Ketononestrol aziridine, an agonistic estrogen receptor affinity label: Study of its bioactivity and estrogen receptor covalent labeling

Jonathan F. Elliston, Jeffery A. Zablocki, Benita S Katzenellenbogen, John A. Katzenellenbogen

Research output: Contribution to journalArticle

Abstract

Ketononestrol aziridine [(6R*,7S*)l-(N-aziridinyl) 6,7-bis-(4-hydroxyphenyl)5-nonanone (KNA)], an aziridine derivative of hexestrol, is an estrogenic affinity label for the estrogen receptor (ER). It has an apparent relative binding affinity 8% that of estradiol and shows time-dependent irreversible binding to the ER in uterine cytosol preparations and intact human breast cancer cells (MCF-7). The agonistic activity of KNA is evident in MCF-7 cells in culture, where it increases the cell growth rate and elevates the level of progesterone receptor. KNA was prepared in high specific activity tritium-labeled form by iodination of a methanesulfonate precursor, followed by catalytic tritium-iodine exchange and aziridinylation; the material prepared has high radiochemical purity and a specific activity of 67 Ci/mmol. The covalent attachment of [3H]KNA to the ER can be followed directly by a solvent precipitation assay. In cytosol preparations of uterine ER, labeling with [3H]KNA proceeds in a time-, concentration-, and temperature-dependent manner; labeling is efficient and selective and, by competition studies, was shown to be estrogen specific. ER in intact MCF-7 cells can also be covalently labeled by treatment with [3H]KNA. Receptor covalently labeled with [3H]KNA sediments as a 4S species on high salt sucrose gradients, and its sedimentation position is shifted by treatment with monoclonal antireceptor antibodies. On sodium dodecyl sulfate-polyacrylamide gels, the principal labeled species migrates with a mol wt of 66,000. KNA should prove to be a useful probe for studies on receptor structure, function, and chromatin interactions, particularly when the behavior of a receptor-agonist complex is being investigated.

Original languageEnglish (US)
Pages (from-to)667-676
Number of pages10
JournalEndocrinology
Volume121
Issue number2
DOIs
StatePublished - Aug 1 1987
Externally publishedYes

Fingerprint

Affinity Labels
Estrogen Receptors
MCF-7 Cells
Tritium
Cytosol
Hexestrol
Halogenation
Progesterone Receptors
Sodium Dodecyl Sulfate
Iodine
Chromatin
Sucrose
Estradiol
Estrogens
Cell Culture Techniques
Salts
Monoclonal Antibodies
ketononestrol aziridine
Breast Neoplasms
Temperature

ASJC Scopus subject areas

  • Endocrinology

Cite this

Ketononestrol aziridine, an agonistic estrogen receptor affinity label : Study of its bioactivity and estrogen receptor covalent labeling. / Elliston, Jonathan F.; Zablocki, Jeffery A.; Katzenellenbogen, Benita S; Katzenellenbogen, John A.

In: Endocrinology, Vol. 121, No. 2, 01.08.1987, p. 667-676.

Research output: Contribution to journalArticle

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