Thyroglobulin Preparations Research Articles

An approach to the structure of thyroglobulin. Hormone-forming sequences in porcine thyroglobulin.

The mixture of CNBr peptides obtained by treatment of porcine thyroglobulin with cyanogen bromide was separated into three fractions by Sephadex G-200 gel filtration in 1 M propionic acid. When one of these fractions was reduced and S-alkylated, a hormone-containing CNBr peptide was purified by filtration on Biogel A-1.5 m and ion-exchange chromatography on DEAE-Sephadex. Similarly, two other hormone-containing CNBr peptides were separated from another reduced and S-alkylated fraction by Sephacryl S-200 gel filtration and DEAE-Sephadex chromatography. The three purified CNBr peptides have the same Mr (15000), differ in amino acid composition and contain 60-70% of the hormones present in the initial thyroglobulin. Fragmentation of these peptides into smaller hormone peptides was carried out by trypsin digestion followed by gel filtration. This resulted in the purification of eight discrete hormone-containing tryptic peptides whose homogeneity was established by the study of their N-terminal and C-terminal sequences. At least four sites for the synthesis of thyroxine and two sites for the synthesis of triiodothyronine have been identified in the three CNBr peptides. A possible additional site was recovered in the form of the peptides seryl-triiodothyronine and probably seryl-thyroxine. To gain information on the number of hormone-forming sites in thyroglobulin iodinated in vivo and on the distribution of these sites in the different hormone-containing peptides isolated, estimation of thyroxine and triiodothyronine content of batches of thyroglobulin of iodine content comprised between 0.73% and 1.6% was carried out, together with the estimation of the hormone content of all the CNBr peptides separated from two preparations of thyroglobulin of different iodine contents (0.73% and 1.23%). It was shown that the number of thyroxine and triiodothyronine residues increased linearly with increasing thyroglobulin iodine content in the range 0.73-1.6% and reached six residues of thyroxine and two residues of triiodothyronine/mol protein for a thyroglobulin iodine content of 1.6% with no indication of saturation. This result is at variance with previous findings on porcine thyroglobulin iodinated in vivo which suggested that the thyroxine content reached a plateau of three to four residues/molecule for an iodine content comprised between 0.7% and 1.1%.

Iodine is Essential for Human T Cell Recognition of Human Thyroglobulin

Here we describe for the first time that recognition by human T cells of human thyroglobulin depends upon its iodine content. We have examined the proliferation of lymphocytes from blood of autoimmune thyroiditis patients and normal individuals to thyroglobulin preparations containing different amounts of iodine. A minimal degree of iodination was required to elicit the proliferative response of both patients and normal individuals since thyroglobulin preparations containing no detectable iodine did not induce proliferation. A non-iodinated thyroglobulin preparation that was iodinated in vitro produced significant proliferation of both patient and normal lymphocytes. Addition of IL-2 to the culture medium enhanced proliferation but did not change the pattern of response.

Bispecific thyroglobulin and thyroperoxidase autoantibodies in patients with various thyroid and autoimmune diseases.

Recent evidence indicates that thyroid autoimmune disorders are associated with the presence of circulating autoantibodies (aAb) with dual specificity for thyroglobulin (TG) and thyroperoxidase (TPO). The question of whether these aAb, called TGPO aAb, are of clinical relevance compared to TG and TPO aAb remains to be determined. The availability of purified preparations of human TG and TPO allowed the development of a specific and sensitive RIA for TGPO aAb in serum. In the present study, we compared levels of aAb that cross-react with both TG and TPO (TGPO aAb) and total TG and TPO aAb levels, respectively, in sera from 84 normal controls and 226 patients with various thyroid and autoimmune diseases, including nontoxic goiter (n = 50), toxic nodular goiter (n = 13), thyroid carcinoma (n = 20), primary idiopathic myxedema (n = 15), postpartum thyroiditis (n = 11), Hashimoto's thyroiditis (n = 38), pernicious anemia (n = 27), rheumatoid arthritis (n = 19), and insulin-dependent diabetes mellitus (n = 33). In addition, 16 patients with Hashimoto's thyroiditis were studied before therapy and after more than 3 months of treatment with L-T4. It was shown that TGPO aAb were generally, but not always, present in the serum of patients with Hashimoto's thyroiditis, which also contained TG and TPO aAb. In contrast, TGPO aAb were undetectable in normal controls (excepting a few cases reaching borderline levels) as well as in sera from the majority of the other patients tested. Selecting sera positive for TGPO and either TG or TPO aAb, a statistically significant correlation was found between TGPO and TG (n = 26; P < 0.005), but not TPO aAb. Interestingly, the TGPO aAb level significantly decreased in patients with Hashimoto's thyroiditis after hormonal therapy (P < 0.05), some of them shifting from TGPO aAb positive before treatment to negative after treatment. In conclusion, TGPO aAb determination distinguishes Hashimoto's patients from patients with either thyroid and/or autoimmune diseases. The specific presence of TGPO aAb in a subset of Hashimoto's patients and their variation during T4 therapy remain to be understood. This could give a clue to mechanisms of autoimmune thyroid disease.

