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Glycoprotein Biosynthesis: Studies on Thyroglobulin

CHARACTERIZATION OF A PARTICULATE PRECURSOR AND RADIOISOTOPE INCORPORATION BY THYROID SLICES AND PARTICLE SYSTEMS

From the ¹ From the Departments of Biological Chemistry and Medicine, Harvard Medical School, the Elliott P. Joslin Research Laboratory, and the Massachusetts General Hospital (Thyroid Unit), Boston, Massachusetts

This report presents a study of the biosynthesis of thryoglobulin from which information was obtained regarding several aspects of the biosynthesis of glycoproteins.

A bound protein was released from the thyroid particles with deoxycholate and after isolation was shown to have the carbohydrate composition, the immunochemical properties, and the electrophoretic behavior of soluble thyroglobulin.

Radioisotope studies with thyroid slices with the use of ¹⁴C-labeled glucose or ¹⁴C-labeled leucine as substrate provided evidence that this particle-bound thyroglobulin-like protein is the precursor of the soluble thyroglobulin. In these incubations, the specific activities of all five monosaccharides, as well as of the leucine of the particle-bound protein were found to be considerably higher than those of the isolated soluble protein at all time intervals studied.

Additional evidence for this biosynthetic role of the particles was obtained from their ability to synthesize both the carbohydrate and peptide portions of the particle-bound thyroglobulin when incubations of cell-free particle systems with ¹⁴C-labeled glucose or leucine were performed.

By enzymatic degradation of the radioactive particle-bound and soluble thyroglobulins, it was possible to show that radioactivity was incorporated into both types of carbohydrate units, as well as into various peptide fractions, suggesting that true synthesis of the molecules was taking place.

Incubations carried out in the presence of puromycin, both with slices and particle systems, indicated that the peptide portion of the thyroglobulin molecule is synthesized prior to the attachment of the carbohydrate and moreover supported the possibility that the synthesis of the carbohydrate portion occurs by a stepwise process.

Studies on the iodine of the thyroglobulin molecule indicated that although some iodination takes place at a particulate site, a significant additional portion must take place at another location.

In this report attention is called to the special precautions which have to be taken in the performance of ribonucleic acid determinations in the thyroid in order to avoid the overestimation of this nucleic acid which may result from the large amount of carbohydrate present in this tissue in the form of thyroglobulin. Mention is also made of the observation that thyroglobulin is soluble in ethanol after trichloroacetic acid or perchloric acid precipitation.

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