Biochimica et Biophysica Acta, 490 (1977) 477-488
© Elsevier/North-Holland Biomedical Press BBA 37575 INTERACTION OF PROGESTERONE RECEPTOR WITH IMMOBILIZED ADENOSINE TRIPHOSPHATE
VIRINDER K. MOUDGIL ~ and DAVID O. TOFT Department of Molecular Medicine, Mayo Clinic, Rochester, Minn. 55901 (U.S.A.)
(Received July 19th, 1976)
SUMMARY Affinity chromatography has been used to study the binding of ATP to cytoplasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCI (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone.receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80 ~ recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-ceUulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors.
INTRODUCTION The progesterone receptor of the avian oviduct has been the subject of many recent investigations [1-3]. Upon entering oviduct cells, progesterone binds the receptor protein and apparently initiates a relocation of this complex to sites on the nuclear chromatin where it may act in regulating genetic expression [1, 4]. The physicochemical properties and steroid binding characteristics of this protein have been studied quite extensively [1-6] and recent efforts have been successful in purifying the receptor to near homogeneity [2, 6]. The hormone-receptor complex isolated from tissue cytosol can bind in vitro to isolated nuclei, nuclear chromatin, and to purified DNA [7-10]. This suggests that the receptor acts, at least in part, throughan interaction with DNA and/or a protein Present address: Department of Biological Sciences, Oakland University, Rochester, Mich. 48063, U.S.A. "
478 fraction of chromatin. However, biochemical activities of the receptor of a more functional nature have not yet been established. Recently, we reported our initial observations on an interaction of the progesterone receptor with ATP°Sepharose [11 ]. The binding appeared to be specific for ATP and was reversible under the conditions employed. Since the selective nature of this interaction suggests a role for ATP in some aspect of receptor function, a more thorough characterization of the binding of receptor to immobilized ATP was undertaken. MATERIALS AND METHODS Analytical grade reagents in glass-distilled water were used throughout. All procedures were carried out at 4 °C. ATP was purchased from Schwarz/Mann, Sepharose 4B and Dextran T-70 from Pharmacia, Uppsala, Sweden; diethylaminoethyl-cellulose (DE 52) from Whatman Biochemicals, Ltd.; acrylamide and N,N'methylene-bis-acrylamide from Bio-Rad; (NH4)zSzO8 from Fisher Scientific; N,N,N,'N'-tetramethylenediamine from Eastman Kodak; and activated charcoal and unlabeled progesterone from Sigma. [1,2-3H2]Progesterone (43.8 Ci/mmol) was purchased from New England Nuclear and was repurified by thin-layer chromatography before use. Buffers. Buffer A: 10 mM Tris. HC1, 12 mM monothioglycerol, 1 mM EDTA, and 2 0 ~ (v/v) glycerol, pH 8.0 (supplemented with KCI as indicated in Figure legends). Buffer B: 10 mM Tris.HC1 and 1 mM EDTA, pH 8.0. Preparation of ATP-Sepharose. ATP was covalently bound to Sepharose-4B as previously described [11] following the method of Lamed et al. [12]. Adipic acid dihydrazide was attached to CNBr-activated Sepharose and ATP was oxidized by periodate and then linked to the Sepharose hydrazide. The ribose of ATP was therefore attached to Sepharose through the 6-carbon bridge of adipic acid dihydrazide. Our preparation contained 13/~mol of ATP per ml Sepharose as determined by phosphate analysis [11]. Column Procedures. DEAE-cellulose was precycled, degassed, and equilibrated in buffer B as recommended by Whatman Biochemicals, Ltd. The resins (DEAE-cellulose or ATP-Sepharose) were equilibrated with buffer A and packed into columns. The flow rate was 1.5-2.0 ml/min (unless otherwise indicated) and either 4.8- or 1.8-ml fractions were collected (indicated in Figure legends). Preparation of Progesterone Receptor. Freshly excised oviducts from White Leghorn hens were obtained from a local produce company. The tissue was rinsed with cold 0.9 ~ saline and homogenized first with a Waring blender and then with a "Tissumizer" (Tekmar model SDT) in two volumes (w/v) of buffer A (modified to contain 10~ glycerol and 0.01 M KC1). The homogenate was centrifuged first at 12 000 × g for I0 min and then at 100 000 x g for 90 rain. Saturated (NH4)2SO4 (pH 7.5) was added to the cytosol to give a final concentration of 35 ~ saturation. The suspension was stirred for 20 min at 4 ° and centrifuged at 12 000 x g for 10 min. The resulting pellets containing the progesterone receptor could be stored at --70 °C for as long as 1 month without significant loss of hormone binding activity. Before use, the pellets were dissolved in buffer A plus 0.01 M KCI using one-tenth the original cytosol volume. This solution was centrifuged to remove undissolved material
479 and incubated with [3H]progesterone (as indicated in figure legends); it served as the starting material for all the experiments. Sucrose Gradient Analysis. Linear 5-20 ~o sucrose gradients (4.5 ml) in buffer A minus glycerol were prepared using a Beckman Gradient Former. The gradients also contained 0.01 or 0.3 M KC1. The receptor samples (0.2 ml) were diluted to lower the glycerol concentration and layered onto the gradients which were then centrifuged at 150 000 x g for 16 h. [14C]Ovalbumin [13] was used as a standard marker to determine sedimentation coefficients [14]. Fractions were collected by piercing the bottom of centrifuge tube and were analyzed for radioactivity. Polyacrylamide Gel Electrophoresis. Stock solutions for acrylamide gels were prepared as follows: stock 1 : 18.5 g Tris, 0.12 ml tetramethylenediamine in 1 M HC1 (24 ml), water to make 100 ml (pH 8.9); stock 2:20 g acrylamide, 0.735 g methylenebis-acrylamide, 20 g glycine, water to make 100 ml; and stock 3:25 mg (NH4)2S2Os, 15 ml glycerol, and 35 ml water. Stock solutions 1 and 2 were stored in the dark at 5 °C while stock 3 was made for each experiment. The gels (5 ~ acrylamide) were prepared at room temperature by mixing the three stock solutions in proportions of 1:1:2, respectively. This mixture was poured into glass cylinders (90 × 6 mm) to a height of 70 mm and polymerization was accomplished in approx. 20 min. The gels were placed in an electrophoresis chamber (Buchler) with upper and lower reservoir buffers of 5 mM Tris.HC1, 38.4 mM glycine, 12 mM thioglycerol and 10~ glycerol, pH 8.9. Pre-electrophoresis was done at 5 °C for 2 h using 4 mA/gel, after which the top reservoir was replaced with fresh buffer. The samples (0.1 ml), prepared in buffer A, but with 15 ~ glycerol, were then layered onto the gels. A 0.01 ~ solution (10 #1) of bromophenol blue in 10 ~ glycerol was also layered on the gel as an electrophoretic marker. Electrophoresis was carried out at 3-5 °C for about 2 h at 2 mA/gel. One set of gels was stained for 35 min with 0.1 ~o Coomassie Blue in 7 ~ acetic acid and then destained for 30 min in a Canalco Gel Destainer and stored in 7 ~ acetic acid. A duplicate set of gels was sliced into 2-mm sections for determining the migration of the [3H]progesterone. receptor complex. The sections were placed in counting vials containing 5 ml of toluene-based scintillation fluid (as described below, but without Triton X-100). Charcoal Adsorption Binding Assay. Prior to affinity chromatography, 