Molecular and Cellular Endocrinology, 19 (1980) 69-77 Q Elsevier/North-Holland Scientific Publishers, Ltd.

69

STABILITY OF NUCLEAR THYROID-HORMONE RECEPTOR AND ITS

BEHAVIOR ON HYDROXYLAPATITE

Katsutoshi YOSHIZATO Developmental Biology Laboratory, Department of Plastic Surgery, Kitasato University School

of Medicine, Sagamihara, Kanagawa 228 (Japan)

Received 4 September 1979; accepted 24 March 1980

Rat-liver chromatin was kept under several conditions. The maximum binding capacity (B max) and the apparent association constant (K,) for triiodothyronine (Ta)-binding were determined. Binding activity (Bmax X K,) was unstable at 0 and -2O’C in the reaction buffer. Maximum binding activity was found upon storage at -70°C or in liquid nitrogen where about 70% of the original activity remained after 3 weeks. With storage, K, decreased more rapidly than Bmax which showed little change. These temperature-dependent changes in the stored chromatin were specific for the receptor and not observed for other characteristics of the chromatin, i.e., template activity and the electrophoretic separation pattern of chromatin pro- teins. The elution profile of the receptor on a hydroxylapatite column was unchanged during storage of the chromatin, suggesting that the chemical nature of the binding protein(s) may not change during storage.

Keywords: thyroid-hormone binding; L-triiodothyronine; chromatin; nuclear receptor; hydroxylapatite.

Although its biological significance is not clearly understood, the presence of nuclear receptor(s) for thyroid hormones has been demonstrated in mammals and amphibians (Oppenheimer et al., 1972; Surks et al., 1973; Samuels et al., 1974; Charles et al., 1975; Degroot and Torresani, 1975; Macleod and Baxter, 1975, 1976; Spindler et al., 1975; Torresani and Degroot, 1975; Yoshizato et al., 1975, 1977; Kistler et al., 1975; Doctor et al., 1976; Latham et al., 1976; Gardner, 1978). In order to clarify the physiological role of the receptor, it is important to obtain information on the physico- and biochemical characteristics of the receptor pro- tein(s) in their pure form. Complete purification of the receptor activity has not been reported. Reports of partial purification of a nuclear extract on a QAE- Sephadex (Latham et al., 1976) and on a DEAE-Sephadex column (Silva et al., 1977) have been published recently. Torresani and Anselmet (1978) reported par-

Abbreviations: ATP, adenosine 5’-triphosphate; CTP, cytosine 5’-triphosphate; DTT, dithio- threitol; EDTA, ethylenediaminetetraacetate; GTP, guanosine S’-triphosphate; Ta or triiodo- thyronine, 3,5,3’-triiodo-L-thyronine; UTP, uridine 5’-triphosphate.

70 Katsutoshi Yoshizato

tial purification on DEAE-Sephadex and DNA-Sepharose. One of the difficul- ties of purification seems to be that receptor activity is very unstable during stor- age. Jaffe and Means (1977) reported that the nuclear binding sites are lost rapidly if they are not occupied by hormones.

In the present paper, we compare the receptor activity of rat-liver chromatins which have been kept under various conditions for up to 3 weeks after chromatin preparation. We were interested in which of the following 2 parameters, apparent association constant (K,) or maximum binding capacity (&,,), would be unstable during the course of chromatin storage. By knowing this, we can predict what hap- pens to the receptor during storage. Other characteristics of the chromatin, i.e., template activity and the electrophoretic separation pattern of chromatin proteins, were determined during the course of chromatin storage in order to ascertain whether the changes observed are specific for the thyroid-hormone receptor or are a

general characteristic of stored chromatin. Fractionation of the receptor protein(s) was also investigated on a hydroxylapatite column. The binding characteristics of triiodothyronine (Ta) to chromatin have been reported previously (Yoshizato et al., 1977).

