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Photoaffinity Labeling Identification of a Specific Binding Protein for the Anabolic Steroids Stanozolol and Danazol: An Oligomeric Protein Regulated by Age, Pituitary Hormones, and Ethinyl Estradiol¹

Pharmacology (L.F., R.P.M.), Toxicology (O.P.L), and Physiology (B.N.D.-C.) Sections, Center of Health Sciences and Faculty of Veterinary, University of Las Palmas de Gran Canaria, 35080 Las Palmas de Gran Canaria, Canary Islands, Spain

Address all correspondence and requests for reprints to: Dr. Leandro Fernández, University of Las Palmas de Gran Canaria, Center of Health Sciences, Department of Clinical Sciences, Pharmacology Section, P.O. Box 550, 35080, Las Palmas de Gran Canaria, Canary Islands, Spain. E-mail: leandro{at}cicei.ulpgc.es

	Abstract

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We have demonstrated previously that both rat and human liver microsomes contain a highly specific binding protein for the anabolic steroids stanozolol (ST) and danazol (DA). In this study we solubilized the male rat liver ST-binding protein (STBP) and investigated the following parameters: 1) pharmacological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17-ethinyl estradiol. We found that STBP is an integral protein bound to the endoplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) provided its optimal solubilization without changes in its pharmacological properties, i.e. high specificity for ST and danazol, between natural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STBP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently labeled STBP under nonreducing conditions showed that [³H]ST binds to a 110-kDa protein. The STBP was resolved under reducing conditions into three peptides of 55, 31, and 22 kDa, respectively. STBP increased from immature to adult rats, and it dramatically decreased after hypophysectomy. Unlike the 22-kDa peptide, both the 55- and 31-kDa peptides drastically decreased in both immature and hypophysectomized rats. 17-Ethinyl estradiol administration to immature or hypophysectomized rats induced the 55- and 31-kDa [³H]STBP to a greater extent than the 22-kDa peptide. Thus, STBP appears as an oligomeric protein composed of hormone-regulated peptides. The availability of solubilized STBP and the fact that it can be induced in vivo represent major steps toward the purification and functional significance of this unique steroid-binding protein.

	Introduction

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17-ALKYLATED ANDROGEN derivatives (17-AA) are used in the treatment of several diseases and are abused by athletes and bodybuilders as androgenic-anabolic steroids (1, 2). The liver is an important target for 17-AA, and many of the pathologic conditions associated with prolonged use of these androgenic-anabolic steroids emanate from the adverse effects of these steroids on hepatic gene expression (3).

The mechanism of action of 17-AA is generally thought to be as androgen agonists, acting through their binding to the androgen receptor (AR). It is believed that they may also act as antiglucocorticoids, by competing with endogenous ligands for their binding to the glucocorticoid receptor (3). However, there exists increasing evidence that 17-AA may act through other nonreceptor-mediated mechanisms.

Thus, several differences have been reported between the effects on the liver of two commonly used androgenic steroids, testosterone esters and 17-AA. The 17-AA (but not testosterone or its esters) causes increases in the concentrations of several plasma glycoproteins that are synthesized in the liver, such as some clotting factors and the inhibitor of the first component of the complement (4, 5, 6). These findings suggest that the specific effects exerted by 17-AA on the liver cannot be mediated by the AR. The investigation of their interaction with specific steroid-binding proteins other than the AR could help to clarify the reasons for the different liver responses to these compounds compared with natural androgens. Among the candidates to be considered are the microsomal steroid-binding proteins.

The low affinity glucocorticoid-binding site (LAGS) (7, 8) is a steroid-binding entity present in the microsomal fraction of the male rat liver. It is under endocrine regulation by glucocorticoids, thyroid hormones (9), GH (10), 17-alkylated estrogens (11), 17-AA (8), 17ß-estradiol (12), and vitamin A (13). The LAGS is capable of interacting with glucocorticoids, progestins, and 17-alkylated steroids, with a lower affinity than that exhibited by the respective receptors for these hormones (K_d, 20–300 nM). Interestingly, the LAGS is unable to bind either natural estrogen or natural androgens. Although currently little is known about the possible intracellular role of the LAGS, it is interesting to note that most of the steroids that it is capable of binding have powerful effects on the liver.

Binding analysis as well as determinations of the kinetics of dissociation of the [³H]dexamethasone ([³H]DEX)-LAGS complex in the presence of 17-AA stanozolol (ST) and danazol (DA), revealed that these steroids cause an irreversible inhibition of the [³H]DEX-binding activity through a negative allosteric mechanism (8). We have found experimental evidence of the existence of a microsomal binding site for these 17-AA that differs from both the AR and the LAGS (14). The [³H]ST-binding protein (STBP) is capable of binding [³H]ST with a binding capacity (B_max) of 21 pmol/mg protein and a K_d of 39 nM. This protein is also present in human tissues (Fernández, L., unpublished data), and it can be saturated with the doses of these steroids used for treatment of various diseases and often abused by athletes (2). Thus, this opens the possibility that STBP could play a role in the intracellular action of these 17-AA.

