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Identification of type I iodothyronine 5′-deiodinase as a selenoenzyme
Vol. 173, No. 3,1990 December 31, 1990
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1143-1149
IDENTIFICATION OF TYPE I IODOTHYRONINE 5'-DEIODINASE AS A SELENOENZYME Dietrich Behne 1" , Antonios Kyriakopoulos 1, Harald Meinhold 2, and Josef K6hrle 3 1Department 'q'race Elements in Health and Nutrition", Hahn-Meitner-lnstitut, D-1000 Berlin 39, 2Department of Nuclear Medicine, Klinikum Steglitz, Freie Universit&t Berlin, D-1000 Berlin 45, and 3Department of Clinical Endocrinology, Medizinische Hochschule Hannover, D-3000 Hannover 61, Federal Republic of Germany Received October 9, 1990
A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'deiodinase subunit, affinity-labeled with N-bromoacetyl-[1251]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme. ©199o AcademicPress, Zn~.
Since the discovery of Se as an essential element (1), glutathione peroxidase (GSH-Px) has been the only selenoprotein in animals for which enzymatic functions have been established (2). Recently, in a study on rats, after in vivo labeling with [75Se]selenite and separation of the tissue proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), a 27.8 kDa selenoprotein was found (3). This compound, which was named protein No. 7, was one of the main selenoproteins in the thyroid and was otherwise only detected in the liver and in the kidney. In the liver it was shown to be membrane-bound (4). As these organs have the highest activities of the enzyme type I iodothyronine 5'-deiodinase (5'-D), which catalyzes the deiodination of the prohormone L-thyroxine (T4) to the biologically active thyroid hormone 3,3',5-triiodothyronine (13) (5,6), we suggested that protein No. 7 might have a role in thyroid hormone metabolism (3,4). This hypothesis was supported by the finding of
* To whom reprint requests should be addressed. Abbreviations: GSH-Px, glutathione peroxidase; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; T4, L-thyroxine; T3, 3,3',5-triiodothyronine; 5'-D, type I iodothyronine 5'-deiodinase; BrAcT4, N-bromoacetyI-L-thyroxine; p27, 27 kDa substrate binding type 15'-deiodinase subunit. 0006-291X/90 $1.50 1143
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decreased T3 production in the liver homogenates of Se-deficient rats (7). A 27 kDa integral membrane protein was identified by affinity labeling with N-bromoacetyI-L-thyroxine (BrAcT4) in rat liver and kidney, and in thyroid and kidney cell lines of various species, as the substrate binding 5'-D subunit (p27) involved in the catalytic reaction of 5'-D (8-11). The occurrence of protein No. 7 and p27 in the same organs and subcellular compartments, the similarity in their molecular masses and the effects of Se deficiency on the 5'deiodination strongly suggest that the two proteins are identical. We therefore carried out several experiments on rats to prove this hypothesis and to investigate the effects of the Se status on the 5'-D activity. MATERIALS AND METHODS Animals: Wistar rats (Mus Rattus, Brunnthal, F.R.G.) were fed for 3 generations either a low Se diet with a Se content of 2jug/kg dry matter (Se-deficient animals) or the same diet with 300jug Se/kg added as sodium selenite (Se-adequate animals) and distilled water ad libitum. The composition of the diet has been described elsewhere (12). The animals were killed under appropriate anesthesia.
Experiment 1: The livers of 4 Se-deficient and 4 Se-adequate 4-month-old female rats were homogenized. From the livers of 2 of the Se-adequate animals cytosol and membranes were prepared by centrifugation at 105 000 x g and mixed with equal amounts of one of the Se-deficient liver homogenates. Samples of the homogenates (0.4 mg protein) and of these mixtures (0.4 + 0.4 mg protein) were taken for the determination of the 5'-D activity. Experiment 2: For the in vivo labeling of the selenoproteins doses of 30/uCi [75Se]selenite (10.3/Jg Se) were injected into the vena caudalis of 2 Se-deficient 5-month-old ma!e rats (animals 1 and 2) at weekly intervals. Animal 1 was given six doses and animal 2 seven doses. Liver samples were taken for quantitative Se analysis. The microsomes of the liver of these animals and of 2 non-labeled Se-adequate rats of the same age were prepared by means of differential centrifugation as previously described (4) for affinity labeling with BrAcT4. 5'-D activity: For the determination of the 5'-D activity a modification of the method of Visser et al. (13) was used. The samples (0.4 or 0.8 mg protein) were incubated in a final volume of 0.5 ml 100 mM Tris/HCI buffer (pH 7.4) with 1/uM T4 in the presence of lmM dithiothreitol at 37 C for 60 min. After the addition of 1 ml ethanol the T3 production was determined in the ethanolic extracts by radioimmunoassay (14). Affinity labeling: BrAcT4 and BrAc[1251]T4 (specific activity 3000/uCi//~g) were synthesized as previously described (8) and used for affinity labeling (8) of rat liver microsomal membranes from in vivo 75Se-labeled rats. SDS-PAGE= The affinity labeled microsomal proteins (45/ug) were separated by SDS-PAGE under reducing conditions in two 16% 0.75 mm gels with the modifications previously described (15). The 27 kDa section was cut out in one wet gel for protein fragmentation. The other gel was dried for the radioactivity measurements. The molecular mass was determined by interpolation using as standards bovine serum albumin (68 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa) and cytochrom C (12.4 kDa).
