Relationships of Thyrotropin to Exophthalmic-producing Substance: PURIFICATION OF HOMOGENEOUS GLYCOPROTEINS CONTAINING BOTH ACTIVITIES FROM [3H]-LABELED PITUITARY EXTRACTS

Abstract

Abstract Preparations of thyrotropin which are purified by standard procedures of column chromatography contain several electrophoretically distinct components which have exophthalmogenic activity and contain galactose. Although preparative disc gel electrophoresis can resolve these heterogeneous preparations into individual glycoproteins, several of the resultant components still have both thyrotropic and exophthalmogenic activity. Two such glycoproteins, each with both activities, are homogeneous in the ultracentrifuge, with a molecular weight of 27,000 ± 3,000. They have a similar, if not identical, amino acid composition. Previous suggestions that the determinants for both thyrotropic and exophthalmogenic activity can reside on the same glycoprotein molecule are reinforced by these data. Terminal galactose residues of glycoproteins may be enzymatically labeled with tritium. This procedure involves a preliminary exposure of the glycoprotein to galactose oxidase and subsequent reduction with (3H) sodium borohydride. Application of this technique has established the terminal nature of galactose in the multiactive but homogeneous preparations obtained by preparative gel electrophoresis. Both the thyrotropic and the exophthalmogenic activities are preserved in the tritiated glycoproteins. The procedure is applicable to crude pituitary preparations, the tritium serving as a convenient marker of the individual thyrotropic and exophthalmogenic species once the several contaminant glycoproteins have been removed. Thyroid-stimulating hormone derived from mouse transplantable tumors also contains terminal galactose which can be labeled by tritiation. Distinct differences from bovine thyrotropin can otherwise be shown in its sugar composition.