Thyroglobulin structure-function. Isolation and characterization of a thyroxine-containing polypeptide from bovine thyroglobulin.

Upon reduction and alkylation, 19 S bovine thyroglobulin gave rise to a family of polypeptides ranging in molecular weight from 10,000 to 330,000.T h i s group of polypeptides has been fractionated by chromatography on agarose gel in 6 M urea.After observing significantly increased iodine and thyroxine contents in the lowest molecular weight fraction obtained from the agarose gel-6 M urea chromatography, subsequent fractionation led to the isolation of a single, iodine-rich polypeptide in homogeneous form.While several low molecular weight polypeptides were found to contain iodine, one peptide, designated TG-F, was distinguished by the fact that its iodine content exceeded 2% and virtually all of the iodine found within its structure was in the form of thyroxine.TG-F was purified from pooled normal bovine thyroid tissue with an average yield of 0.9 mol of polypeptide/mol of 19 S thyroglobulin.It contains 2% iodine, only traces of carbohydrates, and 0.4 mol of thyroxine/mol of peptide.The molecular weight of the polypeptide was found to be 10,300 A 1,300 as determined from sodium dodecyl sulfate gel electrophoresis, thin layer gel filtration in 6 M guanidine hydrochloride, and gel filtration on a calibrated column in ammonium bicarbonate.TG-F contains no free NH2-terminal acid as judged from automated sequenator analysis as well as the dansyl-amino acid procedure.Amino acid analysis revealed 2 lysines, 7 arginines, and a single methionine/mol of peptide.Sodium dodecyl sulfate polyacrylamide gels following cyanogen bromide digestion contained two peptide fragments of similar size and near half the size of TG-F.The absence of significant amounts of iodinated tyrosine derivatives except thyroxine in TG-F suggests that this peptide contains a specific site at which thyroxine formation proceeds both immediately and to completion following iodination of this region by thyroid peroxidase.Thyroglobulin, the iodinated glycoprotein of the thyroid gland, forms the matrix within which thyroid hormones are synthesized.While much is known about this protein (1-4), structure-function studies have been difficult due to its very large size as well as the physical and chemical microheterogeneity described by a number of workers (5-7).Newly synthesized thyroglobulin is a substrate for thyroid peroxidase, a membrane-associated glycoprotein (a), which catalyzes both