Robert A. Webster

Role of nuclear proteins as high affinity sites (“acceptors”) for progesterone in the avian oviduct

Abstract Administering multiple doses of progesterone to immature chicks results in the detection of several classes of oviduct nuclear binding sites. Comparisons of serum progesterone levels and responses to RNA polymerase I and II activities with the nuclear binding, indicate that the highest affinity classes of sites are the biologically important ones. In vitro binding studies using isolated progesterone receptor complex also reveal the presence of these multiple classes of sites. The highest affinity class of nuclear sites in the oviduct, representing 6000 to 10,000 sites per cell with a K D ~ 10 −9 M, appear to be tissue specific. These sites are present but completely masked in the chromatin of non-target organs (spleen and erythrocyte), while 70% are masked in the target tissue (oviduct). Further fractionation studies involving DNA affinity chromatography using chromatin-cellulose resins and molecular sieve-chromatography using Agarose-GuHCl resins reveal that the “acceptor” activity is associated with two molecular weight-proteins between 12,000 and 17,000. These proteins are bound with very high affinity to DNA. The “acceptor-proteins” must be reannealed to DNA to achieve binding activity. These results support that acidic proteins determine the high affinity nuclear binding sites for steroids.

Steroid receptor binding to nuclei: effect of assay conditions on the integrity of chromatin.

Abstract 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 [ 3 H]-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.)

Isolation and characterization of the nuclear acceptor that binds the progesterone-receptor complex in hen oviduct.

Progesterone binds to the nuclear chromatin of hen oviduct cells within minutes after its administration. This is followed by alterations in the RNA polymerase activities (15–30 min after treatment) and the appearance of specific messenger RNA (2 hr after treatment). It appears that the first nuclear event resulting in changes in gene expression represents the chromatin binding. Further characterization of this nuclear interaction, involving the administration of multiple doses of progesterone to estrogen‐treated immature chicks, revealed the presence of several classes of nuclear binding sites in the oviduct. Comparisons of serum progesterone levels and responses of RNA polymerase I and II activities with the nuclear binding indicated that the highest‐affinity classes of sites (∼ 10,000 sites per cell) are the biologically important ones. Binding studies in vitro, using isolated progesterone‐receptor complex, also reveal the presence of these multiple classes of sites. The highest‐affinity class of nucle...