Solubilization of a tamoxifen-binding protein. Assessment of its molecular mass.
Recent findings point to a role of Antioestrogen-Binding Site (ABS) in some of the growth-modulatory effects of antioestrogens. In the present study, a method for the solubilization of ABS from rat uterus microsomal fractions by using 3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS; 20 mM) and KCl (0.4 M) is described. Decreasing the CHAPS concentration below the critical micelle concentration led to long-term stabilization of the protein. All of the membrane-bound ABS was recovered in the extract, and only one class of binding site, with a high affinity for [3H]tamoxifen (KA = 5 x 10(8) M-1) was detectable. This binding was time-dependent and reversible: at 4 degrees C, the association rate constant was ka = 7.2 x 10(4) M-1.s-1, and the reverse rate constant was kd = 1.0 x 10(-4) s-1. Solubilized ABS exhibited an affinity and specificity similar to those of the membrane-bound sites. Under disaggregating conditions, solubilized ABS had an apparent sedimentation coefficient, s20,w, of 5.2 S and a Stokes radius of 6.4 nm. From these two values, molecular masses of 160,000 Da for the detergent-ABS complex, and 110,000 for the protein moiety, were estimated. Assessment of the size of the membrane-bound ABS by a radiation inactivation technique is also described. The 'radiation inactivation size', corresponding to the mass of 1 mol of protein structure(s) whose associated tamoxifen-binding activity is abolished after a single hit by ionizing radiation, was estimated to be 80,000 Da.
Fargin
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INSERM U 168, Department of Endocrinology, CHU Rangueil, Université Paul Sabatier, France.
Faye
J C
JC
le Maire
M
M
Bayard
F
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Potier
M
M
Beauregard
G
G
D000818:Animals; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids; D002851:Chromatography, High Pressure Liquid; D003902:Detergents; D005260:Female; D046911:Macromolecular Substances; D008861:Microsomes / Q000378:metabolism; D008970:Molecular Weight; D009256:Nafoxidine / Q000494:pharmacology; D009594:Nitromifene / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011955:Receptors, Drug; D011960:Receptors, Estrogen / Q000187:drug effects / Q000302:isolation & purification*; D012995:Solubility; D013629:Tamoxifen / Q000378:metabolism; D014599:Uterus / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D046911:Macromolecular Substances; D011955:Receptors, Drug; D011960:Receptors, Estrogen; D013629:Tamoxifen; D009256:Nafoxidine; D009594:Nitromifene; D011189:Potassium Chloride; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate