Molecular and Cellular Endocrinology, 11 (1978) 205-211 0 Elsevier/Nortlt-Holland Scientific Publishers, Ltd.

PHYSICOCHEMICAL PROPERTIES OF THE CYTOPLASMIC TRIIODOTHYRONINE BINDING PROTEIN FROM TADPOLE LIVER AND TAIL FIN Randal C. JAFFE department of PhysioIo~ and Biophys~~s~Universityof nlinois at the medical Center, 901 S. WolcottSt., Chicago, Illinois 60612, U.S.A.

Received 6 February 1978; accepted 10 April 1978

The segmentation velocity, gel filtration properties and pattern of elution from ion exchange geis of the cytoplasmic trfodothyronine (T3) binding protein from Rana catesbeiana

liver and tall fin cytosol were determined. The T3 binding protein in liver cytosol had a sedimentation coeftlcient of 4.4s on suerose gradients and a Stokes radius from Sephadex gel filtration of 38.2 f 4.2 A. From these two values a molecular weight of 71,700 * 4100 and a frictional ratio of 1.28 f 0.05 for the TJ binding protein from tadpole liver has been calculated. The liver T3 binding protein was eluted from DEAE-Sephadex gels at a salt concentration of 0.233 f 0.017 M NaCl. The sedimen~tion coefficient, Stokes radius and salt requirement for elution from DEAE-Sephadex of the T3 binding protein from tail tin cytosol were essentially identical (4.4S, 35.2 t 4.2 A. and 0.213 i 0.015 M NaCt respectively). The calculated molecular weight is 66,100 f 7900 and the frictional ratio is 1.21 f 0.10 for the tail fin T3 binding protein. The great similarity in the physicochemic~ properties of the T3 binding protein from the liver and tail fin implies that the T3 binding protein in each tissue is similar if not identical. The possible reason for the differences in the dissociation constants previously reported for the binding of T3 in the two tissues is discussed. Keywords:

~iiodothyronine metamorphosis.

binding

protein;

physicochemic~

properties;

tadpole

Thyroid hormones bind with a high affinity to liver and kidney cytosol proteins from the rat (Dillman et al., 1974; Defer et al., 1975; Visser et aI., 1976) and dog (Davis et al., 1974) and cytosof from various tissues of the tadpole (Yoshizato et al., 1975; Jaffe and Gold, 1977). In the tadpole the apparent dissociation constant for the binding of t~iodothyron~e (T3) to the cytoplasmic proteins in the liver, tail fin, tail muscle and kidney were significantly different (Yoshizato et al., 1975; Jaffe and Gold, 1977). This difference could be due to a different Ts binding protein in each tissue or the presence of a factor(s) in the cytosol which alters the binding of T3 and, thereby, the dissociation constant. In this paper we employ several physicochemical techniques in order to determine and compare various properties of the binding proteins from the liver and tail fin of the tadpole. 205

