compound, pCMB, on the reduction of 3-keto-S/3-cholanoic acid. The enzyme activity was completely inhibited by addition of 100 nM pCMB. However, the inhibition was prevented by addition of 1 mM DTT.
Fig. 6. Effect of pH on reduction of 3-keto-5/3-cholanoic acid (0) and on oxidation of hthochohc acid (0) by a purified enzyme (peak 2). The purified enzyme was incubated at various pH levels with 178 pM 3-keto-5/3-cholanoic acid in 0.1 M sodium-potassium phosphate buffer and 0.8 mM NADPH. The enzyme was also incubated at various pH levels with 178 PM lithocholic acid in 0.1 M phosphate buffer and 0.8 mM NADP. The amount of 3-keto-58-cholanoic acid formed was determined by gas chromatography described in the text. The same results were obtained from the experiments with peaks 1 and 3.
inhibition
(W 0.0 61.2
Discussion
In the previous study [9], we found that the reduction of 3-keto-5/?-cholanoic acid to lithocholic and isolithocholic acids by the enzymes prepared from cytosol of human liver was dependent on pH level and the sort of coenzyme used in the reaction mixture. The enzyme catalyzing the reduction of 3-keto-5/3-cholanoic acid to lithocholic acid, namely 3a-hydroxysteroid dehydrogenase, was active at a pH level of 7.0 or above in the presence of NADPH, and was different from chloral hydrate reductase, a class of aldo-keto reductases in rat. In this study, we have purified and characterized 3a-hydroxysteroid dehydrogenase, as 3-keto bile acid reductase, from human liver cytosol. One major (peak 2) and two minor peaks (peaks 1 and 3) having 3a-hydroxysteroid dehydrogenase activity were found on the present CM-cellulose chromatography. The results were similar to those in cytosol of the liver obtained from different human cadavers (data not shown). The three forms of the enzyme were purified further to homogeneous protein on SDS-PAGE by several chromatographic methods. The differences in the isoelectric points were found on chromatofocusing. Of the enzymes, only peak 1 was found to have proline, an acidic amino acid, which accounts for the lowest isoelectric point of peak 1. The molecular weights of the three enzymes were closely similar and the enzymes were monomeric proteins. The enzymological properties were nearly identical in the three forms of 3a-hydroxysteroid dehydrogenase. In the experiment for substrate specificity, the purified 3a-hydroxysteroid dehydrogenases were active for 7ahydroxy-3-keto-5/3-cholanoic and 3,7-diketo-S/3cholanoic acids but not for 7a,12a-dihydroxy-3-ketoS/3-cholanoic acid. Since the enzyme catalyzing the reduction of 7a,l2a-dihydroxy-3-keto-5/3-cholanoic acid to cholic acid was found to require NADH as the coenzyme [ll], we examined whether the purified en-
18 zymes could reduce 7a,l2a-dihydroxy-3-keto-5/?cholanoic acid in the presence of NADH instead of NADPH. However, the enzymes could not reduce this 3-keto bile acid (data not shown). Therefore, it would appear that the enzymes purified in this study were different from the enzyme catalyzing the reduction of 7ar,l2a-dihydroxy-3-keto-5j%cholanoic acid and had a stereospecific character for some 3-keto steroids. A similar finding had been also shown in 3/Lhydroxysteroid dehydrogenase from human liver. In our previous study [l 11, the 3P-hydroxysteroid dehydrogenase catalyzing the reduction of 7a,l2a-dihydroxy-3-keto-5j%cholanoic acid to isocholic acid was found to be different from that catalyzing the reduction of 3-keto-5@-cholanoic and 7a-hydroxy3-keto-5&cholanoic acids to the corresponding iso-bile acids. In bile acid synthesis, 7a-hydroxy-5P-cholestan-3-one or 7~~,12a-dihydroxy-5/3-cholestan-3-one is thought to be an important intermediate, which is reduced by 3a-hydroxysteroid dehydrogenase in rat [12]. Our preliminary test with crude human liver homogenate also showed that 7a-hydroxy-5P-cholestan-3-one or 7a,12adihydroxy-5/?