Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand.
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.
Buckland
P R
PR
Howells
R D
RD
Rickards
C R
CR
Rees Smith
B
B
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001386:Azides / Q000494:pharmacology*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D046911:Macromolecular Substances; D008956:Models, Chemical; D011956:Receptors, Cell Surface / Q000187:drug effects*; D011989:Receptors, Thyrotropin; D013388:Succinimides / Q000494:pharmacology; D013961:Thyroid Gland / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D008024:Ligands; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D011989:Receptors, Thyrotropin; D013388:Succinimides; C029534:hydroxysuccinimidyl-4-azidobenzoate; D014357:Trypsin; C019358:disuccinimidyl suberate