In the present study we have found optimal conditions to solubilize a functional STBP from male rat liver microsomes. Thereby, we have been able to analyze its hydrodynamic properties by molecular sieving and sucrose density gradient ultracentrifugation. Photoaffinity labeling, a powerful tool for the identification and characterization of steroid-specific mediators of biological and pharmacological phenomena (15), has been used to confirm the existence of three [³H]ST-binding peptides. STBP was further characterized by studying the effects of age, hypophysectomy, and estrogen administration on [³H]ST-binding peptides.

	Materials and Methods

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Reagents
[³H]ST (25 Ci/mmol) was synthesized by SibTech, Inc. (Elmsford, NY). [³H]DEX (36–45 Ci/mmol), R1881 (methyltrienolone), and R5020 (promegestone) were purchased from New England Nuclear (Boston, MA). Ready Safe scintillation cocktail was purchased from Beckman Coulter, Inc. (Palo Alto, CA). Unless otherwise indicated, the rest of the products cited in this work were purchased from the Sigma (St. Louis, MO).

Animals
Adult (2- to 3-month-old) and immature (22-day-old) male Sprague Dawley rats were used throughout these experiments. Hypophysectomized (HYPOX) adult rats were purchased from Charles River Laboratories, Inc. (St. Aubin les Elbeuf, France). These rats received a solution containing 2.03 g NaCl, 0.0833 g KCl, 0.0021 g CaCl₂, 0.0167 g MgCl₂, and 50 g glucose/liter drinking water. They were used for experimentation 21–28 days after hypophysectomy. The appropriate pseudooperated control animals were included in each experiment. The steroids were dissolved in corn oil containing 10% (vol/vol) ethanol and injected sc into the neck at the doses and on the days specified. Control animals received the same volume (0.1 ml) of the appropriate vehicle. All animals were killed by decapitation 24 h after the last injection, and their livers were removed quickly and washed in ice-cold saline. All experiments were performed according to institutional guidelines for the care and use of laboratory animals.

Preparation of microsomes
All steps were carried out at 0–4 C. Liver samples were homogenized in a Teflon-glass Potter-Elvehjem homogenizer (B. Braun, Melsungen, Germany) in TMMDSI buffer [50 mM Tris-HCl, 10 mM sodium molybdate, 5 mM magnesium chloride, 2 mM dithiothreitol (DTT), 0.25 mM sucrose, 1 mM phenylmethylsulfonylfluoride, 1 mM EDTA, and 1 µg/ml soybean trypsin inhibitor, pH 7.5]. Homogenates were centrifuged at 17,000 x g for 15 min to separate nuclear and mitochondrial fractions. The supernatant was centrifuged at 105,000 x g for 1 h, and the pellets were resuspended in TMMDSI buffer and recentrifuged again at 105,000 x g. The supernatant was discarded, and the pellet was resuspended in an appropriate volume of TMMDSI buffer to give a protein concentration of 2–3 mg/ml. These microsomal suspensions were used for the binding assays.

Radioligand binding assays
Two hundred micrograms of microsomal suspensions or solubilized proteins were incubated overnight at 0–4 C in duplicate with increasing concentrations (from 5–500 nM final concentration) of [³H]ST. Nonspecific binding was measured in parallel incubations with a 200-fold excess of unlabeled ST. At the end of the incubation period, a suspension of 200 µl dextran T70-coated charcoal (DCC; 0.08–0.8% final concentration) in TMMDSI buffer was added, and the samples were shaken, incubated for 10 min at 0–4 C, and centrifuged. At this DCC concentration, the nonspecific binding represented less than 10% of the total binding; higher concentrations of DCC did not achieve greater effectiveness. Nonspecific binding showed an excellent linearity in the overall range of concentrations assayed. Aliquots of the supernatant were taken for radioactivity counting. In the control experiments, incubations were carried out in the presence of the vehicle alone (2% ethanol final concentration). No effect of ethanol (1–5%) was observed. When the rats received hormonal treatment, the microsomal samples were treated with DCC before the exchange assay to remove steroids. The DCC was eliminated by centrifugation. Control samples received the same DCC treatment.

