Characterization of the 4-hydroxytamoxifen (4-OHTAM) bound estrogen receptor of MCF-7 cells solubilized by micrococcal nuclease.
In order to get an insight into the molecular mechanism of antiestrogen action at the chromatin level, we characterized the physical-chemical properties of the chromatin fragments released by micrococcal nuclease digestion of nuclei isolated from MCF-7 cells previously exposed to [3H]4-OHTAM. The [3H]4-OHTAM bound solubilized fragments were characterized in a low ionic strength buffer and in a high ionic strength buffer without and with urea. The following parameters were determined: 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. Following mild nuclease digestion, the solubilized [3H]4-OHTAM bound receptor sedimented as an abundant 6-7 S form and a less abundant approximately 12 S species. Increasing the extensiveness of digestion resulted in one receptor form sedimenting at 5.2 S, Rs = 7.25 nm and Mr = 155,000. About 45% of the applied receptor bound to a DNA-cellulose column could be eluted by a high salt concentrated buffer. Dissociation of the micrococcal nuclease solubilized receptor in 0.4 M KCl resulted in a smaller receptor form with a 4.9 S, Rs = 5.87 nm and Mr = 119,000. Further dissociation in the presence of 3 M urea resulted in a receptor with a 3.5 S, Rs = 5.78 nm and Mr = 83,000. These results suggested that the antiestrogen bound estrogen receptor in chromatin, is associated with a tightly bound protein component and with an additional less tightly bound protein, complexes with DNA.
D001943:Breast Neoplasms / Q000032:analysis*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D005260:Female; D006801:Humans; D008836:Micrococcal Nuclease / Q000494:pharmacology; D008970:Molecular Weight; D011960:Receptors, Estrogen / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D013629:Tamoxifen / Q000031:analogs & derivatives* / Q000378:metabolism; D014316:Tritium
D011960:Receptors, Estrogen; D013629:Tamoxifen; D014316:Tritium; C016601:afimoxifene; D004247:DNA; D008836:Micrococcal Nuclease