High-performance hydrophobic interaction chromatography as a means of identifying estrogen receptors expressing different binding domains.
Methodology for high-performance hydrophobic interaction chromatography (HPHIC) of estrogen receptors (ER) was developed, utilizing a polyether-bonded stationary phase, which was non-ionic in nature. Using a descending salt gradient (2 M to 0 M ammonium sulphate in 40 min), ERs from human breast cancer separated into two isoforms, which retained ligand-binding domains. The same isoforms were observed with ER preparations from rat uterus. When sodium molybdate, a stabilizer of receptor structure, was incorporated into the mobile phase, it altered the ER characteristics, producing an earlier elution of one component, while the other one remained unchanged. Treatment of breast cancer cytosol with RNase A did not alter ER elution from either the hydrophobic or size-exclusion (TSK 3000 SW) columns. Modification of cysteine residues with N-ethylmaleimide led to a broad elution pattern of receptor from the hydrophobic column, implying the existence of multiple conformations of ER. Limited trypsin treatment of ER, which removes the DNA binding domain, led to the elution of only one receptor peak from the hydrophobic column. The receptor eluted at 24 min both in the presence and in the absence of sodium molybdate. Thus, at least one mechanism of the sodium molybdate effect must involve its direct interaction with ER to influence the sequence between the DNA-binding domain and the N-terminus. This also indicates that the most hydrophobic species of ER (sodium molybdate sensitive) may arise due to the interaction of the DNA-binding site with the stationary phase. Other possibilities, such as differential post-translational modifications of the receptor protein could also account for the two isoforms of ER, observed in HPHIC analysis.
Hyder
S M
SM
Department of Biochemistry, University of Louisville, KY 40292.
Wittliff
J L
JL
D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide; D005260:Female; D006801:Humans; D008982:Molybdenum; D051381:Rats; D011919:Rats, Inbred Strains; D011960:Receptors, Estrogen / Q000302:isolation & purification* / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism
D011960:Receptors, Estrogen; D008982:Molybdenum; C024687:sodium molybdate(VI); D012260:Ribonucleases; D005033:Ethylmaleimide