-cholestan-3-one could be reduced, although the amount of the cholestane-diol or cholestane-trio1 formed was very small. However, the purified enzymes, could not reduce these two compounds of cholestan-3-one. Thus, the enzymes purified may also be different from that catalyzing the reduction of 7a- and 7a,l2cr-dihydroxy-5&cholestan-3-ones, important precursors in bile acid synthesis. The enzymes purified from human liver was found to catalyze a bidirectional oxidoreduction. The reductase activity was most active in the physiological pH range, whereas the dehydrogenase activity had a high, unphysiological pH optimum. The results suggest that the enzymes purified are active as reductase under physiological in vivo conditions. Therefore, aside from a role in bile acid synthesis, the 3a-hydroxysteroid dehydrogenase may be important for the reduction of 3-keto bile acids, which were produced by bacterial 3a-dehydrogenation in the intestine and returned to the liver, to the corresponding 3a-hydroxy bile acids. Since Ton&ins demonstrated the presence of 3a-hydroxysteroid dehydrogenase acting on 3a-hydroxysteroid hormones in the cytosol of rat liver [13], there have been many studies on 3a-hydroxysteroid dehydrogenase from mammalian liver. However, the enzyme preparation used was impure in the earlier studies. Recently, rat liver 3cu-hydroxysteroid dehydrogenase was purified to a single protein on SDS-PAGE [14-181 and was found to have an activity of benzene dihydrodiol dehydrogenase [15] or chloral hydrate reductase [6,16], a class of aldo-keto reductases and was shown further to
be identical to bile acid binder in the liver cell [17]. Thus, it is thought that 3a-hydroxysteroid dehydrogenase in rat liver promotes the oxidation and reduction of diverse xenobiotics [18], or the transport of bile acids in hepatocytes, rather than 3cu-hydroxysteroids. The properties of the 3a-hydroxysteroid dehydrogenase of human liver differed from those of rat liver, except for a few similarities. For instance, the K, values and isoelectric points of the human enzyme were higher than those from rat liver [17,18] and the amino acid composition was different between the enzymes of human and rat liver [15]. Furthermore, the enzyme of rat liver had a broad substrate specificity for many 3a-hydroxysteroids [15.17,18] including 7cr- and 7a,l2a-dihydroxy-5/3-cholestan-3-ones and also is active for xenobiotics such as dihydrodiols [15] and chloral hydrate [6] as described above. These may be due to the species difference. However, the human enzymes required a sulfhydryl group for activity as the enzyme from rat liver does [12].
References 1 Bjbrkhem, I. (1985) in New Comprehensive Biochemistry (Danielsson, H. and Sjovall, J., eds.), Vol. 12. pp. 231-278, Elsevier, Amsterdam. 2 Aries, V. and Hill, M.J. (1970) Biochim. Biophys. Acta 202, 535-543. 3 Eneroth, P., Gordon, B. and Sjbvall, J. (1966) J. Lipid Res. 7, 524-530. 4 Bjorkhem, I.. Angelin, B., Einarsson, K. and Ewerth, S. (1982) J. Lipid Res. 23, 1020-1025. 5 Soloway, R.D., Hofmann, A.F., Thomas, P.J., Schoenfield, L.J. and Klein, P.D. (1973) J. CIin. Invest. 52, 715-724. 6 Ikeda, M., Hayakawa, S., Ezaki, M. and Ohmori, S. (1981) Hoppe-Seyler’s Z. Physiol. Chem. 362, 511-520. 7 Laemmli, U.K. (1970) Nature 227, 680-685. 8 Lowry, O.H., Rosebrough, N.J.. Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 9 Amuro, Y., Yamade, W., Maebo, A., Hada, T. and Higashino, K. (1985) B&him. Biophys. Acta 837, 20-26. 10 Imai, K., Tamura, Z., Mashige, F. and Osuga, T. (1976) J. Chromatogr. X20,181-186. 11 Amuro, Y., Yamade, W., Yamamoto, T., Kudo, K., Fujikura, M., Maebo, A., Hada, T. and Higashino, K. (1986) B&him. Biophys. Acta 879, 362-368. 12 Berstus, 0. (1967) Eur. J. Biochem. 2, 493-502. 13 Tomkins, G.M. (1956) J. Biol. Chem. 218, 437-447. Biophys. Acta 877. 14 Usui. E. and Okuda, K. (1986) B&him. 158-166. 15 Vogel, K., Bentley, P., Platt, K.L. and Oesch, F. (1980) J. Biol. Chem. 255, 9621-9625. 16 Ikeda, M., Hattori, H., Ikeda, N., Hayakawa, S. and Ohmori. S. (1984) Hoppe-Seyler’s Z. Physiol. Chem. 365, 377-391. 17 Stolz, A., Takikawa, H., Sugiyama, Y., Kuhlenkamp, J. and Kaplowitz, N. (1987) J. Clin. Invest. 79, 427-434. 18 Penning, T.M., Mukharji, I., Barrows, S. and Talalay, P. (1984) B&hem. J. 222, 601-611.
