Proc. Natl. Acad. Sci. USA Vol. 84, pp. 3420-3424, May 1987 Medical Sciences Purification and characterization of rat liver nuclear thyroid hormone receptors (nuclear preparation/DNA-celiulose/heparin-Sepharose/pyridoxal 5'-phosphate/photoaflinity labeling) KAZUO ICHIKAWA AND LESLIE J. DEGROOT* Thyroid Study Unit, Department of Medicine, The University of Chicago, Chicago, IL 60637 Communicated by Leon 0. Jacobson, January 9, 1987 (received for review September 23, 1986)
ABSTRACT Nuclear thyroid hormone receptor was puri- protein too low for practical use. In the present paper, we fied to 904 pmol of L-3,5,3'-triiodothyronine (T3) binding report a purification ofnuclear thyroid hormone receptor that capacity per mg of protein with 2.5-5.2% recovery by sequen- provides the purest receptor yet reported (904 pmol of T3 tially using hydroxylapatite column chromatography, ammo- binding capacity per mg of protein) with abundance of nium sulfate precipitation, Sephadex G-150 gel filtration, 6.4-14.7 ,ug of receptor protein per purification. Partial DNA-cellulose column chromatography, DEAE-Sephadex col- characterization was also performed using this purified re- umn chromatography, and heparin-Sepharose column chro- ceptor. matography. Assuming that one T3 molecule binds to the 49,000-Da unit of the receptor, we reproducibly obtained 6.4-14.7 ,ug ofreceptor protein with 4.24.9% purity from 4-5 MATERIALS AND METHODS kg of rat liver. Elution of receptor from the heparin-Sepharose column was performed using 10 mM pyridoxal 5'-phosphate, L-T3, D-3,5,3'-triiodothyronine (D-T3), L-3,5,3',5'-tetraiodo- which was observed to diminish binding ofreceptor to heparin- thyronine (L-thyroxine; T4), 3,5,3'-triiodothyroacetic acid, Sepharose or DNA-cellulose. This effect was specific for double-stranded calf thymus DNA, pepstatin A, and sper- pyridoxal 5'-phosphate, since related compounds were not mine were from Sigma. Dithiothreitol was from Bethesda effective. Purified receptor bound T3 with high affinity (6.0 x Research Laboratories. [1251]T3 (3400 ,uCi/,ug; 1 Ci = 37 109 liter/mol), and the order of affinity of iodothyronine GBq) was from New England Nuclear. Sephadex G-150 and analogues to purified receptor was identical to that observed DEAE-SephadexA-50werefromPharmacia. CelluloseMunk- with crude receptor preparations [3,5,3'-triodothyroace- tell 410 was from Bio-Rad. Heparin-Sepharose was from tic acid > L-T3 > D-3,5,3'-triiodothyronine (D-T3) > L-thy- Pierce. Hydroxylapatite gel was prepared as described (14). roxine > D-thyroxine]. Purified receptor had a sedimentation DNA-cellulose was prepared as described (15). coefficient of 3.4 S. Stokes radius of 34 A, and calculated Large-Scale Preparation of Nuclear Extract from Rat Liver. molecular mass of 49,000. Among several bands identified by Solutions used for nuclear extract preparation were as silver staining after electrophoresis in NaDodSO4/polyacryl- follows. Solution A: 0.32 M sucrose/3 mM MgCl2/2 mM amide gels, one 49,000-Da protein showed photoaffinity label- EDTA. Solution B: 2.8 M sucrose/i mM MgCl2. Solution C: ing with ['25I]thyroxine that was displaceable with excess 0.32 M sucrose/i mM MgCl2/20 mM Tris'HCl, pH 7.4/1 mM unlabeled T3. The tryptic fragment and endogenous protein- dithiothreitol/0.1 mM phenylmethylsulfonyl fluoride (PhMe- ase-digested fragment of the affinity-labeled receptor showed S02F). Solution D: 0.3 M KCl/1 mM MgCl2/10 mM potas- saturable binding in 27,000-Da and 36,000-Da peptides, re- sium phosphate, pH 8.0/1 mM dithiothreitol. spectively. These molecular masses are in agreement with Nuclei were prepared from quickly frozen rat liver ob- estimates from gel filtration and gradient sedimentation, indi- tained from Rockland (Gilbertsville, PA). Frozen liver (140 g) cating that affinity labeling occurred at the hormone binding was pulverized and 300 ml of solution A that had been domain of nuclear thyroid hormone receptor. This procedure warmed up to 37°C