Protein fragmentation: 1.5 mm gel slices containing the proteins in the 27 kDa region were subjected to CNBr cleavage or trypsin proteolysis followed by SDS-PAGE (18% 1.5 mm gels) of the fragments as previously described (8) with the following modifications: CNBr cleavage was performed in 2% CNBr for 14 h, and fragmentation by 20 ug trypsin was carried out directly in the wet gel slices as described (16). The gels were dried and cut into sections for tracer determination. Radioactivity measurements: The 27 kDa region in the wet gels was localized by determining the 1251 distribution with an automatic scanner (Radio-TLC-analyzer LB 2832 with LB 2811 counting chamber, Berthold, Wildbad, F.R.G.). In the dried gels the labeled proteins and protein fragments were localized by autoradiography (3). The 75Se activity in the gel sections was measured as described (4). For the 1251 determination a germanium detector with a beryllium window coupled to a 4000 channel analyzer (Canberra, Meriden, CT) was used. 1144
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Se content: The liver Se content was determined by hydric atomic absorption spectrometry by means
of a method described elsewhere (17).
RESULTS AND DISCUSSION The results of experiment 1 (Table 1) show that the 5'-deiodination of T4 to T3 in rat liver homogenates is related to a Se-dependent factor. In agreement with the results of other authors (7) the 5'-D activity was found to be considerably lowered in Se-deficiency. In the liver of the Se-depleted rats the content of Se and thus of GSH-Px had decreased to less than 1% of that in the Se-adequate controls (12), and it has been suggested that effects of Se deficiency on thyroid hormone metabolism may be related to the diminished GSH-Px level (18). However, the 5'-D activity in the Se-deficient liver homogenate was reconstituted by addition of the membrane fraction from Se-adequate rats but not by the cytosolic fraction which contains most of the hepatic GSH-Px (4). This indicated that not GSH-Px but a membranebound Se-dependent component was responsible for the changes in the 5'-D activity. The results were consistent with our hypothesis that the membrane selenoprotein No. 7 is identical to p27 and thus the Se-dependent factor necessary for normal T4 deiodination. To establish the identity of the two proteins we compared their mobilities in SDS-PAGE. Fig. 1 shows the autoradiograms of the in vivo 75Se-labeled microsomal proteins affinitylabeled in vitro with BrAc[1251]T4 (left lane) or BrAcT 4 (right lane). BrAc[1251]T4 was predominantly present in two proteins with molecular masses of 27 and 55 kDa, as previously described (8,9). The 27 kDa protein has recently been identified as the substrate binding 5'-D subunit and is not related to the 55 kDa protein (9). Two 75Se-containing bands (right lane) comigrated with these bands. They have previously been localized in liver microsomal membranes (4). The fact that predominant labeling with BrAc[1251 ] T4 was found in the very two bands in which selenoproteins were present suggests that the T4 derivative interacts with the Se-
Table 1. Effects of a Se-dependent factor on the T4 5'-deiodination to T3 in rat liver Preparation
T3 production (pmol/I)
Liver homogenate a) (Se-adequate)
19.9__+3.8
Liver homogenate a) (Se-deficient)
2.6 +__0.4
Liver homogenate + liver cytosol b) (Se-deficient) (Se-adequate)
3.0 + 0.4
Liver homogenate + liver membranes b) (Se-deficient) (Se-adequate)
19.4±2.6
a) Eachsample contained0.4 mg protein. Valuesare mean_+SD of 4 animals. b) Equal protein amounts of homogenate (0.4 mg) and liver fraction (0.4 mg) were mixed. Valuesare the mean_+SD for the liver fractions of 2 animals mixed each time with the same Sedeficient homogenate. 1145
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BrAc[12SI]T 4
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75Se
kDa
1 2 --3 224 __5
68 45
6
z
29 20,5
(Z)
a
12,4
b
c
Trypsin
d CNBr
Fig. 1. Autoradiogram of 75Se-labeled or BrAc[1251]T4 affinity-labeled rat liver microsomal proteins. Rat liver microsomes (0.1 mg protein) prepared from in vivo 75Se-labeled rats were incubated with BrAc[1251]T4 (540 000 epm/190 fmol) (left lane) or with non-radioactive BrAcT4 (450 pmol) (right lane) in 0.1 M Tris/HCl (pH 7.4), 10 mM dithiothreitol, 0.025% Wl detergent and lmM EDTA in a total volume of 0.1 ml for 15 min at 37 C, as described (8), and separated by SDS-PAGE. Autoradiography of the dried gels was carried out for 2 h (left lane) or 10 days (right lane) at -70 C (3). Ficl. 2. Peptide fragments of BrAc[1251]T4 affinity-labeled 27 kDa proteins from 75Se-labeled rats. Affinity-labeled 27 kDa proteins were located in 16% SDS-PAGE gels, cut out and partially digested by trypsin or cleaved by CNBr as described in "Materials and Methods". Lane a, trypsin digestion of p27; lane b, p27 control without trypsin (dimers and oligomers of p27 were formed during the experimental procedure); lane c, p27 control processed in 70% formic acid without CNBr; lane d, CNBr cleavage of p27. After autoradiography (24 h exposure) sections were cut from lane a and d as indicated for 75Se and 1251 measurements (Table 2).
