In vitro formation of thyroid hormones from 3,5-diiodothyronine by supernatant of submaxillary gland

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1 J. Biosci., Vol. 3 Number 3, September 1981, pp Printed in India. In vitro formation of thyroid hormones from 3,5-diiodothyronine by supernatant of submaxillary gland MITALI GUHA, RATHA N. HATI and ASOKE G. DATTA Department of Physiology, Indian Institute of Chemical Biology, Calcutta MS received 1 May 1981; revised 11 July 1981 Abstract. Cell-free extracts of submaxillary glands from rat and goat iodinate exogenously added 3,5-diiodothyronine to form 3,5,3'-triidothyronine and thyroxine. This capacity was elevated after either thyroidectomy or exposure of rats to cold (3 ±1 C) for 5 h. However, there was no further increase of iodination of 3,5-diiodothyronine when the thyroidectomized rats were exposed to cold stress. The submaxillary extracts have the ability to incorporate radioactive iodide into 3,5-diiodothyronine, 3,5,3'-triiodothyronine and thyroxine. The presence of 3,5-diiodothyronine was also detected in the soluble supernatant of submaxillary extract. Keywords. 3, 5-Diiodothyronine; thyroid hormone biosynthesis; submaxillary gland. Introduction Iodination of tyrosine and monoiodotyrosine (monoiodo-tyr) to form monoiodoand diido-tyr are the intermediate steps in thyroxine biosynthesis. These reactions are known to be catalysed by peroxidases of the thyroid and some other extrathyroidal tissues, like, submaxillary, lacrimal., mammary and other glands. However, it has not yet been possible to demonstrate the enzymatic formation of thyroxine from 3,5-diiodothyronine (3,5-T2) in vitro in extrathyroidal tissues. Results from our laboratory have established the existence (Hati and Datta, 1967, 1969) and, later, the purification of KI-stimulated peroxidase from goat submaxillary gland (Mahajani et al., 1973). The enzyme present in rat submaxillary gland was increased several fold on thyroidectomy (Chandra et al., 1977). The increase of the enzyme activity upon thyroidectomy was abolished on administration of thyroxine (T4) and 3,3',5-triiodothyronine (3,3',5-T 3 ). However, 3,5-T 2 which has no thyroid hormonal activity, also abolished the effect, of thyroidectomy on the iodinating enzyme of the submaxillary gland. The effect of 3,5-T 2 as described above was unexpected and suggests that 3,5-T 2 exerted this effect by being iodinated to 3,3',5-T 3 and T 4 in vivo. Abbreviations used: Monoiodo-tyr, Monoiodotyrosine; Diiodo-tyr, Diiodotyrosine; 3,5-T 2, Diiodothyronine; 3.5.3'-T 2 Triiodothyroninej-tyr Thyroxine3,3'-T 2 and 3,5' 3'-T 3 represent reversed diiodothyronine and triiodothyronine respectively. 239

