Physical-chemical properties of the estrogen receptor released by deoxyribonuclease I.
The physical-chemical properties of the nuclear estrogen receptor released by DNase I were characterized. Nuclei were isolated from MCF-7 cells previously exposed to 10-nM-[3H]estradiol. The parameters determined were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding ability to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. The properties of the receptor released by DNase I obtained from Worthington were compared to the properties of the receptor released by DNase I obtained from Sigma. Digestion with DNase I (Worthington) excised a receptor form which could be solubilized from nuclei by EDTA. This form sedimented at 5.2S with a Rs = 7.08 nm and a calculated Mr = 152.000. About 40% of this receptor form bound to a DNA-cellulose column. 0.4 M KCl dissociated this receptor form into a smaller form sedimenting at 4.2S with Rs = 4.64 nm and a calculated Mr = 80.000. The properties of the receptor solubilized by micrococcal nuclease followed by DNase I (Worthington) digestion were identical to the properties of the DNase I (Worthington) released receptor. Digestion with DNase I (Sigma) released a 3.2S receptor form, which diffused through the nuclear membrane and a 4-5S form which could be extracted from nuclei by EDTA. The 3.2S receptor had a Rs = 2.41 nm, a calculated Mr = 32.000 and less than 5% of it bound to a DNA-cellulose column. Digestion with micrococcal nuclease followed by DNase I (Sigma) solubilized a receptor form with identical properties to the 3.2S receptor. These results suggest that DNase I (Worthington) released a receptor form still associated with some molecules, probably chromatin proteins, which complexed it to DNA, while DNase I (Sigma) released the estradiol binding fragment of the receptor (meroreceptor) as a result of a proteolytic activity present in this preparation.
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000032:analysis; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002498:Centrifugation; D002850:Chromatography, Gel; D004247:DNA / Q000031:analogs & derivatives / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004958:Estradiol / Q000378:metabolism; D008836:Micrococcal Nuclease / Q000378:metabolism; D008970:Molecular Weight; D011960:Receptors, Estrogen / Q000032:analysis*
C033190:DNA-cellulose; D011960:Receptors, Estrogen; D004958:Estradiol; D002482:Cellulose; D004247:DNA; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease