ARCHIVES
OF
BIOCHEMISTRY
Solubilization
MASAO F,‘ur2tce
AND
174,
BIOPHYSICS
199-208
(1976)
and Partial Purification of Steroid Sulfatase Liver: Characterization of Estrone Sulfatase’ IWAMORI,
Kennedy Shrlver Massachusetts
HUGO
W. MOSER,
AND
for Mental
Center
02154,
and
YASUO
from
Rat
KISHIMOTO
Retardation. at the Walter E. Fernald State School, of Neurology, Massachusetts General Hospital, Boston, Massachusetts 021 I4
Waltham,
Department
Received
September
23, 1975
Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-&phosphate phophatase activities. According to the elution volume on a Sephadex G-ZOO column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol HZM revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well asp-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, e&one sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogerm
The capacity for the enzymatic hydrolysis of steroid sulfates exists in many mammalian tissues and microorganisms (11. Interest in the physiological role of the steroid sulfatase’ in mammalian tissues increased greatly after demonstrating the existence of direct pathways for the conversion of cholesteryl sulfate to hormonal steroid sulfates (2). The steroid sulfatases
may also play ~. j ole in the liberation of active hormonal steroids or their precursors from the conjugated form present in blood or stored in tissues. In spite of their ubiquitous presence and probable physiological importance, the steroid sulfatases have not been well characterized. So far unresolved are their relationship to the enzyme arylsulfatase C as well as the question of whether the hydrolysis of the various steroid sulfates is catalyzed by a single enzyme or by several distinct enzymes. Arylsulfatase C activity is measured with the artificial substrates p-nitrophenyl sulfate or p-acetylphenyl sulfate and, like the steroid sulfatases, occurs in mammalian microsomal tissue fractions in highly insoluble form (3, 4). From kinetic, stability, and other studies
’ This work was supported in part by Grants No. HD-05515, HD-04147, NS-10473, NS-10741, and NS11899 from the National Institutes of Health, U.S. Public Health Service. :’ The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout. 199 Copyright All rights
t; 1976 by Academic of reproduction
in any
Press, Inc. form
reserved.
200
IWAMORI,
MOSER,
with partially purified preparations from human placenta, French and Warren concluded that a different enzymes are responsible for hydrolyzing estrone sulfate, dehydroepiandrosterone sulfate, and p-nitrophenyl sulfate, respectively (5). Zuckerman and Hagerman also demonstrated that the pH optimum and stability of estrone sulfatase and p-nitrophenyl sulfatase in rat kidney are different (6). However, Dolly et al., on the basis of heat inactivation, mixed-substrate, and competitive inhibition experiments, recently concluded that one enzyme is responsible for the hydrolysis of both estrone sulfate and p-nitrophenyl sulfate in rat liver (7). Although arylsulfatase C from human placenta (8) and human brain (9) has been reported to be highly purified, their ability to hydrolyze estrone sulfate was not tested. The paucity of information about steroid sulfatase and arylsulfatase C no doubt is related to the fact that those enzymes are membrane bound and difficult to dissolve in aqueous systems. However, recently, several authors have reported limited successin solubilizing and purifying these enzymes. Burstein first solubilized steroid sulfatase from rat liver microsomes by digestion with heat-treated snake venom (10). The solubilized activity was associated with material of molecular weight 600,000 and tended to form insoluble aggregates. Further purification of this material led to a threefold increase in specific activity. Bleau et al. solubilized cholesterol sulfatase from rat liver microsomes by brief sonication or by treatment with sodium dodecyl sulfate (11). The dehydroepiandrosterone sulfatase activity was either inhibited or not solubilized under these conditions. The molecular weight of the solubilized enzyme was estimated to be approximately 23,000 from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. In this communication we will report the solubilization of estrone sulfatase from rat liver microsomes with the zitterion detergent Miranol H2M as well as a 33-fold purification of this enzyme. We will describe some of the characteristics of the
AND
KISHIMOTO
partially purified enzyme as well as evidence that estrone sulfatase and arylsulfatase C are identical but that they differ from enzyme(s) which hydrolyze the sulfate esters of dehydroepiandrosterone, testosterone, pregnenolone, and cholesterol. EXPERIMENTAL
PROCEDURES
Materials Miranol HZM, an amphoteric surface active agent whose structure is shown below, was a gift from Miranol Chemical Co., Inc., Irvington, NJ. The 1% Miranol H2M solutions in the appropriate buffers were kept at 4°C overnight and centrifuged to remove small amounts of precipitate. Tween 20 (polyoxyethylene sorbitan monolaurate) was provided by Atlas Chemical Industries, Inc.
