scholarly journals Discordant Measures of Androgen-Binding Kinetics in Two Mutant Androgen Receptors Causing Mild or Partial Androgen Insensitivity, Respectively1

1999 ◽  
Vol 84 (2) ◽  
pp. 805-810
Author(s):  
Dana L. Shkolny ◽  
Lenore K. Beitel ◽  
Jody Ginsberg ◽  
Gary Pekeles ◽  
Laura Arbour ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Dissociation Constants ◽  
External Genitalia ◽  
Scatchard Analysis ◽  
Three Dimensional Model ◽  
Prolonged Incubation ◽  
Androgen Insensitivity ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Androgen Binding

We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (Kd) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (kdiss) for both androgens. In contrast, the MAIS subject’s hAR (R871G) has kdiss values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR’s ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects’ genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function. An increased kdiss despite a normal Kd for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

Preparation and Characterization of Paclitaxel Imprinted Silica Nanoparticles

2011 ◽  
Vol 399-401 ◽  
pp. 1894-1897
Author(s):  
Jian Hua Li ◽  
Zong Jian Zheng ◽  
Shao Ping Fu ◽  
Jing Bo Zhu
Keyword(s):  
Silica Nanoparticles ◽  
Dissociation Constants ◽  
Sol Gel ◽  
Scatchard Analysis ◽  
Binding Properties ◽  
Imprinted Polymers ◽  
Sol Gel Process ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

Highly selective molecularly imprinted layer-coated silica nanoparticles for paclitaxel were synthesized by molecular imprinting technique with a sol–gel process on the supporter of silica nanoparticles. The morphology of the obtained polymers was characterized by scanning electron microscopy (SEM). The binding properties of the imprinted polymers were evaluated through the equilibrium rebinding experiments. Scatchard analysis revealed that two classes of binding sites were formed in the imprinted polymers with equilibrium dissociation constants of 0.0509 g•L-1and 0.0094 g•L-1, respectively. Paclitaxel and its analogue were employed for selectivity tests. The results indicated that the imprinted polymers exhibited good selectivity and specificity toward paclitaxel.

Characterization and Evaluation of Binding Properties of Salvianolic Acid a Imprinted Polymers Prepared by Precipitation Polymerization

2011 ◽  
Vol 239-242 ◽  
pp. 2423-2426
Author(s):  
Lin Min Zhu ◽  
Shao Ping Fu ◽  
Lin Ying Li ◽  
Jing Bo Zhu
Keyword(s):  
Dissociation Constants ◽  
Precipitation Polymerization ◽  
Scatchard Analysis ◽  
Template Molecule ◽  
Binding Properties ◽  
Imprinted Polymers ◽  
Salvianolic Acid ◽  
Salvianolic Acid A ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

Molecularly imprinted polymers (MIPs) were synthesized by precipitation polymerization using salvianolic acid A (Sal A), acrylamide (AA), ethylene glycol dimethacrylate (EGDMA) and acetonitrile as template molecule, functional monomer, cross-linker and solvent, respectively. The morphology of the obtained polymers was characterized by scanning electron microscopy (SEM). The effect of different polymerization conditions (solvent volume, solvent and template amount) on the size and shape of particles was investigated. The binding properties of the imprinted polymers were evaluated through the equilibrium rebinding experiments. Scatchard analysis revealed that two classes of binding sites were formed in the imprinted polymers with equilibrium dissociation constants of 0.33 μmol·mL-1and 0.07 μmol·mL-1, respectively. Besides Sal A, two structurally related compounds, protocatechuic aldehyde (Pra) and salvianolic acid B (Sal B), were employed for molecular selectivity tests. The results indicated that the imprinted polymers exhibited good selectivity and specificity toward Sal A.

ANDROGEN BINDING IN CYTOSOLS AND NUCLEI OF HUMAN BENIGN HYPERPLASTIC PROSTATIC TISSUE

1978 ◽  
Vol 77 (1) ◽  
pp. 101-110 ◽  
Author(s):  
D. A. N. SIRETT ◽  
J. K. GRANT
Keyword(s):  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Nuclear Fraction ◽  
Tissue Samples ◽  
Nuclear Extracts ◽  
Receptor Interactions ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Androgen Binding

Androgen binding sites have been detected in cytosols and nuclear extracts from human benign hyperplastic prostatic (BPH) tissue with exchange assays using [3H]methyltrienolone and [3H]5α-dihydrotestosterone respectively. The concentrations of binding sites and the equilibrium dissociation constants for the [3H]steroid–receptor interactions have been determined and the specificity of the binding has been examined. The observed properties of the binding sites are consistent with those characteristic of androgen receptors. The binding sites are present in nuclear extracts from all BPH tissue samples examined to date. The amount of binding detected in the nuclear fraction is higher than that found in the cytosol.

