Reversible Binding of Labelled and Non-Labelled Thyrotrophin by Intact Thyroid Tissue in Vitro

Abstract

SUMMARY 125I-Labelled thyrotrophin (TSH) showed saturable binding to hyperplastic guinea-pig thyroid slices in vitro, but not to kidney, adrenal, liver, testis or salivary gland slices. Two populations of binding sites were demonstrated: high order sites showed an affinity constant of 3·8 × 1081/mol and a capacity of 8 × 103 molecules of TSH/cell; low order sites, affinity constant of 2·9 × 1071/mol and capacity of 8 × 104 molecules/cell. The highorder sites saturate at about 1 mu./ml, approximately the level producing maximal in-vivo and in-vitro responses. Binding of 125I-labelled TSH was reversible, the released hormone appearing substantially unchanged as assessed by chromatographic behaviour and antiserum binding. Release of unlabelled TSH from pre-incubated thyroid slices was demonstrated by bioassay. Bovine thyroid slices were found to have a lower capacity for binding than hyperplastic guinea-pig slices.

The Thyrotrophin Receptor in Guinea-Pig Thyroid Homogenate: General Properties

“…In a previous communication we reported the binding of 125I-labelled and native TSH to intact slices of hyperplastic thyroid tissue from PTU-treated guinea-pigs (Manley et al 1972). The present studies of the TSH-binding properties of thyroid homogenate provide further evidence of the biological significance of the binding process and form the basis of a radioligand receptor assay for TSH.…”

“…Measured affinity was not changed by increasing the time of incubation from 30 min to 2 h or 4 h at 37°C. There was no evidence, under these conditions, of the low affinity binding observed with intact thyroid tissue slices (Manley et al 1972 The dose-response curve for stimulation of TSH-responsive adenylate cyclase corresponds closely to that for saturation of binding (Fig. 6), indicating that the 125I-labelled TSH binding phenomenon represents the physiologically significant hormone-receptor interaction.…”

“…The primary mechanism appears to be simple dissociation. We have pre¬ viously reported (Manley et al 1972) that native as well as 125I-labelled TSH dis¬ sociates from thyroid slices with unchanged chemical and biological properties. Homogenization of tissue which has been incubated for 16 h with 125I-labelled TSH yields approx.…”

“…Thyrotrophin and 12SI-labelled thyrotrophin Bovine TSH was purified to 25 i.u./mg for iodination as described previously (Manley et al 1972). Bovine TSH (3 and 4 i.u./mg) was prepared by ethanol and ammonium sulphate fractionation of acetone-dried pituitary powder.…”

Iodothyronine Release from the Perfused Canine Thyroid following Cessation of Stimulation

“…The prolonged effect of TSH is compatible with a slow release of receptor-bound TSH (9,10). To circumvent the continued stimulation of colloid uptake by receptor-bound TSH, 5 mM cAMP was used as a stimulator.…”