Other chemicals used in this study were obtained from the following sources: DE-52 (a pre-swollen DEAE-celluloseY from Whatman; Hypatite C (a hydroxylapatite) from Clarkson Chemical Co.; estrone sulfate from Research Plus Laboratories; dehydroepiandroesterone sulfate from Mann Research Laboratories; pregnenolone sulfate from Ikapharm, Ramat-Gan, Israel; testosterone sulfate from Steraloid; and p-nitrophenyl sulfate from Aldrich. Cholesteryl sulfate was prepared in this laboratory (12). l6,7ZH1estrone sulfate, 40 Ci/mmol, [7-:‘Hldehydroepiandrosterone sulfate, 25.1 Ciimmol, 17-:‘Hltestosterone sulfate, 25 Ciimmol, [7-“Hlpregnenolone sulfate, 25 Cilmmol, and ]1,2-:‘H]cholesteryl sulfate, 47 Ci/mmol, were purchased from New England Nuclear. All these radioactive sulfates were at least 98% chromatographically pure at the time of purchase. These substrates were further purified by column chromatography on Dowex AG l-X8 (BioRadl to remove free steroids or p-nitrophenol. Cytochrome c, NADPH, and glucose B-phosphate were obtained from Miles-Seravac, P-L Biochemicals, and Sigma Chemical Co., respectively. A kit of standard proteins of different molecular weights containing cytochrome c, chymotrypsinogen, ovalbumin, serum albumin, aldolase, catalase, and ferritin was obtained from Boehringer.
Methods Assay
of steroid
sulfatase and arylsulfatase.
The
” Abbreviations used: DEAE-, diethyl aminoethyl; Hepes, N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid; SDS, sodium dodecyl sulfate.
CHARACTERIZATION
OF
estrone sulfatase activity was assayed according to Kishimoto and Sostek (13) with slight modification. The assay mixture contained 0.14 M imidazole-HCl buffer, pH 8.0, and 114 PM 16,7-:iHlestrone sulfate containing 100,000 cpm in a final volume of 0.35 ml. The assay was initiated by the addition of an enzyme preparation, incubated for 30 min at 37°C and stopped by the addition of 1 ml of 0.1 M NaPCO,j. The liberated estrone was extracted twice with 4 ml each of diethyl ether, and its radioactivity was measured in a toluene-Triton X-loo-based scintillation mixture. The sulfohydrolase activities for the sulfate esters of dehydroepiandrosterone, pregnenolone, and testosterone were determined similarly with two exceptions: The incubation period was 1 h, and the pH of the added buffer was 6.6. The substrate concentrations used were: 7.32 PM [7-“Hldehydroepiandrosterone sulfate containing 6 x 10’ cpm, 4.44 FLM 17-:‘H]pregnenolone sulfate containing 6 x 10’ cpm; and 3.51 +M 17-“HItestosterone sulfate containing 5 x 10” cpm. Free steroids were dissolved in ethanol and added to the assay mixture when necessary. The final concentration of ethanol was maintained at 5% (v/v); the hydrolytic activity was not inhibited at this concentration. The cholesteryl sulfatase activity was assayed according to Kishimoto and Sostek (13) with modification. The substrate, [1,2-:‘Hlcholesteryl sulfate containing 6 x 10’ cpm, was emulsified in a 0.1% Tween 20 solution in the imidazole buffer, pH 6.6. and added to the assay mixture. The substrate concentration was 3.91 PM. The reaction was stopped by the addition of 2.4 ml of chloroform-methanol (1:2, v/v) containing 30 /*g of carrier cholesterol, and the solution was centrifuged. The supernatant was mixed with 2.4 ml of chloroform and 0.6 ml of water and centrifuged again. The lower layer was evaporated to dryness and fractionated by thin-layer chromatography; hexane-ether (l:l, v/v) was used as the developing solvent. The cholesterol spot was visualized with I, vapor, scraped from the plate, and eluted with chloroform-methanol-water (20:10:1, by volume) in a glass column containing 0.5 g of Unisil. The radioactivity in this eluate was measured. Arylsulfatase C activity was