containing site in these proteins. This was supported for p27 by chemical CNBr cleavage or partial proteolysis with trypsin. These methods generate specific individual sets of peptide fragments representative of the original molecules and allow the distinction or comparison of two different proteins (16). The autoradiograms of the BrAc[1251]T4-1abeled fragments (Fig. 2)
Table 2. Distribution of 75Se and 1251 in protein fragments after trypsin digest or CNBr cleavage of the proteins in the 27 kDa range labeled with 758e and BrAc[1251]T4 Trypsin Gel a) 1251 75Se 1251/75Se section (cpsxl02) (cpsxl0 -3) (xl05) 1 2 3 4 5 6
4.2 4.4 2.1 3.4 4.8 7.6
2.0 2.2 1.2 1.8 2.1 3.8
CNBr Gel a) 1251 75Se 1251/75Seb ) section (cpsxl02) (cpsxl0 "3) (xl05)
2.1 2.0 1.8 1.9 2.3 2.0
1 2 3 4 5 6
0.5 1.5 1.4 0.7 0.4 0.5
<0.6 c) 1.5 1.2 0.8 <0.6 <0.6
1.0 1.2 0.9
a)The gel sectionswere cut as indicated in Fig. 2. b)CNBrcleavageof the 27 kDa gel slice in 70% formic acid leadsto a 40% to 60% loss of 1251 comparedwith a >90% recovery in the trypsin fragmentation. C)Limitof detection. 1146
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Table 3. Comparison of the content of the type 15'-deiodinase subunit (p27) and the Se content of the subunit in liver rnicrosomes of rats with different Se status p27 Se (pmol/mg microsomal protein) Animal 1 Animal 2 Controls
0.6 1.2 2.0 + 0.6a)
0.4 0.9 1.6b)
a)Mean + SD of 2 animals, b)Valuefrom a Se4Jeficientrat which after replenishmentwith oral doses of 75Se-selenite,equivalentto the Se amounts ingestedfrom a diet with a Se content of 0.3 mg/kg, had almost reachednormal Se status (Behne,D., unpublisheddata).
reveal a pattern similar to that previously described for p27 in the liver and kidney of rats (8). The data for the gel sections of double-labeled samples (Table 2) show that 758e and 1251had identical quantitative distribution patterns and that in each set the ratio of the activities of the two radionuclides in the fragments was constant. This indicates that p27 and protein No. 7 are identical and that Se and BrAcT 4 are present at the same site in the 5'-D subunit. With the finding that p27 is a Se-containing compound it seems likely that the second predominant BrAcT4-1abeled protein with a molecular mass of 55 kDa may also be identical to the comigrating selenoprotein (Fig. 1). It has recently been suggested that this 55 kDa protein is protein disulfide isomerase (19-21) which thus might be a further selenoprotein. A consideration of new selenoenzymes should also include the other iodothyronine deiodinase isozymes. The type II 5'-deiodinase activity in rat brain was likewise found to decrease in Se deficiency (22). The in vivo 75Se and in vitro BrAc[1251]T4 double-labeling technique also yielded information on the molar Se content in p27. With the covalent BrAcT 4 labeling of microsomal membranes the p27 content could be titrated assuming a 1:1 stochiometry and complete BrAcT 4 incorporation into the subunit (9,11). The p27 Se content was calculated directly from the 75Se activity. At the start of the experiment the liver Se pool of the Se-depleted rats was less than 1% of that of the Se-adequate animals (12). It was then replenished almost exclusively with 75Se-labeled selenite of known specific activity. The validity of this approach was shown by comparing the liver Se contents determined by atomic absorption spectrometry (0.16 and 0.23 mg Se/kg wet weight for animals 1 and 2, respectively) with the values calculated from the 75Se activity (0.16 and 0.22 mg Se/kg wet weight). Table 3 shows the results of the comparison of the p27 content and the p27 Se content for rats which differed with regard to their Se status. The ratio of the two parameters indicates that p27 contains one Se atom per molecule. The microsomal p27 content rose with increasing replenishment of the liver Se pool. In the liver of Se-adequate rats fed a diet with 0.3 mg Se/kg a level of about 2 pmol p27/mg microsomal protein was found which corresponds to previous estimates of the 5'-D content (9,23). It will be of interest to find out whether additional Se supplementation can further increase the p27 content and the 5'-D activity in the liver and other tissues. Previous results indicated that BrAcT 4 binds covalently to the hydrophobic thyroid hormone substrate binding site and rapidly inactivates 5'-D by alkylating a hyperreactive reduced cysteine residue assumed to be present in the active site of 5'-D and essential for the catalysis 1147