Biochimica ef Biophysics Acta, 1046 (1990) 12-18 Elsevier
BBALIP
53464
Purification and properties of 3whydroxysteroid dehydrogenase as a 3-keto bile acid reductase from human liver cytosol Kei Kudo, Yoshiki Amuro,
Toshikazu
Hada and Kazuya Higashino
The Third Department of Internal Medicine, Hyogo College of Medicine, Nishinomi.va, Hyogo (Japan)
(Revised
Key words:
(Received manuscript
30 October 1989) received 1 February
Bile acid; Keto bile acid; Keto bile acid reduction;
1990)
3n-Hydroxysteroid
dehydrogenase;
(Human
liver)
The NADPH-dependent 3a-hydroxysteroid dehydrogenases (peaks 1, 2 and 3) acting on 3-keto-5@holanoic acid separated by carboxymethyl-cellulose chromatography from human liver cytosol were purified to homogeneous protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using Affi-Gel blue, phenyl-Sepharose CL4B, TSKgel G3000 SW chromatography and chromatofocusing. The overall purifications of the enzymes from cytosol were 316-fold (peak l), 232-fold (peak 2) and 345fold (peak 3) and the recoveries of the enzymes were 0.4% (peak l), 7.1% (peak 2) and 3.7% (peak 3). The isoelectric points of the enzymes were found to be 7.34,7.46 and 7.88 by chromatofocusing. The molecular weights of the enzymes were similar and estimated to be about 32000 by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzymological properties were nearly identical among the three forms. The reaction was reversible and the optimum pH of the enzymes for oxidation was about 8.4 and that for reduction was about 7.4. The enzymes could not reduce 7a,12a-dihydroxy-3-keto-5/3-cholanoic acid, 7a-hydroxy5/3-cholestan-3-one and 7u,l2a-dihydroxy-5@-cholestan-3-one to the corresponding 3a-hydroxysteroids, whereas the enzymes could reduce 3,7-disubstituted 3-keto bile acids. Thus, the enzymes purified in this study were found to have a stereospecific character for some 3-ketosteroids. The enzyme activity was inhibited by p-chloromercuribenzoate; however, the inhibition was prevented by addition of dithiothreitol to the reaction mixture, indicating that the enzymes required a sulfhydryl group for activity.
Introduction Cholic and chenodeoxycholic acids are synthesized from cholesterol in human and animal liver and deoxycholic and lithocholic acids are formed by bacterial ;rcY-dehydroxylation in the intestine from their respective precursor bile acids [l]. The bile acids are probably oxidized further to the corresponding keto bile acids by intestinal bacteria [2], as several keto bile acids have been found in the feces [3] and portal venous blood [4]. However, keto bile acids were not found in appreciable amounts in the bile, peripheral venous blood and urine, suggesting that keto bile acids could be reduced to the corresponding hydroxy bile acids during passage through the liver. Thus, a previous in vivo study with man [5] showed that radioactive 3,7,12_triketocholic acid could be reduced to 3a-hydroxy-7,12-diketo-S/3-cholanoic, 301,7a-
dihydroxy-12-keto-5P-cholanoic and 3a,7cq12a-trihydroxy-5/?-cholanoic (cholic) acids in the liver. These results indicate the presence of the enzymes catalyzing the reduction of keto-forms of cholanoic acid to the corresponding hydroxy-forms in human liver. Of the reducing enzymes, 3a-hydroxysteroid dehydrogenase would appear to be an enzyme catalyzing the reduction of 3-keto- to 3cY-hydroxy bile acids. The properties of the 3a-hydroxysteroid dehydrogenase as 3-keto bile acid reductase have, however, not been intensively studied, especially in human liver cytosol, although the enzyme from rat liver was found to be able to reduce the 3-keto group in a number of 3-keto steroids of the C19, C21, C24 and C27 series [6]. In this paper, we describe the purification and properties of the 3a-hydroxysteroid dehydrogenase, as 3-keto bile acid reductase, from human liver cytosol. Materials and Methods Chemicals
Correspondence: Medicine, Hyogo 0005-2760/90/$03,50
Y. Amuro, The Third Department of Internal College of Medicine, Nishinomiya, Hyogo, Japan. 0 1990 Elsevier Science Publishers
B.V. (Biomedical