measured as described by Clendenon and Allen 114). The assay mixture contained 10 mM p-nitrophenyl sulfate and 0.2 M imidazole-HCl buffer, pH 8.0, in a final volume of 0.5 ml. Acrylylnmide-gel electrophoresis. Acrylamide-gel electrophoresis under nondenaturing conditions was carried out with a running buffer of 0.1 M phosphate, pH 7.4, which contained 0.1% Miranol H2M. The concentrations of acrylamide and of bisacrylamide were 5.0 and 0.14%, respectively. Fifty micrograms of protein were applied to the gel, and the electrophoresis was run at 4°C for 4 h at a current of 6 mA per tube. To locate the position of the sulfatase, the gels were sliced into 3.mm segments and separately
STEROID
201
SULFATASE
incubated at 37°C for 4 h in 0.3 ml of 0.17 M imidazole-HCl buffer, pH 8.0, containing 0.1% Miranol H2M. Substrate was then added and the sulfatase activity determined. To detect protein bands, another identical gel was stained with 0.2% Coomassie blue, and the destained gels were scanned at 600 nm as described by Dowhan et al. (15). Electrophoresis in the presence of sodium dodecyl sulfate (SDS) on 10% acrylamide gels was performed according to Weber and Osborn (16). The electrophoresis was carried out for 6 h at a current of 8 mA per tube. Analytical procedures. Protein was determined either by the biuret method (17) or by ultraviolet absorption (18) with bovine serum albumin as the standard. The presence of Miranol H2M necessitated small corrections for both procedures. To make the determination as accurate as possible, 500 pg of protein was measured routinely by the former method. Miranol H2M interfered severely with protein determination by the method of Lowry et nl. 119), and this method was used only when the specimen did not contain the detergent. Lipid phosphorus was determined according to Bartlett (20) after wet digestion by H1O, and HCIO,. Cholesterol and cerebroside were measured by gas-liquid chromatography using cholestane and o-mannitol as respective internal standards (21, 22). NADPH-cytochrome c reductase was measured according to the method of DeDuve et al. (23) with a Unicam SP 1800 spectrophotometer. Glucose-Bphosphatase, was assayed by the method of Hers and DeDuve (24) with modification: The incubation medium contained 60 mM maleate buffer, pH 6.5, and 20 mM glucose 6.phosphate in a total volume of 0.25 ml. The incubation was carried out at 37°C for 20 min and stopped by the addition of 1 ml of 3% trichloroacetic acid. After centrifugation, the liberated phosphorus was measured in 0.5 ml of the supernatant from each tube (20). RESULTS
Purification
of Steroid
Sulfatase
Purification of estrone sulfatase was performed in eight steps as described below. Typical results of the procedure are given in Tables I and II, and Fig. 1. All procedures were performed at 4°C unless otherwise mentioned. Step 1: preparation of microsomes. Young rats, 30-40 days old, were sacrificed by decapitation, and the livers were removed immediately. They were homogenized with nine volumes of ice-cold 0.32 M sucrose in a Potter-Elvehjem homogenizer with 20 strokes, and the homogenate was centrifuged at 12,500g for 20 min. The su-
202
IWAMORI, TABLE
PURIFICATION
I
OF ESTRONE FROM
Preparation assayed
1. Microsome 2. Solubilization 3. (NH&SO, precipitate 4. Ethanol precipitate 5. Hydroxyapatite treatment 6. DEAE-cellulose 7. First Sephadex G200 Second Sephadex G-200 8. Sephadex G-100 ” Yields are expressed in each step/total activity
MOSER,
SULFATE
SULFATASE
Liver
RAT
Total protein (mg)
Specific ac- Yield” tivity (5%) (rim01 (mg of protein)-‘h-‘)
3523 3432 525
162 165 740
100 99 69
405
755
54
123
1952
42
56.6 34.3
3405 4635
34 28
26.6
5233
24
25.2
5226
23
as (total activity in microsomes)
recovered x 100.
