Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form.

Abstract

Extracellular storage of thyroglobulin (TG) is an important prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of large amounts is made possible by compactation of TG in the follicle lumen with concentrations of at least 100-400 mg/ml. We recently observed that the luminal content from bovine thyroids can be isolated in an intact state and be separated from soluble TG. For this purpose, bovine thyroid tissue was homogenized and subjected to various steps of purification. This procedure resulted in a pellet of single globules measuring 20-120 microns in diameter. Scanning electron microscopy revealed a unique cobblestone-like surface pattern of isolated globules, showing in detail the impressions of the apical plasma membranes of thyrocytes which had formerly surrounded the luminal content before tissue homogenization. Isolated thyroid globules were rapidly digested by trypsin but extremely resistant to various protein solubilization procedures. Homogenization of isolated globules resulted in the release of approximately 3% of total protein, showing that only a minor proportion of TG was loosely incorporated in thyroid globules whereas approximately 22% appeared to be interconnected with the globule matrix by disulfide bridges. Analysis by SDS-gel electrophoresis and immunoblotting confirmed that the protein released by this procedure consisted of TG. The vast majority (approximately 75%) of the globule matrix protein was found to be covalently cross-linked by non-disulfide bonds. TG in isolated globules was highly iodinated (approximately 55 iodine atoms per 12-S TG subunit) suggesting that the covalent nondisulfide cross-linking occurs in part during the iodination of TG and that this process involves the formation of intermolecular dityrosine bridges. Mechanisms must exist which solubilize or disperse the insoluble luminal content prior to endocytosis of TG.