MATERIALS AND METHODS

Chemicals T3 was purchased from Sigma Chemical Co. and [’ “I]Ta, the specific activity

range of which was from 1060 to 1318 pCi/pg, was obtained from New England Nuclear. According to the accompanying technical data unbound iodine was less than 3% and radiochemical purity was more than 95%. [5-3H]UTP (1 .O mCi/0.026

mg) was obtained from New England Nuclear. Nucleotide triphosphates were pur- chased from Boehringer, Mannheim. Hydroxylapatite was purchased from Bio-Rad

Laboratories. All other chemicals were reagent grades.

Preparation of chromatin The procedure of rat-liver ,chromatin preparation has been described previously

(Yoshizato et al., 1977). The final chromatin pellet was suspended in the solution

for storage. 3 solutions were tested: 1 mM Tris-HCI buffer, pH 8.0; Buffer A (20 mM Tris-HCI, 0.25 M sucrose, 1 mM MgClz, 5% glycerol, 2 mM EDTA and 0.1 mM DTT, pH 7.6); and 2 times diluted Buffer A (l/2 Buffer A). l/2 Buffer A has the same composition as the solution used for the binding of [rzs1]T3 to chromatin as previously reported (Yoshizato et al., 1977). Chromatin thus obtained was stored under 8 different conditions as follows: (1) at 0°C in 1 mM Tris-HCI, pH 8.0 (chromatin concentration was 500 1.18 DNA/ml); (2) at 0°C in l/2 Buffer A (250 pg/ml); (3) at 0°C in Buffer A without sucrose (500 fig/ml); (4) at 0°C in Buffer A (500 pglml); (5) at -20°C in Buffer A (500 E.cg/ml); (6) at -20°C in Buffer A con- taining 50% glycerol as antifreeze (500 Erg/ml); (7) at -70°C in Buffer A (500 pg/ ml); (8) at -196’C (liquid nitrogen) in Buffer A (500 pg/ml).

Stability of nuclear triiodothyronine receptor 11

Binding of T3 to the chromatin Aliquots of the chromatin which had been stored as described above were

removed and the binding activity toward [ r251]Ta was determined as follows. 10 d of [“‘I]Ta were added to 1 ml of the chromatin suspension (l/2 Buffer A) con- taining 250 pg of DNA and the suspension was incubated for 3 h at room tempera- ture (25’C). Chromatin-bound Ts was measured as described previously (Yoshizato et al., 1977). Nonspecific binding of [ r2’I]T3 was determined by incubating the chromatin in the presence of lOOO-fold excess amounts of unlabeled Ts. In all bind-

ing assays 2 parameters (K, and B,,) were determined by a Scatchard analysis (Scatchard, 1949) as indices of the receptor stability.

Determination of template activity of the chromatin Template activity of the chromatin was determined by the modified method of

Ihara and Kawakami (1977). 50 fi of chromatin suspension containing 25 pg of

DNA in Buffer A, 75 ~1 of the reaction mixture which contained 0.4 mM of each of nucleotide triphosphate (ATP, GTP, CTP), 40 mM Tris-HCl, 0.3 M ammonium sul- fate, 1.6 mM MnCl*, 0.4 mM NasEDTA, 4 mM DTT, 20% glycerol and 0.04 mM [3H]UTP (1.25 /Xi), pH 7.5 were combined in a small conical test tube. The reac- tion was started by incubating the tube at 37°C with shaking. After varying lengths of incubation time, 100 ~1 of the content was transferred to a glass fiber disc GF/C

(24 mm, Whatman) by a micropipette. The discs were dipped in 5% trichloro- acetic acid (TCA) (5 ml/disc) for at least 15 min with occasional shaking. Rinse with TCA was repeated 4 times, followed by washes with ethanol and ether. The radioactivity of the dried disc was counted in 5 ml of toluene-based scintillation cocktail.

SDS-polyaclylamide gel electrophoresis of chromatin, Chromatin (500 pg DNA/ml) suspended in Buffer A was diluted with an equal

volume of a solution containing 10 mM Tris-HCl, pH 8.6, 25% glycerol and 1% sodium dodecyl sulfate (SDS). Gel electrophoresis was carried out on 8.75% disc gels in the presence of SDS according to the method of Laemmli (1970).