The sources of the chemicals used in this study were as follows: trifluoroacetic acid anhydride was obtained Division)
13
park2
0.2 -T
0.1 ; J 2
100
Fraction number Fig. 1. CM-cellulose (CM-52) chromatography of the 4670% saturated ammonium were collected and analyzed for enzyme activity (0) and for protein concentration
from Tokyo Kasei (Tokyo, Japan); pyridine nucleotides, 3-keto-S/3-cholanoic acid, S/3-cholestan-3-one, S/3-pregnane-3,20-dione, SP-androstane-3,17-dione from Sigma Chemicals (St. Louis, MO); 7a,l2cr-dihydroxy-3-ketoS/3-cholanoic acid from Calbiochem-Behring (La Jolla, CA); 3,7-diketo-S/3-cholanoic acid, 7a-hydroxy-3-ketoSj&cholanoic acid from Steraloids (Wilton, NH); CMcellulose (CM-52), DEAE-cellulose (DE-52) from Whatman (U.K.); Affi-Gel blue from Bio-Rad (Richmond, CA); phenyl-Sepharose CL4B, Polybuffer 96, PBE 94 from Pharmacia Fine Chemicals (Sweden); Calibration proteins from Boehringer-Mannheim (F.R.G.) and Sigma. 7a-hydroxy-S/3-cholestan-3-one and 7a,l2a-dihydroxy-5fl-cholestan-3-one were kindly supplied by Dr. Kiyohisa Uchida (Shionogi Research Laboratory, Osaka, Japan). S/3-cholestane-3a,7a-diol and SP-cholestane3a,7a,12a-triol were synthesized as follows. The mixture in a total volume of 5 ml contained 2 units of 3ar-hydroxysteroid dehydrogenase (from Pseudomonas testosteroni, Sigma), 400 pg of 7a-hydroxy-SP-cholestan-3-one or 7a,l2cY-dihydroxy-5/I-cholestan-3-one in 100 ~1 of acetone, 0.1 M phosphate buffer (pH 7.4) and 2 mg of NADH was incubated at 37 o C for 20 min. The product formed was extracted with ethyl ether. All other chemicals were obtained from Wako Pure Chemicals (Osaka, Japan). Purification of 3a-hydroxysteroid dehydrogenase Normal human liver was obtained from a human cadaver several hours after death. The liver (200 g wet weight) was homogenized with 3 vol. of 0.25 M sucrose in a Teflon homogenizer. The homogenate was centrifuged at 10000 X g for 15 mm. The cytosol fraction was obtained by centrifuging the 10000 x g supernatant fluid at 105000 X g for 1 h. The precipitate obtained from 40-70% saturated ammonium sulfate fractionation of the cytosol was dissolved in 200 ml of 20 mM
sulfate fraction of the human liver cytosol. Fractions of 10 ml (0). The arrow indicates the start of a linear NaCl gradient.
sodium-potassium-phosphate buffer (pH 6.0) containing 20% (w/v) glycerol and 0.5 mM dithiothreitol (DTT) (buffer A) and dialyzed against the same buffer overnight. The fraction was centrifuged at 10000 X g for 10 min, and the supernatant was applied to a column of CM-cellulose (5.0 x 15.0 cm), previously equilibrated with buffer A. The column was washed with 300 ml of buffer A, and the protein was eluted with a linear gradient of concentration from 0 to 0.2 M NaCl in buffer A. Three peaks containing 3a-hydroxysteroid dehydrogenase activity were found. These three peaks were designated peaks 1, 2 and 3 (Fig. 1). The active fractions of peak 2 or 3 were applied to Affi-Gel blue column (3.2 x 6.0 cm), previously equilibrated with buffer A. The column was washed with the same buffer and the protein was eluted with a linear gradient of concentration from 0 to 2.0 M NaCl in buffer A. The fractions containing enzyme activity were dialyzed against 20 mM phosphate buffer (pH 7.4) containing 20% (w/v) glycerol, 0.5 mM DTT and 1.0 M ammonium sulfate (buffer B). The fractions were applied to phenyl-Sepharose CL-4B column (2.2 x 8.0 cm) equilibrated with buffer B. The column was washed with buffer B and the protein was eluted with a linear gradient of concentration from 1.0 to 0 M ammonium sulfate. The fractions containing enzyme activity were concentrated using Amicon Diaflo membrane and applied to Pharmacia Fast Protein Liquid Chromatography equipped with TSKgel G3000 SW column (7.5 mm X 60 cm), equilibrated with 20 mM phosphate buffer (pH 7.4) containing 20% glycerol and 0.5 mM DT-T. The active fractions of peak 1 from CM-cellulose column were dialyzed against 20 mM Tris-HCl buffer (pH 8.1) containing 20% (w/v) glycerol and 0.5 mM DTT (buffer C), and applied to a column of DEAE-cellulose (5.0 X 15.0 cm) equilibrated with buffer C. The