0.1-ml portions of the receptor sample were analyzed in duplicate for Jail]progesterone binding [15]. The samples were diluted with buffer A to a volume of 0.5 ml. Since these samples initially contained bound hormone, background determinations were made in replicate tubes by adding excess unlabeled progesterone and incubating for 1 h at 37 °C. Bound hormone.receptor complex was quantitated by adding an equal volume of charcoal suspension (0.5 ~ charcoal, 0.05~ dextran T-70, 10 mM Tris, 1 mM EDTA, pH 8.0) to each tube for 5 min. The suspensions were centrifuged for 10 min at 600 x g to yield clear supernatants which contained the receptor-bound [3H]progesterone. The supernatants were decanted into counting vials and analyzed for radioactivity. When receptor preparations were chromatographed in the absence of progesterone, receptor content in the eluted fractions was determined by the capacity to bind added [aH]-progesterone. Aliquots (0.2 ml) from each fraction were incubated with 12 nM [3H]progesterone for 3 h in an ice bath. After adding 0.5 ml ovalbumin (10 mg/ml), each tube was treated with charcoal as described above. With some
480 preparations, the binding affinity and concentration of receptor sites were determined by Scatchard analysis [16]. Other Methods. Radioactivity was determined by combining aqueous samples with 5 ml of a cocktail consisting of toluene (Baker), Triton X-100 (RPI), and spectrafluor (Amersham-Searle), 100:521:42 (v/v/v). The counting efficiency was 33 ~o in a Beckman LS-250 liquid scintillation counter. The protein concentration in fractions after affinity chromatography was determined by the method of Lowry et al. [17]. The protein was first precipitated with I0 ~ trichloroacetic acid and collected on Millipore filters [18] to remove interfering substances such as thioglycerol. RESULTS
Methods of Affinity Chromatography The [3HI-progesterone.receptor complex can be selectively adsorbed on columns of ATP-Sepharose and eluted stepwise with buffer containing high salt B A
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Fig. 1. Affinity chromatography of [3H]progesterone. receptor complex from hen oviduct on ATPSepharose. Panel Ao The [3H]progesterone.receptor sample (9 ml) was applied to a column (10 ml) with a flow rate of 0.2 ml/min. The column was then washed with buffer A containing 0.15 M KC1 with a flow rate of 0.4 ml/min and 20 4.15-ml fractions were collected. The adsorbed material was eluted with buffer A containing 1 M KCI, collecting 20 1.8-ml fractions. Samples (0.1 ml) were removed from each fraction for determination of radioactivity and protein concentration. The peak fraction in this experiment was purified 38-fold (by comparison of bound cpm/mg protein) and total recovery of the receptor complex was 90 ~ . Panel B. The sample was applied as in Panel A and the column was washed with buffer A containing 0.2 M KCI and 20 4.5-ml fractions were collected. The column was then eluted with a salt gradient (0.2-1 M KC1) and 1.8-ml fractions were collected with a flow rate of 0.5 ml/min. Radioactivity ( © - - © ) and protein (0---0) were determined from 0.1-ml samples from each fraction. The KCI concentration ( © - - © ) was determined by conductivity measurements. The same scale is used for both protein and KC1 concentration.
481 (Fig. 1A). The first peak of radioactivity is composed primarily of free hormone while the peak of [3H]progesterone eluted with 1 M KC1 is essentially all bound to protein [11]. Before chromatography, the receptor sample was routinely assayed for both protein concentration and amount of bound hormone. Upon chromatography, the peak eluted with high salt (1 M KCI) contained 90 ~o of the [3H]progesterone bound in the original sample but less than 5 ~o of the total protein in that sample. This procedure routinely provided a 20-40-fold purification of the receptor and recovery of 80-95 70 of the complex applied to the column. ATP is a necessary constituent of this column chromatography. The selective adsorption of the hormone-receptor complex by the resin was evident only when ATP was linked to the Sepharose. Under the salt conditions normally employed, the progesterone receptor was not retained to any significant extent on columns of either Sepharose 4B or Sepharose with linked adipic acid dihydrazide. The binding of receptor to ATP-Sepharose in relation to ionic strength is indicated by the gradient elution shown in Fig. 1B. A single peak of bound progesterone was eluted at a rather high salt concentration (0.5-0.6 M KC1). This result suggests that the binding of ATP to the receptor is of high affinity and also indicates a homogeneity of the receptor with respect to its association with ATP. The integrity of the [3H]progesterone. receptor complex following affinity chromatography was tested by sucrose gradient centrifugation. The complex sedimented as a 4 S unit (Fig. 2B) as did the original receptor complex before chromatography .... :
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A
Low salt - High salt
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Fig. 2. Sucrose gradient centrifugation of [3H]progesterone.receptor complex before and after affinity chromatography. Samples (0.2 ml) were layered onto 5-20~ sucrose gradients containing 0.01 M (O---O) or 0.3 M (0---0) KC1. The gradients were centrifuged for 16h at 150000 × g. [14C]Ovalbumin was the sedimentation standard (3.7 S). (A) The sample before purification consisted of ammonium sulfate-fractionated receptor incubated for 3 h with laH]progesterone (6 nM). (B) The receptor sample was from the peak fraction eluted from an ATP-Sepharose column with buffer A containing 1 M KC1. All samples were diluted with three volumes of buffer B to lower the density before layering onto gradients.
482 (Fig. 2A). While the major component had a sedimentation coefficient of approx. 4 S under both high (0.3 M KC1) and low (0.01 M KCI) salt conditions, the original sample obtained by ammonium sulfate fractionation exhibited a tendency to aggregate at the lower ionic strength. This aggregation did not occur following purification on ATP-Sepharose. Progesterone receptor in the cytosol fraction from chick oviducts has been reported to sediment as a 6-8 S complex under low ionic conditions [4, 15] which is also the case for the receptor from the mature hen oviduct (data not shown). However, during the initial steps of purification, the receptor appeared to lose most of its ability to aggregate to the 6-8 S form. A reduction in receptor aggregation following purification of progesterone receptor from chick oviduct has also been demonstrated [6, 19]. The semipurified receptor was also analyzed by polyacrylamide gel electrophoresis (Fig. 3). The gels contained glycerol (20 ~) which appeared to stabilize the receptor. Most of the progesterone.receptor complex which entered the gel migrated as a single, well-defined band. However, the region between this band and the origin also contained bound hormone of a rather heterogeneous mobility. This pattern is quite similar to that reported by Miller et al. [20] for progesterone receptor from the chick oviduct. ATP-Sepharose chromatography did not detectably alter the mobility of the receptor complex, but it did remove many of the protein bands observed in the electrophoresis of the ammonium sulfate fraction.
