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A C Notides

Publications and source records attributed to A C Notides.

At least 55 records · Page 3Linked to original sources
PubMed1985

Modulation of the estrogen receptor's affinity for DNA by estradiol.

The binding constant for estrogen receptor-DNA interaction when measured in the presence and absence of estradiol revealed a distinct difference dependent upon whether the receptor was hormone-bound or hormone-free. The binding constant of estrogen receptor-DNA interaction was determined by analysis of the exponential elution profile of the estrogen receptor from DNA-Sepharose columns using Tris buffer at a constant salt concentration. The binding constant of the hormone-bound estrogen receptor for DNA in Tris buffer, pH 7.4, containing 0.2 M KCl was 10.1 +/- 0.8 X 10(6) M-1, 5-fold higher than the value for the hormone-free estrogen receptor. Analysis of the number of ionic bonds between the estrogen receptor and DNA indicates that the hormone-free receptor establishes eight salt bridges, while the hormone-bound estrogen receptor establishes 10-13. The affinity of the hormone-bound estrogen receptor for DNA in Tris buffer at pH 7.4 in 0.2 M KCl is 10-fold greater than at pH 8.0, suggesting that ionic bonding between the receptor and DNA may involve histidine residues of the receptor. The concentration-dependence of the hormone-bound receptor's affinity for DNA emphasizes the receptor's associative state as an influence on the receptor's DNA binding characteristics. Our results demonstrate that estradiol modifies the conformation of the estrogen receptor to a state having an increased affinity for DNA.

Animals↗
PubMed1984

The estriol-induced inhibition of the estrogen receptor's positive cooperativity.

The effect of estriol on the positive cooperativity of [3H]estradiol binding to the partially purified calf uterine estrogen receptor was investigated using the kinetic analysis of Sasson and Notides (J. biol. Chem. 257 1982, 11540). The receptor was titrated with variable concentrations of [3H]estradiol with or without estriol; the estriol was maintained in a constant molar ratio to the [3H]estradiol concentration. A 4-fold molar excess of estriol above the [3H]estradiol concentrations inhibited the receptor's cooperative [3H]estradiol binding. In the absence of estriol, the [3H]estradiol receptor interaction was highly cooperative, the Scatchard plot was convex and the Hill coefficient was 1.61 +/- 0.02. In the presence of sufficient estriol to reduce the maximally bound [3H]estradiol to 77%, the Scatchard plot was linear and the Hill coefficient was 1.04 +/- 0.04. The inhibition of the cooperative [3H]estradiol binding by estriol was not due to isotope dilution of the specifically bound [3H]estradiol by the unlabeled estriol. These data demonstrate that the cooperative binding of [3H]estradiol by the receptor that is characteristic of the equilibrium between the two states of the receptor (active and nonactive) is eliminated by the presence of estriol. This finding is consistent with the agonist/antagonist activity of estriol observed in vivo.

Animals↗
PubMed1984

Inability of [3H]estriol to induce maximal cooperativity of the estrogen receptor.

The interaction of [3H]estriol with the partially purified estrogen receptor from calf uterus shows positive cooperativity that is dependent upon receptor concentration and temperature. At a receptor concentration of 1 nM and 25 degrees C the [3H]estriol-receptor cooperativity was low, the Hill coefficient (nH) was 1.03 +/- 0.02 however, with increasing receptor concentrations the receptor's cooperativity increased until at approximately intracellular receptor concentration (20 nM) the nH = 1.20 +/- 0.04. At 0 degrees C and a receptor concentration of 10 nM the [3H]estriol-receptor interaction was highly cooperative, the Scatchard plot was convex and nH = 1.58 +/- 0.04 while at 30 degrees C the Scatchard plot approached linearity and nH = 1.03 +/- 0.02. In comparison, [3H]estradiol was capable of inducing, at 0 or 30 degrees C and at a receptor concentration of 1 nM or greater, maximal receptor cooperativity, nH = 1.63. These data demonstrate: (a) the receptor's conformation and binding mechanism change in a specific manner with temperature, so that receptor analysis at 0 degrees C does not necessarily reflect the receptor's properties at biologically relevant temperatures; (b) the dependence of the receptor's cooperativity on receptor concentration, which suggests interaction between dissociable subunits; and (c) the lower cooperativity induced by estriol, in comparison with estradiol, which indicates estriol is less efficient in shifting the receptor toward a higher affinity or the activated state of the receptor.

Animals↗
PubMed1984

Characterization of the calf uterine progesterone receptor and its stabilization by nucleic acids.

