Solubilized human thyrotrophin receptors behave as one class of high-affinity binding sites.

Evidence is presented justifying the conclusion that, in contrast to the dextran-coated charcoal technique, the widely used technique of separating bound and free TSH with polyethylene glycol is inadequate and yields inaccurate results. Optimum values for the concentration of Triton X-100, pH, salts, temperature and time of incubation were established for the TSH-TSH receptor interaction. According to Scatchard analysis, soluble TSH receptors behaved as one class of binding sites. The affinity constant for this class of binding sites (Ka 1.3 X 10(9) M-1) is identical to that for the high-affinity binding sites found in human thyroid membranes (Ka 1.2 X 10(9) M-1). No low-affinity binding sites could be detected after solubilization of membrane receptors. Chromatography experiments on Sepharose CL-6B indicated that, in excess TSH, each micelle containing TSH receptors (molecular weight 150 000) binds 4 [125I]TSH molecules. These data, together with the absence of low-affinity binding sites, led to the hypothesis that high-affinity TSH binding sites may be formed by the clustering of 4 low-affinity binding sites. Cross-reactivity experiments showed that both alpha- and beta-subunits are involved in the binding of TSH to its receptor; the TSH beta-subunit showed an increased cross-reactivity with soluble receptors.