12
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Since the progesterone receptor binds reversibly to ATP-Sepharose, the use of free ATP as an effective eluent was also studied. When an ATP gradient (0-0.1 M ATP) was used for elution, the receptor was removed uniformly in a single peak
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Fig. 4. ATP gradient elution of progesterone receptor from ATP-Sepharose column. The receptor sample was layered on a 5 ml ATP-Sepharose column which was washed with buffer A containing 0.15 M KC1 and 15 4.8-ml fractions were collected. The receptor complex was eluted with an ATP gradient (0-0.1 M) prepared in buffer A plus 0.15 M KCI and 1.8-ml fractions were collected. Radioactivity ( O - - O ) and protein (11--4) were measured in 0.1-ml portions of each fraction. ATP concentration ( - - . - - . - - ) of the gradient fractions was measured by diluting aliquots 1000-fold in water and reading absorbance at 260 nm.
(Fig. 4). A subsequent elution with 1 M KC1 did not remove any additional receptor complex (see Fig. 5A), and the recovery and purification of receptor by this method were generally comparable to the results achieved by salt elution. At the peak of elution, the buffer concentration of ATP was approx. 30 raM, while the concentration of ATP linked to the column was about 13/~mol per ml of packed Sepharose. As shown above for the salt-eluted material, the progesterone receptor remained intact following elution of affinity columns by ATP (data not shown).
Comparison of A and B receptor species Progesterone receptor can be resolved into two distinct species, A and B, on DEAE-cellulose chromatography [19]. While these forms show identical steroid binding characteristics, they differ somewhat in their stability, sedimentation properties, and interaction with DNA and chromatin. Our studies would suggest that both receptor components have comparable affinities for ATP. A receptor sample was first chromatographed on an ATP-Sepharose column using free ATP for receptor elution (Fig. 5A). The peak fractions were then pooled and applied to a column of DEAE-cellulose. A stepwise elution of the adsorbed protein by 0.15 M KCI and 0.3 M KC1 revealed the presence of both receptor components (Fig. 5B). In a separate experiment, the identity of A and B receptors on DEAE-cellulose was verified using a salt gradient elution (data not shown). The above observations were confirmed using the reverse protocol (Fig. 6). In this experiment, the receptor preparation was first fractionated on DEAE-cellulose. The A and B receptors were then applied separately to columns of ATP-Sepharose.
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Fig. 6. Chromatography of receptor components A and B on ATP-Sepharose. A receptor sample (2.5 nal) containing 7.5 n M [3H]progesterone was fractionated on a DEAE-cellulose column (5 ml) using a salt gradient (0.01-0.5 M KCI) for the elution (not shown). Fractions containing A (DE-A) and B (DE-B) components with maximum radioactivity were pooled separately. To enhance receptor binding, the pooled fractions were supplemented with 0.12 n M [3H]progesterone and incubated at 23 °C for 30 min. They were then chromatographed separately on 1-ml ATP-Sepharose columns. The columns were washed and eluted with buffer A containing first 0.01 M KCI, and then, 1.0 M KCI. 15 fractions were collected in each case and [~H]progesterone was measured in 0.1-ml aliquots.
485 While both preparations contained some non-adsorbed [3H]progesterone (probably representing dissociated hormone), it is clear that the A and B receptor fractions still have the capacity to bind ATP-Sepharose.
Analysis of hormone requirements Since A T P binding may be of functional significance to hormone action, it was of importance to determine whether the presence of progesterone influenced this interaction. To test this, two receptor samples, identical except for the presence of [3H]progesterone in one, were chromatographed on ATP-Sepharose columns (Fig. 7). Following chromatography of the sample without hormone, [all]progesterone was added to the column fractions which were then analyzed for hormone binding. The sample containing [3H]progesterone was applied to the column and binding was detected by a direct measurement of radioactivity in eluted fractions. In both cases a major portion of the receptor was adsorbed to the column, leading to the conclusion that the receptor will bind to ATP-Sepharose even in the absence of hormone. To confirm that the charcoal assay was measuring progesterone receptor, a preparation (fractionated as in Fig. 7, without progesterone) was tested for progesterone affinity by Scatchard analysis (data not shown). A high affinity for progesterone was demonstrated with a constant (Kd) of about 5- 10 -1° M. This affinity is comparable to previous studies [15, 19] and appears to be unchanged following affinity chromatography. 4" HORMONE
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Fig. 7. ATP-affinity chromatography of progesterone receptor in the presence or absence of hormone. Identical receptor samples (1 ml) were incubated for 3 h at 4 °C with and without 6 nM [3H]progesterone. The samples were then chromatographed on 1-ml columns of ATP-Sepharose. The columns were washed with buffer A plus 0.01 M KCI and then with buffer A plus 1 M KCI. 15 0.66-ml fractions were collected in each case. Radioactivity was measured in 0.2-ml aliquots from each fraction obtained from the sample that was preincubated with [3H]progesterone (left panel). Aliquots (0.2 ml) from the fractions obtained after chromatography in the absence of hormone were incubated with 12 nM [3H]progesterone for 3 h at 4 °C and the extent of [3H]progesterone binding was measured by charcoal adsorption assays (right panel).
