Biochem. J. (1 977) 163, 401-407 Printed in Great Britain
401
Purification and Properties of a New Testosterone 17P-Dehydrogenase (NADP+) from Guinea-Pig Liver By ECHIKO KAGEURA and SATOSHI TOKI Faculty ofPharmaceutical Sciences, Fukuoka University, Nanakuma, Fukuoka 814, Japan (Received 6 October 1976) As a result of studies of guinea-pig liver testosterone 17/J-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17f8-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105000g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17fl-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pHoptima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5a-Androstanes, such as 5a-androstane-3a, 1 7fi-diol, 5a-androstane3fi,17fi-diol and 17f8-hydroxy-5a-androstan-3-one, and 5fi-congeners, such as 51J-androstane-3a,17fi-diol, 5fl-androstane-3f6,17fi-diol and 17f6-hydroxy-5fl-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax. values. In a previous paper we demonstrated that testosterone 17fi-dehydrogenase (NADP+) (EC 1.1.1.64), obtained from guinea-pig liver cytosol, participates
in the reversible oxidation of 3-hydroxyhexobarbital [5-(3'-hydroxy-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid], the microsomal oxygenation product ofhexobarbital (Kageura & Toki, 1971). This enzyme was subsequently purified to homogeneity as 3hydroxyhexobarbital dehydrogenase (Kageura & Toki, 1975). During investigation of 3-hydroxyhexobarbital dehydrogenase as a testosterone 171)dehydrogenase (NADP+) (EC 1.1.1.64), another
17.8-dehydrogenase was isolated from the same source (Kageura & Toki, 1971, 1974). The present paper describes the purification and properties of the new testosterone 17,B-dehydrogenase (referred to as the 'new enzyme' throughout). The role of this enzyme in the metabolism of 17,Bhydroxy steroids has been discussed in comparison with that of classical testosterone 17/1-dehydrogenase (NADP+) (referred to as the 'EC 1.1.1.64 enzyme' throughout).
testosterone
Materials and Methods Materials
Testosterone was supplied from Teikoku Hormone Manufacturing Co., Tokyo, Japan. 1718-Hydroxy5fl-androstan-3-one was purchased from Ikapharm,
Vol. 163
Ramat-Gan, Israel; 5fl-androstane-3fi,17.8-diol, 17ahydroxyandrost-4-en-3-one and androst-5-ene-3fl,17,8-diol were from Steraloids, Pawling, NY, U.S.A.; 17fi-hydroxy-5a-androstan-3-one and other steroids were from Sigma Chemical Co., St. Louis, MO, U.S.A.; the oxidized and reduced forms of NAD and NADP were from Oriental Yeast Co., Tokyo, Japan; cytochrome c (horse heart), myoglobin (sperm-whale), chymotrypsinogen A (ox pancreas), ovalbumin and albumin (bovine serum) were from Mann Research Laboratories, Orangeburg, NY, U.S.A.; alcohol dehydrogenase (horse liver and yeast) was from Boehringer, Mannheim, Germany; Sephadex G-25, Sephadex G-75 (superfine grade) and Sephadex G-100 were from Pharmacia Fine Chemicals AB, Uppsala, Sweden; DEAE-cellulose (DE-32) was from W. and R. Balston, Maidstone, Kent, U.K.; hydroxyapatite was from Seikagaku Kogyo Co., Tokyo, Japan; Ampholine carrier ampholites were from LKB Produkter AB, Bromma, Sweden. Methods
Enzyme assay. Testosterone 17,8-dehydrogenase activity was measured by the change in A340 (1cm light-path) in a Shimadzu (Kyoto, Japan) Double40 spectrophotometer. The reaction mixture was 1.5ml containing 0.1 M-Na2HPO4/0.01 M-NaOH buffer, pH 10.7, 0.15,umol of testosterone (in methanol), 0
E. KAGEURA AND S. TOKI
402
0.5,umol of NADP+ and enzyme solution (0.020.05ml). The reaction was carried out at 25°C. A unit of activity was defined as the amount of enzyme that forms 1 umol of NADPH/min at 25°C. Specific activity is expressed in terms of units/mg of protein. Protein concentration was determined by the method of Lowry et al. (1951) after all the protein was precipitated by the method of Folin & Wu (1919); bovine serum albumin was the standard. Purification of testosterone 17fi-dehydrogenase. The
