THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 256, No. 23, Issue of December 10. pp. 12094-12101.1981 Printed In U. S. A.

Affinity Chromatography of Thyroid Hormone Receptors BIOSPECTFIC ELUTION FROM SUPPORT MATRICES, CHARACTERIZATION OF THE PARTIALLY PURIFIED RECEPTOR*

(Received for publication, January 23, 1981, and in revised form, June 22,1981)

James W. Aprilettis, Norman L. Eberhardt, Keith R. Latham@, and John D. Saxterq From the Howard Hughes Medical Institute Laboratories, The Metabolic Research Unit, and the Departments of Medicine and Biochemistry and Biophysics ut the University of California, Sun Francisco, California 94143

In the preceding paper we reported the synthesis of affinity matrices containing 3,5,3'-triiodo-~-thyonine (T3) Linked via its amino group and the diactivated ester of glutaric acid to the free amino groups of diamino- hexane-Sepharose. This report describes the optimiza- tion of this system for the purification of the intranu- clear thyroid hormone receptor.

The thyroid hormone receptors (Kd for T3 -50 to 200 PM) were solubilized from nuclei obtained from rat, sheep, or steer liver. These receptors were partially purified by Sephadex G-100 chromatography and then bound to the affinity gel. After the receptor-containing gel was washed, the receptors were eluted with free TI. An exchange assay was developed for measuring the binding capacity of the eluted receptor; after most of the unlabeled T3 in the affinity-gel eluate had been removed by chromatography on Sephadex G-26, the small amount of nonradioactive Ta remaining was dis- placed from the receptor by incubation with ["'1]T3. The recovery of the receptors from the affinity gel was stimulated approximately 5-fold by the addition of pu- rified core histones (HzA, HZB, H3, and H4, themselves devoid of T3-binding activity) to the wash, elution, and assay buffers, but was not enhanced by the addition of several other acidic or basic proteins. In the presence of 25 to 50 pg/ml of core histones, recovery of receptor from the gel was 10 to 35%, with a purification of more than 500-fold.

Scatchard analysis of hormone binding by the affin- ity-purified material showed an apparent Kd of 50 p~ for Ta and 1 nM for thyroxine (3,5,3',5'-tetraiodo-~-thy- ronine). The relative binding affinities of the affinity- purified receptor €or various hormones and analogs was TB 2 isopropyl T2 (3-'-isopropyl-3,5-diiodothyro- nine) > thyroxine > reverse Ta (3,3',5'-triiodo-~-thyro- nine), which is the same as observed for whole nuclei or crude nuclear extract. The affinity-purified receptor also behaved the same as the crude receptor during chromatography on Sephadex G-100 or DEAE-Sepha- dex. Thus, characteristic properties of the thyroid hor- mone receptors are retained after affinity chromatog- raphy purification, and, importantly, the affinity-puri- fied receptors retain the capacity for reversible hor- mone binding.

* This research was supported by National Institutes of Health Grant 1-R01-AM-18878. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$National Institutes of Health Postdoctoral Fellow, 1 F32

8 Present address, Department of Medicine, Uniformed Services

7 Investigator of the Howard Hughes Medical Institute.

AM05863-01.

University of the Health Sciences, Bethesda, MD 20014.

Thyroid hormones appear to mediate certain of their ac- tions through chromatin-localized receptors (for a review see Ref. 1). To further understand the function of these receptors, we have begun to purify them. Due to the apparent need to obtain a 25,000- to 50,000-fold purification from the initial nuclear extract to achieve homogeneity (2), we have focused on the technique of affinity chromatography. In the previous paper (2), we reported the preparation of an affinity matrix in which the diactivated ester of glutaric acid is used to link T3' to the hydrocarbon spacer arms of diaminohexane-Sepharose. The binding of thyroid hormone receptor to this T3 affinity gel was found to be biospecific (e.g. excess free hormone blocked receptor adsorption by the gel). The results also suggested that the receptor could be eluted from the gel with the use of free T3 as counter ligand ( 2 ) . In the present communication, we report the results of studies performed to elute receptors from the gel such that reversible, high affinity receptor binding properties could be maintained. To optimize the purification and yield of receptor, we took advantage of previous findings that a protein factor(s), possibly histone-like in nature, could help maintain the high affinity TB-binding properties of the receptors (3). We also report some other characteristics of the partially purified receptors that dem- onstrate their similarities to those in crude solubilized ex- tracts.

EXPERIMENTAL PROCEDURES

Reugent~"['*~I]T3 (high specific activity, 500 to 1200 mCi/mg, or carrier free, 2400 to 3400 mCi/mg) and ["51]T4 (600 to 1500 mCi/mg) were purchased from New England Nuclear. Nonradioactive T3 and T, were obtained from Sigma Chemical Co. Reverse T:j and isopropyl T, were generously provided by Dr. Eugene C. Jorgensen. These hormones were monitored for purity as described previously (4). T:] antibody wm obtained from Calbiochem. Coomassie Brilliant Blue G- 250 was obtained from Eastman Kodak Co. Dithiothreitol and Triton X-100 were obtained from Sigma Chemical Co.

Preparation of T.3 Affinity GeZs-The gels used for affinity chro- matography were prepared by coupling LT:, to the primary amines of AH-Sepharose (Phamacia) via a glutarate linkage, as described pre- viously (2). The gels used in these studies contained 1.7 to 2.5 mM bound TB as monitored by incorporation of ['"IJT:3. Prior to use, the gel was washed extensively with 1 M NaCl and with distilled water. To ensure removal of any free T3 which may arise by decoupling of hormone from the gel during storage, T3 leakage was monitored both by following the release of ("51]T3 from the gel and by measuring the ability of buffer incubated with the gel to block binding of ['2,51]T:j by nuclear extract, as described previously (2).

