Estrogenicity of Coumestrol in the Mouse: Fluorescence Detection of Interaction with Estrogen Receptors

Katherine Nelson, Edward J. Pavlik, John R. van Nagell, Michael B. Hanson, Elvis S. Donaldson, Robert C. Flanigan

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

The estrogenicity of coumestrol, a fluorescent phytoestrogen, has been examined in murine uteri. Coumestrol competed with 17β-[3H] estradiol for binding to cytoplasmic estrogen receptors, caused cytoplasmic estrogen receptors to associate with chromatin in the nucleus, and induced progesterone receptors. By use of size-exclusion high-performance liquid chromatography (SEHPLC), the interaction of coumestrol with estrogen receptors was examined directly by monitoring the fluorescence associated with macromolecules having properties characteristic of estrogen receptors. These analyses were made possible by the addition of dimethyl-formamide to the elution buffer, at a concentration (7.5%) which improved recoveries but did not interfere with estrogen receptor binding. It was possible to detect fluorescent coumestrol at approximately 0.5 nM. All determinations were performed with preparations in which estrogen receptor activity was 3-10 nM. Exposure of these preparations to coumestrol (50 nM) resulted in the elution of increased fluorescent activity in the regions where estrogen receptors eluted during SEHPLC. This fluorescent activity was reduced when di-ethylstilbestrol, 17β-estradiol, hexestrol, or tamoxifen was present as a competitor (2 μM) but was unaffected by testosterone or progesterone. Diethylstilbestrol reduced fluorescence below endogenous base lines and thereby displayed a fluorescence quench property which was not observed with other ligands. When hepatic and renal estrogen receptor preparations were used, the injected receptor activity was observed to be the major limiting factor in detecting the interaction of coumestrol with estrogen receptors. These observations are relevant to attempts to visualize estrogen receptors in tumor cells and demonstrate that accepted biochemical criteria for ligand-receptor interaction can be satisfied when fluorescent ligands are examined.

Original languageEnglish
Pages (from-to)2565-2572
Number of pages8
JournalBiochemistry
Volume23
Issue number12
DOIs
StatePublished - Jun 1984

ASJC Scopus subject areas

  • Biochemistry

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