17fi-dehydrogenase was purified modification of the procedure for 3-hydroxyhexobarbital dehydrogenase (Kageura & Toki, 1975). All steps were carried out at 0-4°C and each enzyme fraction was concentrated in a Diaflo PM-10 filter (Amicon Corp., Lexington, MA, U.S.A.). Standard sodium phosphate buffer (0.005M-, 0.05M- or 0.1 MNaH2PO4/Na2HPO4), pH 8.0, containing 0.05% 2-mercaptoethanol was used. Step 1: Tissue extraction. The livers obtained from Hartley male guinea pigs (about 700g body wt.) were homogenized with 2vol. of 0.1M-NaH2PO4/ Na2HPO4 buffer, pH7.4, and centrifuged successively at 9000g for 20min in a Kubota (Tokyo, Japan) KR-200B refrigerated centrifuge, and at 105000g for 60min in a Martin Christ (Osterode am Hartz, Germany) Omega I ultracentrifuge. Step 2: (NH4)2S04 fractionation. The 105000g supernatant was treated with saturated (NH4)2SO4 solution adjusted to pH7.4 with aq. NH3, and the precipitate between 45 and 80% saturation was collected by centrifugation. The precipitate was dissolved in 47.5 ml of standard 0.1 M-sodium phosphate buffer, and the solution was subdivided into 12ml portions, which were stored at -20°C. Step 3: Sephadex G-100 gel filtration. The (NH4)2SO4 fraction (12ml) was applied to a Sephadex G-100 column (2.5 cmx90cm), and eluted with standard 0.05M-sodium phosphate buffer at a flow rate of 15ml/h. The active fractions were combined and concentrated to about 2ml. Step 4: Sephadex G-75 gel filtration. The Sephadex G-100 fraction was applied to a Sephadex G-75 (superfine) column (2.5cmx 90cm). Separation was carried out with the same buffer as in step 3, at a flow rate of 3ml/h. The active fractions (about 96ml) were combined and concentrated to 2ml. To the concentrated solution, 10ml of standard 0.005Msodium phosphate buffer was added and concentrated again. This process was repeated three times. Step 5: DEAE-cellulose DE-32 column chromatography. The concentrated Sephadex G-75 fraction was applied to the DEAE-cellulose column (1.5cmx 75cm), and eluted with a linear gradient formed from 300ml each of standard 0.005M- and 0.05Msodium phosphate buffer. Fractions (10ml) were collected at a flow rate of 5ml/h. During this step, the enzyme activity catalysing the dehydrogenation new by a
testosterone
of testosterone was separated into two peaks, fraction A (tubes 9-14) and fraction B (tubes 20-30). Fraction A (30ml) was combined and concentrated to 4mi. It was confirmed that fraction B corresponds to classical testosterone 17f8-dehydrogenase (NADP+) (EC 1.1.1.64), which also possesses 3-hydroxyhexobarbital dehydrogenase activity. Step 6: Hydroxyapatite column chromatography. The concentrated fraction A was applied to a hydroxyapatite column (1 .5cm x 13 cm). Protein was eluted from the column with a linear gradient formed from 100ml each of standard 0.05M-sodium phosphate buffer and the same buffer containing 0.5M-NaCl. Fractions (10ml) were collected at a flow rate of 15ml/h. The active fractions (tubes 14-19) were collected and used for the study of the new enzyme. The concentrated fraction B (5.4ml) was also applied to a hydroxyapatite column (1.5cmx 10cm) and eluted with a linear gradient formed from 100ml each of standard 0.05M- and 0.1M-sodium phosphate buffer. The active fractions (tubes 13-18) were collected and used for the study ofthe EC 1.1. 1.64 enzyme. Electrophoresis. Polyacrylamide-gel disc electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel disc electrophoresis were performed by the methods of Davis (1964) and of Weber & Osbom (1969) respectively. Density-gradient isoelectric focusing was performed as despribed by Haglund (1971). In each case, the purified enzyme solution was desalted by passage through a Sephadex G-25 column (1.5cmx40cm) with 0.05M-NaH2PO4/ Na2HPO4 buffer, pH 8.0, or I % glycine solution. All other procedures were carried out as reported previously (Kageura & Toki, 1975). Results
