Various incubation conditions previously used in cell-free assays of steroid receptor binding to nuclei were evaluated on the basis of their effect on the integrity of the nuclear chromatin. Incubation of nuclei for 60 min in whole cytosol containing no added salt at 25°C resulted in significant DNA degradation and some proteolysis. This deterioration in the integrity of the chromatin increased its capacity to bind the [3H]-progesterone-receptor complex, possibly via exposure of previously "masked" acceptor sites. Decreasing the temperature and/or increasing the salt concentration reduced or eliminated these changes in the chromatin. Incubation of nuclei in an ammonium sulfate-fraction of the cytosol, which contained the partially purified progesterone receptor, resulted in no detectable deterioration of the chromatin integrity irrespective of the temperature (≤25°C) or the added salt concentration (0-0.15 M KCl). These results suggest that some of the conflicting reports on the nature of the nuclear acceptor sites for steroid receptors could be due to the use of inappropriate assay conditions, which permit degradation of the nucleo-protein and result in altered levels of steroid receptor binding. (Steroid/ Progesterone/Nuclei/Chromatin.).
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