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of 5'-deiodination (5,6,9). We show here by chemical CNBr cleavage at methionine residues and by trypsin proteolysis at lysine or arginine residues that the different labeled fragments contain both BrAcT4 and Se. It therefore seems very likely that Se is situated in the active site of 5'-D and that the hyperreactive cysteine is in fact selenocysteine. With these findings, after GSH-Px a second selenoenzyme with important regulatory functions in animal physiology has been identified. The relationship between Se status and the formation of the biologically active thyroid hormone T3 from the prohormone T4, established in this way, will open up new areas of research on the interactions between the two essential trace elements, selenium and iodine, and the impact of these interactions on the thyroid hormone system. It will also be of significance in understanding the etiology and improving the treatment of thyroid hormone related diseases where the Se status of patients will have to be taken into consideration as an additional factor besides iodine supply. ACKNOWLEDGMENTS This work was supported in part by grants from the Deutsche Forschungsgemeinschaft. We would like to thank Mr. J. Franke, Kiinikum Steglitz, for his help with the animal experiments, Mrs. H. Gessner, Hahn-Meitner-lnstitut, for her most valuable technical assistance, Dr. D. Gawlik, Hahn-Meitner-lnstitut, for his help with the low level 75Se measurements, and Dr. H. Rokos, Henning Berlin GmbH, for generously providing HPLC-purified BrAcT4 and BrAc[ 1251]T4.
REFERENCES 1. Schwarz, K., and Foltz, C.M. (1957) J. Am. Chem. Soc. 79, 3292-3293. 2. Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G., and Hoekstra, W.G. (1973) Science 179, 588-590. 3. Behne, D., Hilmert, H., Scheid, S., Gessner, H., and Elger, W. (1988) Biochim. Biophys. Acta 966, 1221. 4. Behne, D., Scheid, S., Kyriakopoulos, A., and Hilmert, H. (1990) Biochim. Biophys. Acta 1033, 219225. 5. Hesch, R.D., and K6hrle, J. (1986) in Werner's "The Thyroid" (S.H. Ingbar and L.E. Braverman, eds.) 5th ed. pp. 154-200, J.P.Lippincott, Philadelphia. 6. Leonard, J.L., and Visser, T.J. (1986) In Thyroid Hormone Metabolism (G. Hennemann, ed.) pp. 189229, Marcel Dekker, New York. 7. Beckett, G.J., Beddows, S.E., Mortice, P.C., Nicol, F., and Arthur, J.R. (1987) Biochem. J. 248, 443447. 8. K~hrle, J., Rasmussen, U.B., Rokos, H., Leonard, J.L, and Hesch, R.D. (1990) J. Biol. Chem. 265, 6146-6154. 9. K6hrle, J., Rasmussen, U.B., Ekenbarger, D.M., Alex, S., Rokos, H., Hesch, R.D., and Leonard, J.L. (1990) J. Biol. Chem. 265, 6155-6163. 10. Oertel, M., Hesch, R.D., and K~hrle, J. In Abstracts of the Internat. Thyroid Symp. "150th Anniversery of Basedow's Disease", Halle, GDR, May 2-6, 1990. 11. Safran, M., K5hrle, J., Braverman, L.E., and Leonard, J.L. (1990) Endocrinology 126, 826-831. 12. Behne, D., Kyriakopoulos, A., Scheid, S., and Gessner, H. (1991) J. Nutr. (in press). 13. Visser, T.J., Fekkes, D., Docter, R., and Hennemann, G. (1978) Biochem. J. 174, 221-229. 14. Meinhold, H. (1986) In Thyroid Hormone Metabolism (G. Hennemann, ed.) pp. 133-186, Marcel Dekker, New York. 15. Kolbe, H.V.J., Costello, D., Wong, A., Lu, R.C., and Wohlrab, H. (1984) J. Biol. Chem. 259, 9115-9120. 16. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., and Laemmli, U.K. (1977) J. Biol. Chem. 252, 11021106. 1148
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17. 18. 19. 20.
Sch&fer, K., and Behne, D. (1984) Landwirtsch. Forsch. 37, 58-65. Golstein, J., Corvilain, B., Lamy, F., Paquer, D., and Dumont, J.E. (1988) Acta Endocr. 118, 495-502. Obata, T., Kitagawa, S., Gong, Q-H., Pastan, I., and Cheng, S-Y. (1988) J. Biol. Chem. 263, 782-785. Horiuchi, R., Yamauchi, K., Hayashi, H., Koya, S., Takeuchi, Y., Kato, K., Kobayashi, M., and Takikawa, H. (1989) Eur. J. Biochem. 183, 529-538. 21. Schoenmakers, C.H.H., Pigmans, I.G.A.J., Hawkins, H.C., Freedman, R.B., and Visser, T.J. (1989) Biochem. Biophys. Res. Commun. 162, 857-868. 22. Beckett, G.J., MacDougall, D.A., Nicol, F., and Arthur, J.R. (1989) Biochem. J. 259, 887-892. 23. Mol, J.A., Docter, R., Kaptein, E., Jansen, G., Hennemann, G., and Visser, T.J. (1984) Biochem. Biophys. Res. Commun. 124, 475-483.
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IDENTIFICATION OF TYPE I IODOTHYRONINE 5'-DEIODINASE AS A SELENOENZYME Dietrich Behne 1" , Antonios Kyriakopoulos 1, Harald Meinhold 2, and Josef K6hrle 3 1Department 'q'race Elements in Health and Nutrition", Hahn-Meitner-lnstitut, D-1000 Berlin 39, 2Department of Nuclear Medicine, Klinikum Steglitz, Freie Universit&t Berlin, D-1000 Berlin 45, and 3Department of Clinical Endocrinology, Medizinische Hochschule Hannover, D-3000 Hannover 61, Federal Republic of Germany Received October 9, 1990
A 27.8 kDa membrane selenoprotein was previously identified in rat thyroid, liver and kidney, the tissues with the highest activities of type I iodothyronine 5'-deiodinase. This membrane enzyme catalyzes the deiodination of L-thyroxine to the biologically active thyroid hormone 3,3',5-triiodothyronine. A decrease in the activity of this enzyme, observed here in the liver of selenium-deficient rats, was found to be due to the absence of a selenium-dependent membrane-bound component. By chemical and enzymatic fragmentation of the 75Se-labeled selenoprotein and of the 27 kDa substrate binding type I 5'deiodinase subunit, affinity-labeled with N-bromoacetyl-[1251]L-thyroxine, and comparison of the tracer distribution in the peptide fragments the identity of the two proteins was shown. The data indicate that the deiodinase subunit contains one selenium atom per molecule and suggest that a highly reactive selenocysteine is the residue essential for the catalysis of 5'-deiodination. From the results it can be concluded that type I iodothyronine 5'-deiodinase is a selenoenzyme. ©199o AcademicPress, Zn~.
Since the discovery of Se as an essential element (1), glutathione peroxidase (GSH-Px) has been the only selenoprotein in animals for which enzymatic functions have been established (2). Recently, in a study on rats, after in vivo labeling with [75Se]selenite and separation of the tissue proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE), a 27.8 kDa selenoprotein was found (3). This compound, which was named protein No. 7, was one of the main selenoproteins in the thyroid and was otherwise only detected in the liver and in the kidney. In the liver it was shown to be membrane-bound (4). As these organs have the highest activities of the enzyme type I iodothyronine 5'-deiodinase (5'-D), which catalyzes the deiodination of the prohormone L-thyroxine (T4) to the biologically active thyroid hormone 3,3',5-triiodothyronine (13) (5,6), we suggested that protein No. 7 might have a role in thyroid hormone metabolism (3,4). This hypothesis was supported by the finding of
* To whom reprint requests should be addressed. Abbreviations: GSH-Px, glutathione peroxidase; SDS-PAGE, sodium dodecyl sulfatepolyacrylamide gel electrophoresis; T4, L-thyroxine; T3, 3,3',5-triiodothyronine; 5'-D, type I iodothyronine 5'-deiodinase; BrAcT4, N-bromoacetyI-L-thyroxine; p27, 27 kDa substrate binding type 15'-deiodinase subunit. 0006-291X/90 $1.50 1143
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decreased T3 production in the liver homogenates of Se-deficient rats (7). A 27 kDa integral membrane protein was identified by affinity labeling with N-bromoacetyI-L-thyroxine (BrAcT4) in rat liver and kidney, and in thyroid and kidney cell lines of various species, as the substrate binding 5'-D subunit (p27) involved in the catalytic reaction of 5'-D (8-11). The occurrence of protein No. 7 and p27 in the same organs and subcellular compartments, the similarity in their molecular masses and the effects of Se deficiency on the 5'deiodination strongly suggest that the two proteins are identical. We therefore carried out several experiments on rats to prove this hypothesis and to investigate the effects of the Se status on the 5'-D activity. MATERIALS AND METHODS Animals: Wistar rats (Mus Rattus, Brunnthal, F.R.G.) were fed for 3 generations either a low Se diet with a Se content of 2jug/kg dry matter (Se-deficient animals) or the same diet with 300jug Se/kg added as sodium selenite (Se-adequate animals) and distilled water ad libitum. The composition of the diet has been described elsewhere (12). The animals were killed under appropriate anesthesia.