The molecular and steroid hormone-binding properties of the calf uterine progesterone receptor and its interaction with nucleic acids were investigated. A positive cooperative binding interaction of [3H]progesterone with the receptor was evident from a nonlinear Scatchard plot and a Hill coefficient of 1.22 +/- 0.02. The range of progesterone receptor concentrations was 0.73-1.04 pmol/mg protein, approximately twice that of the estrogen receptor. Competitive binding assays revealed a high specificity for progesterone: R5020 greater than or equal to progesterone greater than deoxycorticosterone greater than 5 alpha-pregnane-3,20-dione much greater than 17 alpha-hydroxyprogesterone greater than or equal to 20 alpha-dihydroprogesterone greater than or equal to testosterone greater than or equal to estradiol greater than cortisol. Thus, a progesterone-specific receptor of high affinity and concentration is obtainable from calf uterus in large quantities without estrogen pretreatment. Thermal inactivation of the unoccupied progesterone receptor is inhibited by 10 mM sodium molybdate, whereas thermal inactivation of the ammonium sulfate-purified progesterone receptor is not. Thermal inactivation of the ammonium sulfate-purified receptor is inhibited by nucleic acids and polynucleotides; polyguanylate (poly G) is the most effective. DNA and poly G also effectively restore the progesterone-binding ability of the ammonium sulfate-purified receptor which had been lost due to heat inactivation. After incubation of the unoccupied receptor from 5-30 min at 25 C, the addition of poly G restored the receptor's [3H]progesterone-binding ability to control levels. These data suggest that the progesterone receptor's steroid-binding site is more readily inactivated by heat than is the DNA-binding site, and that nucleic acid binding induces a conformational change, which consequently restores the receptor's progesterone-binding site to functional activity.

Animals↗
PubMed1983

Estriol and estrone interaction with the estrogen receptor. I. Temperature-induced modulation of the cooperative binding of [3H]estriol and [3H]estrone to the estrogen receptor.

We investigated the calf uterine estrogen receptor's interaction with [3H]estriol and [3H]estrone and found that the receptor's cooperative binding is markedly dependent upon temperature and receptor concentration. With increasing temperature, the intensity of the estrogen receptor's cooperativity decreases: at 0 degrees C the Scatchard plot was convex and the Hill coefficient was 1.58 +/- 0.04 (n = 5), while at 30 degrees C the Scatchard plot approached linearity and the Hill coefficient was 1.03 +/- 0.02 (n = 3) for the binding of [3H]estriol with a receptor concentration of 10 to 12 nM. With increasing receptor concentration, the receptor's cooperativity gradually increased. At a receptor concentration of 1 nM, at 25 degrees C, with [3H]estriol the Hill coefficient was low, 1.03 +/- 0.02 (n = 3), while at approximately intracellular receptor concentration (20 nM) the Hill coefficient increased to 1.20 +/- 0.04 (n = 4). Similar results were observed with [3H]estrone. The cooperative interaction of the estrogen receptor with [3H]estriol or [3H]estrone is reversible and dependent upon temperature. The van't Hoff analysis of the apparent dissociation constant of the [3H]estrone- and [3H]estriol-receptor complexes indicates that a transition in the receptor's binding mechanism occurs at 15 degrees C. Therefore, measurements of ligand interactions with the estrogen receptor at 0 to 15 degrees C, may not reflect the binding mechanism of the receptor at more biologically relevant temperatures. The reduced, positively cooperative interactions of [3H]estriol and [3H]estrone with the estrogen receptor at 30 degrees C, in comparison with estradiol, decrease the presence of the activated receptor, which correlates with the partial agonist-antagonist activities of estriol and estrone observed in vivo.

Animals↗
PubMed1983

Estriol and estrone interaction with the estrogen receptor. II. Estriol and estrone-induced inhibition of the cooperative binding of [3H]estradiol to the estrogen receptor.

Kinetic analysis of the estrogen receptor's cooperative equilibrium [3H]estradiol binding (Sasson, S., and Notides, A. C., (1982) J. Biol. Chem. 257, 11540-11545) provides a sensitive method for probing the binding of partial agonists to the estrogen receptor. We studied the effects of estriol and estrone on the positive cooperativity of [3H]estradiol binding to the partially purified, calf uterine estrogen receptor. The receptor was titrated with variable concentrations of [3H]estradiol in combination with estriol or estrone, while maintaining a constant molar ratio of the estriol or estrone to the [3H]estradiol. With either a 4-fold molar excess of estriol or a 25-fold molar excess of estrone above the [3H]estradiol concentrations, the receptor's positive cooperative [3H]estradiol binding was inhibited. The Scatchard plot showed a transition from a convex to a linear curve and a decrease in the Hill coefficient value from 1.61 +/- 0.02 (n = 7) in the absence of estriol or estrone to 1.04 +/- 0.04 (n = 4) in the presence of estriol and 0.99 +/- 0.03 (n = 4) in the presence of estrone. The inhibition of the positive cooperativity of [3H]estradiol binding by estriol or estrone was shown not to be due to isotope dilution of the specifically bound [3H]estradiol by the unlabeled estriol or estrone. These kinetic analyses demonstrate that the positively cooperative equilibrium binding of [3H]estradiol by the receptor, which is characteristic of the receptor's activation process, is eliminated by estriol and estrone and consistent with their partial agonist-antagonist activities observed in vivo.

