Characterization of human progesterone receptor by high performance hydrophobic interaction chromatography.
High performance hydrophobic interaction chromatography has been used to separate progestin receptors (PRs) from human uterus and from the T47D human breast cancer cell line. Reproducible separations of high resolution were achieved using a TSK Phenyl-5PW column and a reverse salt gradient of 400 mM to 0 mM sodium sulfate in phosphate buffer, pH 7.4. Peaks of radioactivity exhibiting hydrophobic behaviour were isolated, as well as a smaller proportion of specific bound receptors located in the void volume fraction. No differences in retention times were observed between uterine and breast cell line samples. When the technique was used in conjunction with rapid vertical tube sucrose density gradient centrifugation, the 8S sedimenting PR from fresh, low-salt cytosol always eluted with a retention time of 24 min. The natural 4S receptor chromatographed as a single peak at 29 min while the 4S receptor species from high-salt cytosol appeared as two distinct peaks of radioactivity with retention times of 29 and 33 min. While specific binding was shown to occur in the void volume of the column, the origin of these receptors were indeterminate. These results would suggest that under these conditions the 8S receptor occurs as a single hydrophobic class of protein, whereas the data provides evidence that transformed 4S receptor may be proportioned into two unequal entities as a function of exposure to salt.
Levy
A
A
Department of Chemical Pathology, School of Pathology, South African Institute for Medical Research, Johannesburg.
Boyle
D M
DM
van der Walt
L A
LA
D001943:Breast Neoplasms; D002460:Cell Line; D002845:Chromatography / Q000379:methods; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D005260:Female; D006801:Humans; D011282:Pregnenediones / Q000378:metabolism; D045167:Progesterone Congeners / Q000378:metabolism; D011869:Radioligand Assay; D011980:Receptors, Progesterone / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D014599:Uterus / Q000737:chemistry* / Q000378:metabolism
D011282:Pregnenediones; D045167:Progesterone Congeners; D011980:Receptors, Progesterone; C020175:16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione