Thyroglobulin Synthesis Research Articles

MISE EN EVIDENCE, PURIFICATION ET PROPRIETES D'UNE IODOALBUMINE, CONSTITUANT PHYSIOLOGIQUE DE LA GLANDE THYROIDE DE RAT

ABSTRACT The presence of an iodinated albumin in the soluble thyroid proteins of rat has been shown after labeling with radioactive iodine the thyroid glands in vivo or in vitro. The purification of this protein fraction was carried out by centrifugation on sucrose gradient followed by filtration on Sephadex G-200. The purified iodoprotein migrates as an albumin in starch-gel or disc electrophoresis. It is immunoelectrophoretically identical to serumalbumin. It contains MIT, DIT and the hormones (T4, T3). After a short pulse with radioactive iodine in vivo or in vitro, its specific radioactivity is much higher than that of S19 thyroglobulin or other thyroglobulin-like proteins. It is concluded that the thyroid gland of normal rat contains (and most probably synthetizes) an albumin which is able to be iodinated and to contribute to the synthesis of thyroid hormones. In conditions where the synthesis of thyroglobulin is impaired, this iodoalbumin might represent the main substrate for thyroid hormone biosynthesis.

Synthèse acellulaire de la thyroglobuline et site d'iodation

Cell-free synthesis of thyroglobulin and the iodination site 1. 1. Thyroid microsomes and polysomes synthesize thyroglobulin (approx. 19 S) in cell-free systems. If it is a true protein synthesis, membranes appear therefore to be unnecessary. 2. 2. The 12-S precursor, observed in cellular systems, is absent in cell-free systems. Thyroglobulin is totally free in the medium. The ribonucleoprotein particles contain only the light labelled fraction apparently not related to thyroglobulin. The processes of chain liberation and polymerization are therefore different in cellular and cell-free systems. In the latter case it seems that the newly synthesized chains are freed by hybridization with the preformed thyroglobulin which is present in the cell sap. 3. 3. Thyroid microsomes iodinate thyroglobulin but also other proteins present in the medium. The specificity observed in cellular systems is lost in cell-free systems. Polysomes are devoid of iodinating activity. The iodination enzyme seems to be on the membranes. These results agree with the fact, previously established, that biosynthesis and iodination are accomplished on two different sites in cellular systems; a specific transport mechanism thus allows the thyroglobulin molecule to reach the iodination site.

Studies on the protein synthesis in the guinea pig thyroid: II. In vivo labeling of thyroglobulin with 3H-leucine

Guinea pigs were given ~H-leucine at different intervals, ranging from 10 minutes to 6 hours, before sacrifice. The thyroids were excised and homogenized, and the homogenate was divided into nuclear, mitochondrial, microsomal, and supernatant fractions by differential centrifugation. The soluble proteins of the supernatant and of the particulate fractions, from the latter released by vigorous rehomogenization of the pellets in buffered saline, were analyzed by centrifugation in a 10-25 % sucrose gradient. About 40 fractions were collected from each gradient, and their optical density at 280 m/~ and radioactivity were determined. Similar analyses were performed on the material solubilized by digi tonin from the particulate fractions after the extraction of soluble proteins with buffered saline. The results show that the 19 S thyroglobulin of the microsomal fraction is labeled before that of the supernatant fraction. At early observation times the microsomal 12 S and 19 S proteins have a higher specific activity than the supernatant 12 S and 19 S, respectively. During the subsequent hours there is a shift in the relation between microsomal and supernatant 12 S and 19 S specific activities. In principle, the same results are obtained from TSHstimulated and nonstimulated thyroids with the exception that in the latter case there is a lag period before the appearance of any labeling of the microsomal 19 S prote in . In the course of the last observation hours the supernatant 12 S labeling decreases while the supernatant 19 S labeling increases. T h e soluble proteins released from the nuclear and mitochondrial fractions show no labeling or only traces of labeling in the 12 S and 19 S regions. Digitonin treatment solubilizes from all particulate fractions labeled components of low sedimentation rate but no labeled 12 S or 19 S proteins. The principal conclusions drawn from these data are: (i) The formation of 12 S protein and 19 S thyroglobulin is microsome associated. No significant labeling of these components occurs in the nuclear or mitochondrial fractions. (ii) The 12 S protein is probably a subunit precursor of 19 S thyroglobulin. (iii) A large proport ion of the labeled substances found in the thyroid after administration of ~H-leucine represents other processes than thyroglobulin synthesis.