Thyroid peroxidase in dyshormonogenetic goiters with organification and thyroglobulin defects.

Thyroid peroxidase (TPO) iodide and guaiacol oxidation activities were evaluated in eight dyshormonogenetic goiters. Two of these had a defective thyroglobulin; the TPO iodide oxidation (431 and 316 U/g ptn) and iodination (31 and 8.6 nmol I/mg ptn) activities were within the normal ranges. The goiters from two siblings with positive perchlorate iodide discharge tests also had normal TPO iodide oxidation (602 and 299 U/g ptn) and iodination activities (44 and 11 nmol I/mg ptn). No TPO iodide oxidation activity was found in the goiters from the other four patients with positive perchlorate iodide discharge tests, and TPO iodide oxidation inhibitory activities were detected in both their TPO and thyroglobulin preparations. Three of them had some TPO guaiacol oxidation activity and did not inhibit normal guaiacol oxidation. The TPO preparation immunoblot of these three goiters showed a faintly visible band of normal 100 kDa TPO. However, in the other patient no guaiacol oxidation activity was detected, and only two bands of low-molecular-weight TPO (72 and 43 kDa) were found, again showing that iodine organification defects in dyshormonogenetic goiters can be due to either qualitative or quantitative TPO defects. The TPO inhibition diminished when iodide was increased in the assay, but was not altered by increasing cofactor (H2O2). Our results, so far, suggest that the TPO-inhibitory substance may interact reversibly with a specific iodide site on the enzyme or with the oxidized form of iodide, and/or could bind free iodide, making it unavailable for enzymatic oxidation.

Pitfalls in the use of ELISA to screen for monoclonal antibodies raised against small peptides

Small peptides are often conjugated to large carrier proteins such as thyroglobulin to increase their immunogenicity. Antibodies are raised against peptide, thyroglobulin and contaminants, such as immunoglobulins or BSA in the thyroglobulin preparation, but anti-contaminant antibodies will not be revealed if immune serum is diluted in buffer containing immunoglobulin-contaminated BSA. This may lead to the development of an inappropriate hybridoma-screening assay which detects more anti-contaminant than anti-peptide antibodies if ELISA plates are coated with a capture antibody or blocked with BSA. Dilution of culture supernatants in buffer containing BSA and Tween 20 minimises the risk of false positive results and makes plate-blocking unnecessary. The high affinity peptide-specific antibodies in immune serum, which are more readily detected than lower affinity monoclonal antibodies, may result in an inadequately sensitive hybridoma-screening ELISA.

Thyrotropin alters the utilization of thyroglobulin's hormonogenic sites.

We injected rabbits and guinea pigs with bovine thyrotropin (TSH) daily for 3 days, while controls received saline. All animals received sodium [125I]iodide on the second day, and thyroglobulin was purified from the thyroids of each group by gel filtration. Hormonogenic tryptic peptides from each S-cyanoethylated thyroglobulin preparation were isolated by high performance liquid chromatography, and their amino acid sequences were determined, permitting their localization within the thyroglobulin polypeptide chain by comparison with cDNA-derived sequences from bovine and human thyroglobulins. Thyroglobulins from the saline-injected rabbits and guinea pigs contained the same four major hormonogenic sites, designated A-D, previously described (Dunn, J. T., Anderson, P. C., Fox, J. W., Fassler, C. A., Dunn, A. D., Hite, L. A., and Moore, R. C. (1987) J. Biol. Chem. 262, 16948-16952). In both species, sites A and C were the major loci for thyroxine and triiodothyronine, respectively. However, site D in the guinea pig had a greater ratio of [125I]thyroxine to [127I]thyroxine than did site A, whereas the reverse was true in the rabbit. TSH administration produced the following changes in thyroglobulins of both species, relative to controls: 1) an increase in the ratio of [125I]triiodothyronine to [125I] thyroxine (rabbit, 0.29 versus 0.17; guinea pig, 0.19 versus 0.08), with the increase in triiodothyronine principally at site C; 2) a marked increase in 125I/127I and in thyroxine formation at site D (14.1% of thyroglobulin's thyroxine versus 9.8% in rabbits, 24 versus 13% in guinea pigs); 3) a corresponding decrease in thyroxine formation at site A (33 versus 43% in rabbits, 30 versus 46% in guinea pigs); and 4) a sharp increase in conversion of thyroglobulin's N-terminal 125I-labeled approximately 20 kDa hormone-rich iodopeptide, which contains site A, to a 125I-labeled approximately 15-kDa (rabbit) or 125I-labeled approximately 13-kDa (guinea pig) form, reflecting probable peptide bond cleavage. Our results show that TSH alters both the structure of the thyroglobulin molecule and the priority of utilization of its hormonogenic sites. We conclude that these changes are important to TSH's enhancement of thyroid hormone synthesis.