Chromatography on hydroxylapatite Chromatin was incubated with [12’I]T3 for 3 h at 25’C and concentrated KC1

and phosphate (potassium salt) solutions were added to the final 1 M and 1 mM, resp. Insoluble materials were removed by a short low-speed centrifugation and the supernate was applied to a hydroxylapatite column which was equilibrated with

1 M KCl and 1 mM phosphate (pH 7.6). Chromatography was carried out at 4°C.

The hydroxylapatite column was washed with the equilibration buffer and eluted with the linear gradient of phosphate (l-400 mM). Phosphate was determined according to Fiske and Subbarow (1925). Unbound free [12’1] T3 was eluted by 1 mM phosphate.

72 Katsutoshi Yoshizato

RESULTS AND DISCUSSION

During storage, aliquots of the stored chromatin were removed to determine K, and B,, for Ts binding by a Scatchard method. Some of these analyses are shown in Fig. 1. The parameters obtained from a Scatchard plot are listed in Table 1. The K, for fresh chromatin (Day 0 in Table 1) is slightly different in each vertical col- umn of Table 1 (2.5-3.6 X 10’ M-l) because separate chromatin preparations

exhibit some variations in this parameter. Different chromatin preparations were employed for the data in each column of Table 1. The range of variation of B,, of the fresh chromatin was from 0.34 pmoles/mg DNA to 0.71 pmoles/mg DNA.

K, and B,, reported previously from our laboratory (Yoshizato et al., 1977) were

4.4 X 10’ M-’ and 0.2 pmoles/mg DNA, resp. From Table 1, it is apparent that during storage the affinity constant decreased

0 20 40 60 80 100 ‘0 40 80 120 160 200

BOUND b ( moles x IO”) BOUND b (moles x IO”)

B xld50r D

BOUND TS ( moles x IO”) BOUND T. (moles x IO”)

Fig. 1. Scatchard plots of Ta binding to the chromatins which were stored under various condi- tions. (A) Chromatin stored at 0°C in l/2 Buffer A (chromatin concentration: 250 fig/ml). 0, fresh chromatin; 0, stored for 1 day; q , stored for 2 days; n , stored for 4 days. (B) Chromatin stored at -20°C in Buffer A (500 pg/ml). o, fresh chromatin; l , stored for 2 days; 0, stored for 4 days; n , stored for 14 days. (C) Chromatin stored at -7O’C in Buffer A (500 pug/ml). o, fresh chromatin; l , stored for 2 days; q , stored for 6 days; n , stored for 14 days; A, stored for 21 days. (D) Chromatin stored at -196°C in Buffer A (500 fig/ml). o, fresh chromatin; 0, stored for 2 days; q , stored for 14 days; n , stored for 21 days.

Stability of nuclear triiodothyronine receptor 13

Table 1 Changes in the binding characteristics of chromatin during storage under various conditions

Conditions of storage

Length of 0°C storage in l Buffer A (days)

o”c -20°c -70°C -196°C in Buffer A in Buffer A in Buffer A in Buffer A

0

1

2

4

6

14

21

K, 3.64 x log 3.00 x 109 3.00 x 109 2.49 X lo9 2.49 x lo9 B max o.34 0.65 0.65 0.71 0.71

2.41 X lo9 2.29 x lo9 - - _ 0.41 0.71 - _ _

2.65 x log 2.01 x 109 2.21 x 109 2.34 x lo9 2.23 x lo9 0.34 0.61 0.62 0.69 0.68

2.22 x 109 2.07 x log 2.63 X log - - 0.27 0.45 0.60 - -

- - 2.01 x 109 - - - 0.75 -

_ -/ 1.87 x lo9 1.74 x 109 1.93 x 109 - - 0.56 0.11 0.75

- - - 1.68 x 109 1.63 X 10’ _ - 0.70 0.72

K,, Apparent association constant, M-l. B max, Maximal binding capacity, pmoles/mg DNA.

much more than did the maximal binding capacity. For example, with storage in liquid nitrogen for 3 weeks, the former parameter decreased to about 70% of the fresh value, while the latter parameter did not change significantly. This would im- ply that hormone-receptor binding is not being lost in an “all or none” fashion, but rather through intermediates which bind hormones less avidly than freshly isolated receptor.