Experiment 1: The livers of 4 Se-deficient and 4 Se-adequate 4-month-old female rats were homogenized. From the livers of 2 of the Se-adequate animals cytosol and membranes were prepared by centrifugation at 105 000 x g and mixed with equal amounts of one of the Se-deficient liver homogenates. Samples of the homogenates (0.4 mg protein) and of these mixtures (0.4 + 0.4 mg protein) were taken for the determination of the 5'-D activity. Experiment 2: For the in vivo labeling of the selenoproteins doses of 30/uCi [75Se]selenite (10.3/Jg Se) were injected into the vena caudalis of 2 Se-deficient 5-month-old ma!e rats (animals 1 and 2) at weekly intervals. Animal 1 was given six doses and animal 2 seven doses. Liver samples were taken for quantitative Se analysis. The microsomes of the liver of these animals and of 2 non-labeled Se-adequate rats of the same age were prepared by means of differential centrifugation as previously described (4) for affinity labeling with BrAcT4. 5'-D activity: For the determination of the 5'-D activity a modification of the method of Visser et al. (13) was used. The samples (0.4 or 0.8 mg protein) were incubated in a final volume of 0.5 ml 100 mM Tris/HCI buffer (pH 7.4) with 1/uM T4 in the presence of lmM dithiothreitol at 37 C for 60 min. After the addition of 1 ml ethanol the T3 production was determined in the ethanolic extracts by radioimmunoassay (14). Affinity labeling: BrAcT4 and BrAc[1251]T4 (specific activity 3000/uCi//~g) were synthesized as previously described (8) and used for affinity labeling (8) of rat liver microsomal membranes from in vivo 75Se-labeled rats. SDS-PAGE= The affinity labeled microsomal proteins (45/ug) were separated by SDS-PAGE under reducing conditions in two 16% 0.75 mm gels with the modifications previously described (15). The 27 kDa section was cut out in one wet gel for protein fragmentation. The other gel was dried for the radioactivity measurements. The molecular mass was determined by interpolation using as standards bovine serum albumin (68 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa) and cytochrom C (12.4 kDa).
Protein fragmentation: 1.5 mm gel slices containing the proteins in the 27 kDa region were subjected to CNBr cleavage or trypsin proteolysis followed by SDS-PAGE (18% 1.5 mm gels) of the fragments as previously described (8) with the following modifications: CNBr cleavage was performed in 2% CNBr for 14 h, and fragmentation by 20 ug trypsin was carried out directly in the wet gel slices as described (16). The gels were dried and cut into sections for tracer determination. Radioactivity measurements: The 27 kDa region in the wet gels was localized by determining the 1251 distribution with an automatic scanner (Radio-TLC-analyzer LB 2832 with LB 2811 counting chamber, Berthold, Wildbad, F.R.G.). In the dried gels the labeled proteins and protein fragments were localized by autoradiography (3). The 75Se activity in the gel sections was measured as described (4). For the 1251 determination a germanium detector with a beryllium window coupled to a 4000 channel analyzer (Canberra, Meriden, CT) was used. 1144
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Se content: The liver Se content was determined by hydric atomic absorption spectrometry by means
of a method described elsewhere (17).