486 When the receptor samples prepared in the presence or absence of [3H]progesterone were chromatographed on ATP-Sepharose columns and eluted with a KC1 gradient, the salt concentration needed for receptor elution was identical in both cases (data not shown). This would indicate that the affinity of receptor for ATPSepharose is not altered significantly by the presence of progesterone. DISCUSSION The present studies define more clearly the interaction between the progesterone receptor and ATP, and demonstrate the utility of ATP-affinity chromatography in the partial purification of the receptor. In the cytosol, the receptor protein constitutes on the order of 0.02 ~ of the total protein mass [5, 6]. ATP-Sepharose chromatography yields a preparation that is still only approx. 1-2 ~o pure at best. However, the ease and capacity of this technique make it an attractive step that can be easily combined with other methods of purification. Receptor prepared from 50 g of oviduct tissue can be readily adsorbed to a I ml column of ATP-Sepharose and the entire fractionation can be performed within a few hours. Receptor elution has been demonstrated using either higher salt or free ATP, both of which provide high yields of receptor. In our experience, gradient elution with KC1 generally provides the greatest purification (50-100-fold). Chromatography of the hormone.receptor complex on ATP-Sepharose did not alter its physicochemical properties as judged by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. Also, the steroid binding affinity of the receptor appeared to be unchanged following the chromatography. Even though many proteins are known to interact with nucleotides, only a very minor fraction of the total proteins was bound to ATP-Sepharose. However, the conditions employed here are suboptimal for many enzyme-ATP interactions, particularly because of the absence of divalent cations which are needed for the activity of these enzymes. Our preliminary studies indicate that divalent cations (Ca 2÷, Mg z+ or Mn 2+) have little effect on the chromatography of the progesterone receptor on ATP-Sepharose. Elution of the progesterone receptor as a single peak from ATP-Sepharose columns would suggest homogeneity in relation to ATP binding. However, by other criteria, the receptor is known to be composed of two similar hormone-binding components (A and B) that are distinguishable by chromatography on DEAEcellulose, phosphocellulose or hydroxyapatite columns [21, 22]. The two components appear to differ slightly in size and in their ability to bind to DNA and chromatin [6,10]. The present results would indicate, however, that the two forms bind ATP in a similar manner. It seemed possible that ATP binding could be related to the known interaction of steroid receptors with phosphate (phosphocellulose) and with polynucleotides such as DNA. While some relationship among these interactions may exist, there are also clear differences. Chromatography on phosphocellulose distinguishes between the A and B receptor components and this interaction is disrupted by salt elution conditions that are below the effective ionic strength required to disrupt ATP binding. The binding of receptor to DNA is also more sensitive to ionic strength, and this interaction appears to be a property of only receptor component A [10].
487 The receptor-ATP interaction seems to be independent of the presence of hormone under the conditions employed here. In addition, earlier studies showed that A T P had little or no affect on the binding of [3H]progesterone to receptor or on the sedimentation of the complex in sucrose gradients [11]. However, subtle interactions between the steroid and nucleotide binding sites may not have been apparent since the present method of affinity chromatography using high concentrations of immobilized A T P is not easily applied to the analysis of binding kinetics. Efforts are now under way to study the kinetics and specificity of nucleotide binding more directly using purified receptor and radioactive ATP. The present studies were carried out using receptor that had been subjected to a m m o n i u m sulfate precipitation. This preparation has been shown to be in an "activated state" in the sense that it has the ability to bind to oviduct nuclei in a cellfree system without temperature elevation [7]. The receptor in the original cytosol fraction can also be activated for nuclear uptake by a process which requires the hormone plus an incubation period at elevated temperature (e.g. 30-60 rain at 23 °C) [7]. Recent studies from this laboratory have shown that the [3H]progesterone. receptor complex in freshly prepared cytosol does not bind to ATP-Sepharose [23]. However, the ability to bind ATP is acquired when the receptor preparation is activated by elevated temperature. A T P may therefore participate in some function of the receptor once it is activated or may actually be involved in the activation process. The latter possibility is suggested by recent studies which show that the addition of various nucleotides can either inhibit or accelerate progesterone receptor activation, depending upon the concentration of nucleotide used [24, 25]. However, this effect is not specific for ATP and may or may not be related to the present binding studies. These and other possibilities will be the subject of future investigations. ACKNOWLEDGEMENTS The technical assistance of Nancy McMahon, Vernon Summerlin, and Bridget Stensgard is greatly appreciated. This work was supported by National Institutes of Health contract HD-3-2769 and by the Mayo Foundation.
REFEFENCES 10'Malley, B. W. and Means, A. R. (1974) Science 183, 610-620 2 Schrader, W. T., Buller, R, E., Kuhn, R. W. and O'Malley, B. W. (1974) J. Steroid Biochem. 5, 989-996 3 Tort, D. (1973) Obstet. Gynecol. Annu. 2, 405~,30 4 O'Malley, B. W., Sherman, M. R. and Toft, D. O. (1970) Proc. Natl. Acad. Sci. U.S. 67, 501-508 5 Sherman, M. R., Corvol, P. L. and O'Malley, B. W. (1970) J. Biol. Chem. 245, 6085-6096 6 Kuhn, R. W., Schrader, W. T., Smith, R. G. and O'Malley, B. W. (1975) J. Biol. Chem. 250, 4220~228 7 Bullet, R. E., Toft, D. O., Schrader, W. T. and O'Malley, B. W. (1975) J. Biol. Chem. 250, 801-808 8 Spelsberg, T. C., Steggles, A. W. and O'Malley, B. W. (1971) J. Biol. Chem. 246, 4188~,197 9 Spelsberg, T. C., Steggles, A. W., Chytil, F. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 1368-1374 10 Schrader, W. T., Toft, D. O. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 2401-2407 11 Moudgil, V. K. and Toft, D. O. (1975) Proc. Natl. Acad. Sci. U.S. 72, 901-905 12 Lamed, R., Levin, Y. and Wilcheck, M. (1973) Biochim. Biophys. Acta 304, 231-235
488 13 14 15 16 17 18 19 20 21 22 23 24 25
Rice, R. H. and Means, G. E. (1971) J. Biol. Chem. 246, 831-832 Martin, R. G. and Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379 Toft, D. O. and O'Malley, B. W. (1972) Endocrinology 90, 1041-1045 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660--672 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Bennet, T. P. (1967) Nature 213, 1131-1132 Schrader. W. T. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 51-59 Miller, L. K., Diaz, S. C. and Sherman, M. R. (1975) Biochemistry 14, 4433-4443 Schrader, W. T. (1975) Methods Enzymol. 36, 187-211 Schrader, W. T., Heuer, S. S. and O'Malley, B. W. (1975) Biol. Reprod. 12, 134-142 Miller, J. B. and Tort, D. O. (1976) Fed. Proc. 35, 1365 Lohmar, P. H. and Toft, D. O. (1975) 57th Ann. Meet. Endo. Soc., New York, Abstract 25 Toft, D., Moudgil, V., Lohmar, P. and Miller, J. (1976) Ann. N.Y. Acad. Sci., in the press
© Elsevier/North-Holland Biomedical Press BBA 37575 INTERACTION OF PROGESTERONE RECEPTOR WITH IMMOBILIZED ADENOSINE TRIPHOSPHATE
VIRINDER K. MOUDGIL ~ and DAVID O. TOFT Department of Molecular Medicine, Mayo Clinic, Rochester, Minn. 55901 (U.S.A.)