206

MATERIALS

R.C. Jaffe

AND METHODS

materials Rana catesbe~ana tadpoles, stages I to X (Taylor and Kollros, 1946) were purchased from Mogul-Ed. They were maintained at 22°C and fed canned spinach until used. Unlabeled T3, Tricaine, bovine serum albumin (BSA) and Norite A were purchased from Sigma; Dextran T-70, Sephadex G-150, DEAE-Sephadex A-25, blue dextran 2000 and molecular weight standards (aldolase, ovalbumin, chymotrypsinogen A and ribonuclease A) were from Pharmacia Fine Chemicals; and protamine sulfate was obtained from Eli Lilly and Co. [iz51]Ta was purchased from Industrial Nuclear Co. and initially had a specific activity of 100 Cilmmol. All other chemicals were obtained from commercial suppliers. ~epa$a~on of cy tosol Animals were anesthetized by placing them in ice-cold 0.1% Tricaine. The liver and tail fin were removed, washed, and then homogenized in a glass-Teflon homogenizer in either ice-cold buffer A (0.5 M sucrose, 2 mM MgCls, 12 mM thioglycerol, 10 mM Tris-HCl, pH 7.4) or buffer B (1 mM MgCla, 12 mM thio~ycerol, 10 mM Tris-HCl, pH 7.4) as indicated. After centrifugation at 12,000 g for 10 min the supernatant was centrifuged at lOS,OOOg for 1 h. The lipid layer was removed and the supernatant (cytosol) placed on ice until used. Sucrose gradient centrifugation Linear 4.8 ml gradients of S--2% sucrose were prepared in buffer B. A 0.2 ml aliquot of cytosol or BSA (5 mg/ml) prepared in buffer B was layered on the top. The gradients were centrifuged for 16 h at 45,000 rpm (average force of 189,000 g) at 0-4°C in an SW 50.1 rotor. Following centrifugation, the bottoms of the tubes were pierced and 16 drop fractions collected. The location of the BSA standard, 4.4s (Sober, 1968) was obtained by measuring the absorbance at 280 nm. Gel filtration chromatography A Sephadex G-l 50 column (1 .S X 90 cm) was poured and washed extensively with buffer B at a flow rate of 10 ml/h. The column was then calibrated using blue dextran 2000, “‘1 , and proteins of known Stokes radius (Sober, 1968): ribonuclease A, 18.4 A; chymot~psinogen A, 23.0 A; ovalbumin, 28.1 8; aldolase, 43.7 A. Samples of cytosol (0.5 ml) were applied to this calibrated column and 1.5 ml fractions collected. The protein elution pattern was determined by measuring the absorbance at 280 nm and the position of the Ta binding protein was obtained as described below. Ion exchange chromatography DEAE-Sephadex A-25 was equilibrated with 1 mM MgC12, 10 mM Tris-HCl, pH 7.4, then poured into a 1.5 X 20 cm column and washed extensively with

Physicochemical properties of T3 binding proteins

207

buffer B at a flow rate of 20 ml/h. The cytosol was then applied and 1.5 ml fractions collected. The column was eluted with buffer B until 20 fractions were collected, then a O-O.5 M NaCl gradient in buffer B was applied. The protein elution profile was determined by measuring the absorbance at 280 nm and the salt concentration was determined using a Radiometer ~onducti~ty meter. Measurement of ( 12‘IJ T3 binding

The location of the T3 binding protein was determined by measuring how much [ 12’I]T3 was specifically bound by each fraction. This approach was necessary since the rapid dissociation of T3 from the binding protein (Jaffe and Gotd, 1977) precluded the more convenient method of chromatographing cytosol preincubated . with labeled hormone and then counting the individual fractions directly. Aliquots of individual fractions were incubated with [‘251]T3 (3.04 X 10m9to 1.11 X lo-* M) or an identical concentration of [1251]T3 plus a lOOO-foldexcess of unlabeled T3 for 2 h at ‘0°C in a total volume of 0.3 ml. The bound was then separated from the free hormone using protamine sulfate as previously described (Jaffe and Gold, 1977) when the fractions from sucrose gradient centrifugation or gel filtration chromatography were being assayed. In brief, 0.2 ml of a 1 mgjml solution of protamine sulfate was added to each tube and the contents mixed. After 5 min at 0°C the tubes were centrifuged at 2000g and the supernatant was removed. The precipitate was then washed 3 times in buffer B. Salt was found to interfere with the precipitation of the [ ’ 2‘I] Tj-binding protein complex by protamine sulfate. Therefore, the separation of bound and free hormone when the fractions were from ion exchange chromatography was carried out using dextrancoated charcoal (DCC). A 0.3 ml aliquot of DCC (0.5% Norite A and 0.05% Dextran T-70 in 1.5 mM EDTA and 10 mM Tris-HCl, pH 7.4) was added. After 5 min at 0°C the tubes were centrifuged at 2000g for 10 min and the supernatant was transferred and counted. Only 16 fractions were assayed in a group in order to preclude spuriously low values due to significantly different periods of exposure to DCC (Jaffe and Gold, 1977). With either method, the amount of specifically bound [12’I]T3 was determined by subtracting the amount of [12’1] Ts bound in the presence of a 1000-fold excess of unlabeled T3 from the amount bound in the absence of excess unlabeled Ta. Other analytical methods