14 column was washed with 300 ml of buffer C and the protein was eluted with a linear gradient of concentration from 0 to 0.2 M NaCl in buffer C. The fractions of enzyme activity from DEAE-cellulose column were purified further with Affi-Gel blue, phenyl-Sepharose CL-4B, TSKgel G3000 SW chromatography described above and chromatofocusing. Chromatofocusing The enzyme preparations were dialyzed against 0.025 M monoethanolamine-acetate buffer (pH 9.4) and was applied to a chromatofocusing column (1.0 X 20.0 cm) packed with Polybuffer exchanger 94 equilibrated with the same buffer. The protein was eluted with 160 ml of a 1 : 10 dilution of Polybuffer 96-acetate (pH 6.0) at a flow rate 30 ml/h. Amino acid analysis The enzymes purified (70-200 pg/rnl) were dialyzed two times overnight against 2000 ml of distilled water and then hydrolyzed in 6 M HCl at 110 ’ C for 24 and 72 h. Amino acid analysis was performed using a Hitachi L-8500 amino acid analyzer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to
Laemmh [7] using a 15% acrylamide gel. The proteins were located using a commercially available silver stain kit (Wako Pure Chemicals, Osaka, Japan). Determination of protein concentration Protein concentration was determined by the method of Lowry et al [8] or from the absorbance at 280 nm with bovine serum albumin as standard. Incubation procedure and analysis of the product formed Activity of 3ar-hydroxysteroid dehydrogenase catalyzing the reduction of 3-keto-5P-cholanoic acid to lithocholic acid was assayed by the method as described previously [9]. Briefly, the standard incubation mixture in a total volume of 3.0 ml contained 178 PM 3-ketoSfi-cholanoic acid in 100 ~1 of ethanol, an appropriate amount of enzyme preparation, 0.1 M phosphate buffer (pH 7.4), and 0.8 mM NADPH. The mixture was incubated at 37” C for 10 min and the reaction was stopped by adding 1.0 ml of 4 M hydrochloric acid. Then 100 pg of 5P-cholanoic acid was added as an internal standard. The bile acids extracted with diethyl ether from the incubation mixture were converted to the hexafluoroisopropyl-trifluoroacetyl derivatives [lo] and were applied to gas chromatography (Shimadzu GC9A) on a glass column (3.0 mm x 1.6 m) of 3% Silicon DC QF-1 at 220’ C. The amount of lithocholic acid formed
TABLE I Purification of 3a-hydroxysteroid
dehydrogenase Protein (mg)
Peak 1 cytosol (N&)$0., 4&70% CM-52 DE-52 Affi-Gel blue phenyl-Sepharose TSK-3000SW Chromatofocusing Peak 2 cytosol (NH&Q 40-704; CM-52 Affi-Gel blue phenyl-Sepharose TSK-3000SW Peak 3 cytosol (f’+)& 40-7046 CM-52 Affi-gel blue phenyl-Sepharose TSK-3000SW
Total enzyme activity (nmol/min)
3936
3818
1866 266 89.7 25.5 7.8 0.3 0.05
2221 2012 1947 1626 997 57.3 15.3
3936
12534
1866 150 25.9 5.5 1.2
8417 6318 5 286 2197 892.5
3936
6303
1866 156.7 23.8 7.4 0.42
4232 3 183 1815 630.6 231.7
Spec. act. (nmol/min per mg)
Yield (%)
Purification (-fold)
1.0
100.0
1.0
1.2 7.6 21.7 63.8 127.3 184.8 306.0
58.2 52.7 51.0 42.6 26.1 1.5 0.4
1.2 7.8 22.4 65.8 131.1 190.5 315.5
3.2
100.0
1.0
4.5 42.1 203.8 402.1 743.8
67.1 50.4 42.1 17.5 7.1
1.4 13.2 64.1 126.4 232.4
1.6
100.0
1.0
2.3 20.3 76.4 85.8 551.7
67.1 50.5 28.8 10.0 3.7
1.4 12.7 47.8 53.7 344.8
15
2.0
0.4
1.0
0.2
0.5
0
0 ^
0
0.2
'T‘ :
1.0
j &
E, %E.c
L 2.0
50
35
0.1
u
I.
SE ga
-
_ Y
0.5
z
0
0
O
0 0.2
20
2.0
10
0
10
20
30
40
Fraction
50
60
70
0
number
%
2
z
10
20
30
Fraction
40
50
60
70
is v v
0 1.0
0.1
0.5
0 Lt
0
;i 5
number
Fig. 2. Affi-Gel blue chromatography of peak 1 (A) from DEAE column and of peak 2(B) and peak 3(C) from CM column. Fractions of 10 ml were collected and analyzed for enzyme activity (0) and for protein concentration (0). The arrow indicates the start of a linear NaCl gradient.
Fig. 3. Phenyl-Sepharose chromatography of peak 1 (A), peak 2 (B) and peak 3 (C) from Affi-Gel blue column. Fractions of 6.0 ml were collected and analyzed for enzyme activity (0) and for protein concentration (0). The arrow indicates the start of a linear ammonium sulfate gradient.
was determined by measuring the peak areas of lithocholic acid and the internal standard.