Preparation of Core Histones-Core histones were prepared from fresh steer thymus by the procedure of Dr. R. D. Cole (University of California, Berkeley)' as modified by Eberhardt et al. (3). Briefly, the

' The abbreviations used are: Ta, LTa, or triiodothyronine, 3,5,3'- tniodo-L-thyronine; T,, 3,5,3',5'-tetraiodo-~-thyronine, RTa or reverse T3, 3,3',5'-triiodothyronine; IT, or isopropyl TP, 3,5-di1odo-3'-isopro- pyl-L-thyronine.

__l__l___

* Et. D. Cole, personal communication.

22094

Affinity Chromatography of Thyroid Hormone Receptors 12095

procedure consists of extracting histone HI from isolated nuclei with 5% trichloroacetic acid, followed by extracting the core histones with 0.25 N HCI. The core histone extract was dialyzed against water, lyophilized, and redissolved in buffer G. These preparations were occasionally contaminated by a small amount of Tj-binding material. This was removed by chromatography on Sephadex G-100. The T:I- binding material and aggregated histones eluted in the void volume, and core histones eluted as two included protein peaks. The included peaks of core histones were dialyzed against buffer E and chromato- graphed on a column of DEAE-Sephadex A-25 equilibrated with the same buffer. The histones passed through the column, while a small amount of protein (less than 1% of the total protein applied) bound to the column and could be eluted by 0.2 M NaCI. I t was important to remove this protein contaminant, as its presence was found to inter- fere with the determination of protein concentrations in the experi- ments in which DEAE-Sephadex chromatography was used to re- move the histones from the affinity-purified receptor.

Buffers-Buffer A: 10 mM Tris.HC1 (pH 7.6),0.32 M sucrose, 2 mM MgCl, 0.24 mM spermine. Buffer B, 20 mM Tris.HC1 (pH 7.6), 2 mM CaC12, 1 mM MgC12, 0.5% Triton X-100. Buffer C, 20 mM Tris.HC1 (pH 8.0), 0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol. Buffer D, 50 mM sodium phosphate (pH 7.6), 1 mM EDTA, 30% glycerol, 1 mM dithiothreitol. Buffer E, 30 mM Tris-HC1 (pH 8.0), 1 mM EDTA, 30% glycerol, 1 mM dithiothreitol. Buffer G, 50 mM sodium phosphate (pH 7.6), 0.2 M ammonium sulfate, 1 mM EDTA, 0.2 mM dithiothreitol, 5% glycerol. All pH measurements were per- formed at 22 "C.

Preparation of Nuclear Extracts-Solubilized receptors were pre- pared from purified rat liver nuclei and stored in liquid nitrogen as previously described (4). A modification of this procedure was used for the preparation of nuclei and solubilized receptor from steer liver and sheep liver. Fresh steer liver was obtained from a local slaugh- terhouse and immediately placed on ice. All procedures were carried out at 4 "C. After being cut into small pieces, 1 kg of tissue was washed with 1.5 liters of buffer A. The tissue was strained with one layer of cheesecloth, combined with 1.5 liters of buffer A, and homog- enized for 1 min in a Waring Blendor. The coarse homogenate was further homogenized with a Tekmar Polytron homogenizer (Cincin- nati, OH) at maximum speed three times for 1 min each, with 2-min cooling between each homogenization. Prior to the third homogeni- zation, buffer A was added to bring the final volume to 4 liters. The homogenate was next filtered through cheesecloth: twice with one layer, followed by single successive passages through two, four, six, and eight layers of cheesecloth. The filtrate was centrifuged at IO00 X g for 10 min and the supernatant was discarded. The pellet was resuspended in three liters of buffer A and centrifuged 10 min a t 700 X g. The nuclear pellet was washed twice in buffer B, resuspended in buffer C to a final volume of 0.2 ml/g of original tissue, and frozen as aliquots in liquid nitrogen. Nuclear extract was prepared as described previously (4), yielding 0.5 ml/g of original tissue.

Sheep livers were removed from pregnant ewes immediately after killing. The liver was quickly cut into small pieces and immediately frozen in liquid nitrogen. Frozen livers were pulverized with a cold pestle and quickly thawed by adding to warm buffer A (37 "C, 2 ml/ g of tissue) with stirring. Then, 250-ml aliquots of the mixture were homogenized for two 1-min periods, separated by 5 min cooling, using a Tekmar Polytron homogenizer a t maximum speed. The homoge- nates were filtered through Miracloth (Calbiochem) and centrifuged 10 min at lo00 X g. The pellet was washed once with buffer A (10 min at 700 X g, 3 ml of buffer/g of tissue) and twice with buffer B. The final nuclear pellet was resuspended in buffer C and stored in liquid nitrogen. Nuclear extract was prepared as described above, yielding 1 ml of extract/g of liver. The Ts-binding capacity of the nuclear extracts was measured as described previously (4).

Sephadex G-IO0 Chromatography-Chromatography of the nu- clear extract on Sephadex G-100 was performed as described previ- ously (3). Fifteen ml of nuclear extract was chromatographed on a column (2.5 X 96 cm) of Sephadex G-100. The column fractions of the included peak of Ts-binding activity were pooled and stored in liquid nitrogen.

Affinity Chromatography of Nuclear Extract-All glassware was siliconized prior to use in affinity chromatography. The thyroid hor- mone receptors in crude nuclear extract could be bound to the affinity gel by either batchwise adsorption or passage through a column. Batchwise adsorption was generally more convenient when working on a small scale (gel bed volume less than 2 ml) or when a number of different adsorption conditions were being examined. However, more receptor was bound per ml of gel bed volume when columnwise

adsorption was used. Thus, unless noted otherwise, the receptors were found to the TR affinity gel by slowly passing nuclear extract, or an extract partially purified by Sephadex G-100 chromatography, through the gel column at room temperature (22 "C). In most exper- iments, 5 to 15 ml of nuclear extract was applied per ml bed volume, at a flow rate which allowed 2 to 3 h total adsorption time. The amount of receptor bound to the gel was estimated by assaying for T:I binding before and after adsorption, and assuming that the decrease in T, binding was equivalent to the amount of bound receptor. The equivalent of 2 to 4 pmol of T:$ binding sites was bound per ml of gel bed volume.

