Intracellular binding of [ 3H]cortisol and its effect on RNA polymerase activity in hypothalamus of the pig

Abstract

Intracellular binding of [ 3H]cortisol in the hypothalamus of pigs and the effects of this binding on RNA polymerase activity were studied. Molecular-sieve chromatography of both the cytosol and the nuclear extract on Sephadex G-100 resulted in a peak of radioactivity eluted with the protein fraction. The very low elution volume of the nuclear steroid-protein complex indicated that the macromolecular receptor of the nuclear extract is larger than that of the cytoplasm. Results from the utilization of two different concentrations of cortisol rerevealed that the binding sites of the cytoplasm and nuclear extract became saturated at a cortisol concentration between 10 −7 M and 10 −4 M. Most of the radioactivity bound in the cytosol and nuclear extract possesed the solubility of cortisol. The nuclear extract possesed a greater percentage of bound radioactivity as cortisol than the cytoplasm. Chromatography of proteins of known molecular weights allowed approximation of molecular weights of the steroid-protein complexes. Most of the [ 3H]cortisol associated with protein in the hypothalamus cytosol was bound to protein with an elution volume which corresponded to a molecular weight of approximately 100,000. The peak of radioactivity in the nuclear extract was associated with protein which was eluted in a volume corresponding to a molecular weight slightly less than 150,000. The influence of the cytosol steroid-protein complexes on RNA polymerase activity in the nucleus was studied. RNA polymerase was significantly increased ( P < 0.05) when nuclei were incubated in the presence of cytosol steroid-protein complexes and compared to nuclei incubated with buffer alone, free cortisol or cortisol in a in albumin.