Parameters influencing the purification of calf uterus estrogen ‘receptor’ by affinity chromatography

Truong, H; Baulieu, Étienne-Émile

doi:10.1016/0014-5793(74)80397-2

Cited by 12 publications

(3 citation statements)

References 6 publications

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“…A better result could theoretically be obtained with the help of affinity chromatography (Failla et al, 1975;Kuhn et al, 1975;Sica et al, 1973;Truong et al, 1974). We have tried to purify the glucocorticoid receptor using the C20 acid derived from corticosterone by periodic acid oxidation.…”

Section: Discussionmentioning

confidence: 99%

Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex

Atger¹,

Milgröm²

1976

Biochemistry

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Steroid-receptor complexes formed at low temperature and ionic strength are unable to bind to target cell nuclei. After a temporary exposure to high ionic strength and/or temperature they become activated (i.e., able to bind to nuclei). However, there exists an equilibrium between activated and nonactivated complexes; thus, mixtures of both populations are obtained. In this paper it is shown that activated [3H]triamcinolone acetonide-rat liver receptor complexes bind strongly to phosphocellulose, whereas nonactivated complexes do not. Thus, it is chromatographically possible to isolate these two populations of complexes. The experimental conditions of the separation have been established. The most important feature is that upon prolonged exposure to phosphocellulose, nonactivated complexes become progressively activated. The separation on phosphocellulose has at least three potential applications. A first application is the possibility of measuring the concentration of activated complexes in incubates. However, when activated complexes were titrated with rat liver nuclei in excess or assayed through binding to phosphocellulose, slightly different results were obtained. This discrepancy was due on one hand to the difficulty of obtaining binding of all the activated complexes and on the other hand to the second activation of some of the complexes during their exposure to phosphocellulose. A second application was the possibility of obtaining a homogeneous population of activated complexes. This was actually achieved, since the complexes eluted from phosphocellulose were demonstrated to be 90--100% activated. The use of such homogeneous preparations simplifies considerably studies on binding of steroid-receptor complexes to nuclear acceptors (nuclei, chromatin, DNA). A third application is the use of phosphocellulose for the purification of receptor. Cytosol containing nonactivated complexes was filtered through phosphocellulose the complexes present in the breakthrough of the column were then activated and bound to phosphocellulose in a second chromatography. Advantage was also taken of the "amphoteric" behavior of the receptor that binds to both anionic (phosphocellulose) and cationic (diethylaminoethylcellulose) resins. Purification (940fold) with 24% yield could be obtained in preparations taking less than 2 days. The partially purified receptor was a heavy aggregate (greater than 12 S) that could be dissociated into 4S subunits by exposure to 0.3 M K C1. It has kept its property of interacting with nuclear acceptor. Preliminary experiments have shown that this technique could be of general application for steroid hormone receptors: activation enhanced binding to phosphocellulose of progesterone, aldosterone, and estradiol receptors.

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Section: Discussionmentioning

confidence: 99%

Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex

Atger¹,

Milgröm²

1976

Biochemistry

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“…Two agarose adsorbents (5,6) with high ER binding affinity were first synthesized and facilitated ER dissociation from columns (Figure 6). 84,85 Bucourt et al continued to use the long aliphatic chain located at different positions of estradiol to explore appropriate absorbents on the column. Compared to other positions, such as C2, C3, C4, C17α, and C17β, estradiol-C7α derivatives showed higher stability, binding specificity, and selectivity and were selected for the purification of ER.…”

Section: Role Of Erα In the Mechanism Of Acquiredmentioning

confidence: 99%

Selective Estrogen Receptor Degraders (SERDs): A Promising Strategy for Estrogen Receptor Positive Endocrine-Resistant Breast Cancer