was added with stirring. Tissue was reproducibly provides classical native rat liver T3 nuclear thawed in a minute with a final temperature of 5°C. The receptor in useful quantity and purity and of the highest folding procedures were performed at 0-4°C. After sediment- specific activity so far reported. ing the tissue, the supernatant was discarded and 200 ml of cold solution A was added with stirring. After sedimenting Nuclear thyroid hormone receptor was first described in 1972 the tissue, the supernatant was discarded and tissue was by Oppenheimer et al. (1), and partially purified receptor divided into two parts. Two hundred milliliters of cold preparations have been characterized. From these studies, solution A with 1 mM PhMeSO2F, 100 nM pepstatin, 1 mM the receptor appears to be a chromatin-associated nonhistone dithiothreitol, and 1 mM spermine was added into each protein (2) with molecular mass of47,000-57,000 (3, 4), which portion. The tissue was homogenized in ice for 20-30 sec binds L-3,5,3'-triiodothyronine (T3) with high affinity in the using a Tekmar homogenizer (Cincinnati, OH) at submaximal presence of sulfhydryl groups (5). An important role of this speed; this was followed by immediate centrifugation at 1000 receptor protein in thyroid hormone action is accepted (6-9). x g for 10 min. After centrifugation, the supernatant was Several laboratories have partially purified receptor using discarded. The pellet was mixed with 200 ml of ice-cold Sephadex G-100 gel filtration, anion-exchange column chro- solution A and was shaken vigorously. After centrifugation at matography, DNA-Sepharose column chromatography, 1000 x g for 10 min, the supernatant was discarded. The high-pressure liquid chromatography, and T3 affinity chro- pellet was mixed with 200 ml ofcold solution A containing 1% matography (3, 10-13). However, these purifications are still far from homogeneity and yielded amounts of receptor Abbreviations: T3, L-3,5,3'-triiodothyronine; T4, L-3,5,3',5'-tetra- iodothyronine (L-thyroxine); PhMeSO2F, phenylmethylsulfonyl flu- oride. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed at: The University payment. This article must therefore be hereby marked "advertisement" of Chicago Thyroid Study Unit, Box 138, 5841 South Maryland in accordance with 18 U.S.C. §1734 solely to indicate this fact. Avenue, Chicago, IL 60637. 3420 Downloaded by guest on September 30, 2021 Medical Sciences: Ichikawa and DeGroot Proc. Natl. Acad. Sci. USA 84 (1987) 3421
Triton X-100, 10 mM Tris HCl (pH 7.4), 1 mM PhMeSO2F, diameter of 2.5 cm with column volume of 500 ml) equili- 100 nM pepstatin A, and 1 mM dithiothreitol and was shaken brated with buffer B. Active fractions after gel filtration were vigorously. After centrifugation at 1000 x g for 10 min, the pooled, diluted 6 times with buffer D, and applied to a supernatant was discarded. The pellet was mixed with 200 ml DNA-cellulose column (50 ml) equilibrated with buffer C. of solution A containing 10 mM Tris-HCl (pH 7.4) and After washing the column with 200 ml of buffer C, receptor centrifuged as above. The supernatant was discarded and the was eluted by a linear gradient of 0.05-1.0 M KCl in a total pellet obtained from 140 g of tissue was mixed with 100 ml of volume of200 ml. Active fractions with sufficient purity were solution B and was shaken vigorously. This mixture was pooled and diluted 10 times with buffer D and applied to a centrifuged at 30,000 rpm for 30 min using a type 30 rotor DEAE-Sephadex column (50 ml). The operative procedures (Beckman). After discarding the supernatant, the pellet was for DEAE-Sephadex were the same as those for DNA- mixed with solution C containing 0.3% Triton X-100 and was cellulose chromatography. Active fractions after the DEAE- incubated for 10 min. After centrifugation at 1000 x g for 10 Sephadex column were diluted 6 times with buffer D and min, the supernatant was discarded, the nuclear pellet was applied to a 10-ml heparin-Sepharose column equilibrated dispersed in a Vortex with solution C, and centrifuged at 1000 with buffer C. The column was washed with 30 ml of buffer x g for 10 min. The nuclear pellet thus obtained was E, and buffer E containing 10 mM pyridoxal 5'-phosphate incubated with solution D at a ratio of 1 ml for 3 g tissue was used for the elution ofreceptors. After passing 7 ml ofthe equivalent nuclei in the presence of 0.2 mM PhMeSO2F for buffer at an elution rate of 2 ml/hr, the column was stopped 1 hr with vigorous dispersion in a Vortex at 10-min intervals. for 9 hr. Elution was then continued slowly using the same Samples were centrifuged at 30,000 rpm for 120 min using a buffer. type 30 rotor. The supernatant (nuclear extract) was "snap- Assay of T3 Binding Activity. T3 binding activity was frozen" in a dry ice/acetone bath and was kept at -70TC for measured for screening of active fractions after column up to 2 wk. This method allowed one to prepare nuclear chromatography and assessment ofpurity ofreceptor in each extracts from 0.8-1.0 kg of rat liver in 1 day. preparation. Receptor preparation was incubated in 0.3 ml of Partial Purification of Rat Liver Nuclear Thyroid Hormone solution D with [1251]T3 in the presence of 50 ,ug ofprotein of Receptor. The following buffers were used. Buffer A: 0.9 M heated nuclear extract for 2 hr at 22°C. Heated nuclear KCI/0.2 mM EDTA/1 mM MgCl2/10 mM potassium phos- extract was prepared by incubating nuclear extract at 100°C phate, pH 7.7/1 mM dithiothreitol/0.1 mM PhMeSO2F. for 15 min; this was followed by centrifugation at 1000 x g for Buffer B: 0.3 M KC1/2 mM EDTA/20 mM potassium 15 min to remove any precipitate. This material had no phosphate, pH 8.0/10mM 2-mercaptoethanol. Buffer C: 0.05 specific T3 binding activity in itself. Incubation temperature M KC1/2 mM EDTA/20 mM potassium phosphate, pH of 22°C was optimum for T3 binding and binding reached a 8.0/10% glycerol/10 mM 2-mercaptoethanol. Buffer D: 20 plateau after 60-90 min. T3 binding activity was stable for up mM potassium phosphate, pH 8.0/2 mM EDTA/20% glyc- to 4 hr. Duplicate tubes contained 0.3 ,M unlabeled T3 to erol/10 mM 2-mercaptoethanol. Buffer E: 0.05 M NaCl/20 determine nonspecific T3 binding. Specific T3 binding was mM potassium phosphate, pH 8.0/2 mM EDTA. All glass- calculated by subtracting nonspecific T3 binding from total ware was siliconized before use. All procedures were per- T3 binding. After the incubation, assay tubes were immedi- formed at 0-4°C. ately cooled and Dowex 1X8, Cl-, 200- to 400-mesh anion- Nuclear extract prepared from 4-5 kg of rat liver adjusted exchange resin was added for separation of bound and free to 0.9 M KCl was applied to a hydroxylapatite column hormones as described (16). (200-ml column volume) equilibrated with buffer A. After the Photoaffinity Labeling of Nuclear Thyroid Hormone Recep- column was washed with 600 ml of the same buffer, receptor tors. Nuclear extract or partially purified receptor was was eluted by a linear gradient of 10-200 mM potassium incubated with 0.5 nM [125I]T4 (5700 ,uCi/,ug, carrier free) phosphate (pH 7.7) in a total volume of 800 ml. Active (New England Nuclear) in the presence or absence of 1 AM fractions were pooled and saturated ammonium sulfate (pH unlabeled T3. After the incubation, samples were cooled and 7.4) with 5 mM dithiothreitol/2 mM EDTA was added to 43% ultraviolet irradiation was performed from 1.5 cm distance for saturation. Precipitate was collected by centrifugation and 60 min using a long-wave ultraviolet lamp (model UVL-56, dissolved in buffer B to a total volume of 15-20 ml. This was Ultra-Violet Products, San Gabriel, CA), which has a wave- applied to a Sephadex G-150 gel filtration column (internal length peak of 366 nm. After irradiation, sodium deoxycho- [30
-I 20