Enzyme purification In the present purification procedure an effort was made to eliminate contaminating protein which persistently accompanied the new enzyme. A summary of the purification procedure of the guinea-pig liver testosterone 17f8-dehydrogenases is given in Table 1. The two enzymes were separated from each other by DEAE-cellulose DE-32 column chromatography. At the final step, the new enzyme was purified 48-fold from the supematant fraction with a yield of 11%, and the EC 1.1.1.64 enzyme was purified 171-fold with a yield of 33%. A quarter of the total testosterone dehydrogenating activity was due to the new enzyme. Purity of enzyme The purified preparation of the new enzyme was examined by polyacrylamide-gel disc electrophoresis. At pH9.4, the enzyme migrated as a single zone 1977
GUINEA-PIG LIVER TESTOSTERONE 17,B-DEHYDROGENASE
403
Table 1. Summary ofpurification of the new testosterone 1 7,8-dehydrogenase and the EC 1.1.1.64 enzyme The enzymes were assayed with testosterone as described under 'Methods'. The supernatant was obtained from 123.5g of guinea-pig liver. Total protein Total activity Purification Yield Fraction Specific activity (units) (units/mg) (mg) 1 7831 0.014 106 100 Supernatant 2 3306 0.027 89 84 (NH4)2SO4 430 13 0.178 77 72 Sephadex G-100 13 427 0.180 77 73 Concentrated 63 143 33 0.439 Sephadex G-75 59 32 60 141 0.428 Concentrated 57 DEAE-cellulose DE-32 12 66 14 0.189 New enzyme 12 78 39 37 1.049 EC 1.1.1.64 37 Concentrated 12 0.186 65 14 New enzyme 11 74 37 EC 1.1.1.64 37 0.992 35 Hydroxyapatite 18 11 48 0.647 New enzyme 11 35 2.316 15 172 EC 1.1.1.64 33
and did not contain any minor contaminating protein. The enzyme also appeared as a single sharp band by polyacrylamide-gel disc electrophoresis in sodium dodecyl sulphate. Substrate specificity
The relative rates of oxidation, apparent Km values and apparent Vmax. values for various types of 17,8-hydroxy steroids are listed in Table 2. In contrast with Sa-androstane derivatives, all of the 5,8-isomers showed higher activity than testosterone even at lower substrate concentration. Among these compounds, 5,B-androstane-3a,17,B-diol exhibited extremely high activity, 12 times that of testosterone at three-fifths of the substrate concentration. On the contrary, 5a-isomers showed lower activity than testosterone. The relative rate for 19-nortestosterone was about 90% of that for testosterone. Several 3-hydroxy steroids having a 17-oxo group were examined; however, the rate of oxidation of these compounds was less than 10% of that of testosterone or zero. Oestradiol-17,Band 17a-hydroxy steroids (e.g. 17a-hydroxyandrost4-ene, 17ahydroxy-5fi-androstan-3-one and oestradiol-17a) did not act as substrates. The apparent Km value and apparent Vmax. value were calculated by the method of Lineweaver & Burk (1934). Most of the Km values obtained from various steroids rangedfrom Ito 1OpM. 17,B-Hydroxy5,6-androstan-3-one showed the lowest Km value and 17fi-hydroxy-5a-androstan-3-one the highest. The Vma. values almost parallel the relative rates of oxidation of the steroids. The KmlVmax. values Vol. 163
indicate that the new enzyme utilizes 5,8-androstane3.,17,B-diol and 17,B-hydroxy-5,6-androstan-3-one most efficiently as substrates; 17.8-hydroxy-5aandrostan-3-one and 19-nortestosterone are not good substrates for the enzyme. Isoelectric density-gradient electrofocusing Fig. 1 shows the results of the isoelectric-focusing study. In this experiment, three substrates, testosterone, 17,8-hydroxy-5,8-androstan-3-one and 17,Bhydroxy-5a-androstan-3-one, all gave the same isoelectric point of pH8.94. The homogeneity of the enzyme preparation was also confirmed, because only the third peak monitored by the A280 contained protein, and this coincided with enzyme activity.