Subcellular fractionation of rat liver membranes
For examining the subcellular distribution of the [³H]ST-binding activity, livers obtained from two adult male rats were perfused with ice-cold physiological saline, as previously described, and they were homogenized in 4 vol ice-cold TMMDSI buffer. All subsequent steps were performed at 4 C. The homogenate was filtered through a nylon bolting cloth (pore size, 50 µm), and the filtrate was centrifuged twice at 1,000 x g for 10 min each time. The nucleus- and plasma membrane-enriched fractions were obtained from this pellet (16), according to the procedure using a Percoll gradient (17). The 1,000 x g supernatant was fractionated as described for rat liver (18). The 1,000 x g supernatant previously obtained was centrifuged at 10,000 x g for 15 min. The roughly middle part of this pellet was suspended in TMMDSI buffer (total volume, 6 ml), and 1.5 ml of suspension was layered on 10 ml isoosmotic 40% Percoll solution ( = 1.08 g/cm³). This was then centrifuged at 60,000 x g (70.1Ti fixed-angle rotor, Beckman Coulter, Inc., Fullerton, CA) for 30 min. Thus, the three fractions, an upper half portion (peroxisome fraction, < 1.070 g/cm³), a lower half portion (mitochondria-enriched fraction, 1.070 < 1.128 g/cm³), and a portion adjacent to the bottom (lysosome-enriched fraction, = 1.123 g/cm³), were roughly separated. Each fraction was washed three times with TMMDSI by means of centrifugation at 16,000 x g for 20 min. The 10,000 x g supernatant was centrifuged at 105,000 x g for 60 min, and the resultant supernatant was recentrifuged under the same conditions and was used as the cytosolic fraction. The first 105,000 x g pellet was used to obtain smooth and rough endoplasmic reticulum in accordance with the modified Rostchild method (19). Briefly, the pellet was resuspended in TMMDSI containing sufficient sucrose to give a 0.15-M final concentration. A portion (7.5 ml) of this solution was layered onto a 2-ml cushion of TMMDSI-1.3 M sucrose and centrifuged for 100 min at 320,000 x g. After this, endoplasmic reticulum fraction could be recovered as a sediment in the bottom of the tube, whereas the smooth endoplasmic reticulum formed a band at the interface that, once diluted 1:3 with ice-cold TMMDSI, was recovered by means of centrifugation at 105,000 x g for 60 min. All fractions above obtained were flash-frozen in liquid nitrogen and stored at -80 C. [³H]ST-binding activity and the activity of the following marker proteins were determined in all fractions: NADPH cytochrome c reductase (endoplasmic reticulum), succinate dehydrogenase (mitochondria), 5'-nucleotidase (plasma membrane), and lactate dehydrogenase (cytosol) (16).

Solubilization of STBP
Unless otherwise indicated, extraction of proteins was accomplished by incubating microsomes (2–3 mg/ml) in the presence of either 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or 6.6 mM Triton X-100 in TMMDSI-0.1 M KCl buffer for 2 h and 30 min, respectively, by gentle agitation at 4 C. Insoluble material was removed by ultracentrifugation at 105,000 x g for 60 min at 4 C, and the resulting supernatant was filtered through a 0.22-µm pore size Millex-GS sterile filter (Millipore Corp., Bedford, MA). For some experiments peripheral membrane proteins were removed by pretreating microsomes with 0.75 M KCl-TMMDSI for 30 min at 0 C, and the extracted microsomes were collected by ultracentrifugation and solubilized with detergents, as described above.

Phase separation of integral membrane proteins in Triton X-114 solution
Phase separation of integral membrane proteins in Triton X-114 solution was accomplished according to the method described by Bordier (20). Briefly, for the separation of proteins a cushion of 6% sucrose (wt/vol) in TMMDSI-0.03% Triton X-114 (500 µl) was placed at the bottom of a 1.5-ml conical Eppendorf microfuge tube. The clear sample was then overloaded on this sucrose cushion, and the tube was incubated for 3 min at 30 C for condensation. After centrifugation, the detergent phase was found as an oily droplet at the bottom of the tube. The upper aqueous phase was removed from the tube and received 0.3% fresh Triton X-114. After a dissolution of the surfactant at 0 C, the mixture was again overloaded on the sucrose cushion used previously, incubated for 5 min at 30 C for condensation, and centrifuged on the previous detergent phase. At the end of the separation, the aqueous phase was rinsed with 1% Triton X-114 in a separate tube without a sucrose cushion. The detergent phase of this last condensation was discarded. After separation, Triton X-114 and buffer were added, respectively, to the aqueous and detergent phases to obtain equal volumes and approximately the same salt and surfactant content for both samples. Aliquots of the separated phases were analyzed by radioligand binding assays.

Gel filtration chromatography of STBP
About 2–3 mg solubilized proteins were applied to a Sephacryl S-300 column (50 x 1.5 cm; Pharmacia LKB, Uppsala, Sweden). The column was equilibrated with TMMDSI and either 10 mM CHAPS or 6 mM Triton X-100. The flow rate of the column was maintained at 18 ml/h, and 1-ml fractions were collected to measure specific [³H]ST-binding activity by the method described above and to determine protein concentration. Two sets of separate experiments were conducted: 1) solubilized proteins (2–3 mg protein/ml) were analyzed for [³H]ST-binding activity and then submitted to gel filtration; or 2) no solubilized proteins (microsomes) were analyzed for [³H]ST-binding activity, solubilized, and submitted to gel filtration. The gel filtration column was calibrated using proteins of known Stokes radius under conditions identical to those used for STBP. The following marker proteins were employed: carbonic anhydrase (1.60 nm), BSA (3.55 nm), alcohol dehydrogenase (4.75 nm), and apoferritin (6.10 nm). Blue dextran 2000 and K₃Fe(SCN)₆ were used to determine the void volume (V_o) and the total liquid volume (V_t) of the column, respectively. Retention of proteins in the matrix was evaluated by the partition coefficient (K_AV), defined as K_AV = (V_e - V₀)/(V_t - V₀), with V_e being the elution volume of the protein. Calibration curves were constructed by plotting the Stokes radii vs. 1 - K_AV, in accordance with Ackers’ method (21).

