Thyrotropin Receptors on Bovine Thyroid Membranes Two Types with Different Affinities and Specificities*

Abstract

We have studied the effects of pH, temperature, and ionic strength on the characteristics of bovine TSH (bTSH) binding to bovine thyroid membranes. Although maximal bTSH binding occurred at low pH, temperature, and ionic strength, the maximal sensitivity [defined as the amount of bTSH yielding 50% inhibition of tracer binding (B/B_o = 50%)] of the assay was observed at physiological pH and temperature in the presence of 50 mM NaCl. Under the latter conditions, the TSH binding assay was highly specific, with less than 0.01% cross-reactivity of crude hCG, pure hCG, hCG-α, hCG-β, bovine thyroglobulin, human γ-globulin, and cholera toxin. Different TSH preparations displayed binding activities proportional to their bioactivities in the range of 10^-11-10^-8 M, whereas TSH subunits had little intrinsic binding activity, compatible with the TSH contamination of the subunit preparations. When assay conditions were changed toward less physiological conditions by decreasing pH to 6.0, temperature to 4 C, and ionic strength to 10 mM by omitting NaCl from the buffer, there was an increased total TSH binding but a decreased TSH sensitivity and specificity. Thus, the bTSH sensitivity (B/B₀ = 50%) was lowered 1000-fold (from 2 × 10 ¹⁰ to 3 × 10 ⁷ M) and the cross-reactivity of certain proteins was greatly enhanced to a maximum of 100% for crude hCG, 10% for bovine thyroglobulin and cholera toxin, and 1% for human γ-globulin. At highly unphysiological conditions, Scatchard plots were linear, with an apparent affinity constant (Ka) of 1 × 10⁷ M ¹, whereas at more physiological conditions, the Scatchard plots were curvilinear, with apparent limiting Ka values of 1-7 × 10⁹ and 0.3-1 X 10⁷ M^-1 The ratio of the number of high affinity to low affinity sites was highest at close to physiological conditions. At physiological pH and temperature in the presence of 50 mM NaCl, the TSH membrane radioreceptor assay predominantly measures TSH binding to a class of sites with high affinity and specificity. This binding site, rather than the site predominantly measured under unphysiological incubation conditions, probably reflects the biologically relevant TSH receptor.