A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using “differential dissociation” techniques

Richard-Foy, Hélène; Redeuilh, Gérard; Richard-Foy, Robert

doi:10.1016/0003-2697(78)90434-7

Cited by 12 publications

(5 citation statements)

References 12 publications

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“…The results obtained indicate that up to high ribosome concentrations Kn varies linearly (Figure 4). This behavior seems to be general and has been described for some ligand-protein interactions (Truong & Bailieu, 1971; Richard-Foy et al, 1978). At the concentrations of 80S ribosomes and 40S ribosomal subunits (5 and 1.7 OD2eo units, respectively) used in the experiments for the determination of the values of both Kd and n of the specific component, the values of Kn were 0.48 and 0.17, respectively (Figure 4).…”

Section: Resultssupporting

confidence: 73%

“…This type of phenomenon is well documented since binding of a number of radioactively labeled ligands (i.e., catecholamines, muscarinic acid, steroid hormones, etc.) to both their specific and nonspecific receptor sites is inhibited by chemically related antagonistic drugs (Cuatrecasas et al, 1974;Levitzki et al, 1975;Beld et al, 1980;Richard-Foy et al, 1978). Obviously under these experimental conditions any attempt to calculate the apparent dissociation constant of the interacting ligand (on the basis of the amount of cold antagonist that displaces 50% of the labeled ligand) could result in serious errors (Jacobs et al, 1975;Levitzki et al, 1975).…”

Section: Discussionmentioning

confidence: 99%

“…The determination of Kn was based on the principle that, when the specific binding does not contribute to the binding measurement, it is verified that Kn = B/U. The technique used to measure Kn requires a concentration of [14a-3H]cryptopleurine such that the specific receptor is completely saturated by nonradioactive ligand and therefore its contribution to the binding measurement is negligible (Richard-Foy et al, 1978). The experimental de-termination of Kn will be described under Results.…”

Section: Methodsmentioning

confidence: 99%

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Quantitation of the specific interaction of [14a-3H] cryptopleurine with 80S and 40S ribosomal species from the yeast Saccharomyces cerevisiae

Doelz¹,

Vázquez²,

Jiménez³

1982

Biochemistry

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Section: Resultssupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Quantitation of the specific interaction of [14a-3H] cryptopleurine with 80S and 40S ribosomal species from the yeast Saccharomyces cerevisiae

Doelz¹,

Vázquez²,

Jiménez³

1982

Biochemistry

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“…This technique can be used with diluted extracts without causing the loss of activity that occurs in the presence of charcoal when the protein concentration is lower than 1 mg/ml. In both cases the background was determined by either the isotopic dilution or the double concentration measurement method [12].…”

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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“…Because the [3H] -170-estradiolchromium tricarbonyl complex shows higher levels of nonspecific binding than [3H]E2, it is important to correct for this nonspecific binding in the concentration of the free ligand in binding affinity experiments. A method for making such corrections has been described (Katznellenbogen et al, 1978;Richard-Foy et al, 1978;. According to these authors, the free ligand concentration which is normally considered to be the difference between total added ligand and the receptor-bound ligand must be corrected by a 1 + nk factor.…”

Section: Methodsmentioning

confidence: 99%

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

Vessières¹,

Top²,

Ismail³

et al. 1988

Biochemistry

117

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As an integral part of the development of a new technique using organometallic markers for the detection of hormone receptors by FT-IR spectroscopy, a series of estradiol derivatives labeled with Cr(CO)3 or Cr(CO)2CS fragments on the A ring has been synthesized. The stereochemistry of one of these steroids, alpha-[3-(dimethyl-tert-butylsiloxy)-17 beta-estradiol]dicarbonyl(thiocarbonyl)chromium(0), has been established by X-ray diffraction. The organochromium-labeled steroids are stable in aqueous methanol solution, and their relative binding affinities to estrogen receptor have been determined; these values vary from 0.4 to 28%. The complex exhibiting the strongest affinity, [3-O-(3-hydroxypropyl)-17 beta-estradiol]-chromium tricarbonyl complex, has been prepared in a tritiated form with a high specific activity (4.1 Ci/mmol). This tritiated hormone binds reversibly to the estradiol receptor in lamb uterine cytosol with an affinity (Kd = 0.85 nM) and number of binding sites (n = 770 fmol/mg of protein) close to the values observed for estradiol itself. The level of nonspecific binding is low, and the hormone is not bound significantly to other nontarget tissues. The observation that the binding affinity of the steroid depends on which side of the steroidal A ring the organometallic label is bound demonstrates the nonequivalence of the two sides of the A ring with respect to the receptor site. The FT-IR spectra of the organochromium markers in the v(CO) region can be used for the detection of the estradiol receptor in lamb uterine cytosol.

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A simple, rapid, and precise method for calculating the steroid hormone-receptor complex concentration in the presence of saturating levels of hormone by using “differential dissociation” techniques

Cited by 12 publications

References 12 publications

Quantitation of the specific interaction of [14a-3H] cryptopleurine with 80S and 40S ribosomal species from the yeast Saccharomyces cerevisiae

Quantitation of the specific interaction of [14a-3H] cryptopleurine with 80S and 40S ribosomal species from the yeast Saccharomyces cerevisiae

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

Organometallic estrogens: synthesis, interaction with lamb uterine estrogen receptor, and detection by infrared spectroscopy

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