Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

Leslie Myatt, James L. Wittliff

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at {difference between}90 mM and {difference between}155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4°C or addition of 10 mM GTP increased the proportion of the {difference between}90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 μM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.

Original languageEnglish (US)
Pages (from-to)1041-1048
Number of pages8
JournalJournal of Steroid Biochemistry
Volume24
Issue number5
DOIs
StatePublished - 1986
Externally publishedYes

Fingerprint

Estrogen Receptor Modulators
Ion Exchange Chromatography
Chromatography
Estrogen Receptors
Ion exchange
Estrogens
Ligands
Estradiol
Phosphates
Tamoxifen
Guanosine Triphosphate
Cytosol
Uterus
Rats
Buffers
Protein Isoforms
Salts
Chemical activation
Association reactions

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

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title = "Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography",
abstract = "The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at {difference between}90 mM and {difference between}155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4°C or addition of 10 mM GTP increased the proportion of the {difference between}90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 μM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.",
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T1 - Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

AU - Myatt, Leslie

AU - Wittliff, James L.

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N2 - The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at {difference between}90 mM and {difference between}155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4°C or addition of 10 mM GTP increased the proportion of the {difference between}90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 μM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.

AB - The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at {difference between}90 mM and {difference between}155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4°C or addition of 10 mM GTP increased the proportion of the {difference between}90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 μM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.

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