Studies of Double-Labeled Mouse Thyrotropin and Free α-Subunits to Estimate Relative Fucose Content

Abstract

The composition and structure of the complex oligosaccharides of thyrotropin (TSH) and free α-subunits are not well established, but are believed to be important determinants of the biological properties of these glycoproteins. We employed a simple double-label technique to learn the relative fucose content of mouse thyrotropin and free α-subunits. Thyrotropic tumor minces were incubated simultaneously with [³⁵S]methionine and [³H]fucose. Thyrotropin and free α-subunits were labeled with both isotopes, and the ratio of ³H/³⁵S was higher in free α-subunits than in thyrotropin; free α-subunits were approximately fivefold richer in fucose than was thyrotropin. The ³H/³⁵S ratio was not substantially altered in TSH or free α-subunits secreted after a brief incubation with 10^-7 M thyrotropin-releasing hormone. Species which incorporated [³H]fucose were resistant to endoglycosidase H. Thus, mouse free a-subunits secreted by thyrotropic tumor are relatively rich in fucose. Double-isotope labeling using an amino acid and a sugar appears to be a useful technique for studies of the glycoprotein hormones.