Sucrose density gradient ultracentrifugation
The sedimentation coefficient (s_20,w) and the partial specific volume of STBP-detergent complexes were determined by centrifugation through sucrose density gradients in H₂O or D₂0 (22). Linear gradients (4 ml) of 2–20% (wt/vol) sucrose in TMMDSI containing 6.6 mM Triton X-100 or 18–34% (wt/vol) sucrose in TMMDSI containing 10 mM CHAPS were prepared in H₂O or D₂0. The gradients were allowed to cool at 4 C, and 100 µl (200 µg protein) of incubated samples were layered on top of the gradients. On parallel gradients the following standard proteins were sedimented in the same run: cytochrome c (s_20,w = 1.17), carbonic anhydrase (s_20,w = 2.75), BSA (s_20,w = 4.6), and alcohol dehydrogenase (s_20,w = 7.4). Centrifugation was carried out at 4 C for 16 h with a Beckman Coulter, Inc., SW60 rotor at 225,000 x g for sucrose/H₂O gradients or 325,000 x g for sucrose/D₂O gradients. After centrifugation, 25 150-µl fractions were collected from the bottom of the tube. [³H]ST-binding activity, protein concentration, and sucrose density in the different fractions were determined. The migration of standard proteins was measured by determining the protein concentration in each of the fractions. The refractive index of each fraction was checked to assure the linearity of the gradient. The density of each fraction was determined by direct weighing of fixed volumes of sample. The s_20,w and the partial specific volume of STBP-detergent complexes were calculated as described by Clarke (23), assuming that the partial specific volume of the protein component is 0.73 cm³/g, and that the same amount of detergent is bound to the protein in both H₂0 and D₂0. The molecular masses of the STBP-detergent complexes were calculated from their Stokes radius, partial specific volume, and s_20,w values using Svedberg’s equation (23). Data are given as the mean ± SD.

Photoaffinity labeling of STBP
[³H]ST (300 nM) was incubated in the dark with 200 µg microsomal membranes or CHAPS-solubilized proteins in TMMDSI buffer for 2 h at 25 C in the absence or presence of a 200-fold higher concentration of nonlabeled ligand. Samples of 50 µl were removed in triplicate to measure reversible binding before irradiation. The incubation mixture was then transferred to wells of 24-well plates (id, 15 mm) and irradiated for 22 min at 0 C with a 254 nm UV lamp (Stratagene, La Jolla, CA) at a distance of 100 mm above plate. Nonspecific binding and photolabeling were defined with 60 µM ST. Photolabeled samples were trichloroacetic acid (TCA)-precipitated (0.5% TCA) and collected by centrifugation. The pellets were resuspended in 100 µl TMMDSI containing 0.5% (wt/vol) SDS and analyzed by SDS-PAGE or were quick-frozen with liquid nitrogen and stored at -70 C until needed.

SDS-PAGE of photolabeled STBP
SDS-PAGE was carried out on polyacrylamide gel gradients by the method of Laemmli (24). Samples were diluted with an equal volume of sample buffer [60 mM Tris-HCl buffer, pH 6.8, containing 10% (vol/vol) glycerol, 2% (wt/vol) SDS, 100 mM DTT, and 0.001% (wt/vol) bromophenol blue] and then heated at 95 C for 5 min. The gels were fixed and stained with Coomassie Brilliant Blue R-250, followed by destaining in 45% methanol and 7% acetic acid. Each sample lane was cut into 2-mm slices. The slices were digested in 900 µl 30% H₂O₂ at 50 C in capped vials overnight. Twenty milliliters of scintillation fluid (Ready-Safe, Beckman Coulter, Inc.) were then added to each vial before determination of total radioactivity by a Packard scintillation counter (Downers Grove, IL). The yield of photolabeling was calculated by adding the total radioactivity in the [³H]ST-labeled peak (minus baseline background) and expressing it as a percentage of total [³H]ST-binding complexes present in the initial nonphotolabeled sample as previously determined. Gels were calibrated using the following ¹⁴C-labeled proteins: ß-galactosidase (116.2 kDa), phosphorylase b (97.4 kDa), BSA (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21.5 kDa), and lysozyme (14.4 kDa). The molecular masses of radioactive macromolecules were estimated by their mobilities relative to standard proteins of known molecular masses.

Protein measurement
Proteins were measured by using the Bio-Rad Laboratories, Inc., D_c (detergent compatible) method (Bio-Rad Laboratories, Inc., Richmond, CA), using BSA as standard.

Data analysis
Mathematical analysis of the data were performed by using the KINETIC, EBDA, and LIGAND-PC curve-fitting programs (25). Statistical comparisons for each of the binding and kinetic parameters with the control sample were made using paired Student’s test. Significance of differences among groups was tested using the Student-Newman-Keuls test. The data are expressed as the mean ± SE. Statistical significance was reported if P < 0.05 was achieved.

	Results

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The STBP is an integral membrane protein from liver endoplasmic reticulum
Subcellular localization of STBP was studied by determining [³H]ST-binding activity in the cellular fractions of adult male rat liver obtained by differential centrifugation. Enrichment of [³H]ST-binding activity in individual fractions was compared with the distribution of specific marker proteins. As shown in Table 1, binding activity was enriched 4-fold in the endoplasmic reticulum together with NADPH cytochrome c reductase activity. No enrichment was found in mitochondria, nuclei, or plasma membrane. The presence of [³H]ST-binding activity in the plasma membrane and nuclear fractions can be explained by contamination with endoplasmic reticulum.