Size exclusion chromatography
Results Enzyme purification The overall purifications
When the enzymes from phenyl-Sepharose chromatography were applied to high performance size exclusion chromatography on a column of TSKgel G3000 SW, the peaks of the three enzymes appeared in the
of 3a-hydroxysteroid dehydrogenases are summarized in Table I. The peaks 1, 2 and 3 (Fig. 1) from CM-cellulose chromatography of 40-708 saturated ammonium sulfate fractionation were purified further about 316-, 232- and 345fold as compared to the original cytosol with yields of about 0.4, 7.1 and 3.78, respectively. The specific activities of the purified enzymes were 306 (peak l), 744 (peak 2) and 552 (peak 3) nmol lithocholic acid formed/mm per mg protein. Affi-Gel blue chromatography
Fig. 2 shows the elution profiles of the enzymes on Affi-Gel blue chromatography. Peak 1 eluted with 0.88 M NaCl (Fig. 2A), peak 2 with 0.96 M NaCl (Fig. 2B) and peak 3 with 1.04 M NaCl (Fig. 2C). Phenyl-Sepharose
chromatography
Fig. 3 shows the elution profiles of the enzymes on phenyl-Sepharose Chromatography. Peak 1 eluted with 0.4 M ammonium sulfate (Fig. 3A), peak 2 with 0.51 ammonium sulfate (Fig. 3B) and peak 3 with 0.48 M ammonium sulfate (Fig. 3C). Chromatofocusing
Fig. 4 shows the elution profiles of peaks 1, 2 and 3 on chromatofocusing. Peak 1 eluted at pH 7.34, peak 2 at pH 7.46 and peak 3 at pH 7.88.
Fraction number
Fig. 4. Chromatofocusing of the enzymes. A, 40-70X saturated ammonium sulfate fraction of the cytosol(lO0 mg of protein); B, peak 1 (50 pg of protein) from TSKgel G3000 SW; C, peak 2 (150 cg of protein) from phenyl-Sepharose; D, peak 3 (150 cg of protein) from phenyl-Sepharose. Fractions of 3.0 ml were collected and analyzed for enzyme activity (0).
16 TABLE
II
TABLE
Amino acid composition of 3a-hydroxysteroid
dehydrogenase
Substrate specificity
Values represent the average of values after 24 and 72 h hydrolysis time, except for four amino acids indicated in the footnotes. Amino acid
Aspartic acid Threonine a Setine a Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine b Leucine Tyrosine a Phenylalanine Histidine Lysine Arginine
(mol amino acid/m01
protein)
peak 1
peak 2
peak 3
22.3 16.7 27.9 30.4 16.2 31.2 25.5 19.1 3.6 12.5 28.3 3.8 11.2 6.9 11.9 13.1
21.8 10.3 26.5 24.3 0.0 27.8 19.1 17.4 7.2 10.0 22.6 4.6 7.7 6.7 11.5 8.3
22.2 12.4 24.7 25.4 0.0 29.6 20.4 15.9 6.4 11.6 21.7 5.6 8.5 7.7 12.8 8.5
a Values obtained by extrapolation to zero hydrolysis b Values after 72 h hydrolysis time.
time.
same region of molecular weight of about 32000. These results were similar to those from size exclusion chromatography in the absence of DTT and glycerol. Sodium dodecyl sulfate-polyactylamide gel electrophoresis The electrophoretogram of SDS-PAGE of the purified enzymes showed single bands in the molecular weight range of about 32000 for each (Fig. 5). Amino acid composition The amino acid compositions of the enzymes are shown in Table II. Peaks 2 and 3 had closely similar amino acid composition except for tryptophan and cysteine, which were not determined in this study. Peak 1 had a significant amount of proline, which was not contained in peaks 2 and 3.
