19S Thyroglobulin Research Articles

Evidence that thyroglobulin has an associated protein kinase activity correlated with the presence of an adenosine triphosphate binding site

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo-labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.

Evidence that thyroglobulin has an associated protein kinase activity correlated with the presence of an adenosine triphosphate binding site.

19S Thyroglobulin (Tg), a dimeric glycoprotein with an M(r) of 660,000, was extracted from rat, bovine, and human (goitrous) thyroid tissues, as well as from culture medium of FRTL5 rat thyroid cells. Subjected to rigorous purification procedures, all Tg preparations showed a protein kinase activity that is able to phosphorylate serine residues in vitro. Further characterization of this enzymatic activity revealed that Tg has the specificities of a protein kinase A when Kemptide was used as specific substrate and after analysis of cAMP-stimulated phosphotransferase activity in all Tg preparations tested. Furthermore, Tg contains a specific and saturable ATP binding site, as evidenced by specific binding with the ATP affinity analog p-fluorosulfonylbenzoyl 5'-p-adenosine (FSO2BzAdo). Limited proteolysis of FSO2[14C]BzAdo-labeled human goiter Tg with clostripain gave rise to a M(r) 64,000 amino terminal polypeptide carrying almost all of the label. The analysis of this fragment revealed two sequences that may play an analogous phosphate-loop (P-loop) function, even though their primary sequences differ slightly from the classical A consensus sequence or P-loop. The ATP binding site of Tg may be functionally associated with the presence of a protein kinase A phosphorylating activity linked to the Tg itself, because the addition of the analog is able to inhibit the enzymatic activity in a dose-dependent fashion. Contamination with non-Tg phosphorylase cannot be ruled out at present, even though the amount of ATP that Tg was able to bind did not significantly change during the various purification steps. Together these findings favor the hypothesis that the Tg molecule contains a protein kinase activity that is capable of autophosphorylation.

Characterization of five monoclonal antibodies obtained after immunization in vitro with a synthetic 19-amino acid peptide of thyroglobulin.

A 19-amino acid synthetic peptide representing the highly conserved amino terminus of thyroglobulin was used for the production of monoclonal antibodies after immunization of splenocytes in vitro. The properties of five of the antibodies were studied. One reacted only with the synthetic peptide. The others reacted with both the synthetic peptide and with thyroglobulin from all species tested so far, confirming that the amino terminus of thyroglobulin is highly conserved. Two of the five antibodies showed a positive reaction when tested on frozen sections of thyroid tissue, but with different reaction patterns. Monoclonal antibody F4 gave a positive reaction in the colloid, which contains mainly 19S thyroglobulin. In contrast, monoclonal antibody G4 gave a positive reaction only in the follicular cells. Monoclonal antibody G4 binds primarily to low molecular weight compounds in thyroglobulin preparations, possibly representing breakdown products of the protein.

Physiological role of thiol proteases in thyroid hormone secretion.

To determine the physiological role of the thiol proteases in T4 and T3 release from thyroglobulin, experiments were performed with 131I-prelabelled rat thyroid lobes incubated in vitro in the presence and absence of leupeptin, an inhibitor of thiol proteases. Basal secretion of [131I]T4 and [131I]T3 from rat thyroid lobes prelabelled in vivo was quite low, but in the presence of 10 mU/ml bovine TSH a marked stimulatory effect was observed. The stimulatory effect of TSH was completely abolished by leupeptin. This was associated with marked inhibition of lysosomal proteolytic activity, suggesting that the inhibitory effect of leupeptin on T4 and T3 secretion could be attributed to its inhibitory action on proteolysis of thyroglobulin. Further evidence for an inhibitory effect of leupeptin on intralysosomal hydrolysis of thyroglobulin was obtained when thyroid lobes were incubated with 131I- in the presence and absence of leupeptin and TSH. The crude lysosomal preparation was fractionated on a Percoll density gradient, which separates 131I-containing particles into a dense peak containing purified lysosomes and a buoyant peak containing pinocytotic vesicles. A marked increase in the 131I-content of the dense peak was observed in the presence of TSH + leupeptin. Analysis of the 131I in the dense fraction by sucrose density gradient centrifugation and by SDS-polyacrylamide gel electrophoresis demonstrated that leupeptin inhibited degradation of 19S thyroglobulin, especially the formation of [131I]peptides of MW less than 14K.

Ultrastructural morphology and evidence of iodide uptake and protein synthesis in normal human thyroid transplants in nude mice.

