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G Redeuilh

Publications and source records attributed to G Redeuilh.

30 records · Page 2Linked to original sources
PubMed1985

The use of the biotinyl estradiol-avidin system for the purification of "nontransformed" estrogen receptor by biohormonal affinity chromatography.

Several biotinyl estradiol derivatives have been prepared by coupling estradiol 7 alpha-carboxylic acid to biotin via different linear linkers. All these compounds exhibit a high affinity for the estrogen receptor as determined by competitive binding assays against [3H]estradiol. These compounds also displaced the dye 4-hydroxyazobenzene-2'-carboxylic acid from the biotin-binding sites of avidin free or immobilized on agarose. It was demonstrated that only the derivatives bearing a long spacer chain (greater than 42 A greater than) between estradiol and biotin were able to bind receptor and avidin simultaneously, suggesting some steric hindrance. The biotin-avidin system has been investigated for the purification of the cytosoluble "nontransformed" estrogen receptor stabilized by sodium molybdate. The method relies on: 1) high biohormonal affinity of receptor for biotinyl estradiol derivative; 2) the specific selection by avidin-agarose column of biotinyl estradiol-receptor complexes; and 3) the biohormonal elution step by an excess of radioactive estradiol. Starting from unfractionated cytosol containing molybdate-stabilized nontransformed 8S estrogen receptor with estradiol 7 alpha-(CH2)10-CO-NH-(CH2)2-O-(CH2)2-O-(CH2)2-NH-CO-(CH2)3-NH-biotin, preliminary experiments using avidin-agarose chromatography and then a specific elution step by exchange with free [3H]estradiol, allowed a 500-1,500-fold purification. Further purification of estrogen receptor was obtained by ion exchange chromatography through a DEAE-Sephacel column and led to a congruent to 20% pure protein, assuming one binding site/65,000-Da unit. The hydrodynamic parameters of the purified receptor were essentially identical to those of molybdate-stabilized nontransformed receptor present in crude cytosol. The advantages of this double biotinyl steroid derivative-avidin chromatographic technique over more conventional affinity procedures are discussed and make it applicable to the purification of minute amounts of steroid receptors in a wide variety of tissues.

Animals↗
PubMed1983

Physicochemical and genetic evidence for specific antiestrogen binding sites.

In rat uterus and human breast cancer MCF-7 cell cytosol, the antiestrogens tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) bind to "antiestrogen binding sites" (ABS), which do not bind estradiol (E). Demonstrated in total cytosol by binding studies with radioactive antiestrogens in the presence of a large concentration of E, ABS can be physically separated from E-binding estrogen receptor (ER) by removing the latter with an E-containing bioaffinity adsorbent or with heparin-Sepharose gel. ABS concentration is 10-20% of that of ER; the Kd for Tam and OH-Tam is 1-2 x 10(-9) M, whereas the Kd of OH-Tam binding by ER (approximately equal to 1 x 10(-10) M) is approximately equal to 1/50 that of Tam. Other triphenylethylene antiestrogens compete against Tam for binding to ABS, contrary to steroid hormones. Sucrose gradient ultracentrifugation analyses of total cytosol and of affinity gel effluents show a heterogenous pattern of ABS from 10 to 40 S, unchanged by 0.4 M KCl and limited trypsinization (which however provoke transitions of ER from 8S to 4S forms) and by 20 mM molybdate (which stabilizes the 8S form of ER and prevents large aggregates). Preliminary results suggest that ABS may be associated with particulate components of the cell. RTx6 cells of a clone selected from MCF-7 cells for resistance to the antigrowth effect of Tam have ER in the same concentration and have similar affinity for E and antiestrogens as do unselected MCF-7 cells. However, RTx6 cells have virtually no ABS detectable by binding and gradient ultracentrifugation studies. It is proposed that the double binding of Tam and OH-Tam to ER and ABS in estrogen target cells may be related to the complex double series of estrogenic and "antiestrogenic" activities displayed by nonsteroidal triphenylethylene derivatives.

Animals↗
PubMed1982

Progesterone receptor from chick oviduct: purification of molybdate-stabilized form and preliminary characterization.