0 FIG. 1. Sequential column chromatography of nuclear thyroid hormone receptor. (A) Hy- It0Ix -10 ? droxylapatite column chromatography; 6.8-ml fractions were collected during the phosphate 6 gradient. (B) Sephadex G-150 column chroma- -0 a tography; 5-ml fractions were collected. (C) z % DNA-cellulose column chromatography; 2-ml 30 z were the z w fractions collected during NaCl gradi- im- . ent. (D) DEAE-Sephadex A-50 column chroma- ° tography; 2-ml fractions were collected during 20 a- the NaCl gradient. (E) Heparin-Sepharose col- umn chromatography; 1-ml fractions were col- C.YU- lected during elution with pyridoxal 5'-phos- phate (Pyr 5'-P) or with 1 M NaCl; 8-ml fractions were collected during sample loading (1) and w washing (w). Aliquots (1 Al for A, 5 ,ul for B-D, Cll 0 and 3 ,ul for E) of fractions were assayed for specific [1251I]T3 binding (.*-) and protein con- tent (0- ). Total ['251]T3 used was 30,000 FRACTION NUMBER cpm. Downloaded by guest on September 30, 2021 3422 Medical Sciences: Ichikawa and DeGroot Proc. Natl. Acad. Sci. USA 84 (1987) RESULTS
0 Partial Purification ofNuclear Thyroid Hormone Receptors. z Fig. 1 shows the column profiles during purification of z~~~~~~ nuclear thyroid hormone receptors. In all experiments, two peaks with T3 binding activities were obtained after DNA- cellulose column chromatography, suggesting at least two DNA were 50- forms of receptors with different affinities for present in our preparation. We pooled the second peak for _.10 further column chromatography. Finally, we used pyridoxal 5'-phosphate to elute nuclear receptor from a heparin- resulting in -1.5-fold further purification 5-P Sepharose column, Z Pyridoxal and concentration of receptor. Fig. 2 demonstrates the inhibitory effect ofpyridoxal 5'-phosphate on nuclear thyroid 0 0 10 20 hormone receptor binding to DNA-cellulose. About 97% of DNA binding activity of receptor was inhibited by 10 mM of INHIBITOR CONCENTRATION (mM) pyridoxal 5'-phosphate. Other pyridoxal analogues did not on to DNA- FIG. 2. Effect ofpyridoxal analogues on DNA binding ofnuclear show such strong effect receptor binding thyroid hormone receptor. Eighty-four femtomoles of [11I]T3- cellulose. Less than 10 mM pyridoxal 5'-phosphate did not receptor complex deprived of free hormone by Dowex resin was cause significant dissociation of [1251I]T3 from receptor. incubated with DNA-cellulose (25 ,ug ofDNA) in 0.09 M NaCl/1 mM About 3% of [125I]T3 dissociated from receptor in 20 mM EDTA/0.3 mM MgCl2/10 mM sodium phosphate, pH 8.0/1 mM pyridoxal 5'-phosphate. This effect ofpyridoxal 5'-phosphate dithiothreitol in the presence of indicated concentrations (abscissa) is similar to that shown for steroid receptors and suggests of pyridoxamine 5'-phosphate (o-o), pyridoxal (o*-), or pyridoxal involvement of a Schiffbase in the receptor-DNA interaction 5'-phosphate (A-A). After 30 min ofincubation at 4°C, samples were (22). The same inhibitory effect of pyridoxal 5'-phosphate centrifuged and the supernatant was removed. DNA-cellulose was washed twice with ice-cold 0.05 M NaCl/10 mM Tris HCl, pH 7.4/1 was demonstrated on receptor binding to heparin-Sepharose. was Results of mM EDTA, and radioactivity was measured for the determination of This effect utilized in receptor purification. receptor binding to DNA-cellulose. Receptor binding to cellulose purification calculated from T3 binding capacity are shown in was determined as background binding. Specific binding to DNA was Table 1. The final purified receptor preparation gave 904 ± 58 calculated by subtracting background binding from total DNA- pmol ofT3 binding capacity per mg ofprotein with 2.5-5.2% cellulose binding and was expressed as percent of receptor binding recovery. Starting with 255 + 33 jig of receptor protein in to DNA in the absence ofpyridoxal analogues (ordinate). Each point 3650 ± 417 mg of total protein, we reproducibly obtained is the mean of duplicate determinations that did not vary more than 6.4-14.7,g ofnuclear receptor in 140-300,g oftotal protein. 