<i>In vitro</i> Nuclear ³H-Hydrocortisone Binding in the Hypothalamus of the Pig

Abstract

Hypothalamic cell nuclear uptake and binding of ³H-hydrocortisone(³H-HC)-receptor complexes were studied in a cell-free system. Greater net concentrations of radioactivity were found in the 0.4 MKC1 non-extractable fraction (sediment) in the first 20 min of incubation and in the 0.4 MKC1 extractable fraction (supernatant) from 30 to 60 min. Incubation in low ionic strength medium favored localization of radioactivity in the supernatant, whereas high ionic strength promoted greater localization in the sediment. Specific binding in the supernatant was non-saturable, but reached a plateau in the sediment at about 5 × 10^–8 M ³H-HC. The observed value of K_d for binding in the nuclear sediment was 4.88 × 10⁹ M^–1 and the maximum number of binding sites was 8.8 femtomoles/mg DNA. RNA polymerase activity increased by 59% in the presence of hormone-receptors over that in the presence of free hormone in buffer.