2 240 Mitali Guha et al. The present study was undertaken to investigate the formation of 3,3',5-T 3 and T 4 from 3,5-T 2 by the cell-free extracts of the submaxillary gland. Our study indicates that the soluble supernatants of both goat and rat submaxillary glands catalyze the iodination of both endogenous and exogenous 3,5-T 2 to form 3,3', 5-T 3 and T 4. Materials and methods Materials Fresh goat submaxillary glands were collected in ice from the local slaughter house and stored at -10 C before use. 3,5-T 2, 3,3',5-T3 and T4, protease (type VI from S. Streptomyces griseus), and bovine serum albumin were the products of Sigma Chemical Co., St. Louis, Missouri, USA. Tertiary-amyl alcohol was procured from Riedel-Dettäen A. G. Seelze-Hannover, Germany. Dowex-50WXB (H + form, mesh) and H 2 O 2 were obtained from British Drug House, Bombay. Na 131 I was supplied by Bhabha Atomic Research Centre, Bombay. Other chemicals used were of Reagent grade. Animals To study the effect of cold stress, male rats (MRC strain) weighing g, maintained on normal diet, were divided into two groups; the control group was kept at room temperature (28 ±2 C) and the experimental group was exposed to a temperature of 3 ± 1 C for 5 h. The submaxillary glands were then removed for the preparation of extracts as described below. Thyroidectomy was performed by the procedure described by Chandra et al. (1977). Preparation of extracts The submaxillary glands from goat or rat (normal or thyroidectomized) were collected and a 10% homogenate was prepared in isotonic KCl at 0 C in a Potter- Elevhjem homogenizer. The extract was centrifuged at 700 g in a Sorvall RC-2B refrigerated centrifuge for 15 min and fatty material floating on the supernatant was removed. The supernatant was centrifuged again for 1 h at g in a Spinco Model L Preparative uitracentrifuge and the resulting supernatant solution was used as the source of iodinating activities Assay of iodinating activity The activity was assayed by measuring the incorporation of 131 I into 3,5-T 2 to form 3,3',5-T 3 and T 4. The incubation mixture contained in a total volume of 3 ml, 133 mm citrate-phosphate buffer of ph 5.0; 0.2 mm 3,5-T 2 ; 0.13 mm KI; 20 µci of Na 131 I (carrier-free); mm H 2 O 2 and a suitable volume of the extract containing about 100 μg of protein. The reaction was initiated by the addition H2O2 incubated for 3 min at 37 C and terminated by the addition of 0.2 ml of 10 mm sodium thiosulphate. Other details of experimental conditions are given in the legends of the respective figures or tables. For quicker measurement of the iodinating activity, half of the thiosulphate -treated incubation mixture was deproteinized with 0.3 ml of 50% trichloroacetic acid and centrifuged. A portion of the

3 Biosynthesis of thyroid hormones 241 deproteinized supernatant was applied on a Dowex 50W 8 (H + form) column (0.8 6 cm) and the unreacted free inorganic iodide was removed by washing the column with 40 ml of deionized water. The amount or organically-bound iodine was measured by determining radioactivity adsorbed to the column by holding it at a fixed point in a solid scintillation gamma counter. To determine the exact amounts of 3,3'5-T 3 and T 4 formed, 100 μl of the thiosulphate -treated reaction mixture from the other half was applied on one corner of a Whatmann 3 MM chromatographic paper without deproteinization and chromatographed in two dimensions with two different solvents. First the chromatogram was developed in a descending way in tertiary amyl alcohol: ammonia (30:70) solvent system for 24 h and then in the second direction in butanol: 7 Ν ammonia (1:1) for 9 h in an ascending mode. Iodocompounds were detected and measured by autoradiographic technique as mentioned earlier by Hati and Datta (1969). Demonstration of the presence of iodothyronines in submaxillary extract The soluble supernant of submaxillary extract was incubated with 131 I and H 2 O 2 in a final volume of 3 ml (as described in the assay procedure) in the absence of any added 3,5-T 2. After 2 h of incubation at 37 C, the reaction was stopped by the addition of thiosulphate as mentioned before. The incubation mixture was adjusted to ph 7.4 with Na 2 H PO 4 and half of the incubation mixture was subjected to proteolytic digestion (the amount of type VI protease from S. griseus being onethird that of the enzyme protein) for 5 h at 37 C. The mixture was extracted thrice with equal volumes of n-butanol. The other half (protease untreated) of the incubation mixture was extracted with butanol in a similar way. The collected butanol layers were concentrated and applied on paper for chromatographic separation and detection of the individual iodothyronines as described in the assay procedure. Protein was estimated by following the method of Lowry et al. (1951). Results Formation of triiodothyronine and thyroxine Figure 1 demonstrates the chromatographic separation of 3,3',5-T 3 and T 4 which were formed from 3,5-T 2 using a goat submaxillary enzyme preparation. Rat, hamster and human submaxillary extracts also catalysed the iodination of 3,5-T 2 (data not presented). Iodide incorporation into 3,5-T 2 increased linearly with time as well as with increasing concentrations of protein and the rate of incorporation of I - was about 230 n mol/min/mg protein. ph Optimum The ph profile of 3,5-T 2 iodination (using citrate-phosphate buffer) is biphasic in nature with a sharp trough in between the two crests. Figure 2 shows a drop in iodinating activity between the two ph optima (5.23 and 5.7, respectively) with a ph difference of only 0.47 in the citrate-phosphate buffer. A similar biphasic. nature was also obtained in other buffers like acetate and citrate (data not shown). However, we cannot afford any explanation for such a ph profile.