Altered plasma catecholamines and numbers of α- and β-adrenergic receptors in platelets and leucocytes in hyperthyroid patients normalized under antithyroid treatment

1985 ◽  
Vol 110 (1) ◽  
pp. 75-82 ◽  
Author(s):  
Dieter Ratge ◽  
Sabine Hansel-Bessey ◽  
Hermann Wisser
Keyword(s):  
Dissociation Constants ◽  
Plasma Catecholamines ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Plasma Norepinephrine ◽  
Thyroid State ◽  
Antithyroid Treatment ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Hyperthyroid Patients

Abstract. We measured plasma catecholamines, α- and β-adrenoreceptor numbers and the accumulation of cyclic adenosine monophosphate (cAMP) in the unstimulated state and in response to 10 μmol/l (-) isoproterenol in blood cells from 29 euthyroid controls and from 18 patients with spontaneous hyperthyroidism. In the thyrotoxic patients plasma norepinephrine (1.14 ± 0.5 nmol/l) and epinephrine (0.3 ±0.14 nmol/l) were significantly decreased compared with plasma norepinephrine (1.87 ± 0.7 nmol) and epinephrine (0.41 ± 0.19 nmol/l) in the controls (P < 0.01 and P < 0.05, respectively) and the values obtained in subjects rendered euthyroid by antithyroid treatment (P < 0.001, respectively). α-adrenoceptor density in platelet membranes obtained from patients in the hyperthyroid state (114 ± 38 sites per cell) was significantly decreased when compared with controls (159 ± 48 sites per cell, P < 0.01) and the values from patients under effective antithyroid treatment (136 ± 35 sites per cell, P < 0.01). On the contrary, a significant increase in β-adrenoceptor density in mononuclear leucocyte (MNL) membranes was found in hyperthyroid patients (1751 ± 237 sites/cell) when compared with controls (1510 ± 351 sites/cell, P < 0.05) and the same patients following antithyroid treatment (1455 ± 260 sites/cell, P < 0.001). The equilibrium dissociation constants (KD) did not change in hyperthyroidism. Basal cAMP concentrations in MNL were higher in untreated thyrotoxicosis (45 ± 18 pmol/106 cells/10 min) than in patients in the euthyroid state (35 ± 9 pmol/106 cells/10 min, P < 0.05). Our data support the hypothesis that the balance of α- and β-adrenoceptors depends on the thyroid state. However, before the reputed catecholamine supersensitivity in hyperthyroid man can be accepted, the relationship between alterations in adrenoceptors and the biological responsiveness to catecholamines has to be demonstrated in different human tissues.

Sex difference in the cytosolic and nuclear distribution of androgen receptor in mouse submandibular gland

1986 ◽  
Vol 108 (2) ◽  
pp. 267-273 ◽  
Author(s):  
S. Kyakumoto ◽  
R. Kurokawa ◽  
Y. Ohara-Nemoto ◽  
M. Ota
Keyword(s):  
Androgen Receptor ◽  
Nuclear Receptors ◽  
Binding Sites ◽  
Androgen Receptors ◽  
Dissociation Constants ◽  
Scatchard Analysis ◽  
Male And Female ◽  
Short Period ◽  
Almost All ◽  
Androgen Binding

ABSTRACT Cytosol and nuclear androgen receptors in submandibular glands of male and female mice were measured by an exchange assay at 0 °C. The binding of [3H]methyltrienolone to cytosol receptors in females was mostly saturated within a short period of incubation (3 h), whereas the saturation was much slower in males; suggesting that almost all of the cytosol receptors were unoccupied in females and the receptors were partially occupied in males. Nuclear receptors were extracted with pyridoxal 5′-phosphate (5 mmol/l) from nuclear fractions with 93–95% efficiency. The exchange of the bound steroids occurred by 24–48 h at 0 °C, suggesting that most of the nuclear androgen receptor was occupied. The binding was low at higher temperatures, probably due to inactivation of the receptor. Scatchard analysis showed that the apparent dissociation constants of cytosol and nuclear receptors were similar (0·8 and 0·9 nmol/l respectively) in both sexes. On the other hand, the number of androgen-binding sites in the nucleus was much higher in males than in females (1052 fmol/mg DNA and 32 fmol/mg DNA respectively), while the number in the cytosol was higher in females than in males (512 fmol/mg DNA and 368 fmol/mg DNA respectively). These observations show that androgen receptors exist mainly (74%) in the nuclei of males, while they exist mostly (94%) in the cytosol of females. J. Endocr. (1986) 108, 267–273

scholarly journals Circular dichroism determination of class I MHC-peptide equilibrium dissociation constants

1997 ◽  
Vol 6 (8) ◽  
pp. 1771-1773 ◽  
Author(s):  
Chantal S. Morgan ◽  
James M. Holton ◽  
Barry D. Olafson ◽  
Pamela J. Bjorkman ◽  
Stephen L. Mayo
Keyword(s):  
Circular Dichroism ◽  
Dissociation Constants ◽  
Class I ◽  
Class I Mhc ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation ◽  
Circular Dichroïsm

