Purification of a human progesterone receptor

Abstract

The cytoplasmic progesterone receptor form human uterus has been purified to apparent homogeneity by a combination of ammonium sulfate fractionation and affinity chromatography. Affinity resins prepared by conventional means were compared to those prepared by a modified method. The latter give more reproducible results. A consistent finding was that low capacity resins gave the highest fold purification of the receptor. The pure receptor sedimented at 3.6 S on sucrose density gradient centrifugation, was eluted as a single band by 0.2 M KCl from DEAE-cellulose, and migrated as a single band of molecular weight 42 000 on NaDodSO4-polyacrylamide gel electrophoresis. Molecular weight determinations, obtained from Strokes' radii and sucrose gradient centrifugation, the receptors' behavior on ion exchange resins, and hormone binding specificity were all similar to those of the receptor found in crude cytosol. When the crude cytosol receptor was photoaffinity labeled by using 3H-labeled 17,21-dimethyl-19-norpregna-4,9-diene-3,20-dione followed by NaDodSO4-polyacrylamide gel electrophoresis, only protein of Mr 42 000 was labeled. This is consistent with our previous findings that alkylation of the pure receptor using 11-deoxycorticosterone bromo[3H]acetate showed labeling of a single protein of Mr 42 000. These properties confirm that the identity and integrity of the receptor have been maintained throughout its purification.

Effect of Carbamates on mRNA Encoding Lipid Enzymes in Hamster Flank Organs

“…However, 8 and 9 decreased the size of the pigmented spots of gonadectomized females treated with T. Since both steroids inhibited the activity of 5α‐reductase, DHT was not formed and as result of this, the diameter size was not increased. Similar results had been previously reported using different inhibitors of 5α‐reductase and was also previously found by our group using 7 and 10 carbamate derivatives of progesterone 5 .…”

Molecular interactions of natural and synthetic steroids in female hamsters’ flank organs

“…protease inhibitors (2 mM PMSF, 10 mg/mL antipain, 5 mM leupeptin [16]; 40 mM tris-HCl, 3 mM EDTA and 20 mM sodium molybdate, dithiothreitol 0.5 mM, 10% glycerol at pH 7.4) [20]. The homogenate was centrifuged at 140,000 Â g for 1 h at 0 8C in a SW 60 Ti rotor (Beckman Instruments, Palo Alto, CA).…”

“…After incubation, 0.21 mL saturated ammonium sulfate in TEMD buffer (30%) was added [20,24]. The mixture was further incubated for 1 h with occasional shaking for the precipitation of the [ 3 H] R5020-complex.…”

Biological Evaluation of Androstene Derivatives

“…The difference between the study of Palmer et al 10 and our study is that we could precipitate the PR with ammonium sulfate 11. This procedure permitted us to obtain a purified fraction of the PR 12 and to improve the competition analysis.…”