After binding was completed, the column was washed for 0.5 to 1 h with 10 to 20 column volumes of buffer D containing 0.4 M ammo- nium sulfate and 25 to 50 pg/ml of core histone. Batchwise elution of the receptor was performed by placing the gel in a test tube and adding an equal volume of buffer D containing 0.1 M ammonium sulfate, 50 pg/ml of core histone, 50 p~ Ts, and 5 nM ['*'II]Ta. The gel and buffer were incubated for 3 h at room temperature with occasional mixing. At the end of the incubation, the gel was removed by centrif- ugation and the supernatant containing the eluted receptor was either used for further studies or frozen in liquid nitrogen. Due to adsorption of T1 by the gel (2), the supernatant generally contained approxi- mately 10 PM T3, as determined by counting the tracer levels of ['2sIJ T3 present. Columnwise elution was performed using buffer D con- taining 0.1 M ammonium sulfate, 25 pg/ml of core histone, 0.1 p~ TJ, and 1 nM [1251]Tn applied to the affinity column at a flow rate of 2 to 3 column volumes/h. Fractions (4.5 to 5.5 ml) were collected, 0.5 ml was removed for assays, and the remainder was frozen in liquid nitrogen. The amount of Ta present in the eluate was determined by monitoring the tracer levels of ['251]T3 present. Protein concentrations were determined by the method of Bradford (5) using bovine serum albumin and core histones as protein standards.

Assay of Affinity-purified Receptor-Free Tj was removed from the affinity-purified material by passing 0.4 to 0.6 ml over a small column of Sephadex G-25 fine that had been prepared in a Pasteur pipette (1.8 ml bed volume) and equilibrated at room temperature with the same buffer used for eluting the receptor from the affinity gels. The first 0.8 ml of elute was discarded and the excluded peak containing the Ts-binding activity was collected in the next 0.8 ml. For multiple assays requiring more receptor, 4 ml of affinity purified material was applied to a 12-ml column, the first 4 ml eluting was discarded and 5 ml was collected. After measuring the amount of T.3 remaining in the eluate (by counting the tracer ['251]T3 present), the specific ['2sI]T:3 binding was measured using the normal assay proce- dure (4). Assay samples were incubated 2 to 4 h a t 22 "C followed by incubation overnight a t 4 "C, prior to final chromatography on Sephadex G-25. Dilution of [12*1]T3 by endogenous unlabeled hormone was calculated assuming complete exchange between labeled and unlabeled hormone. Unless noted, core histone was present at 50 pg/ ml in all assay buffers.

DEAE-Sephadex Chromatography-Chromatography on DEAE- Sephadex was performed using a modification of the procedure of Silva et at. (6). [1251]T:l-binding assays were performed as described above, except that the final Sephadex G-25 column was equilibrated with buffer E. The Sephadex G-25 eluate (containing receptor labeled with ['*'I]T, and less than 0.02 M NaC1) was applied to a small column of DEAE-Sephadex A-25 (0.5 to 1.0 ml bed volume) equilibrated with buffer E. After washing with several column volumes of buffer E, the receptor was eluted with either a linear gradient from 0 to 0.4 M NaCl in buffer E or a single step of 0.2 M NaCl in buffer E. The radioactivity was measured and specific binding was calculated by subtracting nonspecifically bound TS (determined in parallel incubations contain- ing excess unlabeled hormone) from the total bound ["51]Tl eluted from the column. Protein concentrations were determined by the method of McKnight (7) using bovine serum albumin as the protein standard. The NaCl concentrations were measured by taking a 10-pl aliquot, mixing with 1 ml of distilled water, measuring the conductiv- ity, and comparing with standards of known NaCl concentration.

RESULTS

Character izat ion of Nuclear Receptors from Rat, Sheep, and Steer Liver-Our initial work using affinity chromatog- raphy to purify the thyroid hormone receptor was performed using receptors solubilized from rat liver nuclei. However, due to the time and expense involved in obtaining kilogram quan- tities of rat liver, this source may be impractical for larger

12096 Affinity Chromatography of Thyroid Hormone Receptors

scale purifications. Therefore, sheep and steer livers were also used as sources of thyroid hormone receptors for the studies reported here, since they offer an opportunity for obtaining large quantities of starting material rapidly and inexpensively. Fig. 1 shows the results of Scatchard analyses of the binding of T3 and T4 by nuclear extracts obtained from these three sources. All three contained high affinity T3 binding sites, with equilibrium dissociation constants of 89 PM & 38 (S.D., n = 7) for rat liver nuclear extract, 103 PM 5 41 (S.D., n = 3) for sheep liver, and 116 PM It- 45 (S.D., n = 6) for steer liver. The Scatchard plot for steer liver nuclear extract also indicates the

2ot RAT LIVER NUCLEAR EXTRACT

I

p i 0 5 \ 0 50 100 150 200

0 4K SHEEP LIVER NUCLEAR EXTRACT

0 10 20 30 40

NUCLEAR EXTRACT

O o 3 3 20 40 60 80 100

BOUND (pM)

FIG. 1. Scatchard analysis of the binding of [Iz6r]T3 (a) and [12sI]T4 (0) by rat liver (upper panel), sheep liver (middle panel), and steer liver (lower panel) nuclear extract. Nuclear extracts were prepared and assayed as described under “Experimental Procedures,” except that the concentrations of [“‘I]Tg and [’251]T, were varied from IO to 5000 and 6 to 15000 PM, respectively. Nonspe- cific binding was subtracted from all assays. The assay incubations contained 50 p1 of nuclear extract diluted to a final volume of 500 pl, with a final protein concentration of 265 p g / d for rat liver, 115 pg/ ml for sheep liver, and 350 pg/ml for steer liver. The apparent equilibrium dissociation constants were calculated by linear regression analysis. The maximum binding capacity for T.1 was 683 fmol/mg of protein for rat liver nuclear extract, 222 fmol/mg for sheep liver, and 84 fmol/mg for steer liver.