Counting was performed in a Packard autogamma counter (efficiency 67%). Protein was measured by the method of Lowry et al. (1951) using bovine serum albumin as the standard. RESULTS AND DISCUSSION The results of typical sedimentation velocity dete~inations using liver and tail fin cytosol are shown in figs. 1A and 1B respectively. Only a single peak of binding

R.C.Jaffe

208

activity was observed in gradients run with liver or tail fin cytosol. Using the method of Martin and Ames (1961) the sedimentation coefficient for the Ta binding protein of liver and tail fin cytosol is 4.4s. The position of the T3 binding protein relative to the position of the BSA peak is the same in sucrose gradients containing 0.3 M KC1 as it is in gradients without added KC1 (data not shown). The cytoplasmic steroid hormone binding proteins normally exhibit a change in sedimentation coefficient in the presence of KCl, the sedimentation rate being slower in the presence of salt (King and Mainwaring, 1974).

3-

6-

FRACTION

NUMBER

Fig. 1. Sucrose gradient centrifugation profile of liver (A) and tail fin (B) cytoplasmic T3 binding protein. Cytosol (0.2 ml) from either tadpole liver (6.6 mg protein/ml) or tail tin (5.0 mg protein/ml) was layered on top of a S-20% sucrose gradient (4.8 ml) prepared in buffer B. After centrifugation the position of the T3 binding protein was determined as described in Materials and Methods. Gradients containing BSA were run simultaneously and the position of the BSA (indicated by an arrow) determined by the absorbance at 280 nm.

Physicochemical properties of TJ binding proteins

209 16

08

ci ,201

-

110

FRACTION

EE

NUMBER

Fig. 2. Sephadex G-150 gel filtration of liver (A) and tail fin (B) cytoplasmic proteins. A 1.5 X 90 cm column of Sephadex G-150 was equilibrated in buffer B. A 0.5 ml aliquot of each cytosol (liver, 17 mg protein/ml; tail fin, 4.4 mg protein/ml) was chromatographed and 1.5 ml fractions collected; 0.2 ml aliquots were assayed for specific binding of [1251]T3 as described in Materials and Methods. The protein profile was determined by measuring the absorbance at 280 nm.

Gel filtration chromatography on Sephadex G-150 revealed only a single major peak of Ta binding activity in liver (fig. 2A) and tail fin (fig. 2B) cytosol. There was a variable amount of binding activity at or near the excluded volume of the column. This is felt to be due to aggregated material. Porath (1963) has demonstrated that there is a linear relationship between the Stokes radius of a protein and the cube root of its distribution coefficient. From such a plot, using the protein standards of known Stokes radius listed in Materials and Methods, the Stokes radius for the liver Ts binding protein was found to be 38.2 rt 2.2 A and for the tail fin Ts binding protein it was found to be 35.2 f 4.2 w (mean f SD of 3 determinations). The difference between the two values is not statistically significant. Molecular weight determination using the Stokes radius and sedimentation coefficient (Siegel and Monty, 1966) produced a molecular weight for the Ts

05

I

“;

O41

a3

-

02

‘d 2

ai

E

0

Fig. 3. DEAE-Sephadex A-25 column chromatography of liver (A) and tail fin (B) cytosol. Columns (1.5 X 20 cm) of DEAE-Sephadex A-25 were equilibrated in buffer B. Cytosol (34.6 mg of liver protein or 15.3 mg of tail fin protein) was then applied and the column washed in starting buffer. The columns were then eluted with a O-O.5 M NaCl gradient in buffer B. Ahquots (0.2 ml) of the 1.5 ml fractions were then assayed for Ts binding as described in Materials and Methods, using JXC to separate bound and free hormone.

