Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite.
1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.
D000818:Animals; D001665:Binding Sites; D002645:Chickens; D002843:Chromatin / Q000378:metabolism*; D002845:Chromatography; D004958:Estradiol / Q000378:metabolism; D006882:Hydroxyapatites; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008297:Male; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D013696:Temperature
D002843:Chromatin; D006882:Hydroxyapatites; D011506:Proteins; D004958:Estradiol