CRUDE EXTRACT AFFINITY PURIFIED

0 a

... STEER

401 20 T\k I. 1”

10-9 IO-* IO-^

STEER

COMPETITOR (MI FIG. 2. Competition for [1251]triiodothyronine binding in

crude nuclear extract and affinity-purified thyroid hormone receptor. Standard binding reactions were prepared containing 1 nM [‘251]T3 and either 50 p1 of crude nuclear extract from rat, sheep, or steer liver, or 100 p1 of receptor which had been purified from nuclear extract by affinity chromatography and passed over Sephadex G-25, as described under “Experimental Procedures.” Various concentra- tions of competing unlabeled Ts (a), Tq (A), isopropyl T2 (A), and reverse TS (0) were added, and, after incubation for 2 h a t 22 “C then overnight at 4 “C the reactions were assayed for bound radioactive hormone by the standard procedures.

presence of a second class of lower affinity T:3 binding sites. The maximum binding capacities for the high affinity T3 binding sites correspnds to a yield of approximately 50 pg of receptor/kg of tissue for rat liver, 15 ,ug/kg for sheep liver, and 10 ,ug/kg for steer liver. Tq binding sites which had higher dissociation constants than the TR binding sites were also present in these nuclear extracts from all three sources. It is noteworthy that in the steer liver extract, the content of T4 binding sites greatly exceeds the number of T3 binding sites. The nature of these was not evaluated. Since they do not have a high affinity for T3, it is unlikely that they are receptors. However, their presence underscores the need to characterize the binding activity in any purified preparations to verify its identity.

When the ability of nonlabeled hormones to compete with [1251]T3 for binding in the nuclear extracts was examined (see left half of Fig. 2 ) , the rank order of competition obtained for all three sources was: TB 2 isopropyl T.2 > Tq > reverse T3. Isopropyl-T2 appeared to be a weaker competitor for [’2sI]T3 binding in the steer or sheep liver nuclear extracts than it was in the rat liver nuclear extract. When chromatographed on Sephadex G-100, nuclear extracts from all three sources yielded an excluded peak of T3-binding activity and an in- cluded peak with a molecular weight of approximately 50,000 to 60,000 (data not shown). These findings are similar to our previous results with extracts from rat liver (4). Thus, al- though some differences were observed between nuclear ex- tracts from sheep, steer, or rat liver, all three sources appear to contain the putative high affinity thyroid hormone receptor.

Exchange Assay of Affinity Purified Receptors-In the current studies, procedures were developed for using the affin- ity support matrices described in the preceeding ( 2 ) paper to

Affinity Chromatography of Thyroid Hormone Receptors 12097

purify the receptors. Solubilized nuclear receptors are reacted with an affinity matrix containing immobilized hormone. After nonspecifically bound proteins are removed from the gel by washing, the specifically bound receptors are eluted by adding an excess of free hormone. In principle, it would be most efficient to elute with radioactively labeled hormone, since this should facilitate determination of ['251]T3-binding activity. However, in practice this became both prohibitively expensive and impractical because of the high concentrations of T3 required to effect elution from the affinity gel and the exces- sive nonspecific binding at these high concentrations of hor- mone (2). Therefore, it was advantageous to employ condi- tions whereby nonradioactive hormone could be used to elute the receptor from the affinity gel, provided that exchange of the receptor-bound nonradioactive hormone with radiolabeled hormone could subsequently be achieved.

The feasibility of such an approach was first examined in studies employing crude nuclear extract. After nonradioactive T3 was bound to the receptor in nuclear extracts, the free hormone was removed by passing the extract over a small column of Sephadex G-25 and collecting the excluded protein peak. The amount of T3 that remained associated with the excluded protein peak was monitored with tracer ['251]T3 or T3 radioimmunoassay (8). The eluates from columns of fine grade Sephadex G-25 (particle size 20 to 80 pm) contained less T3 than eluates from columns of medium grade Sephadex (50 t o 150 pm), probably due to the slower flow rate (and thus increased time for hormone dissociation) of the fine grade gel. The eluate was assayed for specific T3-binding capacity using ['"I]T3 in the standard binding assay. The samples passed over Sephadex G-25 after preincubation with Ts showed greater than 80% of the specific ['251]T3-binding capacity of control samples preincubated at 4 "C in the absence of unla- beled hormone, and had approximately the same binding capacity as control samples preincubated at 22 "C (Table I). This demonstrates that added [1251]T,3 is capable of exchange with unlabeled hormone bound to the receptor. Thus, this exchange assay is shown to be a feasible method of approxi- mating the specific T:3-binding capacity of the receptors in T3 affinity gel eluates.

Effect of Washing and Counter Ligand on Elution-A

TABLE I Assay of T3 binding after removal of free TJ by Sephadex G-25

chromatography Crude extract from rat liver nuclei (0.1 ml of extract diluted to 0.5

ml with buffer G) was incubated for 3 h at 22 "C in the presence of various concentrations of Ts. Following incubation, 0.4 ml was filtered on Sephadex G-25 fine and 0.8 ml of eluate was collected, as in the standard T,-binding assay. The amount of hormone remaining in the eluate was monitored by adding tracer levels of ["51]T3 to the Ta used for the fwst incubation. The eluate from sample 3 contained 0.04 nM TJ and the eluate from sample 4 contained 0.09 nM TI. As a control, Sop1 of crude extract was diluted to 0.4 ml with buffer G and incubated 3 h at 4 "C or 22 "C in the absence of Ta. At the end of the incubation, buffer G was added to give a final volume of 0.8 mi. Ta binding was assayed in all the samples by the standard assay procedure (4), after incubating 0.4 ml of the sample with 1 nM [iZSI]TJ at 22 "C for 3 h. The binding by sample 1 (incubated a t 4 "C without Ta before assay) was assumed to be 100% binding, and binding by the other samples was normalized to this value.