AND
KISHIMOTO
Step 4: ethanol precipitation. Cold ethanol was added with constant stirring to the solution from step 3 until a 10% ethanol solution was attained; it was then cooled to -20°C. Ethanol, also -2o”C, was gradually added while stirring until a 40% ethanol concentration was reached. The precipitate was immediately removed by centrifugation at 20,OOOg for 20 min at 4°C. The concentration of ethanol in the supernatant was then increased to 50% while maintaining the temperature at -2O”C, and this solution was stirred for 10 min at 4°C. The precipitate was collected by centrifugation at 160,OOOg for 60 min at 4°C and redissolved in buffer B by sonication; small amounts of insoluble material were removed by centrifugation. TABLE COMPARISON COMPOSITION
II
OF ENZYME ACTIVITY BETWEEN MICROSOMES ENZYME
Microsome
pernatant was further centrifuged at 105,OOOg for 60 min. The microsomal pellets were washed in a four-volume (of original liver weight) quantity of 0.02 M imidazole-HCl buffer, pH 7.4 (buffer A), and centrifuged under the same conditions. Step 2: solubilization of microsomes. The freshly centrifuged microsomes were suspended in 0.02 M imidazole-HCl, pH 7.4, containing I% Miranol (buffer B) at a concentration of approximately 3.5 mg of protein/ml and agitated 10 times with a 15s burst of sonic wave produced by a Sonifier cell disruptor Model 1850 (Heat-System-ultrasonics, Inc. 1. Insoluble material was removed by centrifugation at 160,OOOg for 60 min. Step 3: ammonium sulfate precipitation. Solid ammonium sulfate was added to the above clear supernatant at 0°C with constant stirring until a 20% saturation was attained. The pH of the solution was kept at 7.4 by the addition of 5 N NH,OH. After 10 min the precipitate was removed by centrifugation at 20,OOOg for 30 min, and ammonium sulfate was again added to the supernatant to bring the saturation to 30%. The pale brown precipitate was then collected by centrifugation at 160,OOOg for 60 min and redissolved in buffer B.
