19S Thyroglobulin Research Articles

The identification of a specific fragment of dog thyroglobulin responsible for immunoreactivity to parafollicular cells.

This study provides new information related to our earlier finding of a thyroglobulin-like antigen in parafollicular cells. Dog thyroglobulin was saltedout with ammonium sulfate, then chromatographed on a Bio-Gel A-5m column. Four distinct 280-nm-absorbing peaks (I–IV) were observed. Antisera to each of these fractions were studied by immunodiffusion and immunoelectrophoresis and showed that the main antibody produced was against 19S thyroglobulin. Each of the antisera gave distinct immunoreactions with colloid droplets and follicular cells when stained with the immunoperoxidase bridge technique. Only the antiserum to peak I, however, gave a strong positive reaction to the parafollicular cells. Absorption of this antiserum with adequate amounts of the peak II fraction removed the reaction to colloid droplets and follicular cells, but left the reaction with parafollicular cells intact, while absorption with the peak I fraction eliminated the reaction with the parafollicular cells. The results indicate that...

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells.

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I'' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.

Afrikander cattle congenital goiter: characteristics of its morphology and iodoprotein pattern.

The morphology and some properties of the complex iodoprotein pattern of the genetically determined congenital goiter in Afrikander cattle is described. The goiter contained irregularly shaped follicles which were devoid of colloid and the follicular epithelial cells were elongated, measuring about 20 micrometer in length compared to 10 micrometer for normal thyroid cells. The goiter cells contained apical clusters of larger and more numerous lysosomes than normal thyroid cells. Apical vesicles containing electron-dense material which were in contact with the plasma membrane could be seen in most normal thyroid cells, but were extremely scarce in the goiter. In 36 cell profiles studied none was found. The endoplasmic reticulum cisternae of the goiter differed significantly from normal thyroid cells. Fewer ribosomes were seen to be attached to the membranes of goiter cells. Furthermore, unlike normal thyroid cells, many free polysomes were seen in goiter cells. The characteristics of the unusual iodoprotein pattern of the goiter extract, resolved by gel chromatography and sucrose density gradient centrifugation, were qualitatively and quantitatively similar to that described previously (Endocrinology 91, 470, 1972). A relatively small amount of the total soluble protein was iodinated. Of these, only a 12S sedimenting species was precipitated by antithyroglobulin immunoglobulin. When separated on polyacrylamide gels containing sodium dodecyl sulfate and mercaptoethanol, this 12S species was resolved into at least 14 polypeptide components ranging in molecular weights from less than 66,000--330,000. Three of the bands, representing a small percentage of the total protein, seemed to comigrate with the major polypeptides of thyroglobulin and were also precipitated with rabbit antihyroglobulin immunoglobulin. The data indicate that glycosylation of iodoproteins was not affected although 19S thyroglobulin synthesis and subsequent storage were drastically impaired.

Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey ( Lampetra planeri B1.)

1. 1. The biosynthesis of 18–19S thyroglobulin has been studied in a larval and adult freshwater lamprey ( Lampetra planeri Bl.). 2. 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates ( 125I, [ 3H]leucine and various [ 3H]carbohydrates). 3. 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18–19S Tg), containing a small proportion of labeled T 3 and T 4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. 4. In adults, the biosynthesis of 18–19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18–19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. 6. Some 18–19S Tg is apparently stored in the endostyle.

Ultrastructure and biochemistry of thyroid carcinoma.

Thirty-two thyroid tumors, 9 benign, 23 malignant, and 12 samples of normal thyroid tissue were examined by light and electron microscopy. Thyroglobulin content was also measured in the tissues and, in a limited number of cases, enzymatic activities were determined, such as thyroid peroxidase-iodinase, acid protease, and deiodinase. The presence of significant amounts of 19S, 27S and 12S thyroglobulin was well correlated with the ability of the tumors to accumulate radioiodine. It is suggested that the presence of thyroglobulin be used as a marker of potential function of thyroid carcinoma. Two types of ultrastructural changes were observed in thyroid carcinoma. The first one was interpreted as accompanying the progressive loss of function of thyroid tumors, and was represented by the modifications of highly specialized structures such as RER, lysosomal dense bodies, colloid, etc. The second one is suspected to reflect the malignant transformation of the follicular cell. This concerned namely the nuclei, mitochondria, and intracytoplasmic inclusions. These changes may have a diagnostic value since they were not observed in benign conditions.

Translation of thyroglobulin 33S messenger RNA as a means of determining thyroglobulin quaternary structure.