4 242 Mitali Guha et al. Figure 1. Autoradiogram of the products of the 3,5-T 2 iodination. 3,5-T 2 was iodinated in presence of the reagents, described in Methods under 'Assay of iodinating activity'. After termination of the reaction, 100 μl of the reaction mixture was applied on chromatographic paper for separation and analysis of the products as mentioned in Methods. The spot at the origin indicates protein bound iodine. Figure 2. ph optima of the enzyme in citrate-phosphate buffer. In this set of experiments I - incorporation into 3, 3', 5-T 3 (Δ) and T 4 ( ) was measured after paper chromatographic separation of the same as mentioned in Methods.

5 Biosynthesis of thyroid hormones 243 Effect of varying concentrations of substrates on iodination of 3,5-T 2 The rate of iodination increased with increasing concentrations of 3,5-T 2, ΚI and Η 2 Ό 2 up to about 0.2 mm (data not presented). When one substrate was varied, the other two substrates were kept at their optimum concentration. In the next experiment, the products of 3,5-T 2 iodination were analyzed after separating them by paper chromatographic technique and the formation of the firstproduct3,3',5-t 3 was found to be linear with increasing concentrations of 3,5- T 2 (figure 3). Figure 3. Formation of 3,3', 5-T 3 at varying concentrations 3,5-T 2. Formation of 3,3'.5-T 3 was measured after its separation from other iodo compounds by the paper chromatographic procedure described under Methods. In this experiment,270 µg of the enzyme extract was used instead of 100 µg. Effect of various agents on iodination of 3, 5- T 2 Table 1 describes the effect of several compounds on rate of iodination of 3,5-T 2 ; sodium azide was found to be the most potent inhibitor. Formation of iodothyronines in submaxillary extract Diiodothyronine iodination observed in the above experiments prompted us to investigate the presence of iodothyronines in the submaxillary extract. Figure 4 demonstrates the incorporation of 131 I into iodothyronines, using 3,5-T 2 possibly present endogenously in goat submaxillary extract. The zero time incubation mixture or the incubation mixture containing boiled enzyme preparation treated in a similar way showed only one radioactive spot, that of KI, on the autoradiogram (results not shown). The radioactive spot at the 3,5-T 2 region indicates that the compound was formed possibly by coupling of one tyrosine with another labeled

6 244 Mitali Guha et al. diiodo-tyr. Mono- and diido-tyr did not separate well from KI on the paper chromatogram under our experimental conditions. Table 1. Effect of various agents (antithyroid) on diiodothyronine iodination catalysed by goat submaxillary extract. Goat enzyme (0,22 mg) was used in these experiments. The inhibitors, at the concentrations given in the table, were added 10 min before the addition of H 2 O 2. The activities were measured by the quick assay procedure described in Methods. Figure 4. Autoradiogram of the products obtained from endogenous substrates of the submaxillary gland. The figure, shows the formation of 3,3',5-T 3 and T 4 from endogenous substrate, and demonstrates the presence of iodothyronine in the submaxillary extract (see Methods). The autoradiogram was obtained from the incubation mixture, designated as 'protease.untreated' in Methods.