Treatment before assay Specific binding R control

1. Control: 3 h, 4 "C 100 2. Control: 3 h, 22 "C 83 3. 1 nM T.3, 3 h, 22 "C, then filtered with Sephadex 88

4. 10 nM Tar 3 h, 22 "C, then filtered with Sephadex 80 G-25

G-25

TABLE I1 Effect of thyroid hormone concentration on batchwise elution of

TI- binding activity from affinity gel Batchwise adsorption of receptor to the affinity gel was performed

at room temperature by incubating 200 ml of steer liver crude nuclear extract with 5 ml bed volume of gel for 1.5 h with constant stirring. Following adsorption, the gel was removed by centrifugation, resus- pended in diluted crude extract, and divided into several equal sam- ples. The gel samples were either: 1) washed once with nuclear extract diluted 1 : l O with buffer G or 2) washed three times with 10 volumes of buffer G. The washed gels were resuspended in an equal volume of the same wash buffer and unlabeled TS or TI (containing a 1000-fold dilution of ['251]T~ or [?IT4 as tracer) was added at the indicated concentration. After incubation for 25 h at 4 "C with occasional mixing, the gel was pelleted by centrifugation, the hormone concen- tration in the supernatant was measured, and samples were assayed for specific ['251]TJ binding activity, as described under "Experimental Procedures." As a control for [i251]T3 binding by receptor present in the diluted nuclear extract prior to the elution, mock elutions were performed: diluted crude extract was incubated with unlabeled TJ in parallel to the gel elutions into diluted crude extract. The net amount of receptor eluted from the gel was then calculated by subtracting the specific Ts binding measured in these control samples from the binding measured in the supernatants from the gels eluted into crude extract.

Recovery, gel washed with

Hormone added Hormone in eluate Diluted nuclear Buffer G extract

B 9c

1.0 pM TI 0.15 pM TI 2.2 0.4 10 pM TI 1.6 pM TI 14 1.0

100 pM TI 26 ~ L M Ts 59 (N.D.) 10 PM T4 1.9 p~ T4 6 (N.D.)

number of experiments were performed to determine the optimal conditions for using counter ligand to elute receptors from the affinity gels. In the initial experiments, batchwise elution was used in order to facilitate the examination of a large number of variables, such as conditions for washing the affinity gel and eluting the receptor. The results obtained from these experiments were then adapted to allow the use of columnwise elution for purifying larger quantities of receptor.

At first, we obtained only very low recoveries of T3-binding activity from gels that had been washed extensively prior to elution. Thus, to ensure that it was indeed possible to recover a reasonable portion of the receptors bound to the gel, we attempted to minimize possible leakage of receptor from the gel during the washing procedures. In the experiment de- scribed in Table 11, the loaded gel was washed only briefly with a 1 : l O dilution of the nuclear extract prior to adding free hormone to elute the receptor. A much greater recovery of receptor was obtained from gels treated by this procedure than from the gels washed with buffers containing no protein. It was also observed (Table 11) that, consistent with previous formulations (2), the recovery of receptor increased as the amount of free hormone added to the gel was increased, and twice as much binding activity was eluted from the gel in the presence of T3 as was eluted with an equal concentration (10 pM) of Tq. After incubation with the gel, only 15 to 26% of the added free hormone remained in solution, while 74 to 85% of the hormone appeared to be adsorbed to the gel, as monitored by tracer '251-labeled hormone present in the free hormone.

Use of Histones in the Affinity Chromatography Buffers- Other investigations in our laboratory (3) have indicated that purified core histones (H2A, H2B, H3, and H4, themselves devoid of T3-binding activity) can restore T3 binding activity to receptor preparations that have lost their ability to bind hormone. Fig. 3 shows that the addition of core histones (concentration is 7 p~ at 100 pg/ml) to the wash, elution, and

12098 Affinity Chromatography of Thyroid Hormone Receptors

200F-”-” ” ~~ 1

CORE HISTONE (ig/ml)

FIG. 3. Effect of added core histone on recovery of T,-bind- ing activity during affinity chromatography. Crude extract from sheep liver nuclei (46 ml of extract) was bound overnight to 7 ml bed volume of TD affinity gel, as described under “Experimental Proce- dures.” After binding was completed, the gel was resuspended in buffer D containing 0.3 M ammonium sulfate, divided into five equal portions, and packed into small columns. Each sample was washed at 30 “C with 14 ml of buffer D containing 0.3 M ammonium sulfate and the indicated amount of core histones, followed by washing at 22 “C with the buffer D containing 0.1 M ammonium sulfate plus histones. The washed gels were placed in test tubes and 1.2 ml of buffer D containing 0.1 M ammonium sulfate, 50 PM Ts, 50 nM [‘251]T3, and the indicated amount of core histones was added. After incubation for 3 h at room temperature, the supernatant was removed and assayed for T3 binding (0) as described under “Experimental Procedures.” Control assays for Ta binding by histones (0) contained histones but no receptor. Core histone was present in all assay buffers at the concentrations indicated in the graph.

assay buffers increased the amount of receptor recovered from the affinity gel. Ovalbumin (500 pg/ml, 11 p ~ ) or lysozyme (500 pg/ml, 35 p ~ ) did not have any significant effect on the recovery of receptor.

Effect of Time, Temperature, Hormone Concentration, and Salt on The Elution-Experiments were performed to deter- mine the optimum time, temperature, hormone concentration, and salt concentration for batchwise elution of the receptor in the presence of histones. The greatest yield of receptor was obtained when the elution was allowed to proceed at room temperature for 2 to 4 h (Fig. 4) in the presence of 10 p~ TB. The highest specific activity was observed when the wash buffer contained 0.4 M ammonium sulfate and the elution buffer contained 0.1 to 0.2 M ammonium sulfate (Table 111). The high ionic strength buffer used in the first wash minimizes nonspecific ionic interactions of proteins with the gel. In the subsequent low ionic strength buffer, any remaining nonspe- cific binding proteins are bound more tightly to the gel. During this low ionic strength wash, the amount of protein being removed from the gel falls below the limits of detection. TB is then added to this same low ionic strength buffer to elute the receptor.