Liu

2020

J. Med. Chem.

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Estrogen receptor (ER) plays important roles in gene transcription and the proliferation of ER positive breast cancers. Selective modulation of ER has been a therapeutic target for this specific type of breast cancer for more than 30 years. Selective estrogen receptor modulators (SERMs) and aromatase inhibitors (AIs) have been demonstrated to be effective therapeutic approaches for ER positive breast cancers. Unfortunately, 30−50% of ER positive tumors become resistant to SERM/AI treatment after 3−5 years. Fulvestrant, the only approved selective estrogen receptor degrader (SERD), is currently an important therapeutic approach for the treatment of endocrine-resistant breast cancers. The poor pharmacokinetic properties of fulvestrant have inspired the development of a new generation of oral SERDs to overcome drug resistance. In this review, we describe recent advances in ERα structure, functions, and mechanisms of endocrine resistance and summarize the development of oral SERDs in both academic and industrial areas.

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“…Estradiol was attached to the dextran molecule at the 7a position via an eight-atom spacer chain. 7a-Estradiol derivatives have proved to be most appropriate for purification of estrogen receptor by solid-support affinity chromatography in previous work (7,8). Binding of receptor to the estrogen residue covalently linked to the dextran macromolecule yielded a complex (molecular weight >500,000) that was easily separated by gel filtration chromatography from other proteins present in the preparation.…”

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confidence: 99%

Soluble biospecific macromolecule for purification of estrogen receptor.

Hubert¹,

Mešter²,

Dellacherie³

et al. 1978

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

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An alternative to affinity chromotography purification of proteins based on the use of a water-soluble biospecific polymer has been applied to the estrogen receptor from calf uterus. The receptor (4S-sin form) was bound to a dextran-estradiol conjugate (molecuLar weight 500,000) and the complex was isolated by gel filtration. Highly purified receptor was subsequently released from the dextran-estradiol conjugate by exchange with [3Hkestradiol. Biospecific adsorbents, i.e., insoluble materials containing covalently linked radicals capable of binding specific macromolecules (e.g., enzymes, antibodies), have been used extensively for purification of these macromolecules (reviews in refs. 1-3). In comparison with classical methods, affinity chromatography allows for substantially higher purification in a single step and is therefore particularly suitable for biological samples containing very low concentrations of the macromolecule of interest. This is the case with intracellular steroid hormone receptors, which are present in crude target tissue extracts at a concentration about 1/105 that of most other proteins. A biospecific step, therefore, is necessary for their purification (4-6). However, certain difficulties have been encountered due to properties of the insoluble support of the ligand. In particular, such material has a relatively high ion-exchange capacity, which results in considerable retention of nonspecific proteins along with receptor. These proteins subsequently contaminate the receptor preparation at elution. Moreover, the local concentration of ligand residues in the solid support used for affinity chromatography does not depend only on their overall concentration in the column or suspension, but also on the manner in which synthesis was performed. Due to the geometry of distribution of residues within the adsorbent, only a fraction may be accessible for binding to receptor molecules. Because of this, conditions for binding, as well as for release of receptor retained by the affinity adsorbent, must be established empirically.In the present work we describe an alternative to affinity chromatography in which the usual solid support was replaced by a water-soluble macromolecule of dextran (molecular weight -500,000). Estradiol was attached to the dextran molecule at the 7a position via an eight-atom spacer chain. 7a-Estradiol derivatives have proved to be most appropriate for purification of estrogen receptor by solid-support affinity chromatography in previous work (7,8). Binding of receptor to the estrogen residue covalently linked to the dextran macromolecule yielded a complex (molecular weight >500,000) that was easily separated by gel filtration chromatography from other proteins present in the preparation. Receptor was subsequently releasedThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3143by exchange with free [3...

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Parameters influencing the purification of calf uterus estrogen ‘receptor’ by affinity chromatography

Cited by 12 publications

References 6 publications

Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex

Chromatographic separation on phosphocellulose of activated and nonactivated forms of steroid-receptor complex. Purification of the activated complex

Selective Estrogen Receptor Degraders (SERDs): A Promising Strategy for Estrogen Receptor Positive Endocrine-Resistant Breast Cancer

Soluble biospecific macromolecule for purification of estrogen receptor.

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