TY - JOUR
T1 - Estrogenicity of Coumestrol in the Mouse
T2 - Fluorescence Detection of Interaction with Estrogen Receptors
AU - Nelson, Katherine
AU - Pavlik, Edward J.
AU - van Nagell, John R.
AU - Hanson, Michael B.
AU - Donaldson, Elvis S.
AU - Flanigan, Robert C.
PY - 1984/6
Y1 - 1984/6
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0021259769&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0021259769&partnerID=8YFLogxK
U2 - 10.1021/bi00307a005
DO - 10.1021/bi00307a005
M3 - Article
C2 - 6466599
AN - SCOPUS:0021259769
SN - 0006-2960
VL - 23
SP - 2565
EP - 2572
JO - Biochemistry
JF - Biochemistry
IS - 12
ER -