RESULTS AND DISCUSSION The results of experiment 1 (Table 1) show that the 5'-deiodination of T4 to T3 in rat liver homogenates is related to a Se-dependent factor. In agreement with the results of other authors (7) the 5'-D activity was found to be considerably lowered in Se-deficiency. In the liver of the Se-depleted rats the content of Se and thus of GSH-Px had decreased to less than 1% of that in the Se-adequate controls (12), and it has been suggested that effects of Se deficiency on thyroid hormone metabolism may be related to the diminished GSH-Px level (18). However, the 5'-D activity in the Se-deficient liver homogenate was reconstituted by addition of the membrane fraction from Se-adequate rats but not by the cytosolic fraction which contains most of the hepatic GSH-Px (4). This indicated that not GSH-Px but a membranebound Se-dependent component was responsible for the changes in the 5'-D activity. The results were consistent with our hypothesis that the membrane selenoprotein No. 7 is identical to p27 and thus the Se-dependent factor necessary for normal T4 deiodination. To establish the identity of the two proteins we compared their mobilities in SDS-PAGE. Fig. 1 shows the autoradiograms of the in vivo 75Se-labeled microsomal proteins affinitylabeled in vitro with BrAc[1251]T4 (left lane) or BrAcT 4 (right lane). BrAc[1251]T4 was predominantly present in two proteins with molecular masses of 27 and 55 kDa, as previously described (8,9). The 27 kDa protein has recently been identified as the substrate binding 5'-D subunit and is not related to the 55 kDa protein (9). Two 75Se-containing bands (right lane) comigrated with these bands. They have previously been localized in liver microsomal membranes (4). The fact that predominant labeling with BrAc[1251 ] T4 was found in the very two bands in which selenoproteins were present suggests that the T4 derivative interacts with the Se-
Table 1. Effects of a Se-dependent factor on the T4 5'-deiodination to T3 in rat liver Preparation
T3 production (pmol/I)
Liver homogenate a) (Se-adequate)
19.9__+3.8
Liver homogenate a) (Se-deficient)
2.6 +__0.4
Liver homogenate + liver cytosol b) (Se-deficient) (Se-adequate)
3.0 + 0.4
Liver homogenate + liver membranes b) (Se-deficient) (Se-adequate)
19.4±2.6
a) Eachsample contained0.4 mg protein. Valuesare mean_+SD of 4 animals. b) Equal protein amounts of homogenate (0.4 mg) and liver fraction (0.4 mg) were mixed. Valuesare the mean_+SD for the liver fractions of 2 animals mixed each time with the same Sedeficient homogenate. 1145
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75Se
kDa
1 2 --3 224 __5
68 45
6
z
29 20,5
(Z)
a
12,4
b
c
Trypsin
d CNBr
Fig. 1. Autoradiogram of 75Se-labeled or BrAc[1251]T4 affinity-labeled rat liver microsomal proteins. Rat liver microsomes (0.1 mg protein) prepared from in vivo 75Se-labeled rats were incubated with BrAc[1251]T4 (540 000 epm/190 fmol) (left lane) or with non-radioactive BrAcT4 (450 pmol) (right lane) in 0.1 M Tris/HCl (pH 7.4), 10 mM dithiothreitol, 0.025% Wl detergent and lmM EDTA in a total volume of 0.1 ml for 15 min at 37 C, as described (8), and separated by SDS-PAGE. Autoradiography of the dried gels was carried out for 2 h (left lane) or 10 days (right lane) at -70 C (3). Ficl. 2. Peptide fragments of BrAc[1251]T4 affinity-labeled 27 kDa proteins from 75Se-labeled rats. Affinity-labeled 27 kDa proteins were located in 16% SDS-PAGE gels, cut out and partially digested by trypsin or cleaved by CNBr as described in "Materials and Methods". Lane a, trypsin digestion of p27; lane b, p27 control without trypsin (dimers and oligomers of p27 were formed during the experimental procedure); lane c, p27 control processed in 70% formic acid without CNBr; lane d, CNBr cleavage of p27. After autoradiography (24 h exposure) sections were cut from lane a and d as indicated for 75Se and 1251 measurements (Table 2).
containing site in these proteins. This was supported for p27 by chemical CNBr cleavage or partial proteolysis with trypsin. These methods generate specific individual sets of peptide fragments representative of the original molecules and allow the distinction or comparison of two different proteins (16). The autoradiograms of the BrAc[1251]T4-1abeled fragments (Fig. 2)
Table 2. Distribution of 75Se and 1251 in protein fragments after trypsin digest or CNBr cleavage of the proteins in the 27 kDa range labeled with 758e and BrAc[1251]T4 Trypsin Gel a) 1251 75Se 1251/75Se section (cpsxl02) (cpsxl0 -3) (xl05) 1 2 3 4 5 6
4.2 4.4 2.1 3.4 4.8 7.6
2.0 2.2 1.2 1.8 2.1 3.8
CNBr Gel a) 1251 75Se 1251/75Seb ) section (cpsxl02) (cpsxl0 "3) (xl05)
2.1 2.0 1.8 1.9 2.3 2.0
1 2 3 4 5 6
0.5 1.5 1.4 0.7 0.4 0.5
<0.6 c) 1.5 1.2 0.8 <0.6 <0.6
1.0 1.2 0.9
a)The gel sectionswere cut as indicated in Fig. 2. b)CNBrcleavageof the 27 kDa gel slice in 70% formic acid leadsto a 40% to 60% loss of 1251 comparedwith a >90% recovery in the trypsin fragmentation. C)Limitof detection. 1146
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Table 3. Comparison of the content of the type 15'-deiodinase subunit (p27) and the Se content of the subunit in liver rnicrosomes of rats with different Se status p27 Se (pmol/mg microsomal protein) Animal 1 Animal 2 Controls
0.6 1.2 2.0 + 0.6a)
0.4 0.9 1.6b)
a)Mean + SD of 2 animals, b)Valuefrom a Se4Jeficientrat which after replenishmentwith oral doses of 75Se-selenite,equivalentto the Se amounts ingestedfrom a diet with a Se content of 0.3 mg/kg, had almost reachednormal Se status (Behne,D., unpublisheddata).