View larger version (47K): [in this window] [in a new window]   Figure 1. Phase separation of STBP from male rat liver microsomes. Microsomal suspensions (2–3 mg/ml) in the presence of 6 mM Triton X-114 were submitted to phase separation as described in Materials and Methods. Aliquots of the crude microsomes, Triton X-114-solubilized microsomes, and the aqueous and detergent phases were incubated with [3H]ST in the absence (total binding) and presence (nonspecific binding) of a 200-fold excess of ST and analyzed for specific [3H]ST binding as described in Materials and Methods. The results represent the mean ± SEM of three experiments.

To prove that the [³H]ST-binding site present in CHAPS-solubilized proteins represents the previously described STBP (14), its properties were investigated in detailed equilibrium binding studies. Specific binding to CHAPS solubilized STBP was time dependent and reversible. As shown in Fig. 2 (inset), the interaction of [³H]ST with CHAPS-solubilized proteins was rapid, and 4 h of incubation were needed to reach the maximal binding capacity, which remained stable for at least 24 h (data not shown). In these experimental conditions, the association was best fitted following a biexponential model and could be resolved into a fast component (K₊₁₁ = 0.161 ± 0.067 min^-1) and a slow component (K₊₁₂ = 0.0165 ± 0.005 min^-1). Once [³H]ST-binding to solubilized STBP reached equilibrium, the dissociation kinetics of [³H]ST from STBP were carried out by the addition of a 500-fold excess of unlabeled steroid. The incubation was continued at 4 C, and the reaction was stopped by the addition of DCC to the incubation mixture at the times specified (Fig. 2).

View larger version (36K): [in this window] [in a new window]   Figure 2. Association and dissociation kinetics of specific [3H]ST binding to CHAPS-solubilized STBP. The association time course (inset) was started with the addition of [3H]ST (300 nM) to solubilized proteins (•) in the absence (total binding) or in the presence (nonspecific binding) of a 200-fold excess of unlabeled steroid. The incubation was continued at 0 C until it reached equilibrium at 6 h. Then, the kinetics of dissociation were started after the addition of unlabeled ST to give a final concentration of 150 µM. The reactions were stopped by the addition of DCC to the incubation mixture at the times specified, and the supernatant was counted as described in Materials and Methods. Data obtained with crude microsomes () under the same conditions are included as a reference. All data are expressed as a percentage of the maximal capacity, obtained after 6 h of incubation at 0 C. Each point represents the mean ± SEM of three separate experiments.

View larger version (38K): [in this window] [in a new window]   Figure 3. Saturation binding of [3H]ST to CHAPS-solubilized STBP. Solubilized proteins (•; 100 µl) were incubated overnight with 10 increasing concentrations of [3H]ST (final concentration, 5–500 nM) in the absence (total binding) or presence (nonspecific binding) of a 200-fold excess of unlabeled ST. The unbound steroid was removed by adsorption to DCC. Bmax (picomoles per mg protein) and Kd (nM) values were estimated by the nonlinear least square curve-fitting program LIGAND. Data obtained with crude microsomes () under the same conditions are included as a reference. The data, representative of at least six independent experiments performed with similar results, are presented as the Scatchard plots and saturation curves (inset).

Hydrodynamic characterization of the detergent-solubilized STBP
As CHAPS and Triton X-100 proved to be detergents of choice for optimal solubilization of STBP, we next studied the hydrodynamic properties of complexes formed by STBP and these detergents. To determine their molecular sizes, CHAPS or Triton X-100 extracts were chromatographed on a Sephacryl S-300 column. We used Ackers’ method to construct the calibration graphs, getting a good linear relationship between Stokes radius and partition coefficient (Fig. 4, insets). Stokes radii of complexes were determined by interpolation in the calibration plots.

View larger version (33K): [in this window] [in a new window]   Figure 4. Sephacryl S-300 chromatography of detergent-solubilized STBP from rat liver. Before chromatography, detergent-solubilized [3H]ST-binding protein was preincubated with [3H]ST in the absence (total binding) or presence (nonspecific binding) of a 200-fold excess of unlabeled steroid as described in Materials and Methods. Aliquots (2–3 mg protein/ml) of CHAPS (A)- or Triton X-100 (B)-solubilized STBP were loaded on a Sephacryl S-300 column. The [3H]ST-binding activity (•) and protein content () were then measured in each fraction (1 ml). The column was calibrated with proteins of known Stokes radius (inset), and the following protein markers were used: I, carbonic anhydrase; II, BSA; III, alcohol dehydrogenase; and IV, apoferritin. Results are representative of five separate experiments with similar results. V0 and Vt indicate the void volume and total volume of the column, respectively.

View larger version (36K): [in this window] [in a new window]   Figure 5. Hydrodynamic characterization of the detergent-solubilized STBP from rat liver. Before sucrose density gradient ultracentrifugation, detergent-solubilized STBP was preincubated with [3H]ST in the absence (total binding) or presence (nonspecific binding) of a 200-fold excess of unlabeled steroid as described in Materials and Methods. Aliquots (2–3 mg protein/ml) of CHAPS (A)- or Triton X-100 (B)-solubilized STBP were loaded onto 18–34% or 2–20% sucrose gradients for both detergents, respectively, and centrifuged at 350,000 x g for 16 h. Fractions (100 µl) were collected and analyzed for [3H]ST-binding activity (•) and total protein concentration (). The calibration plots were constructed by ultracentrifuging proteins of known s20,w under the same conditions (insets). The s20,w value for STBP was estimated from this standard curve. The following protein markers were used: I, cytochrome c; II, carbonic anhydrase; III, BSA; and IV, alcohol dehydrogenase. Results are representative of five separate experiments with similar results.

