Thyroid hormonogenesis. Identification of a sequence containing iodophenyl donor site(s) in calf thyroglobulin.
The formation of dehydroalanine in thyroglobulin is the result of the side chain elimination of an iodophenyl group during the thyroid hormone formation from two iodotyrosyl residues. This amino acid is easily converted to labeled alanine (upon reduction with [3H] borohydride) or changed to labeled aspartic acid (upon addition of Na14CN and subsequent acid hydrolysis). The cleavage of the protein by CNBr produced many stainable electrophoretic bands, but the autoradiography indicated the presence of a much smaller number of radioactive species. Although three major species raised attention, because they could be all jointly labeled and were present in all preparations, only a species of 15,900 Da was fully studied. It was isolated and its sequence partially determined by Edman degradation. It was established that this species corresponded to the thyroglobulin fragment between methionines 2,432 and 2,578. This peptide contains two hormonogenic sites (positions 2,555 and 2,569) which are either tyrosyl residues or hormone residues arising from them, and five additional tyrosines all potentially involved as donor sites in the hormonogenesis. Upon treatment with N-chlorosuccinimide, the fragment was split into three smaller peptides of about 2,900, 8,500, and 4,600 Da containing 1, 2, and 2 tyrosyl residues, respectively. Only the 8,500-Da subfragment contained [3H]Ala. This finding strongly suggests that at least some of the tyrosines involved as donor sites in thyroid hormonogenesis are within this peptide and possibly map at positions 2,469 and/or 2,522. Moreover, at minimum levels of iodination, when thyroglobulin contains the lowest number of hormone molecules, dehydroalanine is mostly found in the 15,900-Da peptide.
Palumbo
G
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Clinical Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
D000409:Alanine / Q000031:analogs & derivatives / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D013388:Succinimides; D013954:Thyroglobulin / Q000032:analysis*; D013961:Thyroid Gland / Q000032:analysis*
D013388:Succinimides; C015752:N-chlorosuccinimide; D013954:Thyroglobulin; C015102:dehydroalanine; D000409:Alanine