Thyrotropin-Binding Properties of Isolated Thyroid Cells and Their Purified Plasma Membranes.

Plasma membranes have been prepared from porcine thyroid cells stimulated by dibutyryl adenosine 3′:5′-monophosphate in culture. These cells show the histiotypic organization in follicles and the metabolic properties of gland follicular cells. The plasma-membrane fractions have a density in sucrose of 1.18 g/ml (about 80%) and 1.16 g/ml (about 20%). The yield is 2 mg per 6 × 10⁸ cells. Electron microscopy showed vesicular structures and sheets of membranes often joined by membrane junctions. Both showed the triple layer characteristic of the unit membrane ultrastructure. Less than 3% of contamination by microsomes was derived from the assay of NADPH-cytochrome c reductase and NADH oxidase. No contamination by mitochondria was indicated from succinate dehydrogenase activity measurements. NaF- and thyrotropin-stimulated adenylate cyclase, 5′-nucleotidase and alkaline phosphatase are concentrated in the plasma-membrane preparations. Specific binding of ¹²⁵I-labeled thyrotropin of high specific radioactivity (2.5 Ci/μmol) and preserved biological activity was compared to that in intact cells and to the plasma membranes from which they derived. The rate constants of the thyrotropin association with cell or membrane (0.56 and 0.11 nM^-1 min^-1, respectively) and dissociation (0.36 and 0.25 min^-1, respectively) were measured independently at 35 °C (cells) and 27 °C (membranes). The association constants derived from these rate constants (1.5 and 0.4 nM^-1 for cells and membranes) are very similar to that (1.9 and 0.6 nM^-1) determined independently from equilibrium data. Therefore plasma membranes purified from cultured thyroid cells represent an excellent material to study the molecular events linked to receptor activation. The relation between thyrotropin concentration and adenylate cyclase activation of membranes was studied in time conditions of equilibrium binding of thyrotropin. The semilogarithmic plot of thyrotropin concentration correlates sigmoidally with the percentage of adenylate cyclase activation. Half-maximum stimulation is obtained for a thyrotropin concentration of 1.8 nM which correlates remarkably with the dissociation constant of 1.78 nM determined for the binding of the hormone to membranes. [ABSTRACT FROM AUTHOR]