Estradiol-binding kinetics of the activated and nonactivated estrogen receptor.

Weichman, Barry M.; Notides, Angelο C.

doi:10.1016/s0021-9258(17)38319-9

Cited by 182 publications

(52 citation statements)

References 27 publications

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“…Preparation of Estrogen Receptors. Preparation of the calf uterine cytosol has been previously described by Weichman and Notides (1977). Uterine cytosol was made 30% saturated with ammonium sulfate, allowed to sit on ice for up to 1 h, and centrifuged for 10 min at 27000g.…”

Section: Methodsmentioning

confidence: 99%

“…Competitive Binding Assay of Estradiol and Estradiol Analogs. Competitive binding assays were carried out as described by Weichman and Notides (1977). Aliquots (200 pL each) of the dissolved ammonium sulfate fraction were incubated in duplicate for 3 h at 25 °C nM [3H]estradiol plus 9 x 10"12 M to 9 x 6 M unlabeled competitor.…”

Section: Methodsmentioning

confidence: 99%

“…There is evidence that the active form of the estrogen receptor is a homodimer with one hormone binding site per monomer (Notides et al, 1985;Humar & Chambón, 1988) and that the estradiol-estrogen receptor binding interaction is positive cooperative (Weichman & Notides, 1977;Notides et al, 1981). Positive cooperative ligand binding is demonstrated by a concave-downward Scatchard plot and a Hill coefficient of 1.68 ± 0.21 (Notides et al, 1981).…”

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confidence: 99%

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A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

Schwartz

Skafar

1994

Biochemistry

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We have investigated the relation between ligand structure and binding mechanism between the calf uterine estrogen receptor. A series of structurally altered estradiol analogs was used in which either an amino- or a nitro group had been added to the 2 or 4 position on the phenolic A-ring. The binding affinity of both amino analogs and the 4-nitro analog for the estrogen receptor was reduced relative to that of estradiol, as measured by competitive binding assay; the values were between 0.008% and 8% of estradiol's affinity. The slope of the displacement curve for the 4-nitro analog was also significantly different from that of estradiol (p < 0.05), indicating that the binding mechanism of these two ligands was different. The affinity of the 2-nitroestradiol ligand for the receptor was too low to be measured. The binding mechanism was then further investigated by measuring the Hill coefficient of [3H]estradiol binding in the presence of the analog. The presence of a nitro group on C4 eliminated the positive cooperativity of the [3H]estradiol-estrogen receptor interaction; the Hill coefficient of [3H]estradiol binding in the presence of the analog was 0.99 compared with 1.7 for [3H]estradiol alone. Most interestingly, the presence of an amino group on either C2 or C4 brought about a switch from a positive to a negative cooperative binding interaction; the Hill coefficients of [3H]estradiol binding in the presence of the analogs were between 0.6 and 0.7. These results provide additional support for an induced-fit mechanism of ligand-estrogen receptor interactions.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

Schwartz

Skafar

1994

Biochemistry

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Section: Methodsmentioning

confidence: 99%

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

Skafar¹

1991

Biochemistry

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The binding mechanism of the antagonist RU486 to the progesterone receptor was compared with that of the agonists progesterone and R5020. Both progesterone and RU486 bound to the receptor with a Hill coefficient of 1.2, indicating the binding of each ligand is positive cooperative. However, when each ligand was used to compete with [3H]progesterone for binding to the receptor at receptor concentrations near 8 nM, at which the receptor is likely a dimer, the competition curve for RU486 was significantly steeper than the curves for progesterone and R5020 (p less than 0.001). This indicated that a difference in the binding mechanism of RU486 and progesterone can be detected when both ligands are present. In contrast, at receptor concentrations near 1 nM, at which the receptor is likely a monomer, the competition curves for all three ligands were indistinguishable (p = 0.915). These results indicate that RU486 and agonists have different binding mechanisms for the receptor and further suggest that this difference may be related to site-site interactions within the receptor.

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“…We recently demonstrated that the dissociation of [3H(estradiol from the estrogen receptor follows a two-component exponential process which is a property of receptor activation (Weichman & Notides, 1977). The first, or fast, component (k-) results from the dissociation of [3H]estradiol from the nonactivated receptor.…”

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Analysis of estrogen receptor activation by its [3H]estradiol dissociation kinetics

Weichman¹,

Notides²

1979

Biochemistry

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Estradiol-binding kinetics of the activated and nonactivated estrogen receptor.

Cited by 182 publications

References 27 publications

A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

A-Ring Nitro- and Amino-Substituted Estradiol Analogs Produce a Negative Cooperative or Noncooperative [3H]Estradiol-Estrogen Receptor Binding Mechanism

Differences in the binding mechanism of RU486 and progesterone to the progesterone receptor

Analysis of estrogen receptor activation by its [3H]estradiol dissociation kinetics

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