Characteristics of Thyrotropin Binding to Bovine Thyroid Plasma Membranes and the Influence of Human IgG

Abstract

The characteristics of binding of ¹²⁵I-labeled bovine thyrotropin to bovine thyroid plasma membranes were studied. At pH 7.5, 0 C, specific binding was maximal in 3 hr and was progressively inhibited by Na⁺, Mg⁺⁺, Ca⁺⁺ and (NH₄)₂ SO₄ in concentrations of 25 mM and greater. Affinity constants of 1.047 × 10⁸M⁻¹ and 0.57 × 10⁶M⁻¹ were obtained for two binding sites and Hill plots provided a mean slone of 0.86, suggesting negative cooperativity. Addition of 1-thyroxine or 1-triiodothyronine at 0.5 ng to 50 ug per ml had no significant effect but propylthiouracil (10⁻⁷ to 10⁻⁵M) and KC10₄ (10⁻⁷ to 10⁻⁴M) had biphasic effects, first enhancing and then inhibiting binding. Normal human serum IgG, particularly two fractions (3 and 4) obtained by chromatography on columns of cellulose phosphate, and an α-globulin - rich fraction from chromatography of serum proteins on diethylaminoethyl cellulose, inhibited binding at concentrations of 0.5 mg per ml or greater. Thus two possibilities are that normal human serum globulins either compete with thyrotropin for its receptor on thyroid plasma membranes or, in binding to the membranes, cause such perturbation as to affect adversely the conformation of the receptor for thyrotropin.

Thyroid-stimulating antibody (TSAb) is an IgG that circulates in Graves' disease and until recently investigation of this activity has been based on bioassay techniques (1). Since 1974 (2) a radio-receptor assay has increasingly been applied to study of this moiety; as described by several groups (2, 3, 4) TSAb interferes with the binding of thyrotropin to its receptor on the thyroid plasma membrane, and this action can be exploited to develop a radio-ligand assay for the IgG. Despite the apparent successful clinical application of this radio-ligand procedure to measure TSAb, there are some unexplained discrepancies in published reports relevant to the technique. For instance, Smith and Hall (5) showed that IgG from some patients with Graves' disease impaired ¹²⁵-thyrotropin binding to guinea pig thyroid membranes in a biphasic fashion i.e., a higher concentration had a lesser effect than did a lower. Amir et al (6), using bovine thyroid membranes, found no influence of TSAb; Yamashita and Field (7), studying thyrotropin-sensitive adenylate cyclase activity in bovine thyroid membranes described apparent non-competitive inhibition by TSAb, whereas Wolff and Jones, using a similar system, observed no effect (8). Most recently Mukhtar et al (9) reported that 14% of sera from patients with autoimmune thyroiditis contained IgG that inhibited ¹²⁵I-thyrotropin binding to human thyroid membranes.

In view of these data that were not apparently in uniform agreement, we elected to analyze carefully the influence of normal human IgG on the binding of thyrotropin to thyroid membranes, using a homologous bovine-bovine system.