The molecular mass of STBP-detergent complexes were then calculated from gel filtration and ultracentrifugation experiments. The results (Table 2) indicate that in the presence of the zwitterionic detergent CHAPS, the complexes had a molecular mass of at least 118 kDa. However, the complex was dissociated in the presence of the nonionic detergent Triton X-100, yielding two complexes in which molecular masses were 108 kDa for the heavier and 16 kDa for the lighter. These results are compatible with an oligomeric structure for the STBP.

Identification of the [³H]ST-binding peptides by photoaffinity labeling
To further characterize the site of STBP, CHAPS-solubilized microsomes were irradiated in the presence of [³H]ST to covalently label the binding proteins. After removing the excess of [³H]ST using DCC, the samples were exposed to UV light for increasing lengths of time. The maximal yield of photolabeling was achieved at 20 min, and further exposure to UV light did not improve photolabeling yield (data not shown). When photolabeled proteins were resuspended in SDS-sample buffer containing 10 mM DTT followed by SDS-PAGE, we detected three labeled peptides of 55 ± 4, 31 ± 3, and 22 ± 3 kDa (Fig. 6A). Interestingly, the [³H]ST-photolabeled peptides resuspended in sample buffer without DTT revealed the specific labeling of a single protein with an apparent molecular mass of 104 ± 6 kDa (Fig. 6B). These data support the hypothesis that [³H]ST binds to an oligomeric protein, composed by three peptides of different sizes, kept bound by disulfur bonds.

View larger version (29K): [in this window] [in a new window]   Figure 6. SDS-PAGE analysis of [3H]ST photoaffinity-labeled peptides. CHAPS-solubilized proteins from adult male rat liver microsomes were photoaffinity labeled with [3H]ST (300 nM) as described in Materials and Methods. The covalent [3H]ST-binding proteins were precipitated with TCA, and the pellets were redissolved in sample loading buffer. Aliquots (1 mg protein) were electrophoretically separated on a 13–20% polyacrylamide gel gradient under reducing conditions (A) or a 6–16% polyacrylamide gel gradient under nonreducing conditions (B). The positions of 14C-labeled protein markers are shown by arrows; molecular masses for the markers are given in kilodaltons. The data are representative of five independent experiments performed with similar results.

View larger version (35K): [in this window] [in a new window]   Figure 7. Age-dependent expression of [3H]ST-binding peptides. CHAPS-solubilized microsomal proteins from liver of immature () and 2- to 3-month-old (•) male rats were photoaffinity labeled with [3H]ST (300 nM) as described in Materials and Methods. The covalent [3H]ST-binding proteins were precipitated with TCA, and the pellets were redissolved in sample loading buffer. Aliquots (1 mg protein) were electrophoretically separated on a 13–20% polyacrylamide gel gradient under reducing conditions. The positions of 14C-labeled protein markers are shown by arrows. Molecular masses for the markers are given in kilodaltons. The data are representative of five independent experiments performed with similar results.

View larger version (35K): [in this window] [in a new window]   Figure 8. Pituitary-dependent expression of [3H]ST-binding peptides. CHAPS-solubilized microsomal proteins from liver of HYPOX () and 2- to 3-month-old (•) male rats were photoaffinity labeled with [3H]ST (300 nM) as described in Materials and Methods. The covalent [3H]ST-binding proteins were precipitated with TCA, and the pellets were redissolved in sample loading buffer. Aliquots (1 mg protein) were electrophoretically separated on a 13–20% polyacrylamide gel gradient under reducing conditions. The positions of 14C-labeled protein markers are shown by arrows. Molecular masses for the markers are given in kilodaltons. The data are representative of five independent experiments performed with similar results

For that purpose, we first selected groups of immature and HYPOX male rats, both with lower [³H]ST-binding capability than adult rats. By analogy with other studies published (11, 28), we treated the rats for 5 days with EE₂ at a daily dose of 0.5 mg/100 g BW. Control animals received the vehicle only. Administration of EE₂ to immature and HYPOX rats increased the level of [³H]ST-binding activity in both animal models (Fig. 9). Time-course experiments, performed by injecting a daily dose of EE₂ (0.5 mg/100 g BW) into immature male rats, revealed that the induction of the [³H]ST-binding activity by EE₂ required at least 2 days to obtain an appropriate response (Fig. 10). The dose-response experiments, performed by injecting different daily doses of EE₂, (from 0.1–1 mg/100 g BW), showed that pharmacological doses of EE₂ equal to or higher than 0.1 mg/100 g BW were capable of inducing [³H]ST-binding sites (P < 0.05) with respect to the level in the control group. Maximal induction was achieved with 0.5 mg/100 g BW.

View larger version (50K): [in this window] [in a new window]   Figure 9. Effect of EE2 administration to immature and HYPOX males rats. Groups of immature or HYPOX male rats were injected sc with a daily dose of EE2 (0.5 mg/100 g BW). Control animals received vehicle only. The animals were killed 24 h after the last injection, and their livers were processed as described in Materials and Methods. Each value represents the mean ± SEM of at least five separate experiments. **, P < 0.01 vs. vehicle.

