Characterization of estrogen receptors and associated protein kinase activity by high-performance hydrophobic-interaction chromatography.
We have determined that high-performance hydrophobic-interaction chromatography (HPHIC) with weakly hydrophobic columns permit the rapid separation of the labile isoforms of estrogen receptor proteins. Previously we reported the use of the SynChrom propyl 500 column for HPHIC of steroid receptors. However, due to the strongly hydrophobic characteristics of the ligand, [125I]iodoestradiol-17 beta, and the receptor protein, organic solvent was required in the mobile phase for greater recovery of receptor proteins. Here, we report separation of steroid receptors from human breast tumors and rat uteri, using the Beckman CAA-HIC, a non-ionic polyether-bonded column, without the need for organic solvents and with virtually 100% recoveries. Receptors were extracted in 10 mM phosphate buffer (pH 7.4). Maximum resolution and separation were achieved when a descending salt gradient of ammonium sulfate in phosphate buffer (pH 7.4) was used (2-0 M in 30 min). Estrogen receptor (ER) was resolved into two isoforms with tR = 22 +/- 1 min (n = 16, designated as peak I) and 27.5 +/- 0.5 min (n = 14, designated as peak II) and a purification of five- to twenty-fold in a single pass. Free steroid was eluted at tR = 35 +/- 1 min (n = 4). Separation was dependent on adjusting the ionic strength of cytosol to 1.5 M ammonium sulfate. ER, purified by HPHIC, retained ligand binding capacity and exhibited protein kinase activity, which was dominant in the less hydrophobic peak I (tR = 22 min) when immunoprecipitated with the monoclonal antibody D547. This method of rapidly purifying ER with high retention of biological activity may now be applied to the study of the molecular interrelationships of steroid receptor isoforms.
Hyder
S M
SM
Department of Biochemistry, James Graham Brown Cancer Center, University of Louisville, KY 40292.
Sato
N
N
Wittliff
J L
JL
D000645:Ammonium Sulfate; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000032:analysis; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D011233:Precipitin Tests; D011494:Protein Kinases / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D011960:Receptors, Estrogen / Q000032:analysis*; D013997:Time Factors; D014599:Uterus / Q000032:analysis / Q000201:enzymology
D007457:Iodine Radioisotopes; D011960:Receptors, Estrogen; D011494:Protein Kinases; D000645:Ammonium Sulfate