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

Soulignac, O; Secco-Millet, Claude; Rocher, Pierre; Baulieu, Étienne-Émile; Richard-Foy, Hélène

doi:10.1016/0014-5793(77)80769-2

Cited by 11 publications

(4 citation statements)

References 16 publications

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“…In a high salt medium, the molecular weight of the "mi-0 The results obtained with nonpurified R from lamb endometrium are represented. Similar molecular weights have been found for Rc by Soulignac et al (1977) and for Ca2+Rc by Puca et al (1977). Similar elution molarity from the DEAE-cellulose has been reported by Puca et al (1977).…”

Section: Resultssupporting

confidence: 83%

“…The proportion of the "free" RnE2 measured by gel filtration was in agreement with the values obtained by ultracentrifugation analysis. Results were similar when using Sepharose 6B (Figure 3a) or Ultrogel ACA 34 chromatography according to Soulignac et al (1977), and the same Stokes radius (5.2 nm) was found for the Rn localized in NEi and in NE2. However, the free RnE2 contained in NE2 appeared more heterogeneous than that contained in NE], as also shown by ultracentrifugation analysis during the same experiment (Figure lc).…”

Section: Resultssupporting

confidence: 67%

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Characterization of the estradiol receptor extracted from nuclei by micrococcal nuclease

André¹,

Raynaud²,

Rochefort³

1978

Biochemistry

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The estradiol receptor of lamb endometrium, translocated to nuclei in útero and extracted with micrococcal nuclease at 2-4 °C, was compared under low salt conditions to the "native" 8S and to partially proteolyzed cytosol receptor.The degree of extraction of the receptor was roughly parallel to the proportion of digested chromatin and to the yield of extracted DNA. After extensive digestion of chromatin, which produced up to 10% perchloric acid soluble DNA and more nucleosome monomer than dimer, up to 80% of the nuclear receptor was extracted under low ionic strength and subse-

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Section: Resultssupporting

confidence: 83%

Section: Resultssupporting

confidence: 67%

Characterization of the estradiol receptor extracted from nuclei by micrococcal nuclease

André¹,

Raynaud²,

Rochefort³

1978

Biochemistry

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“…Binding specificity ($the purified receptor The affinity chromatography eluate, containing purified 4-S receptor complexed to oestradiol (specific radioactivity of [3H]oestradiol 4 Ci/ mmol), was filtered through a Sephadex G-50 column in order to eliminate unbound hormone. The specific activity of the receptor was then with that we previously estimated by gel filtration and ultracentrifugation for the high-salt non-aggregated native receptor [31] and with that obtained for this receptor purified to homogeneity [32]. From this electrophoretic pattern, purity may estimated to be at least 50 %.…”

Section: Purificution Of the Receptor By Ajjcinity Cizromatogvuphymentioning

confidence: 86%

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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“…These Kd values may be far from the real Kd values since the equilibrium may be broken during the assay of the ternary complex and since several assumptions had to be made. On the basis of a Stokes' radius of 5.5 nm [19] for the ER and by analogy with regulatory proteins in procaryotes, we have assumed that approximately 15 base pairs would be covered by the ER-ligand complex. Moreover, the unbound DNA concentration in our assays is probably much lower than the total DNA concentration, due t,o its interaction with cellulose and cytosol DNA-binding proteins besides ER.…”

Section: Discussionmentioning

confidence: 99%

Estrogen-Receptor-DNA Interaction

Evans

Baskevitch

Rochefort

2005

European Journal of Biochemistry

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We have compared the interaction of the cytosol estrogen receptor with calf thymus DNA after its binding to [3H]estradiol or 4-hydroxy['H]tamoxifen, a high-affinity antiestrogen, in an attempt to find a difference in the nature of these two complexes. The activation of the receptor and its binding to DNA were simultaneously obtained during an 18-h incubation at 0 "C. The binding of a constant concentration of estrogen receptor to increasing concentrations of adsorbed DNA was examined. Scatchard representation of the data showed that the antiestrogen -receptor complex has a twofold lower affinity for DNA than the estradiol-receptor complex, while the proportion ofreceptor able to bind DNA was the same whether bound to estradiol or antiestrogen.Similar results were obtained by using soluble DNA and separation of the unbound and DNA-bound receptor on sucrose gradients.These results indicate that the estrogen receptor binds in v i m to non-specific double-stranded DNA with a lower affinity when it has been activated by an antiestrogen than when activated by estradiol.Antiestrogens are estrogen analogues which prevent estrogen action at the target tissue level by competitive inhibition of the binding ofestrogen to its receptors [l]. The reason for the low biological efficiency of antiestrogens when bound to the estrogen receptor (ER) is, however, not clear. It is known that antiestrogens are able to induce nuclear translocation of estrogen receptor [2,3] and some, but not all, estrogen-specific responses [4]. It is therefore likely that the reason for their weak estrogenic activity is due to an alteration of the ER interaction with chromatin acceptor sites [5]. Since these acceptor sites are likely to consist, at least partly, of non-specific double-stranded DNA [6,7], the interaction between DNA and ER in vitro has also been studied. It has been shown that the ER activated by antiestrogen is able to interact in vitro with DNA [8,9] but the degree of this interaction has not been quantified and compared to that obtained with estrogens.Two major hypotheses are presently available to explain the antagonist activity of these drugs. The first and simplest one is that the binding of antiestrogens is less efficient than estrogen at activating the ER and triggering biological responses. In support of this idea, we have recently found that the rate of dissociation of estrogen and antiestrogen from the ER are differently affected by molybdate, suggesting that ER is differently activated by these two types of ligands [lo]. The second hypothesis is that the antiestrogen behaves as a full estrogen agonist on the ER but blocks estrogen responses through another mechanism resulting from its interaction with other cellular components such as antiestrogen-binding proteins [Ill. To help in discriminating between these two possibilities and in an attempt to find a molecular explanation for the weak or partial estrogenic activity of antiestrogen, we have studied the degree of interaction of the ER with calf thymus DNA.The putative se...

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

Cited by 11 publications

References 16 publications

Characterization of the estradiol receptor extracted from nuclei by micrococcal nuclease

Characterization of the estradiol receptor extracted from nuclei by micrococcal nuclease

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

Estrogen-Receptor-DNA Interaction

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