Thyroglobulin hydrolysis

Figure 42-11. Model of iodide metabolism in the thyroid follicle. A follicular cell is shown facing the follicular lumen (top) and the extracellular space (at bottom). Iodide enters the thyroid primarily through a transporter (bottom left). Thyroid hormone synthesis occurs in the follicular space through a series of reactions, many of which are peroxidase-mediated. Thyroid hormones, stored in the colloid in the follicular space, are released from thyroglobulin by hydrolysis inside the thyroid cell. (Tgb, thyroglobulin MIT, monoiodotyrosine DIT, diiodotyro-sine Tj, triiodothyronine T4, tetraiodothyronine.) Asterisks indicate steps or processes that are inherited enzyme deficiencies which cause congenital goiter and often result in hypothyroidism.

Female guinea pigs are immunised by injection of 200 pg of 3/ -hydroxy-5-chole-noyl-thyroglobulin conjugate. Serum extraction, solvolysis and alkaline hydrolysis of BAs are performed according to the method of Ali and Javitt [46] 3.5 ml of 2,2-dimethoxypropane and 0.4 ml of 1 M hydrochloric acid in methanol are added to... 

Thiocyanate ion, SCN-, inhibits formation of thyroid hormones by inhibiting the iodination of tyrosine residues in thyroglobulin by thyroid peroxidase. This ion is also responsible for the goitrogenic effect of cassava (manioc, tapioca). Cyanide, CN-, is liberated by hydrolysis from the cyanogenic glucoside linamarin it contains, which in turn is biodetoxified to SCN. 

Gross, Leblond, Franklin, and Quastel (1950) have shown that rat thyroids contain small amounts of free iodinated amino acids which can be extracted with butanol from the thyroid without previous hydrolysis. These include monoiodotyrosine, diiodotyrosine, thyroxine, and triiodothyronine, and they are presumably formed by proteolysis of thyroglobulin. Their fate has been the subject of much experimental work in recent years. 

With regard to the fate of the iodinated amino acids in the thyroid, the following concluraons have been reached. Monoiodotyrosine and diiodotyrosine do not leave the thyroid gland after proteolytic hydrolysis of thyroglobulin, but are enzymically deiodinated with the formation of iodide this iodide is available for re-utilization in hormone synthesis. Thyroxine and triiodothyronine are released into the circulation. 

L-Triiodothyronine and L-thyroxine are synthesized in higher animals and humans in the cells of the thyroid gland. These cells accumulate iodide ions from the blood with great efficiency. A halogenoperoxidase (C 2.4.2) reacts with L-tyrosine residues present in thyroglobulin, one of the thyroid proteins, to form L-monoiodo- and L-diiodotyrosine residues (Fig. 293). These residues, w hile still incorporated into thyroglobulin, condense in a radicaHc reaction with each other to L-triiodothyronine and L-thyroxine residues, which are released by hydrolysis of thyroglobulin and secreted in the blood-stream. 

Thyroglobulin release. Under normal conditions, thyroglobulin does not leave the thyroid gland. Instead, the active iodinated derivatives, thyroxine and 3,3, 5-triiodothyronine, are released by proteolysis of thyroglobulin. The enzymes involved and the intermediates formed during hydrolysis have not been identified. It is assumed that several polypeptide intermediates... 

Fig. 7. Radiochromatogram of n-butanol extract (pH 1.0) of the products of hydrolysis of labeled thyroglobulin (40). Ordinate is per cent I . (A) hydrolysis by 6 fV sodium hydroxide. (B) Hydrolysis by 2 N barium hydroxide. (C) Hydrolysis by pancreatic proteases. Solvent, n-butanol-acetic acid-water. I = iodides, II = 3-monoiodotyrosine, III = 3,5-diiodotyrosine, IV = thyroxine and 3,5,3 -triiodothyronine.

MIT. Autoradiography of two-dimensional chromatograms (solvent, phenol-collidine) of alkaline hydrolyzates of whole glands of rats treated with I showed the presence of a few spots of unknown substances (47), one of which was identified as 3-monoiodo-L-tyrosine (8,62,65). The specific activities of the MIT spot (solvent, collidine-ammonia-water) at intervals after injection of labeled iodide showed that MIT is the precursor of DIT and of Tx (65,70). These results were not completely convincing due to the great destruction of DIT by alkaline hydrolysis they have been confirmed, however, by the study of the amino acids freed from labeled thyroglobulin by enzymic hydrolysis under conditions which permitted no deiodination (38). 

MIT, DIT, and Tx in the thyroid extracts of rats injected 48 hours previously with I were first identified as free amino acids along with three unknown substances (16,31). One of the unknowns was later shown to be TRITh (63,67) and traces of MIH were regularly found by the writers. Autography is the best technique for studying the free amino acid of thyroid because the amino acids contain only a very small part of total radioactivity (a few per cent). Since the proteolysis of thyroglobulin seems to be complete and does not cause liberation of iodothyronines faster than iodotyrosines, the composition of the mixture of free iodinated amino acids is similar to that of samples obtained by in vitro hydrolysis of the protein... 

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