5% of the mean. Similar results were obtained from another In making these calculations, we assumed that the nuclear experiment. receptor has a molecular mass of49,000 Da and one receptor molecule has a single binding site for T3. Hormone late was added to 0.017%, and samples were dispersed in a Characterization of Purified Nuclear Thyroid Vortex and incubated for 15 min. Trichloroacetic acid was Receptor. A sedimentation coefficient of 3.4 S by glycerol density gradient centrifugation and Stokes radius of 34 A by added to 8% and samples were centrifuged at 8000 x g for 5 were the min. The supernatant was discarded and the pellet was Sephadex G-150 gel filtration analysis obtained for washed three times with ether/ethanol, 1:1 (vol/vol). Sam- purified receptor (Fig. 3). From these values, a molecular ples were dried under the vacuum, dissolved in 5% NaDod- mass of 49,000 Da, frictional ratio of 1.4, and frictional ratio S04/5% 2-mercaptoethanol/62.5 mM Tris HCl, pH 6.8/10% due to shape of 1.3 were calculated. These values are glycerol/0.004% bromophenol blue and incubated at 4°C identical to those obtained for crude nuclear extract, sug- overnight. Samples were electrophoresed in a NaDodSO4/ gesting that the molecular size of the receptor did not change 10% polyacrylamide gel by the method of Laemmli (17). The during the purification. Scatchard analysis of T3 binding to gel was stained with Coomassie brilliant blue, destained, purified receptor showed an affinity constant of 6.0 ± 1.8 X dried, and autoradiographed. 109 liter/mol and T3 binding capacity of 1163 + 329 pmol/mg Other Methods. Protein content was estimated by a of protein (mean + SD of four separate purifications), Coomassie blue method (18) using a bovine serum albumin whereas crude nuclear extract showed an affinity constant of standard that gives results comparable to the other methods 4.2 ± 1.4 x 109 liter/mol and binding capacity of 1.6 + 0.2 tested [Lowry's method (19) and Biuret reaction (20)]. Silver pmol/mg ofprotein. The potency ofiodothyronine analogues staining of the NaDodSO4/polyacrylamide gel was per- to displace [125I]T3 binding to purified receptor is identical to formed as described (21). that obtained with crude nuclear extract or nuclear extract Table 1. Results of nuclear thyroid hormone receptor purification T3 binding capacity, Total pmol of T3 per mg Receptor, protein, Procedure of protein 9g mg Nuclear extract 1.4 ± 0.3 255 + 33 3650 ± 417 Hydroxylapatite 5.7 ± 1.7 227 ± 46 847 ± 222 Sephadex G-150 31.1 ± 3.4 119 + 27 78.1 ± 18.5 DNA-cellulose 412 ± 96 37.8 + 8.0 1.94 ± 0.56 DEAE-Sephadex 604 ± 86 23.7 ± 6.1 0.81 ± 0.23 Heparin-Sepharose 904 ± 58 9.7 ± 3.4 0.22 ± 0.07 T3 binding capacity was measured by incubating the receptor preparation with 5 nM [wlI]T3 in the absence and presence of 0.3 ,uM unlabeled T3. Specifically bound [1"I]T3 was calculated. Amounts of receptor were calculated assuming that the molecular mass ofthe receptor is 49,000 Da and the receptor has a single T3 binding site. Results are mean ± SD of five separate purifications. Downloaded by guest on September 30, 2021 Medical Sciences: Ichikawa and DeGroot Proc. Nati. Acad. Sci. USA 84 (1987) 3423
kDa M 12 3 4 5 6 kDa MA Uvul m~Oval 92.5- 0 202 68.0- 45-0-
0 31 .0 --
r - AS 21.5- - o
FIG. 5. NaDodSO4/polyacrylamide gel electrophoresis analysis at various stages of nuclear thyroid hormone receptor purification. Samples after salt extraction of nuclei (lane 1), hydroxylapatite column (lane 2), ammonium sulfate precipitation (lane 3), Sephadex 30 40 50 60 70 80 bottom 10 20 30 top G-150 column (lane 4), DNA-cellulose column (lane 5), and heparin- FRACTION NUMBER Sepharose column (lane 6) were analyzed by NaDodSO4/10% polyacrylamide gel electrophoresis; this was followed by silver FIG. 3. Molecular size determination ofpartially purified nuclear staining. Amounts ofprotein were 20 Ag for lanes 1-3, 10 ,ug for lanes thyroid hormone receptor. Nuclear extract (o- - -o) or purified 4 and 5, and 6 Ag for lane 6. Lane M, molecular mass markers. nuclear thyroid hormone receptor eluted from heparin-Sepharose (.