Goitrous cretinism with chromosomal aberration and defect in thyroglobulin synthesis.

A marked discrepancy between the PBI and serum thyroxine values was found in a patient with goitrous cretinism. The iodinated proteins were poorly extracted by butanol, partially resistant to pancreatin hydrolysis, and their iodoamino acid content consisted of MIT, DIT and small amounts of T3 and T4. The predominating iodinated protein in the saline extract of the patient's thyroid gland was found to behave as an albumin during ultracentrifugal, immunological, electrophoretic and salt fractionation procedures. Thyroglobulin was not found by any of these techniques. XO/XY/XXY mosaicism was found in 55 cells counted from peripheral blood. The significance of this finding in relation to the abnormality in the biogenesis of thyroglobulin is unknown.

Loss of organ specific characteristics of hog thyroid cells during growth in vitro.

SummaryNormal hog thyroid glands were treated with enzyme and the dispersed cells cultured in vitro. Radioactive iodide (I125) was added to the growth medium so that the duration of synthesis of thyroglobulin, MIT, DIT, T3, and T4 could be measured. Thyroglobulin production was measured by radio-immunoelectrophoresis and tanned cell hemagglutination inhibition tests. The iodinated amino acids were analyzed by hydrolyzing cell sonicates and separating the iodoamino-acids by thin-layer chromatography. Radioactivity of each amino acid was detected by direct counting in a well scintillation counter and by observation of a radioautograph prepared from a duplicate chromatography plate. Thyroglobulin was synthesized during the first 3 days of in vitro growth. Radioactively labeled T3 and T4 were detected during this same time period. The synthesis of MIT and DIT continued for at least 2 additional days. Serum albumin containing I125 was detected by radioimmunoelectrophoresis during the first 7 days of growth. Ho...

A study of the thyroglobulin, thyroidal protease and iodoproteins in two congenital goitrous cretins

Detailed studies of thyroid functions were carried out in two adult goitrous cretins who were siblings. Both patients had high levels of serum protein-bound iodine, only a fraction of which could be accounted for as iodothyronines. The major circulating iodoprotein possessed many physical characteristics resembling those of serum albumin. Data are presented to indicate that this iodoprotein was not related to thyroglobulin as a precursor or as a degradation product but was rather the product of a collateral pathway of iodine metabolism in the thyroid. Thyroglobulin isolated from these patients showed normal electrophoretic mobility, sedimentation and solubility. The protease from their thyroid glands was able to hydrolyze the thyroglobulin from those glands in an apparently normal manner. The etiology of this disorder may be related to a reduced level of an enzyme in thyroglobulin synthesis with consequent glandular hypertrophy and increased synthesis of other thyroidal iodoproteins.

Defective thyroid hormone biosynthesis with anomalous reaction to thyroid therapy. Report of a case.

A case is presented of an euthyroid woman with an excessively large goitre, present since childhood. A distinct discrepancy was found between total protein bound iodine and butanol extractable iodine in the serum, and an elevated part of the plasma radioactivity after a test dose of 131I was not adsorbed on ion exchange resin. However, this non-adsorbed (»protein-like«) 131I was at least partly extractable with acid butanol. The fractional turnover rate of radiothyroxine was high. The possibility is raised that the pathological component was brought into the circulation as a result of an inborn defect in the thyroidal synthesis or proteolysis of thyroglobulin, and that it was composed of a number of more or less simple peptides, possibly not without some hormonal activity. The uptake of radioiodine by the thyroid was only partly suppressed by desiccated thyroid therapy, but most of the trapped radioactivity did not appear to be incorporated into organic compounds.

Maturation of the rat fetal thyroid.

