Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand

Buckland, Paul Robert, Howells, Roger David, Rickards, Carole and Rees Smith, Bernard 1985. Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand. Biochemical Journal -London- 225 (3) , pp. 753-760.

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Abstract

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine MRC Centre for Neuropsychiatric Genetics and Genomics (CNGG)
Subjects:	R Medicine > R Medicine (General)
Uncontrolled Keywords:	Affinity Labels/pharmacology, Animals, Azides/pharmacology, Cattle, Cell Membrane/metabolism, Cross-Linking Reagents/pharmacology, Electrophoresis, Polyacrylamide Gel, Humans, Ligands, Macromolecular Substances, Models, Chemical, Receptors, Cell Surface/drug effects, Receptors, Thyrotropin, Succinimides/pharmacology, Thyroid Gland/metabolism, Trypsin/pharmacology
Publisher:	Portland Press
ISSN:	0264-6021
Last Modified:	12 Dec 2022 08:52
URI:	https://orca.cardiff.ac.uk/id/eprint/57854

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