View larger version (26K): [in this window] [in a new window]   Figure 10. Time course of EE2 administration to immature male rats on STBP. Groups of immature male rats were injected sc with a daily dose of EE2 (0.5 mg/100 g BW; •). The animals were killed 24 h after the last injection, and their livers were processed as described in Materials and Methods. Control animals received vehicle () only. Each value represents the mean ± SEM of at least five separate experiments. *, P < 0.05; **, P < 0.01 (vs. vehicle alone).

View larger version (27K): [in this window] [in a new window]   Figure 11. Effect of EE2 administration to immature and HYPOX male rats on [3H]ST-binding peptides. Groups of immature (A) or HYPOX (B) male rats were injected sc with a daily dose of EE2 (0.5 mg/100 g BW) for 5 days (•). Control animals received the vehicle only (). The animals were killed 24 h after the last injection, and their livers were processed as described in Materials and Methods. CHAPS-solubilized microsomal proteins from control or EE2-treated rats were photoaffinity labeled with [3H]ST (300 nM) as described in Materials and Methods. The covalent [3H]ST-binding proteins were precipitated with TCA, and the pellets were redissolved in sample loading buffer. Aliquots (1 mg protein) were electrophoretically separated on a 13–20% polyacrylamide gel gradient under reducing conditions. The positions of 14C-labeled protein markers are shown by arrows. Molecular masses for the markers are given in kilodaltons. The data are representative of four separate experiments performed with similar results.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

First, the STBP can be solubilized in a functional form. The Scatchard plot of [³H]ST binding to solubilized proteins revealed a higher B_max level in CHAPS extract than in membranes, e.g. 75 vs. 37 pmol/mg protein, respectively. As it occurs with solubilization of other membrane proteins (29), this indicates 2-fold purification of STBP during solubilization with CHAPS. The estimated K_d value of STBP is similar in CHAPS extracts than in membranes, e.g. 41 vs. 37 nM, respectively. It suggests that modification of the phospholipid environment of STBP does not affect protein-binding affinity. This has been observed for some binding proteins, but not all (30).

The Scatchard plot yielded a straight line ranging from 10–500 nM, which indicates that STBP is composed of a unique class of binding sites. However, at low [³H]ST concentrations the points tended to deviate increasingly from the straight line, as occurs with the membrane-bound STBP (14). This suggests the existence of a positive cooperativism in the interaction of [³H]ST with STBP, in which the binding site acquires higher affinity for [³H]ST with its increased ligand occupancy. This hypothetical model is reinforced by the fact that both association and dissociation kinetics fit with a biphasic interaction of [³H]ST with the STBP.

Gel permeation chromatography of CHAPS extract indicated that STBP is solubilized in a single form with a Stokes radius of 5.05 nm. Prelabeling of STBP in CHAPS extracts or even in membranes by [³H]ST before solubilization (data not shown) resulted in an identical elution profile as [³H]ST labeling after elution of proteins. This finding supports the conclusion that occupation of binding sites does not significantly modify the apparent size of solubilized STBP.

A sucrose density gradient ultracentrifugation of CHAPS-solubilized STBP also showed a single [³H]ST-binding component, with a sedimentation coefficient of 5.40 S. In contrast, hydrodynamic studies with Triton X-100 extracts gave two binding components, suggesting that the [³H]ST-binding activity is associated with an oligomeric protein. These experiments did not evidence any high molecular mass forms of solubilized protein that would be eluted in the V_o of the Sephacryl column or would be pelleted during ultracentrifugation. Thus, it may be suggested that protein aggregation does not occur in our experimental conditions despite the fact that these hydrodynamic studies were carried out in the presence of detergent concentrations below their critical micellar concentration (30). As no significant difference is observed between the sedimentation coefficient values measured in H₂O and D₂O, it has been assumed that the partial specific volume of STBP is similar to that of globular proteins used as markers (23). The partial specific volume of CHAPS, e.g. 0.81 ml/g, is different from that of protein markers, e.g. 0.74 ml/g (23), which suggests that STBP binds a small amount of CHAPS. Taking together the values of Stokes radius and sedimentation coefficient, the molecular mass of the STBP has been estimated to be at least 118 kDa. This is quite close to the estimated molecular mass of 104–112 kDa obtained with SDS-PAGE of photoaffinity-labeled STBP (see below).

SDS-PAGE of photoaffinity-labeled STBP in reducing conditions yielded molecular mass estimates for three [³H]ST-binding peptides of 55, 31, and 22 kDa, respectively. It is possible that the 31- and 22-kDa peptides may result from the proteolytic cleavage of the 55-kDa peptide despite the cocktail of proteolysis inhibitors used. However, the fact that they do not appear in SDS-PAGE under nonreducing conditions makes that possibility unlikely. It is also possible that the 31- and 22-kDa components were monomers generated through the reducing cleavage of disulfide bonds present within a dimeric 55-kDa component. In consonance with this hypothesis, both kinetic studies and Scatchard analysis gave evidence for the existence of a complex interaction of [³H]ST with solubilized STBP. Furthermore, the endocrine regulation studies shown below provided evidence of a variable amount of each peptide under different physiological conditions. The reason why the 55-, 31-, and 22-kDa polypeptides are observed in photoaffinity-labeling experiments but not in hydrodynamic experiments may be tentatively ascribed to the fact that STBP can be cleaved, yet the fragments will remain associated as a complex due to strong hydrophobic interactions; these fragments can only be separated after denaturation in SDS. Thus, the 118-kDa [³H]ST-binding protein identified by hydrodynamic studies turns out to be very similar to the [³H]ST-binding polypeptide identified by SDS-PAGE analysis after photoaffinity labeling of [³H]ST to membranes (data not shown) or CHAPS extracts, i.e. 104–112 kDa. These data confirm that [³H]ST-binding sites are associated with an oligomeric protein with a molecular mass of at least 104–112 kDa.

Age-dependent expression of hepatic proteins related to steroid binding and steroid metabolism are well correlated with the function of the pituitary-liver axis (9, 27). Here we have found evidence that both age and pituitary hormones operate through selective regulation of [³H]ST-binding peptides. In fact, the electrophoretic pattern of adult rat liver microsomes is characterized by a major component of [³H]ST-labeled 31-kDa peptide, and two minor components of 55 and 22 kDa, respectively. In contrast, immature and HYPOX male rats show a major component of 22 kDa as well as a drastic reduction of 55- and 31-kDa peptides. Interestingly, liver endoplasmic reticulum and LAGS expression are under multihormonal control, with estrogen, glucocorticoids, thyroid hormones, and GH participating in its regulation (9, 12, 31, 32). If these hormones play a role in the regulation of [³H]ST-binding peptides is under current research in our lab.

In addition to the physiological endocrine regulation, the [³H]ST-binding peptides are induced by the estrogen EE₂. In fact, EE₂ increases the level of STBP in immature and HYPOX male rats, and these effects seem to operate through a selective induction of 55- and 31-kDa peptides. Although the exact mechanism by which EE₂ induces the STBP is unknown, the fact that STBP can be induced in HYPOX male rats clearly indicates that EE₂ does not need the participation of pituitary hormones to regulate the level of STBP. EE₂ induction of STBP occurs in immature and HYPOX male rats, two animal models with very low or undetectable ER levels (31, 32), which supports previous reports indicating that EE₂ may act in the rat liver through an ER-independent mechanism (11).

Several steroid-binding sites associated with oligomeric proteins, distinct from nuclear receptors, have been reported in liver membranes: a DEX-binding site in rat endoplasmic reticulum containing two 45-kDa peptides (33), a progesterone-binding protein containing two polypeptides of 28 and 56 kDa, respectively (34), and a cortisol-binding protein from hepatic plasma membrane with two polypeptides of 52 and 57 kDa, respectively (35). At present, we cannot completely discard the idea that these steroid-binding peptides may be related to STBP, but many findings make that possibility unlikely. [³H]ST interacts with microsomes at binding sites that show high specificity for ST and DA, among several steroidal and nonsteroidal compounds tested. These compounds include the ligands for the two closest candidates: the LAGS (11, 14) and the progesterone-binding sites (36).

Although there are many hypotheses that assign functional roles for microsomal binding sites, such as involvement in posttranscriptional actions of steroid hormones (37), our observations cannot clarify either the biological role or the mechanism of action of the STBP, both of which should be elucidated in future research. However, it is interesting to note that [³H]ST-binding activity is associated with an integral membrane protein that discards the possibility that its binding protein could be a secretory protein. Because [³H]ST-binding activity is insensitive to cytochrome P450 inhibitors or sulfated steroids (14) along with its apparent molecular mass (104–118 kDa) and the fact that the 55-kDa [³H]ST-binding peptide is a minor component of STBP, it is unlikely that STBP is a steroid-metabolizing enzyme, such as the cytochrome P-450 family (38) (48–56 kDa) or flavin monooxygenases (39) (55–59 kDa). In addition, members of a family of GTP-binding proteins recently identified in the endoplasmic reticulum membrane possess similar molecular masses (20–30 kDa) (40) to the 31- and 22-kDa [³H]ST-binding peptides. Some of them are also associated in larger structures (100 kDa) (41). Because [³H]ST binding is insensitive to GTP and GTP analogs (Fernández, L., and O. P. Luzardo, unpublished data), a relationship between the 31- and 22-kDa polypeptides and these GTP-binding proteins is unlikely, but it cannot be completely excluded.

In summary, we have found experimental evidence of the existence of an oligomeric STBP that is under endocrine and pharmacological regulation. The selective regulation of [³H]ST-binding peptides and the successful solubilization of a functional STBP reported here pave the way for large scale solubilization and ultimate purification of this protein.

	Acknowledgments

 
The authors thank Mr. A. Lleó for his help with animal management, and Mr. I. Naranjo Acosta for his help with the preparation of the manuscript.

	Footnotes

 
¹ This work was supported by grants from Consejería de Educación del Gobierno Autónomo de Canarias (98/074 to L.F.), Dirección General de Enseñanza Superior e Investigación Científica (PM98/0033 to L.F.), and Fundación Universitaria de Las Palmas (02/98 and 17/98). Some of these results were reported at the 13th International Congress of Pharmacology (Munich, Germany).

Received March 16, 2000.

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