Sulfatase” p-Nitrophenyl sulfate E&one sulfate Dehydroepiandrosterone sulfate Pregnenolone sulfate Testosterone sulfate Cholesterol sulfate Glucose-6-phosphatase” NADPH-cytochrome c reductase’ Lipid phosphorus’ Cholesterol” Gluco-cerebroside” Phosphatidyl ethanolaminec Phosphatidyl choline
OF
BIOCHEMISTRY
Solubilization
MASAO F,‘ur2tce
AND
174,
BIOPHYSICS
199-208
(1976)
and Partial Purification of Steroid Sulfatase Liver: Characterization of Estrone Sulfatase’ IWAMORI,
Kennedy Shrlver Massachusetts
HUGO
W. MOSER,
AND
for Mental
Center
02154,
and
YASUO
from
Rat
KISHIMOTO
Retardation. at the Walter E. Fernald State School, of Neurology, Massachusetts General Hospital, Boston, Massachusetts 021 I4
Waltham,
Department
Received
September
23, 1975
Steroid sulfatase, a membrane-bound enzyme present in many mammalian tissues, was extracted from rat liver microsomes by treatment with Miranol H2M, a zwitterion detergent, and sonication. It has been purified approximately 33-fold. All steps of the purification, which included salt and solvent fractionation, hydroxylapatite treatment, ion-exchange chromatography, and gel filtration were performed in the presence of Miranol H2M, most of which was removed from the final preparation by gel filtration. The final preparation did not contain any detectable NADPH-cytochrome c reductase or glucose-&phosphate phophatase activities. According to the elution volume on a Sephadex G-ZOO column, steroid sulfatase has a molecular weight of approximately 130,000. Polyacrylamide-gel electrophoresis in the presence of Miranol HZM revealed one major protein band which was enzymatically active. Purified steroid sulfatase hydrolyzes all the sulfate esters of estrone, dehydroepiandrosterone, pregnenolone, testosterone, and cholesterol as well asp-nitrophenyl sulfate, the substrate for arylsulfatase C, during the purification. However, e&one sulfatase and arylsulfatase C activities were enriched more than the others. Analysis of kinetic data and the effects of different buffers and of Miranol H2M also suggested that estrone sulfatase and arylsulfatase C are identical but that they are distinct from the other sulfatases. Competitive inhibition studies suggest that estrone sulfatase also catalyzes the hydrolysis of the sulfate esters of other estrogerm
The capacity for the enzymatic hydrolysis of steroid sulfates exists in many mammalian tissues and microorganisms (11. Interest in the physiological role of the steroid sulfatase’ in mammalian tissues increased greatly after demonstrating the existence of direct pathways for the conversion of cholesteryl sulfate to hormonal steroid sulfates (2). The steroid sulfatases
may also play ~. j ole in the liberation of active hormonal steroids or their precursors from the conjugated form present in blood or stored in tissues. In spite of their ubiquitous presence and probable physiological importance, the steroid sulfatases have not been well characterized. So far unresolved are their relationship to the enzyme arylsulfatase C as well as the question of whether the hydrolysis of the various steroid sulfates is catalyzed by a single enzyme or by several distinct enzymes. Arylsulfatase C activity is measured with the artificial substrates p-nitrophenyl sulfate or p-acetylphenyl sulfate and, like the steroid sulfatases, occurs in mammalian microsomal tissue fractions in highly insoluble form (3, 4). From kinetic, stability, and other studies
’ This work was supported in part by Grants No. HD-05515, HD-04147, NS-10473, NS-10741, and NS11899 from the National Institutes of Health, U.S. Public Health Service. :’ The term steroid sulfatase is used as a general name for the enzyme(s) which hydrolyzes the sulfate ester of a steroid. Simplified terms, such as estrone sulfatase, instead of the more formal terms, such as estrone sulfate sulfohydrolase, have been used throughout. 199 Copyright All rights
t; 1976 by Academic of reproduction
in any
Press, Inc. form
reserved.