Normal human thyroid tissue was xenotransplanted to athymic mice. Well preserved structural properties of the transplants were observed at both light and electron microscopical level. Analysis of the soluble proteins in the transplants by polyacrylamide gel electrophoresis showed that the major fractions were the 19S thyroglobulin, the 27S iodoprotein and a larger protein fraction. Protein synthesizing capacity of the transplants, as measured by in vivo 3H-leucine incorporation into the soluble transplants proteins was demonstrated. The observed 3H-leucine incorporation was in the same magnitude as that found in the original donor tissue at the time of transplantation. Four weeks after transplantation a significant 125I-iodide uptake (2.0%) was observed in the transplants. Corresponding value for the host thyroids was 6.6%. The biological half-time of the accumulated 125I-iodide in the transplants was reduced from 97 days to 38 days between 2 and 4 weeks after transplantation. The biological half-time in the host thyroid increased from 27 days to 32 days during the same period. This investigation shows that normal human thyroid tissue can be transplanted to nude mice with preserved tissue organisation and maintained cellular function.

Evidence for homologous repeating segments within the elementary polypeptide chain of guinea pig thyroglobulin.

In this paper we report on the peptide maps of the three polypeptides produced by reduction of denatured 19-S thyroglobulin from guinea pig. These maps were obtained by both chemical cleavage (CNBr) and by limited enzymatic proteolysis (Staphylococcus aureus SV 8 protease). Analysis of these peptides showed a strict correspondence among the electrophoretic bands produced by cleavage of the guinea pig thyroglobulin components. These results support the idea that thyroglobulin, even though it has an apparently complex molecular structure, contains polypeptide sequences which are repeated in the elementary chain. Since similar structures should correspond to similar functions, each of these regions may work as a functional domain for the biosynthesis of the thyroid hormones.

Regulation of Enzymatic Iodothyronine Synthesis in Thyroglobulin by Low Concentrations of Iodide

Evidence is presented that free iodide in the reaction medium during iodination of thyroglobulin by lactoperoxidase or thyroidperoxidase in vitro and a limiting H2O2 supply actively regulates the efficiency of the simultaneously occurring synthesis of iodothyronines. Our arguments are as follows. 1 For any level of iodination achieved in vitro, the amount of iodine incorporated in newly formed iodothyronine residues (but not that of the overall iodination) was linearly and inversely correlated to the logarithm of free iodide remaining in the incubation medium. This finding holds true for iodide concentrations in the μM range, regardless of the native iodine content of a 19-S thyroglobulin sample, of the concentration of the enzymes and of the incubation time. 2 When thyroglobulins, iodinated either in the absence or in the presence of excess iodide, are compared the latter contained (for similar degrees of iodination) more newly synthesized diiodotyrosine residues but fewer iodothyronine residues, although over 50 % of these diiodotyrosine residues were hormonogenic. 3 An additional important increase in hormone synthesis was obtained by extending the reaction time beyond the point were all iodide was organified. The latter phenomenon is but another demonstration that coupling always lags behind iodination. 4 The lag between the onset of iodination and that of coupling is a function of the amount of newly organified iodine. Iodothyronine synthesis starts as soon as 8–10 atoms of iodine are incorporated per molecule of thyroglobulin. It is suggested that the inhibition of the coupling reaction by inorganic iodide in vitro may well have a physiological significance insofar as it could be one of several autoregulatory mechanisms in the thyroid gland preventing excessive intrafollicular storage of iodothyronines in response to an increased iodide supply.

EFFECTS OF TSH ON cAMP LEVELS AND THYROID HORMONE RELEASE IN HUMAN THYROID‘AUTONOMOUS’NODULES: RELATIONSHIP WITH IODOTHYRONINE AND IODINE CONTENT IN THYROGLOBULIN

The effect of TSH on the adenylate cyclase-cAMP system and in vitro iodothyronine release, together with the iodothyronine and iodine content of 19s thyroglobulin, were studied in seven clinically euthyroid patients with autonomous thyroid nodules. Basal cAMP and cGMP content together with phosphodiesterase and protein-kinase activities were normal in nodular, and suppressed in extranodular tissue. TSH-dependent cAMP accumulation was reduced in nodular tissue, but normal in the suppressed extranodular tissue. In vitro TSH-dependent iodothyronine release from nodular and extranodular tissue was absent. Thyroxine and iodine content of thyroglobulin extracted from nodular tissue was reduced, while triiodothyronine content was normal but with a low T4/T3 ratio. In extranodular tissue T3, T4 and iodine contents were reduced. In conclusion, autonomous thyroid nodules produced a poorly iodinated thyroglobulin leading to preferential T3 secretion with increased circulating free thyroid hormones even in clinically euthyroid patients.