Colorimetry of dehydroalanine residues preserved as ‘lost side chains’ in thyroglobulin

We developed a new assay method for dehydroalanine residues in thyroglobulin, which had been proposed to be the ‘lost side chains’ during thyroid hormonogenesis. Thyroglobulin preparations were labeled with 4-aminothiophenol at 30 ° C for 10 days. Under the conditions, the reagent reacted only with dehydroalanine and cysteine residues. The 4-aminothiophenol bound to cysteine was eliminated by reductive cleavage. The 4-aminothiophenol-labeled dehydroalanine (4-aminophenylcysteine) residues were liberated by acidic hydrolysis, converted to a colored derivative by the Bratton-Marshall reaction and quantified colorimetrically. The number of dehydroalanine residues was the same as that of hormone residues in each thyroglobulin preparation. The results indicate that when one hormone residue is produced by the coupling of two iodotyrosine residues, the ‘lost side chain’ is preserved as one dehydroalanine residue in the thyroglobulin molecule.

Antigenic Determinants on Thyroglobulin: Comparison of the Reactivities of Different Thyroglobulin Preparations with Serum Antibodies and T Cells of Patients with Chronic Thyroiditis*

To delineate the antigenic determinants on thyroglobulin (Tg) recognized by serum autoantibodies and peripheral blood T cells from patients with chronic thyroiditis, we studied the reactivities of three different Tg preparations, i.e. enzyme-digested Tg fragments, physically or chemically denatured Tg, or Tg with differing iodine contents. Human Tg was digested with staphylococcal V8 protease, and the fragments were separated by high performance liquid chromatography. The autoantibodies reacted with the larger fragments, but their ability to bind to small fragments was limited. On the other hand, T cells reacted similarly with all fragments, regardless of mol wt. The autoantibodies bound little to denatured Tg after its disulfide bonds were destroyed with dithiothreitol or 2-mercaptoethanol, while the reactivity of heat-denatured Tg was partially decreased, and that of Tg denatured with sodium dodecyl sulfate was conserved. Conversely, T cells reacted with Tg denatured by heating or dithiothreitol treatment. These results indicate that autoantibodies recognize mainly a conformational structure of Tg, presumably containing disulfide bonds, whereas T cells recognize the primary structure of Tg. Variations in the iodine content of Tg were not associated with altered reactivity with autoantibodies or T cells. We propose that variations in Tg conformation related to iodination of the molecule do not contribute significantly to its reactivity with autoantibodies and T cells. In addition, T cells reacted with the smaller Tg fragments containing few T3 or T4 residues to a greater extent than they did with larger Tg fragments with the same amount of T3 or T4 as native Tg. Therefore, it appears that the Tg-reactive T cells predominantly recognize determinants on the Tg molecule that are unrelated to hormone-containing sites.

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.

Characterization of phosphate residues on thyroglobulin.

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.

Investigations on the mechanism(s) of the production of anti-thyroid hormone antibodies 4: the effect of adjuvant on the immune response to human thyroglobulin