Thyroglobulin is a 19S protein of approximately 660,000 daltons and unknown quaternary structure. We have previously shown that a 33S mRNA purified from mammalian thyroids promoted synthesis in the Xenopus oocyte of a peptide immunologically related to thyroglobulin. Chemical identity to the native protein is now presented by means of a tryptic peptide analysis. Moreover, the 33S mRNA is shown to contain all the information required for the synthesis of a complete 19S thyroglobulin molecule. Gel filtration in Sepharose under denaturing conditions indicates that the reduced polypeptide encoded by the 33S mRNA is larger than 210,000 daltons. A model of a dimeric thyroglobulin with about 300,000 dalton subunits is presented.

The structure and properties of 27S and larger iodoproteins in the thyroid gland

Iodoproteins larger than 19S were isolated from hog and calf thyroid glands using gel filtration on Sepharose 6B and one or two centrifugations in glycerol density gradients. Purified protein fractions were analysed in the analytical ultracentrifuge and characterized by the combined use of electrophoresis in continuous polyacrylamide gel gradients and electron microscopy. Three bands migrating more slowly than 19S could be identified in the polyacrylamide gels. Electron microscopy of the fastest of these species, having a sedimentation constant of 27S, showed pairs of 19S thyroglobulin molecules which had the same size and the same ovoid shape as normal, well-iodinated thyroglobulin molecules. The ovoids were randomly attached side to side, end to end and side to end. The more slowly migrating proteins were shown to consist of similar aggregates of three, four or more randomly attached molecules. Iodine and sialic acid determinations in 27S and 19S separated from the same pool of well iodinated protein showed no difference in iodine content but a larger amount of sialic acid in 27S than in 19S.

Thyroglobulin messenger RNA: translation of a 33-S mRNA into a peptide immunologically related to thyroglobulin.

Poly(UC)--Sepharose chromatography of the RNA extracted from a thyroid fraction sedimenting between 800 X g and 27000 X g allows the purification of two RNA fractions amounting each to 1% of the applied material. The first one is loosely bound to the column from which it is eluted at 25 degrees C. It is mainly composed of 16-S and 12-S RNA comprising no poly(A) sequences. This could correspond to mitochondrial rRNA. The second one, which is eluted at 50 degrees C, is poly(A)-rich and represents 33-S and 17--18-S RNA species. The 33-S RNA resists heating at 80 degrees C, suggesting that it is composed of one polynucleotide chain. When injected into Xenopus oocytes, the 33-S RNA specifically promotes the synthesis of a peptide with an apparent molecular weight of 185000 and an apparent sedimentation coefficient of 10-S. This peptide is immunologically related to thyroglobulin and could represent its main precursor. Under the conditions tested it does not polymerize spontaneously into 19-S thyroglobulin, suggesting that assembly of the molecule could require specific, post-translational alterations of the precursor and/or the presence of additional lighter subunits.

Thyroglobulins of cyclostomes and an elasmobranch

As part of a study of the biosynthesis and physicochemical properties of primitive vertebrate thyroglobulins, the thyroid-containing tissues of Pacific hagfish ( Eptatretus stouti) and of Pacific lamprey ( Entosphenus tridentatus), and thyroid glands of Pacific dogfish ( Squalus suckleyi) were used. In the hagfish thyroid tissues labeled with radioiodine, only 3–8S iodoprotein having an approximate molecular weight of 155,000 was detectable. A low proportion of thyroid iodine was in the form of labeled thyroxine (2.5%) 4 days after labeling. In lamprey thyroid tissue radioactivity peaks corresponding to 3–8S and 19S proteins were found in ultracentrifugation patterns. However, the main thyroid iodoprotein component purified by ammonium sulfate fractional precipitation was 12S. The proportion of labeled thyroxine to total radioiodine in lamprey iodoproteins was 2.9%. Total iodine content of lamprey thyroprotein was 0.0022%. In dogfish thyroid glands only 19S thyroglobulin as obtained after purification by the ammonium sulfate procedure. Total iodine content was similar to that of mammalian thyroglobulins, 0.50%. The proportion of total iodine in the form of unlabeled thyroxine in an analysis of unlabeled dogfish thyroglobulin was at a high level of 50.9%. The amino acid compositions of lamprey iodoproteins and dogfish thyroglobulin differed in some respects from higher vertebrate thyroglobulins. There was also some difference in the carbohydrate compositions of lamprey iodoproteins and dogfish thyroglobulin, compared to higher vertebrate thyroglobulins. Moreover, the total carbohydrate content of lamprey iodoproteins and of dogfish thyroglobulin was lower than in higher vertebrate thyroglobulins. On the basis of these results the general properties of thyroglobulins could be considered with respect to the question of the evolution of thyroid function.