Maturation of the rat fetal thyroid was studied with the aid of I(131) and of fluorescence and electron microscopy. The I(131) concentration of the fetal gland increased exponentially from day 17 to day 20 of gestation and was related to the weight of the fetus (and presumably the weight of the thyroid) and also to the quantity of I(131) accumulated by the fetus. In the 17-day gland, thyroglobulin or immunologically similar material was sparsely present in the incipient lumens of some cell clusters. With maturation, this material increased and was also observed within follicular cells on days 18 to 19 of gestation. On day 20, the specifically reacting material was present in the follicular lumens and was absent from the cytoplasm of follicular epithelium. Ultrastructurally, the earliest thyroid cells examined were replete with all the organelles found in the more mature epithelium. No direct correlation could be made between the cytoplasmic structures and the presence of thyroglobulin, although the granular endoplasmic reticulum was most likely the organelle responsible for synthesis of thyroglobulin. Thyroglobulin or a precursor was found in fetal thyroid cells before measurable quantities of I(131) were concentrated and before cytoplasmic droplets appeared.

The role of the colloid in the hormone-forming function of the thyroid gland

Experiments were performed on rats. Using the micro — autoradiographic method the author determined the localization of the protein-combined I 131 and S 35 -methionine compounds in the thyroid gland 10 seconds, 5–20 minutes and 1, 4, 24 and 72 hours after the intravenous injection of the labeled preparations. The iodine-thyroglobulin with radioidine inclusion appears first in the colloid. Inclusion of S 35 -methionine into the thyroglobulin molecule occurs in the follicular epithelium cells. The data obtained demonstrate that thryoglobulin synthesis and its iodization in the thyroid gland occur at different sites. Thyroglobulin synthesis takes place in the follicular cells, while protein iodization — either in the cavity of the follicles or at the border of the apical parts of the cells and colloid. The above led to the convulsion that the thyroid colloid should be regarded not as a place of hormone storage but as a structural unit of the organ which participates in hormone formation.

The syndrome of congenital goiter with butanol-insoluble serum iodine

Clinical and laboratory data on five patients suffering from one variety of congenital hypothyroidism with goiter are reported, and similar cases from the literature are reviewed. In these patients hypothyroidism and goiter develop early in life. They may suffer permanent physical and mental retardation unless the disorder is detected and adequate replacement therapy is given. There is increased RaI 131 uptake by the thyroid and no release of I 131 upon administration of potassium thiocyanate. The deiodinase activity is normal. An abnormal iodinated polypeptide or protein is secreted by the thyroid. It is detected by its failure to extract into n-butanol from acidified serum. A similar complex is present in the urine. Abnormalities in the distribution of I 131 in the thyroid subcellular particulate fractions and in the thyroidal proteins have been detected in the two glands which have been available for analysis. This syndrome may be the expression of a congenital defect in thyroglobulin synthesis. Similar observations in certain patients with adenomatous colloid goiter suggest that this type of congenital goiter and a type of adenomatous colloid goiter may represent different levels of expression of a common genetic defect.

The Adrenal Cortex and the Histological Picture in Thyroid Gland, with Special References to the Proliferation of Follicles and the Occurrence of Huge Colloidal Substance within Glandular Cells

