Unspecific and sequence-specific deoxyribonucleic acid binding of the partially purified human progesterone receptor.
The progesterone receptor (PR) was partially purified from T47D human breast cancer cells by sequential chromatography on phosphocellulose, heparin-Sepharose, and DNA-cellulose. Heparin-Sepharose chromatography resulted in an efficient conversion of the receptor to a DNA-binding form (activation) since more than 85% of the 3H-R5020 labeled eluate from heparin-Sepharose was retained on DNA-cellulose and since the cytosolic 8S receptor was converted to a 4S moiety after chromatography on heparin-Sepharose. The 3H-R5020 labeled human PR eluted from DNA-cellulose as a single symmetrical peak at 0.2 M NaCl; after photoaffinity labeling and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this species was shown to consist of about equal amounts of two proteins of Mr approximately equal to 96,000 and 120,000 (the so called A- and B-subunits, respectively). This partially purified receptor preparation (SA 490 pmol/mg protein) did not contain any glucocorticoid receptor (GR) as shown by immunoblotting with a monoclonal antirat GR antibody that cross-reacts with the human GR. Therefore, this preparation was used to compare the specific DNA-binding properties of the human PR with those of the purified rat GR. The human PR bound specifically to the promoter region of mouse mammary tumor virus (MMTV) at a molar ratio between receptor and DNA similar to the molar ratio between GR and DNA needed for binding of rat GR to MMTV, indicating that the PR was purified in a biologically active form.(ABSTRACT TRUNCATED AT 250 WORDS)
Berkenstam
A
A
Department of Pathology, Karolinska Institutet, Huddinge University Hospital, Sweden.
Glaumann
H
H
Gustafsson
J A
JA
D000345:Affinity Labels; D000818:Animals; D001943:Breast Neoplasms / Q000032:analysis*; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D003600:Cytosol / Q000032:analysis; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D010777:Photochemistry; D011397:Promegestone / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D011980:Receptors, Progesterone / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D004279:DNA, Viral; D011965:Receptors, Glucocorticoid; D011980:Receptors, Progesterone; D004247:DNA; D011397:Promegestone; D003850:Deoxyribonuclease I