Elution of the Receptor from T:j Affinity Column-The optimal salt concentration, histone concentration, and tem- perature for eluting the receptor from a column of affinity gel were found to be the same as determined for batchwise elution. Likewise, elution of the receptor was completed within 3 to 4 h. Increasing the flow rate of the column did not significantly change the time required to elute the receptor. The major difference from batchwise elution was that the receptor could be eluted from the column by 0.1 p~ T a . Increasing the hormone concentration above this did not increase the total yield by more than 10% of the amount

L

TIME (hr)

FIG. 4. Kinetics of elution of T.-binding activity from the TB affinity gel at 22 O C. Crude extract from steer liver nuclei was bound to the T3 affinity gel (22 ml of extract/l.5 ml of gel), the gel was washed, and elution was started, as described under “Experimen- tal Procedures.” A t the indicated times, 0.4 ml of gel suspension was removed, the gel was pelleted by centrifugation, and the supernatant was assayed for TJ-binding activity (0). The maximum recovery achieved was 10.4% of the bound receptor.

TABLE I11 Effect of wash and elution salt concentrations on receptor

purification After binding steer liver nuclear extract (52.6 fmol of Ta bound/mg

of protein) to the T3 affinity gel, the gel was resuspended in buffer D containing 0.2 M ammonium sulfate plus 50 p g / d of core histone and divided into 20 equal portions. The gel was washed and receptor was eluted as described under “Experimental Procedures” with the excep- tion that the indicated concentrations of ammonium sulfate were used. All assays were performed at 0.2 M ammonium sulfate. The eluants contained 50 to 65 pg of protein/ml (including 50 pg of histones/ml of buffer) and the highest yield obtained corresponded to 13.5% of the staring material. Ammonium Specific activity with ammonium sulfate concentration sulfate con- during elution (M) at centration

during wash 0 0.05 0.1 0.2 0.3 0.4 0.5 M fmol T , bound/mgprotein

0.2 130 150 300 330 0.3 120 160 370 340 230 0.4 450 450 370 190 0.5 120 220 270 370 270 220 190

0 ..;’ , J 2 10 20 30

FRACTION NUMBER ’ 40

FIG. 5. Elution of rat liver nuclear thyroid hormone receptor from Ts affinity gel. Affinity chromatography was performed as described under ‘‘Experimental Procedures” and in Table IV, exper- iment 1. Fractions 1 to 31 were 8 ml each and collected at a flow rate of 60 ml/h, and fractions 32 to 48 were 5.5 ml each and collected at a flow rate of 18 d / h .

Affinity Chromatography of Thyroid Hormone Receptors 12099

obtained at 0.1 p~ T3, while lowering the TI concentration to 0.01 p~ decreased the yield by 40%. With batchwise elution, maximal yield was not obtained until the hormone concentra- tion had reached 10 p ~ . The affinity gel elution profie for receptor obtained from rat liver nuclei is shown in Fig. 5. For the experiments shown in Table IV the total amount of receptor eluted from the gel was approximately 15 to 18% of the amount originally bound. In other experiments, the total yield was usually between 10 and 35%. As the size of the column and amount of receptor used as increased, there was also generally an increase in the yield of receptor.

Purification Achieved-Since the protein concentration in the affinity gel eluants was the same as the protein concentra- tion in the elution buffer (which contained 25 pg/ml core histones), the majority of the protein present is probably exogenously added core histone. To estimate the purification obtained over the starting material, the core histones were separated from the receptor by chromatography on DEAE- Sephadex. When applied at a low ionic strength, the T3 receptor binds to DEAE-Sephadex (6) but the core histones pass through the column. As shown in Fig. 6, the receptor elutes at approximately the same salt concentration as the receptor in crude extract, -0.11 M NaCl, when the column is eluted with a linear salt gradient. However, since gradient elution diluted the receptor beyond our limits of protein detection, the purification of the receptor was instead deter- mined after elution with a single step of 0.2 M NaC1, which yields more concentrated receptor. Since the receptor in crude nuclear extract was purified less than 2-fold when subjected to this stepwise DEAE-Sephadex chromatography procedure, stepwise DEAE-Sephadex chromatography of the affinity gel eluate probably does not significantly increase the purity of

TABLE IV Purification of rat liver and sheep liver nuclear receptor

In experiments 1 and 2, 45 ml of rat liver nuclear extract was chromatographed on Sephadex G-100, as described under “Experi- mental Procedures,” and the peak fractions (135 ml) were applied to a T3-affinity gel (10 ml bed volume) at a flow rate of 1 ml/min. The affinity columns were washed, receptor was eluted, and 5-ml samples of the eluate were chromatographed on DEAE-Sephadex, using step elution, as described under “Experimental Procedures” and Fig. 4. In experiment 3, 45 ml of sheep liver nuclear extract was chromato- graphed on Sephadex G-100, and the peak fractions (155 m l ) were applied to a 3-ml T:j affinity gel at a flow rate of 1 ml/min. After washing and elution from the column, 4-ml samples were chromato- graphed on DEAE-Sephadex. All T3-binding assays were performed at 1 nM [1251]Ta.

