Affinity Labelling of the Thyrotropin Receptor
Thyrotropin (TSH) receptors from different species and tissues have been covalently cros s-linked to 125I-labelled TSH, and the mol. wts. of the TSH-TSH receptor complexes elucidated. Two successful methods of cross-linking entail use of the reagents disuccinimidyl suberate (DSS) and N-hydroxysuccin-imidyl-4-azidobenzoate (HSAB). Highly purified bovine TSH (70 u./mg) was coupled to HSAB (which is photoreactive), labelled wi th 125I, then incubated with several different TSH receptor preparations and coupled to the receptor by photolysis. Alternatively, the TSH was labelled with 125I and incubated with solubilized receptors; DSS effected cross-linking.
The TSH-TSH receptor complexes were heated in sodium do-decyl sulphate (SDS) solution and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) with and without reduction by mercaptoethanol; the dried gel was autoradiographed. Porcine, human and guinea pig thyroid TSH receptors and guinea pig fat-cell TSH receptors were all found to contain two subunits linked by one or more disulphide bridges, of mol. wt. x 10–3: por-cine, 45 & 27; human, 50 & 32; guinea pig thyroid, 60 & 31, and fat, 57 & 32. Further information was gained by examining the effects on the cross-linking of chemical reagents and trypsin.
KeywordsReceptor Subunit Disulphide Bridge Porcine Thyroid Disuccinimidyl Suberate Decyl Sulphate
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