(Received July 19th, 1976)
SUMMARY Affinity chromatography has been used to study the binding of ATP to cytoplasmic progesterone receptors of hen oviduct. A resin which selectively binds the receptor protein was prepared by linking ATP covalently to Sepharose 4B through a 6-carbon bridge of adipic acid dihydrazide. Receptor bound to the affinity resin was recovered in a single peak upon gradient elution with KCI (0.2-1 M) or ATP (0-0.1 M). While affinity chromatography was normally accomplished using the [3H]progesterone.receptor complex, the hormone was not necessary for ATP binding under the conditions employed. The chromatography of crude receptor preparations allowed up to 100-fold purification with greater than 80 ~ recovery of the receptor. The semipurified receptor appeared intact when analysed by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and DEAE-ceUulose chromatography. The latter procedure separated the receptor into two components, A and B, both of which were capable of binding ATP. Although a specific biochemical role of ATP in hormone receptor action has not been demonstrated, the present studies support this possibility and, in addition, offer a convenient and reliable step for the purification of progesterone receptors.
INTRODUCTION The progesterone receptor of the avian oviduct has been the subject of many recent investigations [1-3]. Upon entering oviduct cells, progesterone binds the receptor protein and apparently initiates a relocation of this complex to sites on the nuclear chromatin where it may act in regulating genetic expression [1, 4]. The physicochemical properties and steroid binding characteristics of this protein have been studied quite extensively [1-6] and recent efforts have been successful in purifying the receptor to near homogeneity [2, 6]. The hormone-receptor complex isolated from tissue cytosol can bind in vitro to isolated nuclei, nuclear chromatin, and to purified DNA [7-10]. This suggests that the receptor acts, at least in part, throughan interaction with DNA and/or a protein Present address: Department of Biological Sciences, Oakland University, Rochester, Mich. 48063, U.S.A. "
478 fraction of chromatin. However, biochemical activities of the receptor of a more functional nature have not yet been established. Recently, we reported our initial observations on an interaction of the progesterone receptor with ATP°Sepharose [11 ]. The binding appeared to be specific for ATP and was reversible under the conditions employed. Since the selective nature of this interaction suggests a role for ATP in some aspect of receptor function, a more thorough characterization of the binding of receptor to immobilized ATP was undertaken. MATERIALS AND METHODS Analytical grade reagents in glass-distilled water were used throughout. All procedures were carried out at 4 °C. ATP was purchased from Schwarz/Mann, Sepharose 4B and Dextran T-70 from Pharmacia, Uppsala, Sweden; diethylaminoethyl-cellulose (DE 52) from Whatman Biochemicals, Ltd.; acrylamide and N,N'methylene-bis-acrylamide from Bio-Rad; (NH4)zSzO8 from Fisher Scientific; N,N,N,'N'-tetramethylenediamine from Eastman Kodak; and activated charcoal and unlabeled progesterone from Sigma. [1,2-3H2]Progesterone (43.8 Ci/mmol) was purchased from New England Nuclear and was repurified by thin-layer chromatography before use. Buffers. Buffer A: 10 mM Tris. HC1, 12 mM monothioglycerol, 1 mM EDTA, and 2 0 ~ (v/v) glycerol, pH 8.0 (supplemented with KCI as indicated in Figure legends). Buffer B: 10 mM Tris.HC1 and 1 mM EDTA, pH 8.0. Preparation of ATP-Sepharose. ATP was covalently bound to Sepharose-4B as previously described [11] following the method of Lamed et al. [12]. Adipic acid dihydrazide was attached to CNBr-activated Sepharose and ATP was oxidized by periodate and then linked to the Sepharose hydrazide. The ribose of ATP was therefore attached to Sepharose through the 6-carbon bridge of adipic acid dihydrazide. Our preparation contained 13/~mol of ATP per ml Sepharose as determined by phosphate analysis [11]. Column Procedures. DEAE-cellulose was precycled, degassed, and equilibrated in buffer B as recommended by Whatman Biochemicals, Ltd. The resins (DEAE-cellulose or ATP-Sepharose) were equilibrated with buffer A and packed into columns. The flow rate was 1.5-2.0 ml/min (unless otherwise indicated) and either 4.8- or 1.8-ml fractions were collected (indicated in Figure legends). Preparation of Progesterone Receptor. Freshly excised oviducts from White Leghorn hens were obtained from a local produce company. The tissue was rinsed with cold 0.9 ~ saline and homogenized first with a Waring blender and then with a "Tissumizer" (Tekmar model SDT) in two volumes (w/v) of buffer A (modified to contain 10~ glycerol and 0.01 M KC1). The homogenate was centrifuged first at 12 000 × g for I0 min and then at 100 000 x g for 90 rain. Saturated (NH4)2SO4 (pH 7.5) was added to the cytosol to give a final concentration of 35 ~ saturation. The suspension was stirred for 20 min at 4 ° and centrifuged at 12 000 x g for 10 min. The resulting pellets containing the progesterone receptor could be stored at --70 °C for as long as 1 month without significant loss of hormone binding activity. Before use, the pellets were dissolved in buffer A plus 0.01 M KCI using one-tenth the original cytosol volume. This solution was centrifuged to remove undissolved material
479 and incubated with [3H]progesterone (as indicated in figure legends); it served as the starting material for all the experiments. Sucrose Gradient Analysis. Linear 5-20 ~o sucrose gradients (4.5 ml) in buffer A minus glycerol were prepared using a Beckman Gradient Former. The gradients also contained 0.01 or 0.3 M KC1. The receptor samples (0.2 ml) were diluted to lower the glycerol concentration and layered onto the gradients which were then centrifuged at 150 000 x g for 16 h. [14C]Ovalbumin [13] was used as a standard marker to determine sedimentation coefficients [14]. Fractions were collected by piercing the bottom of centrifuge tube and were analyzed for radioactivity. Polyacrylamide Gel Electrophoresis. Stock solutions for acrylamide gels were prepared as follows: stock 1 : 18.5 g Tris, 0.12 ml tetramethylenediamine in 1 M HC1 (24 ml), water to make 100 ml (pH 8.9); stock 2:20 g acrylamide, 0.735 g methylenebis-acrylamide, 20 g glycine, water to make 100 ml; and stock 3:25 mg (NH4)2S2Os, 15 ml glycerol, and 35 ml water. Stock solutions 1 and 2 were stored in the dark at 5 °C while stock 3 was made for each experiment. The gels (5 ~ acrylamide) were prepared at room temperature by mixing the three stock solutions in proportions of 1:1:2, respectively. This mixture was poured into glass cylinders (90 × 6 mm) to a height of 70 mm and polymerization was accomplished in approx. 