Preferential secretion of triiodothyronine in man.

The directly measured secretion rates of thyroxine (T4), 3,5,3'-triiodothyronine (T3), and 3,3',5'-triiodothyronine (rT3) were compared to the tissue concentrations of these hormones and to the distribution of follicular iodoproteins in 15 patients submitted to thyroid or parathyroid surgery. The T4/T3 and rT3/T3 ratios were higher for thyroid tissue than for the secretion rates, indicating a preferential secretion of T3. The concentrations of T4 and T3 were higher in the 27S iodoprotein fraction than in the 19S thyroglobulin fraction.

Changes in the Polypeptide Assembly of Guinea Pig Thyroglobulin Induced by Thyrotropin-Regulated Thyroid Activity*

We have previously reported that the relative proportion of three polypeptide chains in guinea pig thyroglobulin is closely related to the iodine content of the protein. The present work demonstrates that it is not the iodine content per se but, rather, TSH-regulated thyroid activity which modulates the substructure of thyroglobulin. In a first set of experiments, the impact of TSH stimulation on sodium dodecyl sulfate (SDS)-induced dissociation of 19S thyroglobulin into 12S subunits was compared to that of iodination. While in control animals the ratio of 12S to 19S thyroglobulin was 48:52, it changed to 35:65 in glands strongly stimulated with TSH and blocked with MMI. This rise in the relative proportion of 19S thyroglobulin occurred despite a simultaneous drop of iodine content from 0.6% to 0.24%. It was only after TSH suppression that the well known inverse correlation between the level of iodination and dissociability reappeared. In a second set of experiments, SDS-treated thyroglobulin was fully reduced by splitting disulfide bonds with mercaptoethanol. In addition to the previously described three polypeptide chains, A, B, and C, a hitherto neglected nonreducible fraction comigrated with 19S thyroglobulin on polyacrylamide gels. Native thyroglobulin with widely varying iodine contents was obtained from unstimulated glands and from glands strongly stimulated with TSH. Drastic changes in the polypeptide chain assembly, depending on the degree of TSH stimulation but entirely independent of iodination, were observed. There was a strong negative correlation between the nonreducible 19S thyroglobulin fraction and both the B and C polypeptide chains with all experimental manipulations. We conclude that thyroglobulin substructure is highly dependent on the degree of TSH stimulation of the thyroid. TSH, through stimulation of unknown metabolic pathways, is a more important determinant of thyroglobulin substructure than the degree of iodination of the protein.

Uptake of radioiodine in follicles of dog C-cell complexes studied by autoradiograph and immunoperoxidase staining.

C-cell complexes are special cell groups consisting of a mass of C-cells associated with other epithelial elements and cysts. They are remnants of ultimobranchial bodies retaining fetal characteristics. In the C-cell complexes there are follicular cells in various stages of differentiation, i.e., the cell clusters not yet organized into follicles, primordial follicles with small lumens and comparatively enlarged follicles storing plentiful amounts of colloid. They have a morphology similar to follicular cells of fetal thyroid glands and react to antiserum to 19S thyroglobulin. In order to determine whether or not the follicles in these complexes have the ability to incorporate radioiodine, autoradiography after a single injection of 125I was combined with immunoperoxidase staining using specific anti-calcitonin, anti-C-thyroglobulin, and anti-19S thyroglobulin antisera. The 19S-positive cells not yet organized into follicles did not take up radioiodine. Primordial follicles showed a heavy accumulation of silver grains over their follicular lumens storing new 19S thyroglobulin as colloid. Comparatively enlarged follicles revealed a strong autoradiographic reaction and their labeling patterns were identical with those of typical thyroid follicles. These results confirm that the follicles in C-cell complexes, as well as thyroid follicles, can incorporate radioiodine and are related to thyroid hormone synthesis. That is, functional thyroid follicles can arise from the ultimobranchial bodies.