Experiment Specific pzgL- Total T:I Yield %Fit activity binding for step ~x+rar+

1. Rat liver

Sephadex G-100 Nuclear extract

Affinity gel DEAE-Sepha-

dex 2. Rat liver

Nuclear extract Sephadex G-100 Affinity gel DEAE-Sepha-

dex 3. Sheep liver

Nuclear extract Sephadex G-100 Affinity gel DEAE-Sepha-

dex

mg pro- -fold pmoU

tein

0.43 1.4 3.3

220 510

0.43 1.22 2.8

24 7 570

0.094 0.18 1.9

44 460

pmol

46.5 22.4 4.02 2.5

49.9 21.2 3.24 1.99

3.4 0.78 0.115 0.044

w

48 18 62

42 15 61

23 15 37

FRACTION NUMBER

FIG. 6. DEAE-Sephadex chromatography of rat liver nu- clear extract and affinity chromatography-purified TI recep- tor. As described under “Experimental Procedures,” receptor in crude nuclear extract (0) or the T.3 affinity gel eluate from Table IV, experiment 2 (O), was prebound with 1 nM [1261]T,y, loaded onto separate columns of DEAE-Sephadex (1 ml bed volume, 7.5 X 23 mm), and eluted by a linear NaCl gradient (---). The fraction volumes were 2.5 ml each during the loading and wash (fractions 1 to 10) and were 0.2 ml each during the gradient (fractions 11 to 50). The crude nuclear extract applied to the column contained 1065 fmol of bound [‘251]T3 (3.2 X 10’ cpm) and 2.65 mg of protein in a volume of 10 ml (the radioactivity eluting in fractions 4 and 5 is probably due to overloading). Recovery of radioactivity from the column was 62%; 34% of the [‘”I]T3 applied was in the peak eluted by NaCl (fractions 18 to 28). The affinity-purified receptor applied to the column con- tained 291 fmol of bound [”‘II]Ta (8.7 X IO5 cpm) and 0.3 mg of protein (including histones) in a volume of 15 ml; approximately 54% of the applied radioactivity was recovered in the elution peak and the total recovery was 70%. No protein was detectable in the eluted peak. No further radioactivity was recovered by raising the NaCl concentration of both columns to 1.0 M NaCl.

the receptor. However, Silva et al. (6) have reported a 10- to 25-fold puritication of the receptor by using gradient elution from DEAE-Sephadex, and, in larger scale preparations, such treatment may be effective for attaining additional purifica- tion of the affinity-purified receptor.

Table IV shows the results of experiments in which crude extracts from rat or sheep liver nuclei were chromatographed on Sephadex G-100, purified by affinity chromatography (The elution profile for experiment 1 is shown in Fig. 5.), and then subjected to DEAE-Sephadex chromatography to remove the exogenously added core histones. With the rat liver receptor, this procedure resulted in greater than 500-fold purification over nuclear extract, or approximately 200-fold purification over the Sephadex G-100 eluate applied to the affinity column. A similar degree of purification was obtained with receptor obtained from sheep liver nuclei.

Characteristics of the Affinity Purified Material-Fig. 2 (right) shows a study of the hormone-binding specificity of the affinity-purified binding activities of receptors obtained from the three different animal sources. In this experiment, the ability of nonlabeled hormones to compete with [Iz5I]T3 for binding to the affinity-purified receptor was monitored. T3 had the greatest capacity for competing with [‘251]T3 for binding to the affinity-purified receptor. Isopropyl TP also had a high affinity for the purified receptor. T, had a slightly lower affinity, and reverse T3 had a much lower affinity. This rank order of binding potency parallels that of the original receptor preparations (4).

A Scatchard analysis of T3 and T4 binding by the affinity- purified receptor from experiment 1 of Table IV is shown in Fig. 7. The apparent equilibrium dissociation constant (Kd) of the affinity-purified receptor for T3 was 52 PM, while the Kd for T4 was 1.5 nM, approximately 20 times greater than that

12100 Affinity Chromatography of Thyroid Hormone Receptors

ROUND I p M l

FIG. 7 . Scatchard analysis of [lZ6I]T3 (0) and ['251]T4 (0) bind- ing by rat liver nuclear thyroid hormone receptor after affinity chromatography purification. The eluate from the affinity column described in Table IV, experiment 1, was assayed for hormone binding as described under "Experimental Procedures," except that the con- centrations of ["'II]T3 and ["'IlTq were varied from 20 to 2000 and 100 to 10,OOO PM, respectively. The assays also contained a small amount of unlabeled T3 (16 PM) which was not removed from the affinity gel eluate by the Sephadex G-25 chromatography step prior to the assay. Nonspecific binding was subtracted from all assays.

x

E 4 -

BSA Ovalbumm

IO

0

6

4

2

0 40 50 60 70 80

ELUTION VOLUME (ml)

FIG. 8. Sephadex G-100 fractionation of rat liver nuclear extract (0) and affinity-purified receptor (0) bound to ['"IITT~. Rat liver extract or affinity-purified receptor (Table IV, experiment 2) was incubated in a standard 0.5-ml reaction mixture containing buffer G and 1 nM [12"I]T3 for 6 h at room temperature. The samples were chilled and loaded onto a Sephadex G-100 column (1.5 X 90 cm, 130 ml bed volume) equilibrated in buffer G at 4 "C. Fractions (1.6 to 1.7 ml) were collected at 0.3 ml/min and the amount of [1251]T3 was determined in each fraction. The column was standardized with bovine serum albumin (BSA), ovalbumin, blue dextran, and cyto- chrome c (V, = 96 ml).

for T3. In a Scatchard analysis of the affinity-purified receptor from experiment 2, the Kd for TR was determined to be 46 PM, while the Kd for T4 was 1.1 nM. In both experiments, it was observed that the concentration of high affinity T s and T4 binding sites is the same. The Kd values are approximately the same as those of the initial nuclear extract, which had a K d for T3 of 78 PM and a Kd for Tq of 0.67 nM.

The affinity-purified receptor eluted at the same position as the receptor in nuclear extract during chromatography on Sephadex G-100 (Fig. 8). This position corresponds to a glob- ular protein with a molecular weight of 60,000 for the rat and 55,000 for the sheep receptor. The affinity-purified receptor also sedimented through sucrose gradients at the same posi- tion as the receptor in the crude nuclear extract (not shown).

DISCUSSION

In the current studies, we have employed the technique of affinity chromatography to purify the nuclear thyroid hor- mone receptors. Our findings suggest that this method can be utilized to obtain the receptors with a reasonable yield and a substantial purification.