The product of the 2 parameters is used as the stability index of the chromatin for binding to Ts and is plotted against storage periods as shown in Fig. 2. Storage in the Buffer A greatly improved the stability of the chromatin as compared to that in a dilute Tris-HCl solution. This effect would seem to be partially ascribed to DTT, which has been reported to stabilize the nuclear Ts binding sites (Torresani and Degroot, 1975; Torresani et al., 1978). Sucrose had no effect on the chromatin stability (see n+ in Fig. 2). Decreased preservation temperatures greatly im- proved the chromatin stability. On storage at -20°C in Buffer A for 2 weeks, about 55% of the initial activity was retained. At -70°C, 70% of the fresh chromatin activity was still present, and the same activity was found at liquid nitrogen temper- ature after 3 weeks. Storage in Buffer A containing 50% glycerol at -2O’C did not show any difference from that in Buffer A without glycerol. Thus, in the optimal

74 Katsutoshi Yoshizato

02468 IO 12 14 16 18 20 22

DAYS IN STORAGE

Fig. 2. Changes in the receptor activity of chromatin for T3 during storage. Chromatin prepara- tions were stored under various conditions and Scatchard plotting was performed to obtain K, and Bm,,. The product of the 2 parameters is used as the stability index and plotted against days in storage. V, at 0°C in 1 mM Tris, pH 8.0; A, at O’C in Buffer A without sucrose; o, at 0°C in Buffer A; 0, at 0°C in l/2 Buffer A; q , at -20°C in Buffer A; n , at -70°C in Buffer A; A, at -196°C in Buffer A.

storage conditions studied so far we can retain about 70% of the initial activity even after 3 weeks. It is interesting to know whether the temperature-dependent changes in the chromatin described above are specific for the receptor activity or a general characteristic of stored chromatin. Template activity of the fresh chromatin as mea- sured by an incorporation of [3H] UTP increased linearly during time periods of up to 40 min, but that of the stored chromatin tested after a week showed a non-linear increase at all temperatures (Fig. 3). About half of the template activity was lost during a week’s storage even at -70°C, and there was no marked difference

between -70°C and 0°C. After 18 days, about 70% of the activity disappeared at O”C, whereas almost the same activity was found at -70°C as compared to one week’s activity. Another characteristic, the chromatin protein profile, was investi-

gated during the storage of chromatin. No significant changes in the SDS-poly- acrylamide gel electrophoretic profile in fresh chromatin and the chromatin stored at -20°C and -70°C for 7 or 18 days (Fig. 4) could be found. Upon storage at O”C, some changes in the low molecular weight region were found after 7 days. The same profile as at Day 7 was obtained at Day 18. These results show that changes ob-

served in T3-receptor activity during storage are specific for the receptor and are not related to other characteristics such as template activity and protein profiles.

Fractionation of the T3-binding protein was investigated with hydroxylapatite (Fig. 5). T3-binding protein(s) from freshly prepared chromatin was eluted from the column between 25 and 50 mM phosphate and no other binding component was

found. This is in contrast to observations in estrogen receptor bound to chromatin.

Gschwendt (1976) reported that estrogen receptor from chicken-liver chromatin was fractionated into 2 components on hydroxylapatite, one eluting at 20 mM phosphate and the other at 200 mM phosphate. Chromatin stored at -7O’C for 16 days was incubated with [ ‘*‘I]T3 and chromatographed on the hydroxylapatite

column. T3-binding protein(s) was eluted in the same way as fresh chromatin. This was also the case for the chromatin which had been stored for 4 months at -7O’C. These results suggested that the biochemical nature of the binding protein(s) seems

Stability of nuclear triiodothyronine receptor 75

3 XI0

4-

:3 1 l-

‘2 -

I P

Ol-

A

/

“LA A

/ A

,I/ 0 20 40 60 00

INCUBATION TIME(MIN.)

20 40 60 60

xld

INCUBATION TIME (MIN.)

3t-”

0 20 40 60 00

INCUBATION TIME.(MIN.)