Protein composition in single follicles, homogenates and fine-needle aspiration biopsies from normal and diseased human thyroid

The protein composition of the thyroid colloid was analysed by microgel electrophoresis and densitometry in 41 euthyroid patients. The colloid samples were obtained from single follicles by micropuncture, from homogenates of microbiopsies or from aspiration biopsies. Fourteen of the patients had morphologically normal thyroid tissue, 18 had atoxic nodular goitre and 19 of the patients had atoxic adenoma. Ten of the patients with nodular goitre had prior to the investigation received lithium therapy for psychiatric disorders. The main component of the thyroid colloid was 19S thyroglobulin (TG), but larger iodoproteins (S-TG) and smaller protein fractions, an albumin-like protein and a pre-albumin fraction, were also present in varying relative amounts. Analyses of homogenates of microbiopsies from normal thyroid tissue demonstrated the same protein composition as observed in single follicles. In colloid samples from atoxic nodular goitre the lighter protein fractions were absent in most of the samples. Analyses of homogenates or aspiration biopsies could not demonstrate this alteration in the protein composition in nodular goitre. Lithium therapy resulted in a significantly lower amount of the lighter protein fractions but unchanged amounts of the globulin fractions in atoxic nodular goitre.

Electrophoretic analysis of thyroid iodoproteins in highly cross-linked polyacrylamide gels.

The electrophoretic behavior of thyroglobulin and larger thyroglobulin-like iodoproteins prepared from the hog thyroid was studied in polyacrylamide gels over a wide range of cross-linking degrees (C%). When examined at a constant total acrylamide monomer concentration (T%) of 5%, the mobility of 19S thyroglobulin decreased with increase in C below C = 5%, whereas above C = 5%, it increased markedly with increase in C, giving a minimum mobility at C = 5%. Similar biphasic mobility curves were obtained with 27S and 37S thyroid iodoproteins. Furthermore, in a region above C = 15%, at least two additional larger components which escaped detection in the gels with lower degrees of cross-linking appeared as separate bands. Ferguson plots constructed for thyroid iodoproteins at a higher constant C of 20% gave straight lines intersecting at a common point at T = 0%. From the calculated slopes of the Ferguson plots, it has been established that the thyroid contains a series of multimers of 19S thyroglobulin as constituent iodoproteins. Structural parameters of highly cross-linked gels were estimated under the assumption that the gels would be predominantly composed of a random meshwork of gel beads.

Immunohistochemical study of the C-cell complex of dog thyroid glands with reference to the reactions of calcitonin, C-thyroglobulin and 19S thyroglobulin.

Continued from the previous study in fetal animals (Kameda et al. 1980), the development and maturation of C-cell complexes in postnatal dogs from newborn to adult were investigated by use of an immunoperoxidase method using antisera to calcitonin, C-thyroglobulin (C-Tg) and 19S thyroglobulin, respectively. The younger the animals were, the more numerous were undifferentiated cells and high columnar epithelial cells in the complexes. With increasing age, the constituent elements of the complexes progressively differentiated. In one type of complex there are a large number of C-cells in various developmental stages, as well as undifferentiated cells and cysts. C-cell complexes composed mostly of mature C-cells were regarded as the more highly differentiated structures of this type. A second type contains follicular cells in various stages of differentiation in addition to undifferentiated cells and C-cells, i.e., 19S-positive cell masses not yet organized into follicles, primordial follicles with small lacunae and comparatively larger follicles. The follicular cells in the complexes were similar with respect to immunoreaction and folliculogenesis to the cells of fetal thyroids, but they developed very slowly. In conclusion, the present study indicates that follicular thyroid cells can differentiate within C-cell complexes, i.e., they develop from cells of ultimobranchial body origin.

Development and cytodifferentiation of C cell complexes in dog fetal thyroids. An immunohistochemical study using anti-calcitonin, anti-C-thyroglobulin and anti-19S thyroglobulin antisera.

The development of C-cell complexes was investigated in dog fetuses by an immunoperoxidase method with three specific antisera: anti-calcitonin, anti-C-thyroglobulin (C-Tg), and anti-19S thyroglobulin. Ultimobranchial bodies joined with the thyroid anlage and then dispersed into the parenchyma to form large C cell groups. Sparse reaction products of C-Tg initially appeared in C cells with small amounts of cytoplasm. Later at about day 39 of gestation, when the immunoreactivity of calcitonin and 19S thyroglobulin appeared weakly in C cells and follicular cells, C-cell complexes were identified as large cell masses containing numerous undifferentiated cells without no immunoreactivity for any of the antisera. As development proceeded, the undifferentiated cells developed progressively the morphology of C cells. In addition, the undifferentiated cells developed 19S thyroglobulin immunoreactivity, that is, within some of the complexes small clusters of cells filled with material immunoreactive for 19S thyroglobulin. They were not organized into follicles during the fetal period, and were very slow in development. Depending on the degree of development of the undifferentiated cells, several features of the complexes were noted. The present study indicates that not only C cells but also follicular thyroid cells appear to be derived from the ultimobranchial bodies.