6
III
The substrates, 178 pM, were incubated with the enzymes purified in the presence of NADPH under standard incubation conditions described in text. In the experiment in which 3-ketosteroids were used as substrate, the steroids extracted were directly analyzed by gas chromatography (3% QF-1) at 210 o C. Substrates
(Product
(178 PM)
peak 1
peak 2
peak 3
3-Keto-Sb-cholanoic acid 7a-Hydroxy-3-ketoSD-cholanoic acid 3.7-Diketo-SB-cholanoic acid 7a,l2a-Dihydroxy-3keto-5/&cholanoic acid 5/3-Cholestan-3-one 7a-Hydroxy-SFcholestan-3-one 7a,l2a-DihydroxySP-cholestan-3-one 5/?-Pregnane-3,20dione SP-Androstane-3,17dione
374.5
771.5
551.8
135.4
268.8
176.8
126.3
259.0
146.4
0.0
0.0
0.0
0.0 0.0
0.0 0.0
0.0 0.0
0.0
0.0
0.0
0.0
0.0
0.0
96.3
171.5
117.9
formed
nmol/min
per mg)
Substrate specificity The substrate specificity for the purified enzymes is shown in Table III. The enzymes could reduce 7a-hydroxy-3-keto-5/%cholanoic and 3,7-diketo-SP-cholanoic acids to the corresponding 3cY-hydroxy bile acids. However, the enzymes could not reduce 7cql2cY_dihydroxy3-keto-S/3-cholanoic acid, Sfi-cholestan-3-one, 7a-hydroxy-5/3-cholestan-3-one and 7ql2a-dihydroxy-58cholestan-3-one to the corresponding 3a-hydroxysteroids. Effect of pH and reversibility of reaction As shown in Fig. 6, the reaction of the enzymes was found to be reversible. The optimum pH of the enzymes
A
LO-
C
B
X 2 .P f j 2
Mr 45,000 Ire 2.0.
Mr 29,000 26,000 s
-
I
Mr 12,500 -W
s I
1.0 ’ 0.5 Fig. 5. Sodium dodecyl
-
sulfate-polyacrylamide
1.0
1.5
Relative mobility gel electrophoresis of the purified enzymes. Migration B, peak 2; lane C, peak 3 (3 pg of each).
was from top to bottom.
Lane A, peak 1; lane
17 TABLE
IV
Effect of pCMB on reduction of 3-keio-.5b-cholanoic acid to liihocholic acid In
this experiment, 5.6 pg of the enzymes was used.
pCMB added
peak 3
peak 2
peak 1
inhibition
enzyme activity (nmol/min)
(I)
enzyme activity (nmol/min)
(I)
enzyme activity (nmol/min)
None 0.05 pM 0.10 pM 0.10 pM
1.70 0.76 0.00
0.0 55.3 100.0
4.32 0.59 0.00
0.0 86.5 100.0
3.09 1.20 0.00
100.0
+D’l’TlmM
1.79
0.0
4.47
0.0
3.12
0.0
inhibition
for the reduction was about 7.4, whereas that for the oxidation was about 8.4. Effects of concentration of pyridine nucleotides and substrate The apparent K, values for pyridine nucleotides
and 3-keto-S/%cholanoic acid of the enzymes were calculated by nonlinear least squares method using Canon Small Computer CX-1 (Canon, Tokyo, Japan). The values of peaks 1, 2 and 3 for NADPH were 116 PM, 102 PM and 120 PM, respectively. Those for 3-keto-5fi-cholanoic acid of 76 PM, 80 PM and 82 PM, respectively. Effect of pCMB
Table IV shows the effect of sulfhydryl compound, pCMB, on the reduction of 3-keto-S/3-cholanoic acid. The enzyme activity was completely inhibited by addition of 100 nM pCMB. However, the inhibition was prevented by addition of 1 mM DTT.
Fig. 6. Effect of pH on reduction of 3-keto-5/3-cholanoic acid (0) and on oxidation of hthochohc acid (0) by a purified enzyme (peak 2). The purified enzyme was incubated at various pH levels with 178 pM 3-keto-5/3-cholanoic acid in 0.1 M sodium-potassium phosphate buffer and 0.8 mM NADPH. The enzyme was also incubated at various pH levels with 178 PM lithocholic acid in 0.1 M phosphate buffer and 0.8 mM NADP. The amount of 3-keto-58-cholanoic acid formed was determined by gas chromatography described in the text. The same results were obtained from the experiments with peaks 1 and 3.