The adult male rats of WISTAR strain were devided into three experimental groups. The first consisted of 25 adrenalectomized rats which were decapitated 2, 5, 10, 15 and 20 days respectively after the removal; the second of 5 intact and 5 adrenalectomized with the administrations of 2.5mg of cortisone per day for 7 days; the third of the same number of intact and operated with the injections of 2.5mg of DOCA per day for 7 days. The changes in thyroid glands were histologically investigated.Within 2 days the alterations were not profound yet in adrenalectomized animals. However, in 5 days the proliferation took places in follicles principally located at the center of the gland, being probably based on the augementation in hyperplasia of parafollicular cells whose enlarged cytoplasm usually contain, under the normal condition, a number of PAS-positive granules or vacuoles. Such a regeneration developed gradually in 10 and 15 days, finally ceased to advance to gain a subsequent fixed configuration. It was an information in the present observation as to the particular changes caused by the adrenalectomy that the huge colloidal substances of various sizes frequently appeared at manifold areas of the cell-bodies, especially accumulated at the luminal surface lacking the secretory cytoplasmic processes. These substances tended to be vacuolized at the basal part of the cell, associated with the vacancy or extraordinary reduction in amount of the intrafollicular colloid. With regard to their functional significance, an assumption may be drawn that they are not any indices to the intracellular synthesis of thyroglobulin, but rather accounted as the reabsorption picture (YOSHIMURA, IRIYE and YAGI 1958). The frequency in occurrence of these substances was the highest in 5, 10 and 15 days.Following the prolonged administration of cortisone the follicles of intact rats were proliferous because of the advanced differentiation in the common glandular cells. Intrafollicular colloid faintly stained included abundant circumference vacuoles and at the periphery of the lumina the cytoplasmic processes indicating a possible apocrine secretion were often detected. With the administration of cortisone the thyroid glands of the adrenalectomized rats resulted in the serious disorder in the cell arrangement, which was presumably originated in their malignant hyperplasia, although it is impossible for us to determine whether this proliferous phenomenon is ascribed to the activity of the common follicular cells or that of parafollicular ones. The huge colloidal substances following the adrenalectomy completely disappeared with the chemical introduction. The effect of DOCA upon the thyroid glands of intact and adrenalectomized rats were so delicate or minute, but take a moderate action to disperse the huge colloidal substances induced by the adrenalectomy, and also to make a slight proliferation of follicles. The mode of action of coritcoids upon the thyroid gland was discussed on the basis of present morphological aspects.

Thyroglobuline marquée ou artificiellement iodée sans dénaturation. formation dans des conditions expérimentales diverses et propriétés

1. 1. Thyroglobulin marked with radioactive iodine ( 131I) has been prepared from thyroid gland (ox) by perfusion with a solution containing marked iodides, or from the same gland (dog, pig) of animals which received an injection of the same salts. The study of the solubility of this protein in the presence of neutral salts (ammonium sulphate, equimolecular mixture of mono- and dipotassium phosphates) has been continued, based on the determination of the protein-nitrogen and on the measurements of the radioactivity. These latter measurements have allowed the identification of the iodoprotein in complex mixtures, the following of its selective precipitation, and the controlling of its purification. 2. 2. Ox and pig thyroglobulins cannot be distinguished from each other by their solubility in solutions of increasing ammonium sulphate and potassium phosphates concentration, while the preparation from the dog possesses specific properties in this respect. 3. 3. The identification of marked thyroglobulin in mixtures with the help of its radioactivity and of its solubility has made possible the investigation of thyroid extracts from normal animals (dog, pig) and from those animals (dog) the thyroid activity of which was considerably increased by administration of thyreostimulin or which was inhibited by benzyl- or propylthiouracil. Repeated injections of thyreostimulin caused a decrease of iodoprotein in the extracts, but they still had the same properties as those of normal animals. From this it follows that the hypophysis hormone, of which it has already been established that it accelerates the formation of thyroxine, must increase simultaneously the synthesis of thyroglobulin and its enzymatic hydrolysis and that without altering the nature of these processes. Gland extracts of animals treated with derivatives of thiouracil contain not only thyroglobulin of the same solubility as that of normal animals, but also other iodoproteins. These thiouracil derivatives not only decrease the activity of the thyroid gland but also cause the formation of iodoproteins different from thyroglobulin, while at the same time slowing down the formation of the latter. Their action thus no doubt consists in some disturbance of the synthesis of thyroid proteins. 4. 4. The marked thyroglobulin shows the same heterogeneity as the non-radioactive protein and its solubility at various concentrations of neutral salts in general indicates the presence of three fractions. Artificial iodination to a more or less considerable degree without denaturation alters neither the solubility properties nor the electrophoretic homogeneity of the thyroglobulin. The fact that this protein appears in solubility investigations to be heterogeneous does thus not prove the presence of fractions iodinated to a greater or lesser extent; its nature remains to be established.