Binding of the 35-s of 35-s-propylthiouracil by follicular thyroglobulin in vivo and in vitro.

Radioactivity was found bound to follicular thyroglobulin after administration of 35-S-propylthiouracil (PTU) to rats. Denaturation of the thyroglobulin using various procedures could not separate the 35-S from the protein; it was concluded that the 35-S is bound to thyroglobulin covalently. Fractionation of saline-soluble thyroid proteins was performed by ultracentrifugation on sucrose gradients. The PTU-sulphur/thyroglobulin (S/Tg) molar ratio was calculated in all fractions. One hour after the injection of PTU the S/Tg molar ratio was the same for 19S thyroglobulin from rats on stock diet and 18S thyroglobulin from rats on low iodine diet. Injection of KI to the animals before administration of 35-S-PTU significantly reduced the ratio. The highest S/Tg ratio 1.10 was noted at 19S thyroglobulin, 17 h after a single injection of PTU. Daily injection of PTU for six days increased the S/Tg ratio to 3.3. Inverse relationship between dose of PTU and S/Tg ratio was noted at one hour. In animals injected with large dose of 35-S-PTU and sacrificed several hours later the S/Tg was higher at the 12S subunits than at the 19S protein. The amount of PTU bound to 3-8S subunits was minimal. Sulphite liberated 64 percent of the 35-S bound to thyroglobulin which appeared as four compounds on thin layer chromatography plates. The main 35-S compound liberated by sulphite was sulphate.

Incorporation of carbohydrates into abnormal thyroglobulin in an experimental rat thyroid tumor.

Previous findings indicated that in line 1–1C2 of experimental rat thyroid tumor there is decreased secretion of 19S thyroglobulin associated with a lack of incorporation of sialic acid into thyroglobulin and sialyltransferase activity in the tumor. In the present work we studied another tumor line, 1–8, in which particle bound iodopro-tein is also increased. This tumor, however, secretes an abnormal form of thyroglobulin. Soluble and digitonin-solubilized iodoproteins were examined in tumor labeled in vitro with N-acetylmannosamine, galactose, mannose, N-acetyl-glucosamine, leucine and iodine. About half of each label except galactose and N-acetyl-mannosamine was in the pellet after digitonin extraction and the remainder was evenly distrib-uted between soluble and solubilized fractions. After galactose labeling, the soluble fraction con-tained relatively more labeled protein; after N-acetylmannosamine, the pellet content was higher but most of the remainder was in the soluble fraction. The soluble and so...

Properties of an iodoprotein from thyroid tissue of the Pacific hagfish, Eptatretus stoutii

In hagfish, thyroid tissue incorporation of radioiodine into proteins associated with cellular particulate matter reached a level of more than 50% of the total labeled iodoproteins. Only a single 3-8S iodoprotein was found by sucrose density gradient centrifugation of the soluble protein obtained from thyroid tissue by ultracentrifugation. No 19S thyroglobulin was found. Labeled thyroxine amounted to 2.5% of the total 131I in proteins.

Thyroidal morphogenesis and biosynthesis of thyroglobulin before and after metamorphosis in the lamprey, Lampetra reissneri

In larval and metamorphosed adult lampreys, a correlation was made between the structure of the subpharyngeal gland and the thyroid gland and the biosynthesis of thyroid hormones. The incorporation of radioiodine by the larval subpharyngeal gland increased gradually during development, but in metamorphosed adult lampreys it was reduced. No distinctive morphological changes of the subpharyngeal gland were observed throughout the larval stages examined. Radioautographic evidence of localization of radioiodine was found in the epithelial cells of the subpharyngeal gland, and at the apical surfaces of these cells. On the other hand, 24 hr after administration almost all radioiodine visualized by this technique was localized in the lumina of the thyroid follicles of metamorphosed lampreys. From the subpharyngeal gland of larval lampreys (body length 46–70 mm), iodinated proteins having a sedimentation coefficient of 3–5 S, and a small amount of iodinated 19S thyroglobulin, were separable by sucrose density ultracentrifugation. The proportion of iodinated 19S thyroglobulin increased as development proceeded. After metamorphosis 19S thyroglobulin was the principal iodinated protein found in the thyroid gland, and the iodinated 3–5 S proteins were barely detectable. By chromatography it was shown that the iodinated 3–5 S protein obtained from the larval subpharyngeal gland by sucrose density gradient centrifugation contained thyroid hormones.