7 Biosynthesis of thyroid hormones 245 The possibility that 3,5-T 2 is present in the extract of goat submaxillary gland is further strengthened from the data in table 2. It may be concluded from table 2 that most of the 3,5-T 2,3,3' 5-T 3 and T 4 were bound to a protein in the submaxillary gland which were released upon proteolytic digestion. It could further be suggested that the synthesis of thyroid hormones are facilitated on the protein molecule. Table 2. Formation of iodothyronines from endogenous substrate(s) of goat submaxillary extract. Goat submaxillary supernatant 2 ml containing about 9.6 mg protein was used in this experiment. * Averages of results from three experiments. In these experiments all the reagents except 3,5-T 2, as mentioned in "Assay of Iodinating Activity" were added and the products were analyzed after paper chromatograpic separation. Detection of 3, 5-T 2 in submaxillary gland To detect the presence of 3,5-T 2 in the submaxillary gland, 30 ml of soluble supernatant containing 249 mg protein was extracted with butanol and chromatographed as indicated before. Although 3,5-diiodothyronine could not be detected in the soluble supernatant, it was detected after the proteolytic digestion of the submaxillary extract. The compound which was sensitive to ferrichloride-ferricyanidearsenic acid reagent was identical to that of authentic 3,5-T 2 having Rf values of 0.39 and 0.46 in tertiary amyl alcohol: ammonia and butanol: ammonia systems respectively (results not shown). Effect of thyroidectomy and cold stress on3, 5-T 2 iodination by soluble supernatant of rat submaxillary gland A previous report from our laboratory already indicated that in rats the monoiodotyr iodination catalysed by the soluble supernatant of the submaxillary gland increased several fold upon thyroidectomy, (Chandra et al., 1977). It was of interest to study the enzyme activity for 3,5-T 2 iodination under identical conditions. The results of table 3 indicate that, although T 3 formation from 3,5-T 2 catalysed by the submaxillary enzyme preparation from a 7 day thyroidectomized

8 246 Mitali Guha et al. animal was not very significant, T 4 formation increased by about 80% following thyroidectomy. The increase in T 4 formation was observed even 4 days after thyroidectomy (data not presented). Table 3. Effect of thyroidectomy and cold stress on the iodination of diiodpthyronine by rat submaxillary extract Enzyme protein (200 μg) was used in all the systems. In the second experiment, the animals were kept in cold (3 C) for 5 h, killed; and enzyme activity measured in the extract of submaxillary gland as mentioned in Methods. The products of 3,5-T 2 in all the above experiments were analyzed after chromatographic separation. It was reported earlier that there was an increase in peroxidase activity of rat submaxillary gland upon exposure to cold (Bhattacharyya et al., 1972). Since the incorporation of iodide into 3,5-T 2 and the peroxidase activity of the submaxillary glands both increased upon thyroidectomy, we studied the effect of cold stress on 3,5-T 2 iodination by the submaxillary enzyme preparations of normal and thyroiddectomized rats. Table 3 shows that on exposure of a normal rat to cold, the total thyroid hormone formation by the submaxillary enzyme preparation was stimulated significantly over the control group but that when cold stress was applied to thyroidectomized rats, no additional effect was observed (results not shown). Discussion We Have reported earlier (Hati and Datta, 1967,1969) that the microsomal fraction of the goat submaxillary gland, iodinates tyrosine or monoiodo-tyr to produce monoiodo and diiodo-tyr. The evidence presented in this communication indicates that the cell-free extract of goat submaxillary gland, in contrast to thyroid glands (Taurog, 1964) also iodinates free 3,5-T 2 to produce free 3,3' 5-T 3 and T 4. The communication further indicates that the iodinating enzyme present in the submaxillary gland is capable of producing thyroid hormone via iodination of a protein, as proteolysis releases about four-fold more thyroid hormones (table 2). Earlier observations of Taurog and Evans (1967) suggested extrathyroidal thyroxine formation in completely thyroidectomized rats, since they found significant amounts of thyroxine m the plasma, 2-4 weeks after thyroidectomy. Thus, it seems likely that extrathyroidal thyroxine formation occurs via the iodination of