binding protein from liver of 71,700 * 4100 and a molecular weight of 66,100 ?: 7900 for the T’s binding protein from tail fin. The difference between the two molecular weights is not statistically si~i~c~t. Likewise, the calculated frictional ratio (Tanford, 1963) for the tadpole liver T3 binding protein is 1.28 + 0.05 and 1.21 f 0.10 for the tail fin T3 binding protein. Again, the differences between the frictional ratia for the Liver and tail fin T3 binding proteins are not statistically significant. The results for the friction& ratio correspond to a prolate ellipsoid with an axial ratio of 5 and an oblate ellipsoid with an axial ratio of 5 or 6 (Svedberg and Pedersen, 1940). The third property studied was elution from ~EAE-Sephadex ion exchange columns. Only a single peak of Ts binding activity was found in cytosol from tadpole liver and tail fin (figs. 3A and 3B respectively). The salt concentration required for the elution was essentially the same for both liver and tail fin (0.233 * 0.017 M NaCl and 0.213 + 0.015 M NaCI respectively).

Physicochemical properties of T3 binding proteins

211

Previous reports (Yoshizato et al., 1975; Jaffe and Gold, 1977) have shown that the Ta binding proteins in tadpole liver and tail fin have different affinities for Ta. The two binding proteins were shown, however, to bind various thyroid hormone analogues with similar relative affinities (Jaffe and Gold, 1977). This raised the possibility that the binding proteins in the two tissues were similar. The studies reported here indicate (1) that there is only one apparent Ta binding protein in each tissue, and (2) that the pliysicochemical properties of the Ts binding protein from the two tissues are essentially identical. This raises the possibility that the difference in the liver and tail fin cytosols’ afffinity for Ts is due to the presence of a factor which modifies the binding rather than distinct Ts binding proteins in the two tissues. This question can be resolved following purification of the T3 binding protein from the two tissues, The purification of the binding protein would also help to clarify its functional role.

ACKNOWLEDGEMENTS The excellent technical assistance of Ms. Marsha L. Gold is gratefully acknowledged. This work was supported in part by National Institutes of Health Grant AM18985.

REFERENCES Davis, P.J., Handwerger, B.S. and Glaser, F. (1974) J. Biol. Chem. 249,6208-6217. Defer, N., Dastugue, B., Sabatier, M.M., Thomopoulos, P. and Kruh, J. (1975) Biochem. Biophys. Res. Commun. 67,995-1004. Dillman, W., Surks, M.I. and Oppenheimer, J.H. (1974) Endocrinology 95,492-498. Jaffe, R.C. and Gold, M.L. (1977) Mol. Cell. Endocrinol. 8, 1-13. King, R.J.B. and Mainwaring, W.I.P. (1974) In: Steroid Cell Interactions (University Park Press, Baltimore). Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265275. Martin, R.G. and Ames, B.N. (1961) J. Biol. Chem. 236,1372-1379. Porath, J. (1963) Pure Appl. Chem. 6,233-244. Siegel, L.M. and Monty, K.J. (1966) Biochim. Biophys. Acta 112, 346-362. Sober, H.A. (1968) In: Handbook of Biochemistry (Chemical Rubber Co., Cleveland). Svedberg, T. and Pedersen, K.O. (1940) In: The Ultracentrifuge (Clarendon Press, Oxford). Tanford, C. (1963) In: Physical Chemistry of Macromolecules (Wiley, New York). Taylor, A.C. and Kollros, J.J. (1946) Anat. Rec. 94, 7-24. Visser, T.J., Bernard, H.F., Dotter, R. and Hennemann, G. (1976) Acta Endocrinol. (Kbh.) 82,98-104. Yoshizato, K., Kistler, A. and Frieden, E. (1975) J. Biol. Chem. 250,8337-8343.

Physicochemical properties of the cytoplasmic triiodothyronine binding protein from tadpole liver and tail fin.

Molecular and Cellular Endocrinology, 11 (1978) 205-211 0 Elsevier/Nortlt-Holland Scientific Publishers, Ltd. PHYSICOCHEMICAL PROPERTIES OF THE CYTOP...
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