20 min. The gels were placed in an electrophoresis chamber (Buchler) with upper and lower reservoir buffers of 5 mM Tris.HC1, 38.4 mM glycine, 12 mM thioglycerol and 10~ glycerol, pH 8.9. Pre-electrophoresis was done at 5 °C for 2 h using 4 mA/gel, after which the top reservoir was replaced with fresh buffer. The samples (0.1 ml), prepared in buffer A, but with 15 ~ glycerol, were then layered onto the gels. A 0.01 ~ solution (10 #1) of bromophenol blue in 10 ~ glycerol was also layered on the gel as an electrophoretic marker. Electrophoresis was carried out at 3-5 °C for about 2 h at 2 mA/gel. One set of gels was stained for 35 min with 0.1 ~o Coomassie Blue in 7 ~ acetic acid and then destained for 30 min in a Canalco Gel Destainer and stored in 7 ~ acetic acid. A duplicate set of gels was sliced into 2-mm sections for determining the migration of the [3H]progesterone. receptor complex. The sections were placed in counting vials containing 5 ml of toluene-based scintillation fluid (as described below, but without Triton X-100). Charcoal Adsorption Binding Assay. Prior to affinity chromatography, 0.1-ml portions of the receptor sample were analyzed in duplicate for Jail]progesterone binding [15]. The samples were diluted with buffer A to a volume of 0.5 ml. Since these samples initially contained bound hormone, background determinations were made in replicate tubes by adding excess unlabeled progesterone and incubating for 1 h at 37 °C. Bound hormone.receptor complex was quantitated by adding an equal volume of charcoal suspension (0.5 ~ charcoal, 0.05~ dextran T-70, 10 mM Tris, 1 mM EDTA, pH 8.0) to each tube for 5 min. The suspensions were centrifuged for 10 min at 600 x g to yield clear supernatants which contained the receptor-bound [3H]progesterone. The supernatants were decanted into counting vials and analyzed for radioactivity. When receptor preparations were chromatographed in the absence of progesterone, receptor content in the eluted fractions was determined by the capacity to bind added [aH]-progesterone. Aliquots (0.2 ml) from each fraction were incubated with 12 nM [3H]progesterone for 3 h in an ice bath. After adding 0.5 ml ovalbumin (10 mg/ml), each tube was treated with charcoal as described above. With some
480 preparations, the binding affinity and concentration of receptor sites were determined by Scatchard analysis [16]. Other Methods. Radioactivity was determined by combining aqueous samples with 5 ml of a cocktail consisting of toluene (Baker), Triton X-100 (RPI), and spectrafluor (Amersham-Searle), 100:521:42 (v/v/v). The counting efficiency was 33 ~o in a Beckman LS-250 liquid scintillation counter. The protein concentration in fractions after affinity chromatography was determined by the method of Lowry et al. [17]. The protein was first precipitated with I0 ~ trichloroacetic acid and collected on Millipore filters [18] to remove interfering substances such as thioglycerol. RESULTS
Methods of Affinity Chromatography The [3HI-progesterone.receptor complex can be selectively adsorbed on columns of ATP-Sepharose and eluted stepwise with buffer containing high salt B A
5
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, [3HI-progesterone
110
42
42
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44
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Fig. 1. Affinity chromatography of [3H]progesterone. receptor complex from hen oviduct on ATPSepharose. Panel Ao The [3H]progesterone.receptor sample (9 ml) was applied to a column (10 ml) with a flow rate of 0.2 ml/min. The column was then washed with buffer A containing 0.15 M KC1 with a flow rate of 0.4 ml/min and 20 4.15-ml fractions were collected. The adsorbed material was eluted with buffer A containing 1 M KCI, collecting 20 1.8-ml fractions. Samples (0.1 ml) were removed from each fraction for determination of radioactivity and protein concentration. The peak fraction in this experiment was purified 38-fold (by comparison of bound cpm/mg protein) and total recovery of the receptor complex was 90 ~ . Panel B. The sample was applied as in Panel A and the column was washed with buffer A containing 0.2 M KCI and 20 4.5-ml fractions were collected. The column was then eluted with a salt gradient (0.2-1 M KC1) and 1.8-ml fractions were collected with a flow rate of 0.5 ml/min. Radioactivity ( © - - © ) and protein (0---0) were determined from 0.1-ml samples from each fraction. The KCI concentration ( © - - © ) was determined by conductivity measurements. The same scale is used for both protein and KC1 concentration.
481 (Fig. 1A). The first peak of radioactivity is composed primarily of free hormone while the peak of [3H]progesterone eluted with 1 M KC1 is essentially all bound to protein [11]. Before chromatography, the receptor sample was routinely assayed for both protein concentration and amount of bound hormone. Upon chromatography, the peak eluted with high salt (1 M KCI) contained 90 ~o of the [3H]progesterone bound in the original sample but less than 5 ~o of the total protein in that sample. This procedure routinely provided a 20-40-fold purification of the receptor and recovery of 80-95 70 of the complex applied to the column. ATP is a necessary constituent of this column chromatography. The selective adsorption of the hormone-receptor complex by the resin was evident only when ATP was linked to the Sepharose. Under the salt conditions normally employed, the progesterone receptor was not retained to any significant extent on columns of either Sepharose 4B or Sepharose with linked adipic acid dihydrazide. The binding of receptor to ATP-Sepharose in relation to ionic strength is indicated by the gradient elution shown in Fig. 1B. A single peak of bound progesterone was eluted at a rather high salt concentration (0.5-0.6 M KC1). This result suggests that the binding of ATP to the receptor is of high affinity and also indicates a homogeneity of the receptor with respect to its association with ATP. The integrity of the [3H]progesterone. receptor complex following affinity chromatography was tested by sucrose gradient centrifugation. The complex sedimented as a 4 S unit (Fig. 2B) as did the original receptor complex before chromatography .... :
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A
Low salt - High salt
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B
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Top Fraction number
Fig. 2. Sucrose gradient centrifugation of [3H]progesterone.receptor complex before and after affinity chromatography. Samples (0.2 ml) were layered onto 5-20~ sucrose gradients containing 0.01 M (O---O) or 0.3 M (0---0) KC1. The gradients were centrifuged for 16h at 150000 × g. [14C]Ovalbumin was the sedimentation standard (3.7 S). (A) The sample before purification consisted of ammonium sulfate-fractionated receptor incubated for 3 h with laH]progesterone (6 nM). (B) The receptor sample was from the peak fraction eluted from an ATP-Sepharose column with buffer A containing 1 M KC1. All samples were diluted with three volumes of buffer B to lower the density before layering onto gradients.