Forty-five female DDYK mice were divided into three groups and immunized with different preparations of human thyroglobulin (HTg). Group 1 mice were immunized subcutaneously in the back with HTg emulsified with FCA. Group 2 mice were immunized subcutaneously in the back with HTg dissolved in PBS. Group 3 mice were immunized with HTg dissolved in PBS and FCA separately in the right (HTg) and left (FCA) of the back. After the fourth immunization (45 days after the first immunization), antisera as well as thyroid glands were obtained, and titers of anti-HTg, anti-T3, anti-T4 and the presence of lesions of autoimmune thyroiditis were examined. In group 1 mice, titers of anti-HTg and anti-T4 antibodies were significantly higher than those of the other two groups, whereas titers of anti-T3 antibodies were significantly lower than those of the other two groups. In group 1 mice, a significant correlation between titers of anti-T3 and anti-T4 antibodies was observed, whereas in group 2 and group 3 mice, no such correlation was observed. The incidence of lesions of autoimmune thyroiditis was significantly higher in group 2 and group 3 mice than in group 1 mice. Thus emulsification of HTg with FCA had inhibitory effects on the induction of experimental thyroiditis in mice immunized with HTg. These results indicate the possibility that FCA has a modulatory effect both quantitatively and qualitatively on the immune response against HTg in mice.

The Immunochemical Properties of Thyroglobulin in Human Thyroid Tumors in Reaction to Rabbit Anti-Human Normal Thyroglobulin-Serum

Interactions between antiserum (rabbit anti-human normal thyroglobulin-serum) and human thyroglobulin preparations (obtained from the tissues of the normal thyroid gland, thyroid adenoma, and carcinoma) were compared by inhibition effect with the binding between 131I-labeled standard thyroglobulin and antiserum, set up by a double antibody RIA. Thyroglobulins isolated from normal glands (designated as Nor-Tg) have a high affinity to the antiserum. In contrast, thyroglobulins in follicular adenoma or adenomatous goiter (designated as Ad-Tg) decrease the potency of the affinity to the antiserum. Furthermore, thyroglobulins in papillary or follicular carcinoma (designated as Ca-Tg) markedly decrease such a potency. With the t-test, the inhibition curves of Nor-Tgs are almost parallel to each other. Most of the inhibition curves of Ad-Tgs and Ca-Tgs are not parallel to the curve of Nor-Tg 1 (P-value for non-parallelism less than or equal to 0.05). Therefore, it seems that Tg preparations obtained from tumor tissue are heterogeneous in terms of specificity and/or affinity to antiserum, judging from the results of the non-parallel inhibition curves. The present results also show that the contribution of iodine content in Tg has little or no effect on the nature of Tg-immunogenicity.

The identification of terminal α(1→3)-linked galactose in N-acetyllactosamine type of glycopeptides by means of 500-MHz 1H-NMR spectroscopy

500-MHz 1H-NMR spectroscopy was employed to study two N-acetyllactosamine-type glycopeptide fractions which were derived from a bovine thyroglobulin preparation (Cummings, R.D., and Kornfeld, S. (1982) J. Biol. Chem. 257, 11230-11234). By this method, their branches were found to be terminated either by NeuAc in alpha (2----6)-linkage or by Gal in alpha (1----3)-linkage. For the first time, the Gal alpha (1----3) Gal beta (1----4) GlcNAc beta (1----.) sequence is characterized by 1H-NMR to occur in N-glycosidic carbohydrate chains of glycoproteins. Moreover, this approach made possible the branch localization of such a unit. Microheterogeneity with respect to the presence of alpha-linked Gal or NeuAc in terminal position of a certain branch in one of the preparations, could be adequately assessed in terms of structures by 1H-NMR.

Transcytosis in thyroid follicle cells.

Inside-out follicles prepared from pig thyroid glands were used for studies on endocytosis. endocytosis. In this in vitro system, only the apical plasma membranes of follicle cells were exposed to tracers added to the culture medium. Cationized ferritin (CF) bound to the apical plasma membrane and was transferred first to endosomes and to lysosomes (within 5 min). Later, after approximately 30 min, CF was also found in stacked Golgi cisternae. In addition, a small fraction of endocytic vesicles carrying CF particles became inserted into the lateral (at approximately 11 min) and the basal (at approximately 16 min) plasma membranes. Morphometric evaluation of CF adhering to the basolateral cell surfaces showed that the vesicular transport across thyroid follicle cells (transcytosis) was temperature-sensitive; it ceased at 15 degrees C but increased about ninefold in follicles stimulated with thyrotropin (TSH). Thyroglobulin-gold conjugates and [3H]thyroglobulin (synthesized in separate follicle preparations in the presence of [3H]leucine) were absorbed to the apical plasma membrane and detected mainly in lysosomes. A small fraction was also transported to the basolateral cell surfaces where the thyroglobulin preparations detached and accumulated in the newly formed central cavity. As in the case of CF, transcytosis of thyroglobulin depended on the stimulation of follicles with TSH. The observations showed that a transepithelial vesicular transport operates in thyroid follicle cells. This transport is regulated by TSH and includes the transfer of thyroglobulin from the apical to the basolateral plasma membranes. Transcytosis of thyroglobulin could explain the occurrence of intact thyroglobulin in the circulation of man and several mammalian species.

