Purification of estradiol <i>receptor</i> by affinity chromatography. Representative experiments

Truong, H; Geynet, Claudine; Millet, Claude; Soulignac, O; Bucourt, R.; Vignau, M; Torelli, V.; Baulieu, Étienne-Émile

doi:10.1016/0014-5793(73)80306-0

Cited by 40 publications

(7 citation statements)

References 9 publications

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“…Attachment of the steroid to the supporting matrix by an extremely long molecular bridge has been reported to facilitate release of the bound receptor (20,21) but, unless removed by chromatography over heparin-Sepharose (18), enzymes present in target tissue extracts tend to cleave the ester or amide groups in the macromolecular spacer linking the steroid to the supporting medium. The affinity chromatography system described here uses an adsorbent in which estradiol is connected to the supporting matrix by a short molecular bridge that is not susceptible to cleavage by hydrolytic enzymes (Fig.…”

Section: Discussionmentioning

confidence: 99%

Monoclonal antibodies to human estrogen receptor.

Greene¹,

Nolan²,

Engler³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

446

110

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Extranuclear estrogen receptor protein (estrophilin) of MCF-7 human breast cancer celIs was purified by passage of the cytosol fraction of a cell homogenate through an affinity column of estradiol linked to Sepharose by a substituted di-n-propyl sulfide bridge in the 17a position. Elution The preparation of specific antibodies to estrogen receptors has provided a means of recognizing receptor proteins other than by their binding to labeled steroid. Antibodies to calf uterine estrophilin,-generated in either rabbits (1-3) or a goat (3, 4), crossreact with estrogen receptors, but not with androgen or progesterone receptors, from a wide variety of animal species. The hormone specificity and crossreactivity of the rabbit and goat antiestrophilins make them attractive as probes for the study of receptor structure and function, but their use in immunochemical procedures for receptor purification, assay, and cellular localization is limited by the heterogeneity of these antibody preparations. Monoclonal antiestrophilin antibodies secreted by hybridoma cells, obtained by fusion of mouse myeloma cells with splenic lymphocytes from a Lewis rat immunized with calf uterine estrophilin, have been described (4, 5) but, like the immunoglobulin in the serum of the parent rat, they reacted specifically with calf estrophilin and thus are unsuitable for the study of receptors from species other than calf.This Preparation of Estradiol Affinity Adsorbent. 17a-Allylestradiol 3-acetate, prepared by the reaction of estrone with allylmagnesium chloride (9) followed by acetylation of the phenolic group (10), was converted to the side-chain epoxide by treatment with m-chloroperoxybenzoic acid (11). Reaction of the epoxide with reduced thiopropyl-Sepharose (2-hyAbbreviations: E*R, [3H]estradiol-estrophilin complex; i-Ig, immunoglobulin secreted by hybridomas derived from immunized rat; n-Ig, immunoglobulin from serum of nonimmunized rat; HAT, hypoxanthine/amethopterin/thymidine; P3, P3-X63-Ag8; Sp2/0, Sp2/0-Agl4. 5115The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Section: Discussionmentioning

confidence: 99%

Monoclonal antibodies to human estrogen receptor.

Greene¹,

Nolan²,

Engler³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

446

110

View full text Add to dashboard Cite

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“…Cytosol was prepared from calf uterus stored in liquid nitrogen, as previously described [7] . The tissue was homogenized in TE buffer (Tris-HCl) 50 mM, EDTA 1.5 mM, pH 7.4) at 0-4"C, the temperature at which all the subsequent operations were performed.…”

Section: Biological Materialsmentioning

confidence: 99%

Filtration of calf uterine cytosol on ultrogel ACA 34 prevents aggregation of the estradiol receptor

Soulignac¹,

Secco-Millet²,

Rocher³

et al. 1977

FEBS Letters

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“…When non-radioactive ligand was present in the extract prior to the ,addition of ['HIoestradiol, the total number of receptor binding sites was determined using liquid-phase exchange technique [13]. All samples were incubated with 0.1 pM non-radioactive oestradiol until equilibrium was achieved.…”

Section: Receptor-ligand Complex Measurementsmentioning

confidence: 99%

Properties of Biospecific Adsorbents, Obtained by Immobilization of Oestradiol 7α‐Derivatives, for Purification of Calf‐Uterine Cytosol Oestradiol Receptor

Redeuilh

Richard-Foy

Secco

et al. 1980

European Journal of Biochemistry

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The properties of three types of adsorbents obtained by coupling oestradiol 7a-derivatives to agarose were compared. The adsorbents examined were : oestradiol 7x-decamethylene-agarose, oestradiol 7r.-decamethylene-poly(alanyl-lysine)-a~arose and oestradiol 7a-trimethylene-poly(alanyl1ysine)-agarose. The following results were obtained.(1) All these adsorbents are stable at 0 -C for at least a year when stored in water. In the presence of cytosol they are stable for several hours and are reusable after a simple wash. (2) A new method allowing the calculation of the maximal receptor binding capacity of an absorbent was developed. The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5 Y! pure protein (assuming one oestradiol binding site per molecule of M , 60000). When starting from a low salt preparation containing the native 8-S receptor, partially purified by heparin-Ultrogel chromatography, preliminary experiments using affinity chromatography gave a further purification of 250 -500-fold and led to a 50 -90 "( pure protein (assuming one oestradiol binding site per molecule of M , 70000).The use of affinity chromatography has been proposed as a necessary step for purification of hormone receptor. We have attempted to find an adsorbent convenient for the purification of oestradiol receptor from calf uterine cytosol. Two different forms of receptor may be characterized by their sedimentation coefficients: 8-S receptor [l] and 4-5-S receptor [2] in low and high salt concentration, respectively. This oestradiol receptor is difficult to purify because of its non-specific and irreversible aggregation with cytosol proteins. Mild trypsin treatment leads to a nonaggregatable form of the receptor [3] with a sedimentation coefficient of 4 S, whatever the ionic conditions [4]. For this reason the 4-S receptor was used for a systematic investigation of the parameters influencing purification by affinity chromatography. This 4-S Abbreviution. Poly(Ala-Lys), poly(oL-alanyl-L-lysine) copolymer. molecular form of the receptor is probably biologically inactive (unable to translocate to nucleus or to bind DNA), but its binding properties to the hormone remain unchanged.New biospecific adsorbents have been previously synthesized and studied in this laboratory [5]. The affinity of the receptor for the adsorbent was found to depend on the position at which the spacer is fixed onto the steroid, and therefore an oestradiol derivative carrying a long spacer chain i.e. decamethylene, ( C H Z )~~, attached at the 7a position was coupled to an insoluble matrix [5]. Such adsorbents offer many advantages in comparison with some previously described [6,7], in particular: (a) great stability due to the absence of ester bonds in the spacer chain, and (b) high degree of selectivity for the receptor since the 7a position of fixation of the spacer chain does not interfere with the high affinity of the receptor for the immobilized ligand.

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Purification of estradiol receptor by affinity chromatography. Representative experiments

Cited by 40 publications

References 9 publications

Monoclonal antibodies to human estrogen receptor.

Monoclonal antibodies to human estrogen receptor.

Filtration of calf uterine cytosol on ultrogel ACA 34 prevents aggregation of the estradiol receptor

Properties of Biospecific Adsorbents, Obtained by Immobilization of Oestradiol 7α‐Derivatives, for Purification of Calf‐Uterine Cytosol Oestradiol Receptor

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