A molydate-stabilized, 'non-activated' form of the progesterone receptor from the cytosol of oestrogen-stimulated chick oviduct has been purified to homogeneity by a three-step procedure. The first step, affinity chromatography using a N-(12-amino-dodecyl)-3-oxo-4-androsten-17 beta-carboxamide-substituted Sepharose gel, purified the receptor 1500-2700-fold with approximately equal to 50% recovery. In the second step, ion-exchange chromatography through a DEAE-cellulose column, progesterone receptor was eluted as a single peak at 0.1 M KCl. Purification after this step was greater than 6700-fold. The third step was filtration through Ultrogel AcA 34, resulting in overall purification approximately equal to 7400-fold with overall recovery approximately equal to 25% of pure receptor on the basis of 1 binding site/molecule of Mr 85000. The purified molybdate-stabilized receptor had a sedimentation coefficient approximately equal to 7.9S +/- 0.1 (n = 4) in 0.15 M or 0.4 M KCl containing sucrose 5-20% gradient and approximately equal to 8.9S +/- 0.2 (n = 6) in 0.15 M KCl containing glycerol 10-35% gradient, and its Stokes radius was 7.05 +/- 0.10 nm (n = 3) (calculated Mr between 240000 and 280000). Binding specificity of the purified receptor was the same as that found in crude cytosol. SDS-PAGE revealed a single band migrating as a polypeptide of Mr approximately equal to 85000 +/- 2300 (n = 9). PAGE under non-denaturing conditions at total acrylamide concentrations 5%, 7% and 9% showed a single [3H]ORG 2058-protein band (ORG 2058 is a high-affinity analogue more suitable than progesterone for electrophoretic studies). The data suggest that the high molecular weight molybdate-stabilized progesterone receptor purified from oestrogen-primed chick oviduct is composed of only approximately equal to 85000-Mr polypeptide chains.

Animals↗
PubMed1980

Properties of biospecific adsorbents, obtained by immobilization of oestradiol 7 alpha-derivatives, for purification of calf-uterine cytosol oestradiol receptor.

The properties of three types of adsorbents obtained by coupling oestradiol 7 alpha-derivatives to agarose were compared. The adsorbents examined were: oestradiol 7 alpha-decamethylene-agarose, oestradiol 7 alpha-decamethylene-poly(anayl-lysine)-agarose and oestradiol 7 alpha-trimethylene-poly(alanyl-lysine)-agarose. The following results were obtained. (1) All these adsorbents are stable at 0 degrees C for a 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 maximala receptor binding capacity of an absorbent was developed. (3) The geometry of the column and the dynamics of the loading have no influence on the binding capacity of the adsorbents. (4) Binding of the cytosol receptor to the adsorbent depends on whether the receptor had previously been partially purified by heparin-Ultrogel chromatography or treated with low or high salt concentration or trypsin. It was demonstrated that aggregation decreases the binding of the receptor to the adsorbents. (5) A satisfactory recovery of receptor upon elution is possible only with biospecific adsorbents containing low concentrations of coupled steroid (less than or equal to 0.2 mg/ml). The use of these adsorbents for the purification of the trypsin-treated receptor directly from cytosol allowed a 2500-fold purification corresponding to 5% pure protein (assuming one oestradiol binding site per molecule of Mr 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 Mr 70000).

Adsorption↗
PubMed1979

[Two-step purification of estradiol calf uterine cytosol receptor].

The use of two affinity chromatography methods: heparin-Ultrogel and estradiol 7 alpha-(CH2)3-poly (alanine-lysine)-agarose, allows us to purify the calf uterine estradiol cytosol receptor with a 20% recovery. Electrophoresis under denaturing conditions allows us to estimate its molecular weight as 70 000 dalton.

Animals↗
PubMed1979

[Production and detection of antibody against estradiol receptor in the calf uterus. Interaction with the estrogen receptor from the hen oviduct].

The antibodies against estrogen receptor were obtained after injecting Rabbits with a cytoplasmic receptor fraction isolated from Calf uterus. The estrogen receptor was partially proteolysed by the action of trypsin and subsequently purified by affinity chromatography (purification 4,000 to 10,000 fold, to a purity of 5-20%). The affinity of the antibody for the proteolysed receptor is KD approximately 1 nM and serum titres have reached values of approximately 50 nM. The values remained constant after the third injection. Preliminary results indicate that the antibody has approximately the same affinity for "native" cytoplasmic estrogen receptor from Calf uterus, as well as for the "trypsinized" forms of estrogen receptor isolated from Calf uterine cytosol and Hen oviduct nuclei.

Animals↗