inhibition
(W 0.0 61.2
Discussion
In the previous study [9], we found that the reduction of 3-keto-5/?-cholanoic acid to lithocholic and isolithocholic acids by the enzymes prepared from cytosol of human liver was dependent on pH level and the sort of coenzyme used in the reaction mixture. The enzyme catalyzing the reduction of 3-keto-5/3-cholanoic acid to lithocholic acid, namely 3a-hydroxysteroid dehydrogenase, was active at a pH level of 7.0 or above in the presence of NADPH, and was different from chloral hydrate reductase, a class of aldo-keto reductases in rat. In this study, we have purified and characterized 3a-hydroxysteroid dehydrogenase, as 3-keto bile acid reductase, from human liver cytosol. One major (peak 2) and two minor peaks (peaks 1 and 3) having 3a-hydroxysteroid dehydrogenase activity were found on the present CM-cellulose chromatography. The results were similar to those in cytosol of the liver obtained from different human cadavers (data not shown). The three forms of the enzyme were purified further to homogeneous protein on SDS-PAGE by several chromatographic methods. The differences in the isoelectric points were found on chromatofocusing. Of the enzymes, only peak 1 was found to have proline, an acidic amino acid, which accounts for the lowest isoelectric point of peak 1. The molecular weights of the three enzymes were closely similar and the enzymes were monomeric proteins. The enzymological properties were nearly identical in the three forms of 3a-hydroxysteroid dehydrogenase. In the experiment for substrate specificity, the purified 3a-hydroxysteroid dehydrogenases were active for 7ahydroxy-3-keto-5/3-cholanoic and 3,7-diketo-S/3cholanoic acids but not for 7a,12a-dihydroxy-3-ketoS/3-cholanoic acid. Since the enzyme catalyzing the reduction of 7a,l2a-dihydroxy-3-keto-5/3-cholanoic acid to cholic acid was found to require NADH as the coenzyme [ll], we examined whether the purified en-
18 zymes could reduce 7a,l2a-dihydroxy-3-keto-5/?cholanoic acid in the presence of NADH instead of NADPH. However, the enzymes could not reduce this 3-keto bile acid (data not shown). Therefore, it would appear that the enzymes purified in this study were different from the enzyme catalyzing the reduction of 7ar,l2a-dihydroxy-3-keto-5j%cholanoic acid and had a stereospecific character for some 3-keto steroids. A similar finding had been also shown in 3/Lhydroxysteroid dehydrogenase from human liver. In our previous study [l 11, the 3P-hydroxysteroid dehydrogenase catalyzing the reduction of 7a,l2a-dihydroxy-3-keto-5j%cholanoic acid to isocholic acid was found to be different from that catalyzing the reduction of 3-keto-5@-cholanoic and 7a-hydroxy3-keto-5&cholanoic acids to the corresponding iso-bile acids. In bile acid synthesis, 7a-hydroxy-5P-cholestan-3-one or 7~~,12a-dihydroxy-5/3-cholestan-3-one is thought to be an important intermediate, which is reduced by 3a-hydroxysteroid dehydrogenase in rat [12]. Our preliminary test with crude human liver homogenate also showed that 7a-hydroxy-5P-cholestan-3-one or 7a,12adihydroxy-5/?-cholestan-3-one could be reduced, although the amount of the cholestane-diol or cholestane-trio1 formed was very small. However, the purified enzymes, could not reduce these two compounds of cholestan-3-one. Thus, the enzymes purified may also be different from that catalyzing the reduction of 7a- and 7a,l2cr-dihydroxy-5&cholestan-3-ones, important precursors in bile acid synthesis. The enzymes purified from human liver was found to catalyze a bidirectional oxidoreduction. The reductase activity was most active in the physiological pH range, whereas the dehydrogenase activity had a high, unphysiological pH optimum. The results suggest that the enzymes purified are active as reductase under physiological in vivo conditions. Therefore, aside from a role in bile acid synthesis, the 3a-hydroxysteroid dehydrogenase may be important for the reduction of 3-keto bile acids, which were produced by bacterial 3a-dehydrogenation in the intestine and returned to the liver, to the corresponding 3a-hydroxy bile acids. Since Ton&ins demonstrated the presence of 3a-hydroxysteroid dehydrogenase acting on 3a-hydroxysteroid hormones in the cytosol of rat liver [13], there have been many studies on 3a-hydroxysteroid dehydrogenase from mammalian liver. However, the enzyme preparation used was impure in the earlier studies. Recently, rat liver 3cu-hydroxysteroid dehydrogenase was purified to a single protein on SDS-PAGE [14-181 and was found to have an activity of benzene dihydrodiol dehydrogenase [15] or chloral hydrate reductase [6,16], a class of aldo-keto reductases and was shown further to
be identical to bile acid binder in the liver cell [17]. Thus, it is thought that 3a-hydroxysteroid dehydrogenase in rat liver promotes the oxidation and reduction of diverse xenobiotics [18], or the transport of bile acids in hepatocytes, rather than 3cu-hydroxysteroids. The properties of the 3a-hydroxysteroid dehydrogenase of human liver differed from those of rat liver, except for a few similarities. For instance, the K, values and isoelectric points of the human enzyme were higher than those from rat liver [17,18] and the amino acid composition was different between the enzymes of human and rat liver [15]. Furthermore, the enzyme of rat liver had a broad substrate specificity for many 3a-hydroxysteroids [15.17,18] including 7cr- and 7a,l2a-dihydroxy-5/3-cholestan-3-ones and also is active for xenobiotics such as dihydrodiols [15] and chloral hydrate [6] as described above. These may be due to the species difference. However, the human enzymes required a sulfhydryl group for activity as the enzyme from rat liver does [12].
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