INFLUENCE OF IODINE ON THE FORMATION OF 27S AND LARGER IODOPROTEINS IN RAT THYROID FOLLICLES

SUMMARY The importance of the presence of iodine for the formation of iodoproteins larger than 19S thyroglobulin, i.e. 27S and larger iodoproteins (large-TG), was studied. Samples of colloid were collected in vivo by micropuncture of single rat thyroid follicles after treatment with a low iodine diet or potassium perchlorate. The protein composition of the colloid was analysed by microgel electrophoresis on polyacrylamide gels. It was observed that the large-TG fractions were reduced by approximately 50% after treatment with a low iodine diet for 3 months. The large-TG fractions were almost absent after treatment with perchlorate for 3 weeks. Administration of 5 or 10 μg potassium iodide to the animals on a low iodine diet resulted in a significant increase of the relative amount of the large-TG fractions in the colloid within approximately 2 h, the higher dose of KI producing a more pronounced accumulation of the large-TG fractions. Inhibition of protein synthesis by cycloheximide did not prevent the increase of large-TG proteins in animals on a low iodine diet after the administration of 5 μg KI. The administration of iodide to rats treated with potassium perchlorate did not result in the appearance of large-TG fractions. It is concluded that the aggregation of thyroglobulin molecules occurs in the follicle lumen as a consequence of iodine administration. This formation of large-TG fractions does not require exocytosis of newly synthesized protein.

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With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.

Afrikander cattle congenital goiter: characteristics of the defective 19S thyroglobulin synthesis.

The characteristics of the defective thyroglobulin biosynthesis in genetically determined Afrikander cattle goiter were compared with normal thyroglobulin biosynthesis. In vivo biosynthesis was investigated by incubating goiter and normal thyroid slices with radioactive amino acids. The proteins in the incubation medium and in the soluble (100,000 × g supernatant) and solubilized (digitonin extract of the 100,000 × g precipitate) fractions were analyzed by sucrose gradient centrifugation and immunoprecipitation. While 19S thyroglobulin was clearly present and labeled in the fractions from normal thyroid slices, none of the goiter fractions contained a 280 nm-absorbing 19S peak. The radioactivity of all of the goiter fractions, however, overlapped the 19S zone and 20–40% of the counts in the 12 to 19Ssedimenting regions were precipitable by an antithyroglobulin double antibody system. By employing standardized methodology (Biochem Biophys Res Commun 78: 230, 1977), 33S mRNA was isolated from normal thyroid...

Variations in the structure of thyroglobulins from normal and goitrous human thyroids.

Thyroglobulin samples were prepared individually be gel chromatography from the thyroids of five persons without thyroid disease and four with goiters. Gel electrophoresis at different pHs and gel concentrations showed a single major band corresponding to 19S thyroglobulin in rabbits, with occasional faint bands corresponding to 12S and 27S species. The thyroglobulins of the normals differed from each other in electrophoretic pattern on sodium dodecyl sulfate (SDS)-urea gels and in composition of iodine, monosaccharides, and amino acids. Nine amino acids showed significant variation among the five thyroglobulins at the P less than 0.01 level, and only two (lysine and alanine) did not vary. The content of both sialic acid and fucose varied widely, but their sum was similar among the five samples. Thyroglobulin samples from the goiters differed from the normals and from each other in composition and in pattern on SDS-urea gels. The variability itself was more impressive than were differences in any particular component. Relative to the normals, these thyroglobulins showed increases in content of sialic acid (P less than 0.01) and lysine (P less than 0.10), and increases in the faster bands on gel electrophoresis in SDS-urea. Two goiters were from patients with the multiple hamartoma syndrome, and the only metabolic abnormality found was a low content of iodothyronine in thyroglobulin. The other two goiters also showed inadequate coupling of iodotyrosyls. In addition, one contained a soluble iodoprotein of very high molecular weight, which was immunologically identical to 19S thyroglobulin but differed in chemical composition. We conclude from the compositional data that there is not a single structure for "normal" thyroglobulin, but that multiple molecular configurations occur naturally and are compatible with adequate hormone synthesis. Extensive variations in thyroglobulin structure are frequently found with goiter, and we suggest that these may be involved in its pathogenesis.

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine: a microgel electrophoretic study of single rat thyroid follicles.

Prophylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis. It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks. A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.