Test with combined substrates To confirm that the new enzyme dehydrogenates different types of 17,6-hydroxysteroids, testosterone, 17,8-hydroxy-5,8-androstan-3-one and 17,B-hydroxy5ac-androstan-3-one were selected as substrates and the mixed-substrate test was performed by the method of Adams (1949). As shown in Table 3,
the total rate of the reaction was less than the sum of the rates of the reactions measured separately. These results indicate that a single enzyme is responsible for the oxidation of the 17,B-hydroxy steroids. Detection and identification of the reaction product
The reaction product
was
extracted with ethyl
acetate and identified by t.l.c. on silica gel HF254
E KAGEURA AND S. TOKI
404
Table 2. Substrate specificity ofnew testosterone 17,B-dehydrogenase Relative activities were measured with 0.1 mm of substrate at 1 5s intervals. Details are described under 'Methods'. The Km and Vmax. values were calculated from double-reciprocal plots. The maximal substrate concentration used for kinetic studies was 0.06 mM (substrates 3 and 7) and 0.1 mm (other steroids listed). The following compounds showed less than 10%I of the relative activity observed with testosterone: 3a-hydroxy-5a-androstan-17-one, 3,f-hydroxy-5aandrostan-17-one, 3ca-hydroxy-5,8-androstan-17-one, 3,i-hydroxy-5/i-androstan-17-one, androst-5-ene-3,8,17,/-diol and 3,B-hydroxyandrost-5-en-17-one. The following compounds showed no detectable activity: 17a-hydroxyandrost4-en-3-one, 17a-hydroxy-5f8-androstan-3-one, oestradiol-17a and oestradiol-17fl. Km Relative activity Vmax. (.umol/min Km Vmax. per mg of protein) Substrate (gM) 36 27 0.75 100 1. Testosterone 2. 3. 4. 5. 6. 7. 8. 9. 10.
93 63 25 30 3 1180 134 330 117
19-Nortestosterone
5a-Androstane-3a,17/?-diol* 5a-Androstane-3f8,17fl-diol* 17fi-Hydroxy-Sa-androstan-3-one
5a-Androstan-17,8-ol*
5fi-Androstane-3a,17,8-diol* 5fi-Androstane-3fl,17fi-diol 17f8-Hydroxy-5,8-androstan-3-one
5f8-Androstan-17piol*
53 23
0.79 0.61
67 37
77
0.42
183
11 29 6.7
8.9 0.96
1 30 2
3.0
* Because of the low solubility of these compounds, the reaction was performed at 37°C. Substrate concentrations used for the reaction mixture were 0.06mM for 5a-androstane-3a,17,8-diol and 5,8-androstane-3a,17,8-diol, and 0.02mM for Sca-androstane-3,8,17/i-diol, 5a-androstan-17I7-ol and 5.i-androstan-17fi-ol. By comparison with the activity of testosterone assayed at 37°C and 25°C, the results obtained for these substrates were converted into values at 25°C.