The biosynthesis of thyroxine incorporation of (U- 14 C) tyrosine into thyroglobulin by mouse thyroid glands in vivo and in vitro.

SUMMARY The incorporation in vivo of [U-14C]tyrosine into 19S thyroglobulin by mice was achieved to a level of 2·85 c.p.m./μg. This level of incorporation was insufficient to permit the isolation of 14C-labelled iodinated tyrosines or residues. Isolated mouse thyroid lobes were used as an in-vitro system for the synthesis of 19S thyroglobulin. The lobes continued to incorporate 131I into 19S thyroglobulin for at least 48 h and this incorporation of iodine was specifically inhibited by propylthiouracil. The isolated mouse thyroid lobe in-vitro system was used to incorporate 14C-labelled amino acids into 19S thyroglobulin. [U-14C]Tyrosine was incorporated to a level of 1150 c.p.m./μg. Electrophoretic separations of enzymic hydrolysates of [14C]tyrosine-labelled 19S thyroglobulin showed the presence of [14C]tyrosine, [14C]monoiodotyrosine, [14C]di-iodotyrosine and [14C]thyroxine. The presence of [14C]tyrosine, [14C]monoiodotyrosine and [14C]thyroxine was demonstrated by chromatography of the eluates from the electrophoretic separations. The results provide evidence for the utilization of tyrosyl residues within the thyroglobulin molecule for iodination and subsequent coupling to form thyroxine.

A microgel electrophoretic study of the stability of thyroid iodoproteins

The stability of the large iodoproteins in the rat thyroid gland was studied by polyacrylamide gel electrophoresis. 19S Tyroglobulin was stable in phosphate-buffered saline (0.01 M phosphate, 0.15 M NaCl, pH 6.8; PBS) containing 20% (w/w) sucrose. Reduction of the sucrose concentration by dilution with PBS resulted in the appearance of two 12S fractions and a band migrating with the front. After the same treatment of 27S iodoprotein, also stable in PBS + 20% sucrose, no large dissociation products appeared, only a band moving with the front. Urea of 8 M, pH 6.8, and d2 M urea, pH 8.1, partially dissociated 19S thyroglobulin into three species and a band at the electrophoretic front. The main dissociation product probably represents a modified 12S protein. Both treatments further resulted in a reduced gel migration of undissociaetd tyroglobulin. After ürea treatment of 27S iodoprotein, larger amounts of front species were found than after the same treatment of 19S tyroglobulin, where only trace amounts of large dissociation products were observed. The main dissociation product in the front band of 19S tyroglobulin had a molecular weight of 82,000–86,000 (SDS gel electrophoresis). Four other distinct fractions were observed. It was concluded that sucrose has a stabilizing effect on the large thyroid iodoproteins. The different dissociation patterns of 19S thyroglobulin and 27S iodoprotein indicate that the subunits of 27S, to a larger extent than those of 19S, are linked together by non-covalent bonds.

Relationship between thyroglobulin synthesis, lodine metabolism, and histogenesis in the developing rabbit fetal thyroid gland

The biosynthesis of thyroglobulin and its iodination have been studied in the rabbit fetus in relation to the morphogenesis and structure of the thyroid gland. Sixteen- to 28-day-old fetuses were injected intraperitoneally in vivo through the uterine wall with [ 3H]leucine and 125I. Tritium-labeled proteins were analyzed by sucrose gradient centrifugation and dise electrophoresis, in combination with immunological techniques. Thyroglobulin was estimated by radioimmunoassay, and 127I by chemical determination. The first definite follicles were seen in electron micrographs on day 18. Colloid is not stored in large amounts until day 22. Proteins having antigenic sites in common with adult thyroglobulin can be radio-immunogically detected in the thyroids of 16- and 17-day-old fetuses, before the formation of follicles, before the capacity of collecting radioiodine appear, and before detectable amounts of 127I exist in the glands. Low molecular weight precursors of thyroglobulin are present in the 18-day thyroid; they seem to actively polymerize into 19S thyroglobulin on day 22. As soon as microfollicles have differentiated, iodine is concentrated, iodination of thyroglobulin occurs, and thyroid hormones are synthesized. The ability of the rabbit fetal thyroid gland to concentrate iodide and to synthesize thyroid hormones thus seems to be linked to the organization of follicles.

A microgel electrophoretic analysis of the colloid proteins in single rat thyroid follicles. II. The protein concentration of the colloid single rat thyroid follicles.

