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The relationship between the binding of thyrotrophin (TSH) to receptors and the production of cyclic AMP has been investigated using crude human thyroid membranes. Receptor binding was studied by use of 125I-labelled bovine TSH, which had been purified by adsorption to and elution from human thyroid membranes. This 125I-labelled material showed the same biological activity as the unlabelled hormone. Binding studies at equilibrium indicated that the association constant of the TSH-membrane interaction decreased as the amount of TSH bound increased. Kinetic data did not provide definite evidence that this was due to an effect of increasing receptor occupancy on the dissociation rate constant. Detectable stimulation by TSH of cyclic AMP production by the membranes was observed with as little as 30 micromicron. (1 ng) hormone. Increasing amounts of TSH over the range 0-250 micromicron caused increases in the production of cyclic AMP, proportional to the amount of hormone bound. With larger amounts of TSH, increasingly greater amounts of bound hormone were required to give corresponding increases in cyclic AMP formation, and addition of TSH in amounts greater than 16 millimicron resulted in progressive inhibition of cyclic AMP formation. Kinetic studies indicated that receptor binding was not rate-limiting in the stimulation of cyclic AMP production by TSH.

Thyroid-stimulating antibodies (TSAb) were found to inhibit the binding of labelled thyrotrophin (TSH) to thyroid membranes in a dose-dependent manner and this effect was localized in the Fab part of the TSAb molecule. Analysis of the binding data suggested that TSAb and TSH bound to the same receptor site. Production of cyclic AMP by the thyroid membranes was stimulated by TSAb and TSAb-Fab with a similar time course to that observed with TSH. Kinetic studies indicated that the binding of TSAb to the thyroid membranes was not rate-limiting in the process of stimulation of cyclic AMP production.

The cytochemical bioassay is able to detect minute amounts of thyroteopin and thyroid stimulating immunoglobulins. Estimations of serum TSH by the cytochemical bioassay are in good agreement with values obtain by radioimmunoassay. In untreated hyperthyroid patients with Graves' disease the TSH levels are significantly lower than in control subjects. Thyroid stimulating immunoglobulins showed a delayed time corse of action in the bioassy and this provided a voncenient method of distinguishing between the two thyroid stimulators.

Thyrotrophin (TSH) receptors have been extracted from human and porcine thyroid membranes by treatment with Triton X-100. 125I-Labelled bovine TSH was used to monitor receptor activity. Analysis by gel filtration and electrophoresis on acrylamide gels containing sodium dodecyl sulphate suggested that Triton extracts of human thyroid membranes contained TSH receptors with a molecular weight in the region of 50 000 closely associated with Triton micelles of approximate molecular weight 300 000. Isoelectric focusing studies indicated that the Triton-solubilized TSH binding activity had an isoelectric point of pH 4--4.5. The soluble TSH receptors were heat-labile, showed optimum TSH binding at pH 7.4 and reduced hormone binding at high ionic strength. The TSH binding characteristics of membrane-bound and solubilized human TSH receptors were similar and both preparations gave curved Scatchard plots. Solublized porcine TSH receptors appeared to have a similar molecular weight to the human receptors and were also closely associated with Triton micelles of approximate molecular weight 300 000. Scatchard analysis of TSH binding to membrane-bound or solubilized porcine TSH receptors gave approximately linear plots with association constants of 2.8 +/- 0.95 (S.E.M.) X 10(9) and 1.7 +/- 0.27 x 10(9) l/mol respectively. Comparison of the binding capacities of the solubilized and membrane-bound porcine receptors indicated that the 0.5% Triton extracts contained 40% of the original TSH binding activity and that this was present at a concentration of 25 ng/ml.

We describe here two patients with hypothyroidism due to pituitary-hypothalamic disease in whom basal thyrotrophin (TSH) levels measured by radioimmunoassay (RIA) were elevated yet when measured by a cytochemical bioassay (CBA) were found to be normal. This finding and the absence of the normal rise of thyroid hormones in response to thyrotrophin-releasing hormone (TRH) mediated release of TSH confirms for the first time the secretion of TSH with impaired biological activity. Primary thyroid disease as a cause for the elevated immunoreactive TSH was excluded by the absence of circulating thyroid antibodies and by a normal thyroidal radioiodine uptake response to exogenous TSH.

The distribution of TSH receptor antibody activity in the 7S and 19S fractions of Graves' sera has been re-evaluated. Serum fractions were obtained by gel filtration from 12 Graves' sera and assayed for TSH receptor binding activity in a radioreceptor assay. Thyroid stimulating activity was determined in a cultured porcine thyroid cell bioassay. In apparent contrast to the findings of Baker et al. (1983) TSH receptor binding activity was confined to the 7S gel filtration fraction, containing IgG, and was not detected in the 19S fraction, containing IgM. Similarly thyroid stimulating activity was detected only in the 7S fraction. 7S fractions from seven Graves' sera were fractionated by isoelectric focusing and the fractions analysed for TSH receptor binding activity and TSH agonist and antagonist activities. Five of the IgGs showed TSH agonist activity and in all five, the peak thyroid stimulating activity (measured by stimulation of cyclic AMP release from isolated porcine thyroid cells) was in fractions with a pI of between 8.0 and 9.5. In four of these five IgGs, TSH receptor binding activity showed similar isoelectric distribution to the thyroid stimulating activities. High levels of TSH receptor binding activity without associated TSH agonist or antagonist activity were however observed in some isoelectric fractions of the fifth stimulating Graves' IgG studied. All the isoelectric fractions from the fifth IgG with thyroid stimulating activities contained TSH receptor binding activity. Two of the Graves' IgGs showed TSH antagonist activity and both the TSH receptor binding and TSH antagonist activities of these IgGs showed similar isoelectric distribution with the peak activities at a pI of around 9.0. Consequently, it was not possible to separate TSH agonist or TSH antagonist activities from TSH receptor binding activity in seven Graves' sera by isoelectric focusing although in one IgG several isoelectric fractions contained isolated receptor binding activity. These findings are in keeping with the hypothesis that the biological activities of Graves' IgGs are intimately related to their ability to bind to the TSH receptor.