10
0.1 .~-0.50 5I
401
Purification and Properties of a New Testosterone 17P-Dehydrogenase (NADP+) from Guinea-Pig Liver By ECHIKO KAGEURA and SATOSHI TOKI Faculty ofPharmaceutical Sciences, Fukuoka University, Nanakuma, Fukuoka 814, Japan (Received 6 October 1976) As a result of studies of guinea-pig liver testosterone 17/J-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17f8-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105000g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17fl-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pHoptima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5a-Androstanes, such as 5a-androstane-3a, 1 7fi-diol, 5a-androstane3fi,17fi-diol and 17f8-hydroxy-5a-androstan-3-one, and 5fi-congeners, such as 51J-androstane-3a,17fi-diol, 5fl-androstane-3f6,17fi-diol and 17f6-hydroxy-5fl-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax. values. In a previous paper we demonstrated that testosterone 17fi-dehydrogenase (NADP+) (EC 1.1.1.64), obtained from guinea-pig liver cytosol, participates
in the reversible oxidation of 3-hydroxyhexobarbital [5-(3'-hydroxy-1'-cyclohexen-1'-yl)-1,5-dimethylbarbituric acid], the microsomal oxygenation product ofhexobarbital (Kageura & Toki, 1971). This enzyme was subsequently purified to homogeneity as 3hydroxyhexobarbital dehydrogenase (Kageura & Toki, 1975). During investigation of 3-hydroxyhexobarbital dehydrogenase as a testosterone 171)dehydrogenase (NADP+) (EC 1.1.1.64), another
17.8-dehydrogenase was isolated from the same source (Kageura & Toki, 1971, 1974). The present paper describes the purification and properties of the new testosterone 17,B-dehydrogenase (referred to as the 'new enzyme' throughout). The role of this enzyme in the metabolism of 17,Bhydroxy steroids has been discussed in comparison with that of classical testosterone 17/1-dehydrogenase (NADP+) (referred to as the 'EC 1.1.1.64 enzyme' throughout).
testosterone
Materials and Methods Materials
Testosterone was supplied from Teikoku Hormone Manufacturing Co., Tokyo, Japan. 1718-Hydroxy5fl-androstan-3-one was purchased from Ikapharm,
Vol. 163
Ramat-Gan, Israel; 5fl-androstane-3fi,17.8-diol, 17ahydroxyandrost-4-en-3-one and androst-5-ene-3fl,17,8-diol were from Steraloids, Pawling, NY, U.S.A.; 17fi-hydroxy-5a-androstan-3-one and other steroids were from Sigma Chemical Co., St. Louis, MO, U.S.A.; the oxidized and reduced forms of NAD and NADP were from Oriental Yeast Co., Tokyo, Japan; cytochrome c (horse heart), myoglobin (sperm-whale), chymotrypsinogen A (ox pancreas), ovalbumin and albumin (bovine serum) were from Mann Research Laboratories, Orangeburg, NY, U.S.A.; alcohol dehydrogenase (horse liver and yeast) was from Boehringer, Mannheim, Germany; Sephadex G-25, Sephadex G-75 (superfine grade) and Sephadex G-100 were from Pharmacia Fine Chemicals AB, Uppsala, Sweden; DEAE-cellulose (DE-32) was from W. and R. Balston, Maidstone, Kent, U.K.; hydroxyapatite was from Seikagaku Kogyo Co., Tokyo, Japan; Ampholine carrier ampholites were from LKB Produkter AB, Bromma, Sweden. Methods
Enzyme assay. Testosterone 17,8-dehydrogenase activity was measured by the change in A340 (1cm light-path) in a Shimadzu (Kyoto, Japan) Double40 spectrophotometer. The reaction mixture was 1.5ml containing 0.1 M-Na2HPO4/0.01 M-NaOH buffer, pH 10.7, 0.15,umol of testosterone (in methanol), 0
E. KAGEURA AND S. TOKI
402
0.5,umol of NADP+ and enzyme solution (0.020.05ml). The reaction was carried out at 25°C. A unit of activity was defined as the amount of enzyme that forms 1 umol of NADPH/min at 25°C. Specific activity is expressed in terms of units/mg of protein. Protein concentration was determined by the method of Lowry et al. (1951) after all the protein was precipitated by the method of Folin & Wu (1919); bovine serum albumin was the standard. Purification of testosterone 17fi-dehydrogenase. The