Restricted antigenicity of thyroxyls in human thyroglobulin.

Interactions between two human iodothyronine-binding autoantisera and three preparations of human thyroglobulin (Tg) were not proportional to the latter's thyroxyl residue content. Probably only one of the several thyroxyl-containing sites in Tg reacted with the immunoglobulins from both antisera. In the case of one of the antisera, which was thyroxine (T4)-specific, the thyroxyl residue was the immunodominant feature of the antigenic site. The other antiserum, which had a specificity for 3,5,3'-tri-iodothyronine (T3), recognized different determinants around the same thyroxyl residue, but this residue was not itself an important element of the binding site. Thus, despite the specificity for T3 free in solution, the presence of T4 in the complete antigenic site was tolerated, since other structures supplied the bulk of the binding energy. 'Specificity' of this antiserum for T3 in solution is therefore coincidental and need not be ascribed to the presence of T3 in the original immunogen. Some results obtained in these studies may be interpreted as supporting the possibility that a modified Tg was the immunogen for the generation of these naturally occurring human antisera.

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations.

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.

High-Speed Gel Permeation Chromatography of Human Thyroglobulin and Sheep Liver Aldehyde Dehydrogenase

Abstract This paper reports the application of high speed gel permeation techniques for the fractionation of human thyroglobulin preparations on chemically bonded, polar phase microparticulate silicas. Both resolution and recovery were found to be dependent on the ionic strength and pH of the eluent. At high salt concentrations significant hydrophobic interactions may occur between the support matrix and the protein solutes. The sheep liver enzyme, aldehyde dehydrogenase, was found to exhibit similar salt-and pH-dependent effects under these chromatographic conditions.

Fused rocket immunoelectrophoretic analysis of human and sheep thyroglobulins purified by gel chromatography.

The application of fused rocket immunoelectrophoretic methods for the analysis of human and sheep thyroid extracts is reported. Different experimental procedures, evaluating thyroglobulin preparations obtained by gel chromatographic separations with a variety of gel supports and elution conditions, were examined. Adsorption experiments demonstrated that many of the antigenic contaminants in chromatographed thyroglobulin were due to serum proteins. The precipitin profiles obtained with the fused rocket immunoelectrophoretic experiments indicate that considerable tailing of 19S-thyroglobulin occurs when chromatographed on either Sephadex G-200 or Bio-Gel A-1.5 m using low ionic strength buffers. The retention of thyroglobulin on these supports would account, in part, for the observed presence of thyroid proteins with antigenic determinants identical to 19S-thyroglobulin in chromatographed subfractions of notionally lower molecular weight. Because of the case of execution, the described methods provide a useful alternative to existing methods for the assessment of homogeneity of chromatographed thyroglobulin samples.

Evidence that thyroglobulin contains nonidentical half molecule subunits.

Bovine thyroglobulin was extracted from unfrozen glands, purified by sucrose gradient centrifugation, and fractionated into a narrow range in iodine content by RbCl isopycnic centrifugation. The subunit composition of these preparations was studied by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The extent of dissociation of 19 S into 12 S half-molecules followed the known relationship with iodine, i.e. decreased dissociability of 19 S with increased iodine content. The undissociated 19 S band always consisted of three closely spaced, equidistant bands. Reduction of the disulfide bonds of thyroglobulin by mercaptoethanol in SDS solution resulted in the formation of two major and one minor components (S, F, and A). The concentration of A was always less than 10% of the total. The ratio of S to F was, however, about equal in thyroglobulin preparations which ranged in iodine content from 0.2 to 1%. The final ratios were obtained before all the disulfides were reduced. The relative mobilitis of S, F, and A, decreased with increasing extent of reduction. Fully reduced S and F, but not A, migrated slower than unreduced 12 S. The three reduced alkylated polypeptides were purified by preparative SDS-polyacrylamide gel electrophoresis and their molecular weights were determined by sedimentation equilibrium in 8 M urea. Their Mw and Mz values agreed closely with that of the unreduced 12 S half-molecule subunit, thus indicating that reduction of the disulfide bonds changes the shape but not the molecular weights of the subunits.

Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 s thyroglobulin

Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37°C at pH 4.5 and 25°C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol.wt. corresponding to the native 12 S were the major component, strongly suggesting a mol.wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.