482 (Fig. 2A). While the major component had a sedimentation coefficient of approx. 4 S under both high (0.3 M KC1) and low (0.01 M KCI) salt conditions, the original sample obtained by ammonium sulfate fractionation exhibited a tendency to aggregate at the lower ionic strength. This aggregation did not occur following purification on ATP-Sepharose. Progesterone receptor in the cytosol fraction from chick oviducts has been reported to sediment as a 6-8 S complex under low ionic conditions [4, 15] which is also the case for the receptor from the mature hen oviduct (data not shown). However, during the initial steps of purification, the receptor appeared to lose most of its ability to aggregate to the 6-8 S form. A reduction in receptor aggregation following purification of progesterone receptor from chick oviduct has also been demonstrated [6, 19]. The semipurified receptor was also analyzed by polyacrylamide gel electrophoresis (Fig. 3). The gels contained glycerol (20 ~) which appeared to stabilize the receptor. Most of the progesterone.receptor complex which entered the gel migrated as a single, well-defined band. However, the region between this band and the origin also contained bound hormone of a rather heterogeneous mobility. This pattern is quite similar to that reported by Miller et al. [20] for progesterone receptor from the chick oviduct. ATP-Sepharose chromatography did not detectably alter the mobility of the receptor complex, but it did remove many of the protein bands observed in the electrophoresis of the ammonium sulfate fraction.
12
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Since the progesterone receptor binds reversibly to ATP-Sepharose, the use of free ATP as an effective eluent was also studied. When an ATP gradient (0-0.1 M ATP) was used for elution, the receptor was removed uniformly in a single peak
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Fig. 4. ATP gradient elution of progesterone receptor from ATP-Sepharose column. The receptor sample was layered on a 5 ml ATP-Sepharose column which was washed with buffer A containing 0.15 M KC1 and 15 4.8-ml fractions were collected. The receptor complex was eluted with an ATP gradient (0-0.1 M) prepared in buffer A plus 0.15 M KCI and 1.8-ml fractions were collected. Radioactivity ( O - - O ) and protein (11--4) were measured in 0.1-ml portions of each fraction. ATP concentration ( - - . - - . - - ) of the gradient fractions was measured by diluting aliquots 1000-fold in water and reading absorbance at 260 nm.
(Fig. 4). A subsequent elution with 1 M KC1 did not remove any additional receptor complex (see Fig. 5A), and the recovery and purification of receptor by this method were generally comparable to the results achieved by salt elution. At the peak of elution, the buffer concentration of ATP was approx. 30 raM, while the concentration of ATP linked to the column was about 13/~mol per ml of packed Sepharose. As shown above for the salt-eluted material, the progesterone receptor remained intact following elution of affinity columns by ATP (data not shown).
Comparison of A and B receptor species Progesterone receptor can be resolved into two distinct species, A and B, on DEAE-cellulose chromatography [19]. While these forms show identical steroid binding characteristics, they differ somewhat in their stability, sedimentation properties, and interaction with DNA and chromatin. Our studies would suggest that both receptor components have comparable affinities for ATP. A receptor sample was first chromatographed on an ATP-Sepharose column using free ATP for receptor elution (Fig. 5A). The peak fractions were then pooled and applied to a column of DEAE-cellulose. A stepwise elution of the adsorbed protein by 0.15 M KCI and 0.3 M KC1 revealed the presence of both receptor components (Fig. 5B). In a separate experiment, the identity of A and B receptors on DEAE-cellulose was verified using a salt gradient elution (data not shown). The above observations were confirmed using the reverse protocol (Fig. 6). In this experiment, the receptor preparation was first fractionated on DEAE-cellulose. The A and B receptors were then applied separately to columns of ATP-Sepharose.
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Fig. 6. Chromatography of receptor components A and B on ATP-Sepharose. A receptor sample (2.5 nal) containing 7.5 n M [3H]progesterone was fractionated on a DEAE-cellulose column (5 ml) using a salt gradient (0.01-0.5 M KCI) for the elution (not shown). Fractions containing A (DE-A) and B (DE-B) components with maximum radioactivity were pooled separately. To enhance receptor binding, the pooled fractions were supplemented with 0.12 n M [3H]progesterone and incubated at 23 °C for 30 min. They were then chromatographed separately on 1-ml ATP-Sepharose columns. The columns were washed and eluted with buffer A containing first 0.01 M KCI, and then, 1.0 M KCI. 15 fractions were collected in each case and [~H]progesterone was measured in 0.1-ml aliquots.
485 While both preparations contained some non-adsorbed [3H]progesterone (probably representing dissociated hormone), it is clear that the A and B receptor fractions still have the capacity to bind ATP-Sepharose.
Analysis of hormone requirements Since A T P binding may be of functional significance to hormone action, it was of importance to determine whether the presence of progesterone influenced this interaction. To test this, two receptor samples, identical except for the presence of [3H]progesterone in one, were chromatographed on ATP-Sepharose columns (Fig. 7). Following chromatography of the sample without hormone, [all]progesterone was added to the column fractions which were then analyzed for hormone binding. The sample containing [3H]progesterone was applied to the column and binding was detected by a direct measurement of radioactivity in eluted fractions. In both cases a major portion of the receptor was adsorbed to the column, leading to the conclusion that the receptor will bind to ATP-Sepharose even in the absence of hormone. To confirm that the charcoal assay was measuring progesterone receptor, a preparation (fractionated as in Fig. 7, without progesterone) was tested for progesterone affinity by Scatchard analysis (data not shown). A high affinity for progesterone was demonstrated with a constant (Kd) of about 5- 10 -1° M. This affinity is comparable to previous studies [15, 19] and appears to be unchanged following affinity chromatography. 4" HORMONE
- - HORMONE 20
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Fig. 7. ATP-affinity chromatography of progesterone receptor in the presence or absence of hormone. Identical receptor samples (1 ml) were incubated for 3 h at 4 °C with and without 6 nM [3H]progesterone. The samples were then chromatographed on 1-ml columns of ATP-Sepharose. The columns were washed with buffer A plus 0.01 M KCI and then with buffer A plus 1 M KCI. 15 0.66-ml fractions were collected in each case. Radioactivity was measured in 0.2-ml aliquots from each fraction obtained from the sample that was preincubated with [3H]progesterone (left panel). Aliquots (0.2 ml) from the fractions obtained after chromatography in the absence of hormone were incubated with 12 nM [3H]progesterone for 3 h at 4 °C and the extent of [3H]progesterone binding was measured by charcoal adsorption assays (right panel).