17fi-dehydrogenase was purified modification of the procedure for 3-hydroxyhexobarbital dehydrogenase (Kageura & Toki, 1975). All steps were carried out at 0-4°C and each enzyme fraction was concentrated in a Diaflo PM-10 filter (Amicon Corp., Lexington, MA, U.S.A.). Standard sodium phosphate buffer (0.005M-, 0.05M- or 0.1 MNaH2PO4/Na2HPO4), pH 8.0, containing 0.05% 2-mercaptoethanol was used. Step 1: Tissue extraction. The livers obtained from Hartley male guinea pigs (about 700g body wt.) were homogenized with 2vol. of 0.1M-NaH2PO4/ Na2HPO4 buffer, pH7.4, and centrifuged successively at 9000g for 20min in a Kubota (Tokyo, Japan) KR-200B refrigerated centrifuge, and at 105000g for 60min in a Martin Christ (Osterode am Hartz, Germany) Omega I ultracentrifuge. Step 2: (NH4)2S04 fractionation. The 105000g supernatant was treated with saturated (NH4)2SO4 solution adjusted to pH7.4 with aq. NH3, and the precipitate between 45 and 80% saturation was collected by centrifugation. The precipitate was dissolved in 47.5 ml of standard 0.1 M-sodium phosphate buffer, and the solution was subdivided into 12ml portions, which were stored at -20°C. Step 3: Sephadex G-100 gel filtration. The (NH4)2SO4 fraction (12ml) was applied to a Sephadex G-100 column (2.5 cmx90cm), and eluted with standard 0.05M-sodium phosphate buffer at a flow rate of 15ml/h. The active fractions were combined and concentrated to about 2ml. Step 4: Sephadex G-75 gel filtration. The Sephadex G-100 fraction was applied to a Sephadex G-75 (superfine) column (2.5cmx 90cm). Separation was carried out with the same buffer as in step 3, at a flow rate of 3ml/h. The active fractions (about 96ml) were combined and concentrated to 2ml. To the concentrated solution, 10ml of standard 0.005Msodium phosphate buffer was added and concentrated again. This process was repeated three times. Step 5: DEAE-cellulose DE-32 column chromatography. The concentrated Sephadex G-75 fraction was applied to the DEAE-cellulose column (1.5cmx 75cm), and eluted with a linear gradient formed from 300ml each of standard 0.005M- and 0.05Msodium phosphate buffer. Fractions (10ml) were collected at a flow rate of 5ml/h. During this step, the enzyme activity catalysing the dehydrogenation new by a
testosterone
of testosterone was separated into two peaks, fraction A (tubes 9-14) and fraction B (tubes 20-30). Fraction A (30ml) was combined and concentrated to 4mi. It was confirmed that fraction B corresponds to classical testosterone 17f8-dehydrogenase (NADP+) (EC 1.1.1.64), which also possesses 3-hydroxyhexobarbital dehydrogenase activity. Step 6: Hydroxyapatite column chromatography. The concentrated fraction A was applied to a hydroxyapatite column (1 .5cm x 13 cm). Protein was eluted from the column with a linear gradient formed from 100ml each of standard 0.05M-sodium phosphate buffer and the same buffer containing 0.5M-NaCl. Fractions (10ml) were collected at a flow rate of 15ml/h. The active fractions (tubes 14-19) were collected and used for the study of the new enzyme. The concentrated fraction B (5.4ml) was also applied to a hydroxyapatite column (1.5cmx 10cm) and eluted with a linear gradient formed from 100ml each of standard 0.05M- and 0.1M-sodium phosphate buffer. The active fractions (tubes 13-18) were collected and used for the study ofthe EC 1.1. 1.64 enzyme. Electrophoresis. Polyacrylamide-gel disc electrophoresis and sodium dodecyl sulphate/polyacrylamide-gel disc electrophoresis were performed by the methods of Davis (1964) and of Weber & Osbom (1969) respectively. Density-gradient isoelectric focusing was performed as despribed by Haglund (1971). In each case, the purified enzyme solution was desalted by passage through a Sephadex G-25 column (1.5cmx40cm) with 0.05M-NaH2PO4/ Na2HPO4 buffer, pH 8.0, or I % glycine solution. All other procedures were carried out as reported previously (Kageura & Toki, 1975). Results
