Osamu Tarutani

Subunit structure of hog thyroglobulin: dissociation by treatment with sodium dodecyl sulfate.

Abstract 1. 1. In order to investigate the subunit structure of hog thyroglobulin, the effects of sodium dodecyl sulfate on its behavior were studied employing several fractionated preparations which had been obtained by DEAE-cellulose chromatography and had different iodine contents. 2. 2. At lower concentrations of sodium dodecyl sulfate, i.e. , 1 mM or below, in a phosphate buffer of pH 6.5 at ionic strength 0.1, thyroglobulin showed the presence of a slower-sedimenting component (S-12) together with the unaltered protein (S-19) on analytical ultracentrifugation. In increasing sodium dodecyl sulfate concentrations, the conversion of S-19 to S-17 occurred, giving a mixture of S-12 and S-17. With a further increase in sodium dodecyl sulfate concentration, the sedimentation coefficients of both S-12 and S-17 decreased continuously, while their concentration dependence increased. 3. 3. Molecular weight determinations by means of sedimentation equilibrium have revealed that the S-12 component has a molecular size approximately half that of native S-19 thyroglobulin (mol. wt., 670 000). S-17 was estimated to have the same size as that of S-19. Under all conditions studied, neither a complete dissociation into S-12 nor further dissociation of S-12 into smaller units was observed. It was concluded that thyroglobulin (S-19) contains two types of molecules: one consisting of the two S-12 subunits which are linked by noncovalent bonds, and the other in which all the polypeptide chains are linked by covalent bonds and/or by other similarly strong forces. Only the former type of molecule is dissociable in the presence of sodium dodecyl sulfate. 4. 4. Subfractions of thyroglobulin iodinated to lesser degrees had a tendency to form larger amounts of S-12 by sodium dodecyl sulfate than more highly iodinated fractions. Heterogeneity of thyroglobulin with respect to subunit structure was thus indicated. 5. 5. The interaction of thyroglobulin and sodium dodecyl sulfate was further studied by measuring the number of bound sodium dodecyl sulfate, and the effects of pH and ionic strength on S-12 formation and the reversibility of the reaction.

Subunit structure of hog thyroglobulin Dissociation of noniodinated and highly iodinated preparations

Abstract Noniodinated thyroglobulin, usually not contained in the normal thyroid, was prepared in fairly large quantities from goitrogen-treated hogs and subjected to dissociation studies. In contrast to normal thyroglobulin, noniodinated thyroglobulin could be dissociated completely by sodium dodecyl sulfate into subunits (S-12) with a molecular weight one-half that of the native molecule (S-19), as revealed by sedimentation experiments in an analytical ultracentrifuge. In addition, noniodinated thyroglobulin was dissociated more readily in alkaline solutions than normal thyroglobulin. On the contrary, highly iodinated preparations of hog thyroglobulin prepared by iodination of normal thyroglobulin in a cell-free system were markedly resistant to sodium dodecyl sulfate or alkali. These findings, in combination with the results of our previous investigation on normal, fractionated preparations of hog thyroglobulin, indicate the presence of two types of molecules, i.e. , dissociable and nondissociable molecules, in thyroglobulin. It is suggested that fully dissociable, noniodinated thyroglobulin is converted to a nondissociable type of molecule during the course of iodination in the thyroid.

Genetic Control of the Production of Anti-Thyroid Hormone Antibodies in Mice Immunized with Human Thyroglobulin

Various strains of mice with different H-2 and Igh-I allotypes were immunized with human thyroglobulin (HTg) and genetic control of the production of anti-thyroid hormone antibodies was examined. Anti-HTg antisera obtained from different strains of mice were examined for the presence of anti-thyroid hormone antibodies. At least one Ir-gene which controls the production of anti-T4 antibodies by immunization with HTg was identified in the I-A subregion. The presence of Ir-genes outside the H-2 was also suspected. Concerning the production of anti-T3 antibodies, only three strains (B10.A, C3H.SW, BALB/cJ) were high responders and therefore we were unable to identify the Ir-gene for them. These results indicate the presence of Ir-gene which controls the immune response in mice against thyroid hormone and suggest that the anti-thyroid hormone antibodies observed in various thyroidal and non-thyroidal disorders could be anti-HTg antibodies. The significance of genetic control of the production of anti-thyroid hormone antibodies in mice immunized with HTg is discussed.

Properties of carbohydrate-stripped thyroglobulin III. solubility characteristics of thyroglobulin☆

In studying the solubility characteristics of thyroglobulin, it was found that the removal of sialic acid residues from either human or hog thyroglobulin by treatment with neuraminidase markedly decreased the solubility in salt solutions. On the other hand, naturally sialic acid-rich thyroglobulin obtained from human diffuse goiter was slightly more soluble than thyroglobulin in which sialic acid content was within the normal range. However, the desialized preparations prepared from both normal and sialic acid-rich thyroglobulin showed markedly lower solubility and further, showed no significant difference in solubility between them. These results indicate that the surface charge of thyroglobulin which influenced the salting-out property depends largely on the number of sialic acid residues in a thyroglobulin molecule, not on the degree of iodination of thyroglobulin.

Production of anti-human thyroglobulin and anti-thyroid hormone antibodies in rabbits immunized with homogenate of human papillary cancer tissue

Papillary cancer tissue of the thyroid gland removed from each of three patients was homogenized in phosphate buffer followed by centrifugation. Each of three rabbits was immunized with each of the supernatants (TC-1, TC-2, TC-3). These rabbits were immunized on days 0, 7, 14, and 21, and serum from each rabbit, obtained 4 weeks after the first immunization, was examined for the presence of anti-human thyroglobulin (HTg), anti-thyroxine (T4), and anti-triiodothyronine (T3) antibodies. Production of anti-HTg antibodies was observed in all three rabbits. In addition, despite the low content of iodine, T3, and T4 in thyroglobulin that had been purified from the papillary cancer tissues (p-HTg), production of anti-T4 and anti-T3 was observed in two of the three rabbits, and the other immunized with TC-1 showed anti-T4 but no anti-T3 antibodies. The significance of the production of anti-thyroid hormone antibodies in rabbits with respect to the antigenic structure of p-HTg with low content of iodine and thyroid hormone is discussed.