A B C D

Fig. 3. Changes in template activity of chromatin during storage. Chromatin was prepared and stored at 3 different temperatures. Aliquots of the stored chromatin were withdrawn for the determination of template activity by the method described in the Materials and Methods sec- tion. (A) Fresh chromatin. (B) Chromatin stored for 7 days. o, at 0°C; 0, at -20°C; 0, at -70°C. (C) Chromatin stored for 18 days. o, at 0°C; l , at -20°C; 0, at -70°C. Each point represents the average of duplicate determinations.

Fig. 4. SDS-polyacrylamide gel electrophoresis of the stored chromatm. Chromatin was pre- pared and stored at 3 different temperatures for 18 days. Electrophoresis of the fresh and the stored chromatin was performed as described in the Materials and Methods section. (A) Chro- matin stored at 0°C. (B) Chromatin stored at -20°C. (C) Chromatin stored at -7O’C. (D) Fresh chromatin. The migration of standard proteins is indicated by arrows. Tl, trypsin inhib- itor; BA, bovine serum albumin; p, p’, c1 and c, each represents a subunit of RNA polymerase. BPB, bromophospho blue.

Katsutoshi Yoshizato 16

0.7-

0.6 -

f 0.5 ”

E 0.4- c s N 0.3

a

O 0.2 -

2coO

FRACTION NUMBER

Fig. 5. Chromatography of chromatin proteins on hydroxylapatite. Chromatin (5 mg DNA) was incubated with [ 1251]Ta (2.4 X 10eg M) at 25°C. After 3 h the reaction was stopped by cool- ing the tube and concentrated KC1 and phosphate were added to the final 1 M and 1 mM, resp. Insoluble materials were removed by low-speed centrifugation. The supernate was applied to a hydroxylapatite column. The column was washed extensively by a solution containing 1 M KCl, 1 mM phosphate and 0.1 mM DTT to remove unbound free [ ’ *‘I]Ta. Then elution was carried out by a phosphate gradient between 1 and 400 mM. Column, 1.7 X 20 cm. Gradient was formed by mixing 200 ml of 1 and 400 mM phosphate in 1 M KCl and 0.1 mM DTT. Flow rate, 16 ml/h. o, O.D. 280 nm; l , cpm of [1251]Ts; n , cpm of [ ‘25I]Ta from chromatin incubated with [ 1 251]Ts in the presence of 1000 times excess amounts of unlabeled Ta ; phosphate con- centration is indicated by a solid line.

to be unchanged during the course of storage. About 70% of the Ta-binding sites in the chromatin, measured by the method reported previously (Yoshizato et al., 1977), were recovered from the column. Before the chromatographic separation, binding activity of the chromatin was 1.52 X lo-l4 moles of Ts per mg protein. After the hydroxylapatite-column separation, the specific activity of the receptor increased about _I0 times to 1 .I6 X lo-l3 moles per mg protein. Fractionation of the nuclei or chromatin with hydroxylapatite will be a useful step in receptor puri-

fication, where nucleic acid and the major protein can be removed and a consider- ably higher purification rate is obtainable. DNA was not eluted from the column up to 400 mM phosphate. Fig. 5 shows that 2 or more binding protein(s) could be frac- tionated on the column when the elution was performed with a gentle phosphate gradient.

When isolation and purification of the receptor from cell nuclei are undertaken, a considerable amount of nuclei or chromatin is needed as a starting material. The present results show that if the desired amounts of cell nuclei or chromatin cannot be prepared at one time, we may prepare the chromatin from separate batches dur- ing a 2-3-week period with only a 30% loss of initial activity in the first batch or in the early batches of consecutively prepared material.

Stability of nuclear triiodothyronine receptor 71

ACKNOWLEDGEMENTS

We would like to thank Dr. Gordon T. James of the University of Colorado and Dr. Earl Frieden of Florida State University, both of whom read this manuscript and provided helpful suggestions. We would also like to express our gratitude to Professor N. Shioya of Kitasato University, who showed constant interest and encouraged the author during the present study. This work was supported by a grant from the Ministry of Education, Science and Culture of Japan.

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