Enzyme purification In the present purification procedure an effort was made to eliminate contaminating protein which persistently accompanied the new enzyme. A summary of the purification procedure of the guinea-pig liver testosterone 17f8-dehydrogenases is given in Table 1. The two enzymes were separated from each other by DEAE-cellulose DE-32 column chromatography. At the final step, the new enzyme was purified 48-fold from the supematant fraction with a yield of 11%, and the EC 1.1.1.64 enzyme was purified 171-fold with a yield of 33%. A quarter of the total testosterone dehydrogenating activity was due to the new enzyme. Purity of enzyme The purified preparation of the new enzyme was examined by polyacrylamide-gel disc electrophoresis. At pH9.4, the enzyme migrated as a single zone 1977
GUINEA-PIG LIVER TESTOSTERONE 17,B-DEHYDROGENASE
403
Table 1. Summary ofpurification of the new testosterone 1 7,8-dehydrogenase and the EC 1.1.1.64 enzyme The enzymes were assayed with testosterone as described under 'Methods'. The supernatant was obtained from 123.5g of guinea-pig liver. Total protein Total activity Purification Yield Fraction Specific activity (units) (units/mg) (mg) 1 7831 0.014 106 100 Supernatant 2 3306 0.027 89 84 (NH4)2SO4 430 13 0.178 77 72 Sephadex G-100 13 427 0.180 77 73 Concentrated 63 143 33 0.439 Sephadex G-75 59 32 60 141 0.428 Concentrated 57 DEAE-cellulose DE-32 12 66 14 0.189 New enzyme 12 78 39 37 1.049 EC 1.1.1.64 37 Concentrated 12 0.186 65 14 New enzyme 11 74 37 EC 1.1.1.64 37 0.992 35 Hydroxyapatite 18 11 48 0.647 New enzyme 11 35 2.316 15 172 EC 1.1.1.64 33
and did not contain any minor contaminating protein. The enzyme also appeared as a single sharp band by polyacrylamide-gel disc electrophoresis in sodium dodecyl sulphate. Substrate specificity
The relative rates of oxidation, apparent Km values and apparent Vmax. values for various types of 17,8-hydroxy steroids are listed in Table 2. In contrast with Sa-androstane derivatives, all of the 5,8-isomers showed higher activity than testosterone even at lower substrate concentration. Among these compounds, 5,B-androstane-3a,17,B-diol exhibited extremely high activity, 12 times that of testosterone at three-fifths of the substrate concentration. On the contrary, 5a-isomers showed lower activity than testosterone. The relative rate for 19-nortestosterone was about 90% of that for testosterone. Several 3-hydroxy steroids having a 17-oxo group were examined; however, the rate of oxidation of these compounds was less than 10% of that of testosterone or zero. Oestradiol-17,Band 17a-hydroxy steroids (e.g. 17a-hydroxyandrost4-ene, 17ahydroxy-5fi-androstan-3-one and oestradiol-17a) did not act as substrates. The apparent Km value and apparent Vmax. value were calculated by the method of Lineweaver & Burk (1934). Most of the Km values obtained from various steroids rangedfrom Ito 1OpM. 17,B-Hydroxy5,6-androstan-3-one showed the lowest Km value and 17fi-hydroxy-5a-androstan-3-one the highest. The Vma. values almost parallel the relative rates of oxidation of the steroids. The KmlVmax. values Vol. 163
indicate that the new enzyme utilizes 5,8-androstane3.,17,B-diol and 17,B-hydroxy-5,6-androstan-3-one most efficiently as substrates; 17.8-hydroxy-5aandrostan-3-one and 19-nortestosterone are not good substrates for the enzyme. Isoelectric density-gradient electrofocusing Fig. 1 shows the results of the isoelectric-focusing study. In this experiment, three substrates, testosterone, 17,8-hydroxy-5,8-androstan-3-one and 17,Bhydroxy-5a-androstan-3-one, all gave the same isoelectric point of pH8.94. The homogeneity of the enzyme preparation was also confirmed, because only the third peak monitored by the A280 contained protein, and this coincided with enzyme activity.