Our initial work with affinity chromatography was per- formed using thyroid hormone receptors solubilized from rat liver nuclei. However, since this source may be impractical for larger scale purifications, steer and sheep livers were also used as sources of thyroid hormone receptors for the studies re- ported here. These tissues can provide a less expensive source for large quantities of hormone receptor. The purification procedures developed here appear to be effective with recep- tors obtained from any of these sources.

After solubilized receptors were bound to the affinity gel and nonspecifically bound proteins were removed by extensive washing, the specifically bound receptors were eluted from the gel by adding free hormone (counter ligand). The amount of receptor recovered from the gel increased as the concentra- tion of T3 added was increased, and Tq was found to be less effective than Tj in eluting the receptor from the gel. This result is consistent with the receptor-gel interaction being biospecific, that is, due to specific interactions of the receptor with matrix-bound TQ rather than due to nonspecific interac- tions.

The high concentration of unlabeled TR added during the elution prevented direct use of the normal assay for measuring high affinity TR binding by the receptor. Therefore, before performing the binding assay, the free TR was removed by passing the affinity gel eluate over a Sephadex G-25 column. This was done at room temperature and with a low flow rate in order to allow maximal dissociation of protein-bound hor- mone. The level of T3 remaining with the receptor preparation was monitored by tracer levels of ['"IITj in the hormone used to elute the receptor from the affinity gel. The concentration of unlabeled TS could usually be reduced below 1 nM for batchwise eluates containing 10 PM T3 and below 20 pM for affinity column eluates containing 0.1 PM T3. This permitted use of the normal binding assay, in which the receptor is incubated with 1 nM ['2"I]T3 and nonspecific binding is meas- ured in parallel assays tubes to which a 1000-fold excess of unlabeled hormone is added. It also allowed us to perform a Scatchard analysis of hormone binding by the affinity-purified receptor. Model experiments using crude nuclear extract in- dicated that labeled hormone could exchange with unlabeled hormone bound to the receptor. However, since 100% ex- change was not observed and since this assay involves a number of manipulations and incubations (including chro- matography on two Sephadex G-25 columns), we may expect that the actual yield of receptor from the affinity column is probably greater than the values determined using this ex- change technique, especially at the low protein concentrations in the current experiments.

In our initial affinity chromatography studies, it was ob- served that although adsorbed receptor could be eluted from gels that were unwashed or briefly washed with diluted nu- clear extract, only very small quantities of receptor could be eluted from gels that had been extensively washed. Since core

Affinity Chromatography of Thyroid Hormone Receptors 12101

histone-containing preparations have been shown (3) to re- store high affinity T3-binding activity to preparations that have lost this activity, it was considered that loss of core histones or some related factor during affinity chromatogra- phy may have resulted in a loss of the receptor's high affinity Ty-binding capacity. Indeed, it was found that the addition of purified core histones to the affinity chromatography buffers could increase the amount of receptors eluted from the gel. The addition of histones alone, without counter-ligand, did not result in the elution of the receptor. Thus, the increased yield is not simply due to elution of the receptor by histones. Other proteins (lysozyme and ovalbumin) did not increase the yields. This effect of core histones on recovery of Ts-binding activity during affinity chromatography is consistent with the hypothesis that interactions of the receptor with histones or some related protein are important in maintaining high affin- ity T3-binding by the receptor.

It was important to ask if the material eluted from the affinity gel is indeed the high affinity nuclear thyroid hormone receptor. This was first examined in a study of the hormone- binding specificity of the affinity-purified receptor. Fifty per cent of the binding of 1 nM [1251]T3 was blocked by the addition of 1 nM unlabeled TB, indicating that the affinity-purified receptor possesses a high affinity for TS. The rank order of competitive inhibition of ['251]T3 binding in the affinity-puri- fied preparation was T3 2 isopropyl Tz > T4 > reverse T3. This is identical with that of the crude nuclear thyroid hor- mone receptor (4, 6, 9-11). Scatchard analysis of Ts and T4 binding by the affinity-purified material confirmed that it was indeed the high affinity nuclear receptor. The chromato- graphic properties of the affinity-purified receptor on Sepha-

dex G-100 and DEAE-Sephadex, as well as its sedimentation on sucrose gradients, were also identical with those of the crude receptor (4,6).

Of significance is the finding that the purified receptors retain their capacity for reversible hormone binding. The reversible binding by thyroid hormone receptor may prove useful in examining the effects of the presence or absence of thyroid hormone on the functioning of the receptor and its role in the control of gene expression.

REFERENCES 1. Eberhardt, N. L., Apriletti, J. W., and Baxter, J. D. (1980) in

Biochemical Actions of Hormones (Litwack, G., ed) Vol. 7, pp. 311-394, Academic Press, New York

2. Latham, K R., Apriletti, J. W., Eberhardt, N. L., and Baxter, J. D. (1981) J. Biol. Chem. 256, 12088-12093

3. Eberhardt, N. L., Ring, J. C., Johnson, L. K., Latham, K. R., Apriletti, J. W., Kitsis, R., and Baxter, J. D. (1979) Proc. Natl. Acad. Sci. U. S. A . 76, 5005-5009

4. Latham, K. R., Ring, J. C., and Baxter, J . D. (1976) J. Biol. Chem.

5. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 6. Silva, E. S., Astier, H., Takane, U., Schwartz, H., and Oppenhei-

mer, J . H. (1977) J . Biol. Chem. 252,6799-6805 7. McKnight, G. S. (1977) Anal. Biochem. 78,86-92 8. Gharib, H., Ryan, R. J., Mayberry, W. E., and Hockert, T. (1971)

J. Clin. Endocrinol. 33, 509-516 9. Oppenheimer, J . R., Schwartz, H. L., Dillman, W., and Surks, M.

I. (1972) Biochem. Biophys. Res. Commun. 55, 544-550 10. Koerner, D., Surks, M. I., and Oppenheimer, J. H. (1974) J. Clin.

Endocrinol. Metab. 38, 706-709 11. Torresani, J., and DeGroot, L. J. (1975) Endocrinology 96, 1201-

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