Oestradiol binding to nuclei of anterior pituitary cells of the ram.
The methodology to fully characterise nuclear receptor for oestradiol-17 beta (E2) in the ram pituitary has been investigated. Purified nuclei, clean under the electron microscope, were obtained from 2.4 M sucrose ultracentrifugation and were extracted for 2 h at 0 degrees C with 0.6 M NaCl. After centrifugation, the supernatant was incubated with [3H]E2 with or without a 100-fold excess of unlabelled E2. The main results were: the specific binding was maximum at 20 degrees C in 2-3 h and remained constant up to 19 h without significant metabolism; an incubation temperature of 25 degrees C reduces the binding, while at 0 degrees C maximum binding was attained at a much slower rate; the binding was linearly related to the dose of nuclear proteins; the binding was not affected by DNase and RNase but was suppressed by trypsin, pronase or a temperature of 56 degrees C; binding was specific for oestrogens; preincubation of cytosol with [3H]E2 and then coincubation with nuclei showed an uptake of the [3H]E2 receptor complex by nuclei; such a transfer was inhibited if cytosol was previously heated; after a prelabelled cytosol-nuclei coincubation, a specific binding peak was found in the nuclear extract submitted to sucrose gradient sedimentation (4.1S); in vivo injection of 100 micrograms E2 resulted in a sharp increase in nuclear receptor numbers 30 and 60 min later, with a concomitant drop in cytosolic receptor numbers. These results indicate that E2 can bind to pituitary nuclei in the ram.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D003600:Cytosol / Q000378:metabolism; D003851:Deoxyribonucleases / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D006358:Hot Temperature; D008297:Male; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011402:Pronase / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011959:Receptors, Estradiol / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism*; D012260:Ribonucleases / Q000494:pharmacology; D012756:Sheep; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D011959:Receptors, Estradiol; D011960:Receptors, Estrogen; D012965:Sodium Chloride; D004958:Estradiol; D003851:Deoxyribonucleases; D012260:Ribonucleases; D014357:Trypsin; D011402:Pronase