Test with combined substrates To confirm that the new enzyme dehydrogenates different types of 17,6-hydroxysteroids, testosterone, 17,8-hydroxy-5,8-androstan-3-one and 17,B-hydroxy5ac-androstan-3-one were selected as substrates and the mixed-substrate test was performed by the method of Adams (1949). As shown in Table 3,
the total rate of the reaction was less than the sum of the rates of the reactions measured separately. These results indicate that a single enzyme is responsible for the oxidation of the 17,B-hydroxy steroids. Detection and identification of the reaction product
The reaction product
was
extracted with ethyl
acetate and identified by t.l.c. on silica gel HF254
E KAGEURA AND S. TOKI
404
Table 2. Substrate specificity ofnew testosterone 17,B-dehydrogenase Relative activities were measured with 0.1 mm of substrate at 1 5s intervals. Details are described under 'Methods'. The Km and Vmax. values were calculated from double-reciprocal plots. The maximal substrate concentration used for kinetic studies was 0.06 mM (substrates 3 and 7) and 0.1 mm (other steroids listed). The following compounds showed less than 10%I of the relative activity observed with testosterone: 3a-hydroxy-5a-androstan-17-one, 3,f-hydroxy-5aandrostan-17-one, 3ca-hydroxy-5,8-androstan-17-one, 3,i-hydroxy-5/i-androstan-17-one, androst-5-ene-3,8,17,/-diol and 3,B-hydroxyandrost-5-en-17-one. The following compounds showed no detectable activity: 17a-hydroxyandrost4-en-3-one, 17a-hydroxy-5f8-androstan-3-one, oestradiol-17a and oestradiol-17fl. Km Relative activity Vmax. (.umol/min Km Vmax. per mg of protein) Substrate (gM) 36 27 0.75 100 1. Testosterone 2. 3. 4. 5. 6. 7. 8. 9. 10.
93 63 25 30 3 1180 134 330 117
19-Nortestosterone
5a-Androstane-3a,17/?-diol* 5a-Androstane-3f8,17fl-diol* 17fi-Hydroxy-Sa-androstan-3-one
5a-Androstan-17,8-ol*
5fi-Androstane-3a,17,8-diol* 5fi-Androstane-3fl,17fi-diol 17f8-Hydroxy-5,8-androstan-3-one
5f8-Androstan-17piol*
53 23
0.79 0.61
67 37
77
0.42
183
11 29 6.7
8.9 0.96
1 30 2
3.0
* Because of the low solubility of these compounds, the reaction was performed at 37°C. Substrate concentrations used for the reaction mixture were 0.06mM for 5a-androstane-3a,17,8-diol and 5,8-androstane-3a,17,8-diol, and 0.02mM for Sca-androstane-3,8,17/i-diol, 5a-androstan-17I7-ol and 5.i-androstan-17fi-ol. By comparison with the activity of testosterone assayed at 37°C and 25°C, the results obtained for these substrates were converted into values at 25°C.
10
0.1 .~-0.50 5I