200
IWAMORI,
MOSER,
with partially purified preparations from human placenta, French and Warren concluded that a different enzymes are responsible for hydrolyzing estrone sulfate, dehydroepiandrosterone sulfate, and p-nitrophenyl sulfate, respectively (5). Zuckerman and Hagerman also demonstrated that the pH optimum and stability of estrone sulfatase and p-nitrophenyl sulfatase in rat kidney are different (6). However, Dolly et al., on the basis of heat inactivation, mixed-substrate, and competitive inhibition experiments, recently concluded that one enzyme is responsible for the hydrolysis of both estrone sulfate and p-nitrophenyl sulfate in rat liver (7). Although arylsulfatase C from human placenta (8) and human brain (9) has been reported to be highly purified, their ability to hydrolyze estrone sulfate was not tested. The paucity of information about steroid sulfatase and arylsulfatase C no doubt is related to the fact that those enzymes are membrane bound and difficult to dissolve in aqueous systems. However, recently, several authors have reported limited successin solubilizing and purifying these enzymes. Burstein first solubilized steroid sulfatase from rat liver microsomes by digestion with heat-treated snake venom (10). The solubilized activity was associated with material of molecular weight 600,000 and tended to form insoluble aggregates. Further purification of this material led to a threefold increase in specific activity. Bleau et al. solubilized cholesterol sulfatase from rat liver microsomes by brief sonication or by treatment with sodium dodecyl sulfate (11). The dehydroepiandrosterone sulfatase activity was either inhibited or not solubilized under these conditions. The molecular weight of the solubilized enzyme was estimated to be approximately 23,000 from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate. In this communication we will report the solubilization of estrone sulfatase from rat liver microsomes with the zitterion detergent Miranol H2M as well as a 33-fold purification of this enzyme. We will describe some of the characteristics of the
AND
KISHIMOTO
partially purified enzyme as well as evidence that estrone sulfatase and arylsulfatase C are identical but that they differ from enzyme(s) which hydrolyze the sulfate esters of dehydroepiandrosterone, testosterone, pregnenolone, and cholesterol. EXPERIMENTAL
PROCEDURES
Materials Miranol HZM, an amphoteric surface active agent whose structure is shown below, was a gift from Miranol Chemical Co., Inc., Irvington, NJ. The 1% Miranol H2M solutions in the appropriate buffers were kept at 4°C overnight and centrifuged to remove small amounts of precipitate. Tween 20 (polyoxyethylene sorbitan monolaurate) was provided by Atlas Chemical Industries, Inc.
Other chemicals used in this study were obtained from the following sources: DE-52 (a pre-swollen DEAE-celluloseY from Whatman; Hypatite C (a hydroxylapatite) from Clarkson Chemical Co.; estrone sulfate from Research Plus Laboratories; dehydroepiandroesterone sulfate from Mann Research Laboratories; pregnenolone sulfate from Ikapharm, Ramat-Gan, Israel; testosterone sulfate from Steraloid; and p-nitrophenyl sulfate from Aldrich. Cholesteryl sulfate was prepared in this laboratory (12). l6,7ZH1estrone sulfate, 40 Ci/mmol, [7-:‘Hldehydroepiandrosterone sulfate, 25.1 Ciimmol, 17-:‘Hltestosterone sulfate, 25 Ciimmol, [7-“Hlpregnenolone sulfate, 25 Cilmmol, and ]1,2-:‘H]cholesteryl sulfate, 47 Ci/mmol, were purchased from New England Nuclear. All these radioactive sulfates were at least 98% chromatographically pure at the time of purchase. These substrates were further purified by column chromatography on Dowex AG l-X8 (BioRadl to remove free steroids or p-nitrophenol. Cytochrome c, NADPH, and glucose B-phosphate were obtained from Miles-Seravac, P-L Biochemicals, and Sigma Chemical Co., respectively. A kit of standard proteins of different molecular weights containing cytochrome c, chymotrypsinogen, ovalbumin, serum albumin, aldolase, catalase, and ferritin was obtained from Boehringer.
Methods Assay
of steroid
sulfatase and arylsulfatase.
The
” Abbreviations used: DEAE-, diethyl aminoethyl; Hepes, N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid; SDS, sodium dodecyl sulfate.