Samples of the colloid in rat thyroid follicles were obtained by micropuncture of superficial isthmic follicles in vivo and of superficial and deeper follicles in saline perfused thyroids. The protein compositions of the samples were analyzed by microgel electrophoresis. The amount of protein in the different fractions in the colloid samples was determined by microdensitometry. The sample volumes were estimated by measuring the amount of radioactivity in a solution occupying the same space in the tip of the collecting pipette as the colloid sample. Samples of colloid containing 19S thyroglobulin (TG) and proteins migrating more slowly than 19S TG had low protein concentrations, about 5 g/100 ml. Samples containing a prealbumin fraction in addition to the large proteins in the previous group had significantly higher protein concentrations, about IS g/100 ml. Colloid samples containing additional proteins (an albumin- like protein and a globulin) generally had mean protein concentrations around 10 g/100 ml....

Polypeptides non-covalently associated in 19-S thyroglobulin

The present studies were performed on several preparations of mammalian (sheep, ox, rat, horse, pig) and human normal or goiter 19-S thyroglobulin. Homogeneity of the preparations was controlled by analytical centrifugation and disc electrophoresis. Succinylated or native thyroglobulins dissolved in buffers containing urea, guanidine·HCl or sodium dodecyl sulfate release discrete polypeptides (5–8) non-covalently associated with the remainder of the molecule and whose molecular weights are below 330 000. The fastly migrating components have been partially isolated by filtration on Sephadex G-200 equilibrated in buffers containing urea, guanidine·HCl or sodium dodecyl sulfate and their apparent molecular weights determined by electrophoresis in sodium dodecyl sulfate-acrylamide gels. They represent about 20% by weight of the native protein when gel filtration is performed in the presence of a sodium dodecyl sulfate-containing buffer (pH 8.0) and their molecular weights are between 14 000 and 150 000. The amino acid composition of the mixture of fastly migrating components differs from that of native thyroglobulin by a much lower content in cystine (about 1 3 ) and a higher content in isoleucine, tyrosine, histidine, lysine, thyroxine and 3,5,3′-triiodothyronine. Components of identical or almost identical apparent molecular weights, but in larger amounts, are separated from fully reduced and S-carboxymethylated 19-S thyroglobulins by electrophoresis in sodium dodecyl sulfate-acrylamide gels. It is concluded that native 19-S thyroglobulin contains polypeptides non-covalently associated with the bulk of the protein. This observation is discussed in relation to the subunit structure of 19-S thyroglobulin.

Membrane-bound thyroglobulin in an experimental thyroid tumor of rats

Soluble and particulate iodoproteins have been studied in an experimental rat thyroid tumor (Wollman Line 1-1C 2) labeled by intraperitoneal injection of 125I-. The uptake of the tumor was very low (approx. 1% of the dose per g), and most of the radioactivity (75–95%) was particulate. The particulate iodoproteins were solubilized (10–78%) by vigorous rehomogenization and digitonin treatment; the soluble and solubilized proteins were analyzed by density gradient ultracentrifugation, microimmunodiffusion and double immunoprecipitation. Labeled 19-S thyroglobulin comprised 30–60% of the soluble iodoproteins and 10–90% of the total solubilized iodoproteins. In the iodoproteins solubilized by rehomogenization there was 10–23% 19-S at 60 min or less after injection, and most of the 125I sedimented at <6 S. This “light fraction” had a density similar to that of endoplasmic or plasma membranes and yielded 25–75% 19-S thyroglobulin when treated with digitonin. Subcellular fractionation showed that 62% of the total tumor 125I was in the “nuclear” fraction, 35% in the “microsomal” fraction and only 3% in the “mitochondrial” fraction, which should contain lysosomes. In vitro experiments failed to show non-specific adsorption of thyroglobulin as a source of membrane-bound thyroglobulin. The results suggest that in the tumor studied there is decreased synthesis of thyroglobulin and a possible defect in its release from cell membranes.

Bovine 37S iodoprotein: Isolation and characterization

A thyroglobulin-like protein with a sedimentation constant of 36.6S has been isolated from bovine thyroid glands and further characterized. Its molecular weight, 2 × 10 6, is close to three times that of 19S thyroglobulin, the major thyroid iodoprotein. The amino acid composition of this iodoprotein also closely resembled that of 19S thyroglobulin and of the 27S iodoprotein. A reaction of identity in a double diffusion system with 19S, 27S and 36.6S has also been found. The iodine content of the 36.6S iodoprotein was greater than that of 19S thyroglobulin, but similar to that of the 27S species isolated from the same thyroid extract.