486 When the receptor samples prepared in the presence or absence of [3H]progesterone were chromatographed on ATP-Sepharose columns and eluted with a KC1 gradient, the salt concentration needed for receptor elution was identical in both cases (data not shown). This would indicate that the affinity of receptor for ATPSepharose is not altered significantly by the presence of progesterone. DISCUSSION The present studies define more clearly the interaction between the progesterone receptor and ATP, and demonstrate the utility of ATP-affinity chromatography in the partial purification of the receptor. In the cytosol, the receptor protein constitutes on the order of 0.02 ~ of the total protein mass [5, 6]. ATP-Sepharose chromatography yields a preparation that is still only approx. 1-2 ~o pure at best. However, the ease and capacity of this technique make it an attractive step that can be easily combined with other methods of purification. Receptor prepared from 50 g of oviduct tissue can be readily adsorbed to a I ml column of ATP-Sepharose and the entire fractionation can be performed within a few hours. Receptor elution has been demonstrated using either higher salt or free ATP, both of which provide high yields of receptor. In our experience, gradient elution with KC1 generally provides the greatest purification (50-100-fold). Chromatography of the hormone.receptor complex on ATP-Sepharose did not alter its physicochemical properties as judged by sucrose gradient sedimentation and polyacrylamide gel electrophoresis. Also, the steroid binding affinity of the receptor appeared to be unchanged following the chromatography. Even though many proteins are known to interact with nucleotides, only a very minor fraction of the total proteins was bound to ATP-Sepharose. However, the conditions employed here are suboptimal for many enzyme-ATP interactions, particularly because of the absence of divalent cations which are needed for the activity of these enzymes. Our preliminary studies indicate that divalent cations (Ca 2÷, Mg z+ or Mn 2+) have little effect on the chromatography of the progesterone receptor on ATP-Sepharose. Elution of the progesterone receptor as a single peak from ATP-Sepharose columns would suggest homogeneity in relation to ATP binding. However, by other criteria, the receptor is known to be composed of two similar hormone-binding components (A and B) that are distinguishable by chromatography on DEAEcellulose, phosphocellulose or hydroxyapatite columns [21, 22]. The two components appear to differ slightly in size and in their ability to bind to DNA and chromatin [6,10]. The present results would indicate, however, that the two forms bind ATP in a similar manner. It seemed possible that ATP binding could be related to the known interaction of steroid receptors with phosphate (phosphocellulose) and with polynucleotides such as DNA. While some relationship among these interactions may exist, there are also clear differences. Chromatography on phosphocellulose distinguishes between the A and B receptor components and this interaction is disrupted by salt elution conditions that are below the effective ionic strength required to disrupt ATP binding. The binding of receptor to DNA is also more sensitive to ionic strength, and this interaction appears to be a property of only receptor component A [10].
487 The receptor-ATP interaction seems to be independent of the presence of hormone under the conditions employed here. In addition, earlier studies showed that A T P had little or no affect on the binding of [3H]progesterone to receptor or on the sedimentation of the complex in sucrose gradients [11]. However, subtle interactions between the steroid and nucleotide binding sites may not have been apparent since the present method of affinity chromatography using high concentrations of immobilized A T P is not easily applied to the analysis of binding kinetics. Efforts are now under way to study the kinetics and specificity of nucleotide binding more directly using purified receptor and radioactive ATP. The present studies were carried out using receptor that had been subjected to a m m o n i u m sulfate precipitation. This preparation has been shown to be in an "activated state" in the sense that it has the ability to bind to oviduct nuclei in a cellfree system without temperature elevation [7]. The receptor in the original cytosol fraction can also be activated for nuclear uptake by a process which requires the hormone plus an incubation period at elevated temperature (e.g. 30-60 rain at 23 °C) [7]. Recent studies from this laboratory have shown that the [3H]progesterone. receptor complex in freshly prepared cytosol does not bind to ATP-Sepharose [23]. However, the ability to bind ATP is acquired when the receptor preparation is activated by elevated temperature. A T P may therefore participate in some function of the receptor once it is activated or may actually be involved in the activation process. The latter possibility is suggested by recent studies which show that the addition of various nucleotides can either inhibit or accelerate progesterone receptor activation, depending upon the concentration of nucleotide used [24, 25]. However, this effect is not specific for ATP and may or may not be related to the present binding studies. These and other possibilities will be the subject of future investigations. ACKNOWLEDGEMENTS The technical assistance of Nancy McMahon, Vernon Summerlin, and Bridget Stensgard is greatly appreciated. This work was supported by National Institutes of Health contract HD-3-2769 and by the Mayo Foundation.
REFEFENCES 10'Malley, B. W. and Means, A. R. (1974) Science 183, 610-620 2 Schrader, W. T., Buller, R, E., Kuhn, R. W. and O'Malley, B. W. (1974) J. Steroid Biochem. 5, 989-996 3 Tort, D. (1973) Obstet. Gynecol. Annu. 2, 405~,30 4 O'Malley, B. W., Sherman, M. R. and Toft, D. O. (1970) Proc. Natl. Acad. Sci. U.S. 67, 501-508 5 Sherman, M. R., Corvol, P. L. and O'Malley, B. W. (1970) J. Biol. Chem. 245, 6085-6096 6 Kuhn, R. W., Schrader, W. T., Smith, R. G. and O'Malley, B. W. (1975) J. Biol. Chem. 250, 4220~228 7 Bullet, R. E., Toft, D. O., Schrader, W. T. and O'Malley, B. W. (1975) J. Biol. Chem. 250, 801-808 8 Spelsberg, T. C., Steggles, A. W. and O'Malley, B. W. (1971) J. Biol. Chem. 246, 4188~,197 9 Spelsberg, T. C., Steggles, A. W., Chytil, F. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 1368-1374 10 Schrader, W. T., Toft, D. O. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 2401-2407 11 Moudgil, V. K. and Toft, D. O. (1975) Proc. Natl. Acad. Sci. U.S. 72, 901-905 12 Lamed, R., Levin, Y. and Wilcheck, M. (1973) Biochim. Biophys. Acta 304, 231-235
488 13 14 15 16 17 18 19 20 21 22 23 24 25
Rice, R. H. and Means, G. E. (1971) J. Biol. Chem. 246, 831-832 Martin, R. G. and Ames, B. N. (1961) J. Biol. Chem. 236, 1372-1379 Toft, D. O. and O'Malley, B. W. (1972) Endocrinology 90, 1041-1045 Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51,660--672 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Bennet, T. P. (1967) Nature 213, 1131-1132 Schrader. W. T. and O'Malley, B. W. (1972) J. Biol. Chem. 247, 51-59 Miller, L. K., Diaz, S. C. and Sherman, M. R. (1975) Biochemistry 14, 4433-4443 Schrader, W. T. (1975) Methods Enzymol. 36, 187-211 Schrader, W. T., Heuer, S. S. and O'Malley, B. W. (1975) Biol. Reprod. 12, 134-142 Miller, J. B. and Tort, D. O. (1976) Fed. Proc. 35, 1365 Lohmar, P. H. and Toft, D. O. (1975) 57th Ann. Meet. Endo. Soc., New York, Abstract 25 Toft, D., Moudgil, V., Lohmar, P. and Miller, J. (1976) Ann. N.Y. Acad. Sci., in the press