CHARACTERIZATION
OF
estrone sulfatase activity was assayed according to Kishimoto and Sostek (13) with slight modification. The assay mixture contained 0.14 M imidazole-HCl buffer, pH 8.0, and 114 PM 16,7-:iHlestrone sulfate containing 100,000 cpm in a final volume of 0.35 ml. The assay was initiated by the addition of an enzyme preparation, incubated for 30 min at 37°C and stopped by the addition of 1 ml of 0.1 M NaPCO,j. The liberated estrone was extracted twice with 4 ml each of diethyl ether, and its radioactivity was measured in a toluene-Triton X-loo-based scintillation mixture. The sulfohydrolase activities for the sulfate esters of dehydroepiandrosterone, pregnenolone, and testosterone were determined similarly with two exceptions: The incubation period was 1 h, and the pH of the added buffer was 6.6. The substrate concentrations used were: 7.32 PM [7-“Hldehydroepiandrosterone sulfate containing 6 x 10’ cpm, 4.44 FLM 17-:‘H]pregnenolone sulfate containing 6 x 10’ cpm; and 3.51 +M 17-“HItestosterone sulfate containing 5 x 10” cpm. Free steroids were dissolved in ethanol and added to the assay mixture when necessary. The final concentration of ethanol was maintained at 5% (v/v); the hydrolytic activity was not inhibited at this concentration. The cholesteryl sulfatase activity was assayed according to Kishimoto and Sostek (13) with modification. The substrate, [1,2-:‘Hlcholesteryl sulfate containing 6 x 10’ cpm, was emulsified in a 0.1% Tween 20 solution in the imidazole buffer, pH 6.6. and added to the assay mixture. The substrate concentration was 3.91 PM. The reaction was stopped by the addition of 2.4 ml of chloroform-methanol (1:2, v/v) containing 30 /*g of carrier cholesterol, and the solution was centrifuged. The supernatant was mixed with 2.4 ml of chloroform and 0.6 ml of water and centrifuged again. The lower layer was evaporated to dryness and fractionated by thin-layer chromatography; hexane-ether (l:l, v/v) was used as the developing solvent. The cholesterol spot was visualized with I, vapor, scraped from the plate, and eluted with chloroform-methanol-water (20:10:1, by volume) in a glass column containing 0.5 g of Unisil. The radioactivity in this eluate was measured. Arylsulfatase C activity was measured as described by Clendenon and Allen 114). The assay mixture contained 10 mM p-nitrophenyl sulfate and 0.2 M imidazole-HCl buffer, pH 8.0, in a final volume of 0.5 ml. Acrylylnmide-gel electrophoresis. Acrylamide-gel electrophoresis under nondenaturing conditions was carried out with a running buffer of 0.1 M phosphate, pH 7.4, which contained 0.1% Miranol H2M. The concentrations of acrylamide and of bisacrylamide were 5.0 and 0.14%, respectively. Fifty micrograms of protein were applied to the gel, and the electrophoresis was run at 4°C for 4 h at a current of 6 mA per tube. To locate the position of the sulfatase, the gels were sliced into 3.mm segments and separately
STEROID
201
SULFATASE
incubated at 37°C for 4 h in 0.3 ml of 0.17 M imidazole-HCl buffer, pH 8.0, containing 0.1% Miranol H2M. Substrate was then added and the sulfatase activity determined. To detect protein bands, another identical gel was stained with 0.2% Coomassie blue, and the destained gels were scanned at 600 nm as described by Dowhan et al. (15). Electrophoresis in the presence of sodium dodecyl sulfate (SDS) on 10% acrylamide gels was performed according to Weber and Osborn (16). The electrophoresis was carried out for 6 h at a current of 8 mA per tube. Analytical procedures. Protein was determined either by the biuret method (17) or by ultraviolet absorption (18) with bovine serum albumin as the standard. The presence of Miranol H2M necessitated small corrections for both procedures. To make the determination as accurate as possible, 500 pg of protein was measured routinely by the former method. Miranol H2M interfered severely with protein determination by the method of Lowry et nl. 119), and this method was used only when the specimen did not contain the detergent. Lipid phosphorus was determined according to Bartlett (20) after wet digestion by H1O, and HCIO,. Cholesterol and cerebroside