9 Biosynthesis of thyroid hormones 247 both free 3,5-T 2 as well as a protein, although, the site(s) and mechanism of this reaction remain to be elucidated. From different studies it could be suggested that the iodination of tyrosine of monoiodo-tyr in the submaxillary gland has some physiological significance as these reactions are modulated by thyroidectomy (Chandra et al., 1977) or by cold stress (Bhattacharyya et al., 1972). However, none of these studies indicated that thyronine was produced in the submaxillary gland. The stimulation of 3,5-T 2 iodination upon thyroidectomy confirms the observation of Chandra et al. (1977) who showed that monoiodo-tyr iodination catalyzed by a submaxillary preparation increased several fold after thyroidectomy. Thyroid hormone (T 3 +T 4 ) formation from 3,5-T 2 (table 3) also increased after exposure to cold, but there was no further change in T 3 and T 4 formation after exposure of thyroidectomized rats to cold. Increase in the activity under cold stress may depend on the presence of the thyroid gland. These results confirm the observation of Bhattacharya et al. (1972). The scheme suggested by Roche and Michel (1955, 1956) for the synthesis of T 4 from 3,5-T 2 is consistent with our observations with the submaxillary enzyme preparation. However, it has been reported earlier (Alexander, 1961) that thyronine, 3,5-T 2 and 3,3',5-T 3 are not iodinated by rat or human thyroid extracts. Unpublished results from our laboratory indicate that thyronine is readily iodinated by submaxillary gland extracts and that the product obtained is not identical to 3,5-T 2, 3,3',5-T 3 or T 4 as its R f value is different from those of these three iodocompounds in tertiary-amyl alcohol: ammonia (30:70) as well as in a butanol: ethanol: ammonia (5:1:2) system. Another pathway of T 3 and T 4 synthesis is suggested by the observation that 3,3'- T 2 is formed by coupling of two molecules of monoiodo-tyr catalysed by a beef thyroid extract (Fischer et al., 1964). Formation of labeled 3,3'-T 2 and 3,3',5'-T 3 (rt 3 ) in vivo in thyroid glands of rat has also been reported by Taurog et al. (1976) who suggested that 3,3'-T 2 is formed by coupling of two molecules of monoiodo-tyr and is further iodinated to 3,3',5'-T 3. The above results imply that 3,5-T 2 is synthesized within the submaxillary gland by coupling of a tyrosine and a diiodo-tyr molecule. According to Roche and Michel (1956) and Kochupillai and Yalow (1978), the formation of 3,5-T 2 is neither due to iodination of thyronine nor to exchange of 131 I with the non-labelled iodine molecule of 3,5-T 2. Our results indicate that 3,5-diiodothyronine is a metabolite of the submaxillary gland. References Alexander, N. M. (1961) Endocrinology, 68, 671. Bhattacharyya, J., Ghosal., J., Ghosh, D. K. and Datta, A. G. (1972)Environ. Physiol. Biochem., 2,195. Chandra, T., Das, R. and Datta, A. G. (1977) Eur. J. Biochem., 72, 259. Fischer, A. G., Schulz, A. R. and Oliner, L. (1964) Biochem. Biophys. Res. Commun., 14, 39. Gmelin, R. and Virtanen, A. I. (1959) Acta Chem. Scand., 13,1469. Hati, R. N. and Datta, A. G. (1967) Biochem. Biophys. Acta., 14B, 310. Hati, R. N. and Datta, A. G. (1969) J. Endocrinol., 44,177. Kochupillai, Ν. and Yalow, R. S. (1978) Endocrinology, 102, 128.

10 248 Mitali Guha et al. Lowry, O. H., Rosebrough, Ν. J., Farr, Α. L. and Randall, R. J. (1951) J. Biol. Chem., 193, 265. Mahajani, U., Halder, I. and Datta, A. G. (1973) Eur. J. Biochem., 37, 541. Roche. J. and Michel, R. (1955) Fortschr. Chem. Org. Naturstoffe. 12, 349. Roche, J. and Michel, R. (1956) In recent progress in hormone research, 12, 1. Taurog, A. (1964) Proc. Mayo Clin., 39, 569. Taurog, A. and Evans, E. S. (1967) Endocrinology, 80, 915. Taurog, Α., Riesco, G. and Larsen, P. R. (1976) Endocrinology, 99, 281.