reveal a pattern similar to that previously described for p27 in the liver and kidney of rats (8). The data for the gel sections of double-labeled samples (Table 2) show that 758e and 1251had identical quantitative distribution patterns and that in each set the ratio of the activities of the two radionuclides in the fragments was constant. This indicates that p27 and protein No. 7 are identical and that Se and BrAcT 4 are present at the same site in the 5'-D subunit. With the finding that p27 is a Se-containing compound it seems likely that the second predominant BrAcT4-1abeled protein with a molecular mass of 55 kDa may also be identical to the comigrating selenoprotein (Fig. 1). It has recently been suggested that this 55 kDa protein is protein disulfide isomerase (19-21) which thus might be a further selenoprotein. A consideration of new selenoenzymes should also include the other iodothyronine deiodinase isozymes. The type II 5'-deiodinase activity in rat brain was likewise found to decrease in Se deficiency (22). The in vivo 75Se and in vitro BrAc[1251]T4 double-labeling technique also yielded information on the molar Se content in p27. With the covalent BrAcT 4 labeling of microsomal membranes the p27 content could be titrated assuming a 1:1 stochiometry and complete BrAcT 4 incorporation into the subunit (9,11). The p27 Se content was calculated directly from the 75Se activity. At the start of the experiment the liver Se pool of the Se-depleted rats was less than 1% of that of the Se-adequate animals (12). It was then replenished almost exclusively with 75Se-labeled selenite of known specific activity. The validity of this approach was shown by comparing the liver Se contents determined by atomic absorption spectrometry (0.16 and 0.23 mg Se/kg wet weight for animals 1 and 2, respectively) with the values calculated from the 75Se activity (0.16 and 0.22 mg Se/kg wet weight). Table 3 shows the results of the comparison of the p27 content and the p27 Se content for rats which differed with regard to their Se status. The ratio of the two parameters indicates that p27 contains one Se atom per molecule. The microsomal p27 content rose with increasing replenishment of the liver Se pool. In the liver of Se-adequate rats fed a diet with 0.3 mg Se/kg a level of about 2 pmol p27/mg microsomal protein was found which corresponds to previous estimates of the 5'-D content (9,23). It will be of interest to find out whether additional Se supplementation can further increase the p27 content and the 5'-D activity in the liver and other tissues. Previous results indicated that BrAcT 4 binds covalently to the hydrophobic thyroid hormone substrate binding site and rapidly inactivates 5'-D by alkylating a hyperreactive reduced cysteine residue assumed to be present in the active site of 5'-D and essential for the catalysis 1147
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of 5'-deiodination (5,6,9). We show here by chemical CNBr cleavage at methionine residues and by trypsin proteolysis at lysine or arginine residues that the different labeled fragments contain both BrAcT4 and Se. It therefore seems very likely that Se is situated in the active site of 5'-D and that the hyperreactive cysteine is in fact selenocysteine. With these findings, after GSH-Px a second selenoenzyme with important regulatory functions in animal physiology has been identified. The relationship between Se status and the formation of the biologically active thyroid hormone T3 from the prohormone T4, established in this way, will open up new areas of research on the interactions between the two essential trace elements, selenium and iodine, and the impact of these interactions on the thyroid hormone system. It will also be of significance in understanding the etiology and improving the treatment of thyroid hormone related diseases where the Se status of patients will have to be taken into consideration as an additional factor besides iodine supply. ACKNOWLEDGMENTS This work was supported in part by grants from the Deutsche Forschungsgemeinschaft. We would like to thank Mr. J. Franke, Kiinikum Steglitz, for his help with the animal experiments, Mrs. H. Gessner, Hahn-Meitner-lnstitut, for her most valuable technical assistance, Dr. D. Gawlik, Hahn-Meitner-lnstitut, for his help with the low level 75Se measurements, and Dr. H. Rokos, Henning Berlin GmbH, for generously providing HPLC-purified BrAcT4 and BrAc[ 1251]T4.
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