Animals↗
PubMed1982

Characterization of two uterine proteases and their actions on the estrogen receptor.

We have characterized two previously undetected proteases from the calf uterine cytosol and measured their actions on the estrogen receptor. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucylglycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant to exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity. These data indicate that a decrease in the sedimentation rate of the estrogen receptor in sucrose gradients with low salt or a change in the receptor's elution on DEAE-cellulose chromatography is not related to receptor activation but is produced by the receptor-active protease or other proteases.

Animals↗
PubMed1982

The inhibition of the estrogen receptor's positive cooperative [3H]estradiol binding by the antagonist, clomiphene.

The calf uterine estrogen receptor showed positive cooperativity of [3H]estradiol equilibrium binding; the Scatchard plot was convex and the Hill coefficient was 1.69 +/- 0.021 (n = 14). The effects of the estrogenic antagonists, zuclomiphene (cis-2-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate) and enclomiphene (trans-23-(p-[2-chloro-1,2-diphenylvinyl]phenoxy)triethylamine citrate), on the positive cooperativity of [3H]estradiol binding were measured by titrating the receptor with a variable concentration of [3H]estradiol and antagonist while maintaining a constant excess in a specific ratio of the antagonist to the [3H]estradiol. With a 45- to 55-fold molar excess of zuclomiphene or an 820- to 900-fold molar excess of enclomiphene above the [3H]estradiol concentration, the receptor's positive cooperative [3H]estradiol binding was inhibited. A transition from a convex to a linear Scatchard plot and a decrease in the Hill coefficient from 1.69 to 1.10 +/- 0.02 (n = 6) were induced. The specifically bound [3H]estradiol was inhibited 43 to 50% by the zuclomiphene and enclomiphene. The addition of unlabeled estradiol in a 1- or 2.3-fold molar excess above that of the [3H]estradiol concentration produced a 50 to 75% competitive displacement of the specifically bound [3H]estradiol; nevertheless, the Scatchard plot remained convex and the Hill coefficient was 1.74 and 1.80, respectively. Thus, inhibition of the positive cooperativity of [3H]estradiol binding by the clomiphene isomers was not due to dilution of the specifically bound [3H]estradiol by the antagonist. These data demonstrate that there are two molecular mechanisms by which an estrogen antagonist interferes with the function of the receptor: as a competitor, thus blocking the estrogen receptor's binding site to an agonist, and second by inducing conformational changes that inhibit site:site interactions and receptor activation.

Animals↗
PubMed1981

Positive cooperativity of the estrogen receptor.

The equilibrium [3H]estradiol binding by the partially purified estrogen receptor from calf uteri was measured at 25 degrees C. The Scatchard plot of the binding data showed a convex curve characteristic of positive cooperativity and a Hill coefficient of 1.58 +/- 0.21, at receptor concentrations of 1 to 10 nM. Below a receptor concentration of approximately 0.3 nM the Scatchard plot approached linearity, suggesting that the cooperative interactions are dependent upon a monomer--dimer equilibrium. Trypsin pretreatment of the receptor resulted in a loss of dimer formation and of the cooperative interactions. The positive cooperative characteristics of the estrogen receptor were shown not to be produced by receptor inactivation, failure to complete the [3H]estradiol--receptor equilibrium reaction, or radioimpurity of the [3H]estradiol. These findings indicate that the activated 5S estrogen receptor is a homodimer and that its formation is associated with a positive cooperative estradiol-binding reaction.

Allosteric Regulation↗
PubMed1979

The role of ligand-binding as a determinant of the structure and activation of the estrogen receptor.

The dissociation of estradiol from the estrogen receptor occurs in two kinetic phases: a fast component having a half-time of approximately 3 min and a slower, or second, dissociating component having a half-time of approximately 95 min at 28 degrees. The fast component is produced by the dissociation of estradiol from the nonactivated 4 S receptor, a monomer. Thus, the magnitude of the fast component of the [3H] estradiol biphasic dissociation curve is proportional to the fraction of the receptor in the nonactivated state. The slow component is the estradiol dissociating from the activated 5 S receptor, a dimer. The salt-extracted estrogen receptor isolated from uterine nuclei shows a single, slow dissociating component equal to the slower component of the cytoplasmic biphasic dissociation curve. Estradiol binding shifts the receptor equilibrium from the low affinity nonactivated 4 S receptor toward the high affinity activated 5 S receptor. The kinetics of estriol dissociation from the receptor shows a larger fractional magnitude for the fast component and a faster second dissociating component than estradiol. This suggests that estriol transforms a smaller fraction of the receptor to the activated state and that the activated estriol receptor has a shorter half-time than estradiol. The biphasic dissociation kinetics of an estrogen from the receptor provides a new and sensitive criterion for measuring receptor activation.

Animals↗