Characterization of triton-solubilized TSH receptors from human thyroid plasma membranes

Abstract

The TSH receptor from human thyroid plasma membranes was solubilized in 10 mM Tris/HCl, 50 mM NaCl, pH 7.4 containing 0.5% triton X-100. Binding of [125I]TSH to the soluble receptor showed rapid and reversible kinetics and reached a maximum within 30 min at 37.degree. C, by 1 h at 25.degree. C and by 24 h at 4.degree. C. Optimal pH was 7.4. The soluble receptor retained specificity with cross-reactivity only to crude (0.03%). Scatchard plots were curvilinear, indicating the presence of at least 2 binding sites. The high affinity site showed an affinity constant of 1.1 .times. 109 M-1 with binding capacity of 1.3 .times. 10-10 M/mg protein. TSH-binding inhibitor Ig from patients with Graves'' disease inhibited [125I]TSH binding to the soluble receptor in a dose-dependent manner. NaCl inhibited the TSH binding and this was ascribed to the decrease in the receptor capacity. Trypsin, neuraminidase and phospholipase C treatment of the solubilized receptor had no effect on TSH binding. The apparent MW of tle receptor, determined by gel filtration on Sepharose 6B, was .apprx. 300,000.