Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms
10.1002/jmr.300010206
Crossref journal-article
Wiley
Journal of Molecular Recognition (311)
Abstract

AbstractWe have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+ and Ni2+ ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3 M urea‐induced transformation to the DNA‐binding form, and (2) limited trypsin digestion to separate the steroid‐ and DNA‐binding domains. Imidazole (2–200 mM) affinity elution and pH‐dependent (pH 7–3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+ and Ni2+ ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+ > Cu2+ » Zn2+) at neutral pH. Generally, similar results were obtained with IDA–Cu2+ and IDA–Ni2+ in the absence of urea. Receptors were tightly bound and not eluted before 100 mM imidazole or pH 3.6. Different results were obtained using IDA–Zn2+; at least four receptor isoforms were resolved on IDA–Zn2+. Receptor–metal interaction heterogeneity and affinity for IDA–Zn2+ and IDA–Cu2+, but not IDA–Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA–Zn2+ chromatography were not separable using high‐performance size‐exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA‐affinity chromatography. The affinity of trypsin‐generated (mero) receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High‐affinity metal‐binding sites were mapped to the DNA‐binding domain, but at least one of the metal‐binding sites is located on the steroidbinding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor‐metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal‐binding proteins suggests new and potentially powerful receptor immobilization and purification regimes previously unexplored by those in this field.

Bibliography

Hutchens, T. W., & Li, C. M. (1988). Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms. Journal of Molecular Recognition, 1(2), 80–92. Portico.

Authors 2
  1. T. William Hutchens (first)
  2. Chee Ming Li (additional)
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Dates
Type When
Created 20 years, 3 months ago (May 29, 2005, 6:22 a.m.)
Deposited 1 year, 10 months ago (Oct. 20, 2023, 7:20 p.m.)
Indexed 46 minutes ago (Sept. 3, 2025, 5:17 a.m.)
Issued 37 years, 5 months ago (April 1, 1988)
Published 37 years, 5 months ago (April 1, 1988)
Published Online 21 years, 7 months ago (Feb. 3, 2004)
Published Print 37 years, 5 months ago (April 1, 1988)
Funders 0

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@article{Hutchens_1988, title={Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms}, volume={1}, ISSN={1099-1352}, url={http://dx.doi.org/10.1002/jmr.300010206}, DOI={10.1002/jmr.300010206}, number={2}, journal={Journal of Molecular Recognition}, publisher={Wiley}, author={Hutchens, T. William and Li, Chee Ming}, year={1988}, month=apr, pages={80–92} }