-.), labeled with [2-5IJT3, was deprived offree hormone by Dowex could be visualized in this system. Lane b must include 0.24 resin. Samples were applied to a Sephadex G-150 gel filtration Ag of receptor since 6 Ag of total protein was applied (4.2% (internal diameter, 1.5 cm; column volume, 180 ml) with an elution purity according to T3 binding capacity). Thus, we can rate of 5.5 ml/cm2 per hr (A) or on to 5 ml of 8-35% (wt/vol) linear was in glycerol gradient sedimentation using an SW 65 rotor (Beckman) at roughly estimate that the amount of49,000-Da protein 60,000 rpm at 0C for 18 hr (B); 2.4 ml per fraction (A) or 0.156 ml per agreement with its T3 binding capacity. fraction (B) was collected. Radioactivity and absorbance at 280 nm To further confirm that the 49,000-Da protein is nuclear in each fraction were measured. i, Internal standard of ovalbumin. thyroid hormone receptor, photoaffinity labeling of nuclear A, Void volume. receptor preparation was performed using underivatized [1251]T4. As shown in Fig. 6, the 49,000-Da protein showed obtained from other tissues {order of iodothyronine ana- covalent [1251]T4 binding that was displaceable by excess logues to displace [125I]T3 being 3,5,3'-triiodothyroacetic unlabeled T3. This specific labeling was only seen after acid > L-T3 > D-3,5,3'-triiodothyronine (D-T3) > L-thyrox- purification of receptor by DNA-cellulose chromatography. ine (L-T4)> D-thyroxine (D-T4) from potent to weaker} (Fig. Tryptic digestion of affinity-labeled receptor showed the 4). specifically labeled band at 27,000 Da. Recently, we obtained NaDodSO4/Polyacrylamide Gel Electrophoresis Analysis of data indicating that digestion of thyroid hormone nuclear Nuclear Thyroid Hormone Receptor at Steps During the Purification. Receptor preparations pooled after each column receptor by trypsin produced a globular T3 binding fragment step were analyzed by NaDodSO4/polyacrylamide gel elec- kDa I 2 3 4 5 6 7 8 trophoresis followed by silver staining (Fig. 5). After purifi- cation the preparation had five bands at a molecular mass of more than 100,000 Da, 56,000 Da, 49,000 Da, 44,000 Da, and 33,000 Da and a wide stain from 66,000 to 58,000 Da. As mentioned above, our estimation of molecular mass from gradient sedimentation and gel filtration data is compatible with the idea that the 49,000-Da band represents nuclear thyroid hormone receptor. Molecular mass markers (0.2 ,ug of each protein) were applied to assure that receptor protein
- .,. ,.O- 100- 0..--L- 0 ~~~ Excess s~ + + D < ' Unlabeled T3 - + - + - + - + 0 D-T4 0 L-T4 FIG. 6. Photoaffinity labeling of nuclear thyroid hormone recep- >SZ 50 Trioc ' u tor. Samples after salt extraction of nuclei (lanes 1 and 2) and heparin-Sepharose column (lanes 3 and 4) were photoaffinity-labeled with 0.5 nM ['2-I]T4 in the absence (lanes 1, 3, 5, and 7) or presence 50-F T (lanes 2, 4, 6, and 8) of 1 AM unlabeled T3. In lanes 5 and 6, photoaffinity labeling was performed on receptor purified up to the DNA-cellulose column step. Four micrograms of trypsin per mg of 0' &o & 68 0 16 O protein was then added and incubated at 10'C for 30 min. The reaction was stopped by adding a 5-fold excess of soybean trypsin IODOTHYRONINE ANALOGUE CONCENTRATION (M) inhibitor and cooling in ice. In lanes 7 and 8, nuclear extract was prepared in the absence of proteinase inhibitor and purified. Small FIG. 4. Potency of iodothyronine analogues to displace ['1-'I]T3 molecular mass T3 binding activity was separately pooled after binding to partially purified nuclear thyroid hormone receptor. Sephadex G-150 column chromatography and further purified by Partially purified receptor eluted from heparin-Sepharose was incu- DNA-cellulose column chromatography. This sample was used for bated with 0.1 nM ['1-'IJT3 in the presence of various amounts of affinity labeling. Samples were applied on NaDodSO4/1 0 poly- 3,5,3'-triiodothyroacetic acid (a-a), L-T3 (L- *), n-3,5,3'-triiodo- acrylamide gel electrophoresis gels and autoradiographed. Amounts thyronine (D-T3) (a- - -.), L-thyroxmne (L-T4) (u-u), or D-thyroxine of protein applied were 200 ug for lanes 1 and 2, 6 Ag for lanes 3 and (D-T4) (o--- o). Bound [125I]T3 was determined and expressed as 4, and 100 jtg for lanes 5-8. Exposure time was 60 days for lanes 1 percent of[125I]T3 binding in the absence ofunlabeled iodothyronine. and 2, 29 days for lanes 5-8, and 8 days for lanes 3 and 4. Downloaded by guest on September 30, 2021 3424 Medical Sciences: Ichikawa and DeGroot Proc. Natl. Acad. Sci. USA 84 (1987) with molecular mass of 26,000 Da calculated from its sedi- to T3. This finding, together with the finding that KCl mentation coefficient and Stokes radius (unpublished data). concentrations of <0.2 M exacerbate (data not shown), When nuclear extract was prepared in the absence of pro- whereas higher KC1 concentrations ameliorate the loss of T3 teinase inhibitor, we obtained a small T3 binding fragment binding activity at the time of resin test, suggest that receptor that displayed characteristics of nuclear receptor except for is adsorbed onto the resin. This causes a lower estimation of reduced affinity to DNA. Since the addition of proteinase the amount of receptor when small amounts of protein were inhibitor and quick preparation of nuclei significantly re- assayed. For this reason, 50 ,ug of protein of heated nuclear duced the generation of this small fragment, we consider this extract was included in each T3 binding assay mixture. to be an endogenous proteinase digest of nuclear receptor. These improvements in nuclear receptor purification and The molecular mass of this T3 binding fragment was 38,000 assay allowed us to use a six-step purification procedure (five Da when calculated from its Stokes radius of 31 A and columns and an ammonium sulfate precipitation), resulting in sedimentation constant of 2.9 S. Photoaffinity labeling of this considerable purification of nuclear thyroid hormone recep- sample showed saturable labeling at molecular mass of 36,000 tor without appreciable degradation or alteration in charac- Da (Fig. 6). These data suggest that saturable photoaffinity teristics of receptor. labeling by underivatized T4 occurred at the hormone binding This preparation of receptor (6.4-14.7 gg of protein with domain of nuclear thyroid hormone receptor. 4.2-4.9% purity) should allow us to prepare monoclonal antibodies against the nuclear thyroid hormone receptor, DISCUSSION unless this receptor is less immunogenic than steroid hor- mone receptor (23). Our current strategy to achieve complete Purification of nuclear thyroid hormone receptor is very purification of receptor is to make monoclonal antibodies to difficult to achieve because of its extremely low abundance receptor and further purify the receptor by immunoaffinity in target organs. To overcome this problem, we developed a column. method that allows preparation of nuclear extract from Silver staining ofNaDodSO4/polyacrylamide gel identified 0.8-1.0 kg of rat liver in a day. No difference in quality and a band strongly indicative of receptor (molecular mass of quantity of receptor as assessed by thyroid hormone binding 49,000 Da and saturable photoaffinity labeling with [251I]T4). assay and molecular size determination was found in nuclear However, it is still possible that other proteins with the same extracts prepared in this way in comparison to that prepared molecular mass are included in this band. in the conventional way. However, extensive homogeniza- tion used in this method liberated proteolytic enzymes and This research was supported by Public Health Service Grant resulted in fragmentation of nuclear receptor and other AM13377 and the David Wiener Research Fund. proteins in the absence of protease inhibition. We found that addition of PhMeSO2F, EDTA, and pepstatin A at the time 1. Oppenheimer, J. H., Koerner, D., Schwartz, H. L. & Surks, M. I. (1972) J. Clin. Endocrinol. Metab. 