were measured by gas-liquid chromatography using cholestane and o-mannitol as respective internal standards (21, 22). NADPH-cytochrome c reductase was measured according to the method of DeDuve et al. (23) with a Unicam SP 1800 spectrophotometer. Glucose-Bphosphatase, was assayed by the method of Hers and DeDuve (24) with modification: The incubation medium contained 60 mM maleate buffer, pH 6.5, and 20 mM glucose 6.phosphate in a total volume of 0.25 ml. The incubation was carried out at 37°C for 20 min and stopped by the addition of 1 ml of 3% trichloroacetic acid. After centrifugation, the liberated phosphorus was measured in 0.5 ml of the supernatant from each tube (20). RESULTS
Purification
of Steroid
Sulfatase
Purification of estrone sulfatase was performed in eight steps as described below. Typical results of the procedure are given in Tables I and II, and Fig. 1. All procedures were performed at 4°C unless otherwise mentioned. Step 1: preparation of microsomes. Young rats, 30-40 days old, were sacrificed by decapitation, and the livers were removed immediately. They were homogenized with nine volumes of ice-cold 0.32 M sucrose in a Potter-Elvehjem homogenizer with 20 strokes, and the homogenate was centrifuged at 12,500g for 20 min. The su-
202
IWAMORI, TABLE
PURIFICATION
I
OF ESTRONE FROM
Preparation assayed
1. Microsome 2. Solubilization 3. (NH&SO, precipitate 4. Ethanol precipitate 5. Hydroxyapatite treatment 6. DEAE-cellulose 7. First Sephadex G200 Second Sephadex G-200 8. Sephadex G-100 ” Yields are expressed in each step/total activity
MOSER,
SULFATE
SULFATASE
Liver
RAT
Total protein (mg)
Specific ac- Yield” tivity (5%) (rim01 (mg of protein)-‘h-‘)
3523 3432 525
162 165 740
100 99 69
405
755
54
123
1952
42
56.6 34.3
3405 4635
34 28
26.6
5233
24
25.2
5226
23
as (total activity in microsomes)
recovered x 100.
AND
KISHIMOTO
Step 4: ethanol precipitation. Cold ethanol was added with constant stirring to the solution from step 3 until a 10% ethanol solution was attained; it was then cooled to -20°C. Ethanol, also -2o”C, was gradually added while stirring until a 40% ethanol concentration was reached. The precipitate was immediately removed by centrifugation at 20,OOOg for 20 min at 4°C. The concentration of ethanol in the supernatant was then increased to 50% while maintaining the temperature at -2O”C, and this solution was stirred for 10 min at 4°C. The precipitate was collected by centrifugation at 160,OOOg for 60 min at 4°C and redissolved in buffer B by sonication; small amounts of insoluble material were removed by centrifugation. TABLE COMPARISON COMPOSITION
II
OF ENZYME ACTIVITY BETWEEN MICROSOMES ENZYME
Microsome
pernatant was further centrifuged at 105,OOOg for 60 min. The microsomal pellets were washed in a four-volume (of original liver weight) quantity of 0.02 M imidazole-HCl buffer, pH 7.4 (buffer A), and centrifuged under the same conditions. Step 2: solubilization of microsomes. The freshly centrifuged microsomes were suspended in 0.02 M imidazole-HCl, pH 7.4, containing I% Miranol (buffer B) at a concentration of approximately 3.5 mg of protein/ml and agitated 10 times with a 15s burst of sonic wave produced by a Sonifier cell disruptor Model 1850 (Heat-System-ultrasonics, Inc. 1. Insoluble material was removed by centrifugation at 160,OOOg for 60 min. Step 3: ammonium sulfate precipitation. Solid ammonium sulfate was added to the above clear supernatant at 0°C with constant stirring until a 20% saturation was attained. The pH of the solution was kept at 7.4 by the addition of 5 N NH,OH. After 10 min the precipitate was removed by centrifugation at 20,OOOg for 30 min, and ammonium sulfate was again added to the supernatant to bring the saturation to 30%. The pale brown precipitate was then collected by centrifugation at 160,OOOg for 60 min and redissolved in buffer B.
Sulfatase” p-Nitrophenyl sulfate E&one sulfate Dehydroepiandrosterone sulfate Pregnenolone sulfate Testosterone sulfate Cholesterol sulfate Glucose-6-phosphatase” NADPH-cytochrome c reductase’ Lipid phosphorus’ Cholesterol” Gluco-cerebroside” Phosphatidyl ethanolaminec Phosphatidyl choline