35, 330-333. of homogenization and during Triton X-100 treatment of 2. Surks, M. I., Koerner, D., Dillman, W. & Oppenheimer, J. H. crude nuclei was effective in preventing this problem. Triton (1973) J. Biol. Chem. 248, 7066-7072. X-100 treatment of crude nuclei reduced the volume of 3. Latham, K. R., Ring, J. C. & Baxter, J. D. (1976) J. Biol. nuclei, and thus a large quantity of nuclei could be processed Chem. 251, 7388-7397. through heavy sucrose centrifugation by combining nuclei 4. Pascual, A., Casanova, J. & Samuels, H. H. (1982) J. Biol. with 2.8 M sucrose solution and centrifuging in a large- Chem. 257, 9640-9647. capacity fixed-angle rotor. This method allowed us to start 5. DeGroot, L. J., Refetoff, S., Strausser, J. & Barsano, C. from nuclear extract prepared from 4-5 kg of rat liver, which (1974) Proc. Natl. Acad. Sci. USA 71, 4042-4046. allowed us to use more column steps and resulted in isolation 6. Oppenheimer, J. H., Schwartz, H. L. & Surks, M. I. (1974) Endocrinology 95, 897-903. of higher purity of receptor with higher recovery. 7. Samuels, H. H., Stanley, F. & Casanova, J. (1979) J. Clin. Another problem in purification of receptor is rapid loss of Invest. 63, 1229-1240. T3 binding activity during purification. The effects ofglycerol 8. Nyborg, J. K., Nguyen, A. P. & Spindler, S. R. (1984) J. Biol. and phosphate to prevent receptor degradation were utilized Chem. 259, 12377-12381. in the present work. Since receptor irreversibly loses its T3 9. Shupnik, M. A., Ardisson, L. J., Meskell, M. J., Bornstein, J. binding activity at pH below 6.0 or above 10.5 (data not & Ridgway, C. (1986) Endocrinology 118, 367-371. shown), a neutral pH was kept during the purification. 10. Torresani, J. & Anselmet, A. (1978) Biochem. Biophys. Res. Another factor contributing to the apparent loss ofT3 binding Commun. 81, 147-153. activity resides in the T3 binding assay. When a small amount 11. Silva, E. S., Astier, H., Thakare, V., Schwartz, H. L. & of is used in as Oppenheimer, J. H. (1977) J. Biol. Chem. 252, 6799-6805. protein the binding assay, after partial 12. Nikodem, V. M., Cheng, S. Y. & Rall, J. E. (1980) Proc. Natl. purification of receptor, an appreciable amount of T3 binding Acad. Sci. USA 77, 7064-7068. activity is lost at the time of the resin test used to separate 13. Apriletti, J. W., Eberhardt, N. L., Latham, K. R. & Baxter, bound and free hormones. This loss of T3 binding activity J. D. (1981) J. Biol. Chem. 256, 12094-12101. was avoided to some extent by adding proteins or high 14. Muench, K. H. (1971) Nucleic Acids Res. 2, 515-523. molecular mass compounds into binding assay mixture or by 15. Alberts, B. & Herrick, G. (1974) Methods Enzymol. 21, performing the resin test at 0.9 M KCl concentration. We 198-217. found that addition ofheated nuclear extract, which itselfhad 16. Torresani, J. & DeGroot, L. J. (1975) Endocrinology 96, no specific T3 binding activity, to the assay mixture was most 1201-1209. effective in the loss 17. Laemmli, V. K. (1970) Nature (London) 227, 680-685. preventing of T3 binding activity at the 18. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. time of binding assay. A maximal effect was seen with >30 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, gg of heated nuclear extract in 0.3 ml of assay mixture. R. J. (1951) J. Biol. Chem. 193, 265-275. Scatchard analysis showed that loss of T3 binding activity 20. Gornall, A. G., Bardawill, C. J. & David, M. M. (1949) J. Biol. from dilute receptor in the assay mixture was due to the loss Chem. 177, 751-766. ofbinding capacity without a significant change in affinity for 21. Morrisey, J. H. (1981) Anal. Biochem. 117, 307-310. T3, and addition of heated nuclear extract to the assay 22. Cake, M. H. (1978) J. Biol. Chem. 253, 4886-4891. mixture restored binding capacity without changing affinity 23. Milgrom, E. (1985) Pharmacol. Ther. 28, 389-415. Downloaded by guest on September 30, 2021