Adrenal medullary regulation of rat renal cortical adrenergic receptors

1987 ◽  
Vol 253 (5) ◽  
pp. F1063-F1067
Author(s):  
P. R. Sundaresan ◽  
M. M. Guarnaccia ◽  
J. L. Izzo
Keyword(s):  
Adrenal Medulla ◽  
Adrenergic Receptors ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Physiological State ◽  
Plasma Corticosterone ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

The role of the adrenal medulla in the regulation of renal cortical adrenergic receptors was investigated in renal cortical particulate fractions from control rats and rats 6 wk after adrenal demedullation. The specific binding of [3H]prazosin, [3H]rauwolscine, and [125I]iodocyanopindolol were used to quantitate alpha 1-, alpha 2-, and beta-adrenergic receptors, respectively. Adrenal demedullation increased the concentration of all three groups of renal adrenergic receptors; maximal number of binding sites (Bmax, per milligram membrane protein) for alpha 1-, and alpha 2-, and beta-adrenergic receptors were increased by 22, 18.5, and 25%, respectively (P less than 0.05 for each). No differences were found in the equilibrium dissociation constants (KD) for any of the radioligands. Plasma corticosterone and plasma and renal norepinephrine levels were unchanged, whereas plasma epinephrine was decreased 72% by adrenal demedullation (P less than 0.01); renal cortical epinephrine was not detectable in control or demedullated animals. Our results suggest that, in the physiological state, the adrenal medulla modulates the number of renal cortical adrenergic receptors, presumably through the actions of a circulating factor such as epinephrine.

Further studies on the corticosteroid-receptor system of the nasal gland of the domestic duck (Anas platyrhynchos)

1983 ◽  
Vol 61 (7) ◽  
pp. 731-743 ◽  
Author(s):  
Thomas Sandor ◽  
Afzal Z. Mehdi ◽  
John A. DiBattista
Keyword(s):  
Ammonium Sulfate ◽  
Reaction Temperature ◽  
Rate Constants ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Similar Data ◽  
Nuclear Binding ◽  
Ammonium Sulfate Precipitate ◽  
Equilibrium Dissociation Constants ◽  
Equilibrium Dissociation

The interaction of tritiated corticosterone with the nasal gland corticosterone receptor was investigated. Kinetic studies have shown that the association of [3H]corticosterone–receptor followed second-order reaction kinetics and the dissociation of the ligand from the receptor became "pseudo" first order in the presence of large excess of radioinert steroids at 0, 15, 25, and 35 °C. Similar data were obtained with an ammonium sulfate precipitate of the cytosol. Dissociation rate constants varied from 10−5 to 10−3 s−1 and the association rate constants varied from 0.5 × 104 to 3.8 × 105 M−1∙s−1, depending on the reaction temperature and the cytoplasmic receptor preparation. Equilibrium dissociation constants were in 10−8–10−9 M range. Equilibrium dissociation constants, calculated from kinetic data (kd/ka), showed a marked temperature dependence, while those obtained by saturation analysis varied much less with the reaction temperature. Data obtained in these experiments were used to calculate some thermodynamic parameters of the binding of corticosterone to the cytoplasmic receptor. The enthalpy of dissociation was 101.5 and 79.4 kJ∙mol−1 and the entropy of dissociation was 200 and 280 J∙mol−1∙degree−1 for the crude cytoplasmic receptor and the ammonium sulfate precipitate, respectively. From the equilibrium dissociation constants, the enthalpy and entropy of the equilibrium binding was calculated. Polynomial fitting of Ka values versus 1/T yielded enthalpy (ΔH) values from −0.9 to −88.8 kJ∙mol−1, depending on the nature of the receptor preparation. Entropy values were negative for kinetically derived equilibrium association constants from the crude cytosol at all temperatures and for 0 and 15 °C for the precipitate. Entropy values were positive for Ka values obtained from kinetic rates at 25 and 35 °C and for Ka's calculated from saturation analysis. Further experiments with the precipitate confirmed our previous contention that the nasal gland cytoplasmic corticosterone receptor metabolized the bound ligand to 11-dehydrocorticosterone, though the receptor preparation was corticosterone specific. The following hydrodynamic parameters were obtained on the binding macromolecule: molecular weight, 316 000; s20,w, 8.0; Stokes radius (rs), 77.3 Å (1 Å = 0.1 nm); total frictional ratio (f/f0), 1.71. The labeled receptor preparation translocated to homologous nuclear binding sites following heat activation and, at the nuclear binding sites, the ligand was almost exclusively in its oxidized form. Measurement of the nuclear steroid–receptor complex by exchange assay with [3H]corticosterone confirmed the presence of nuclear binding sites. From these studies, it was concluded that the nasal gland of the duck contains specific, glucocorticoid-type corticosterone receptors and that the effector steroid is probably 11-dehydrocorticosterone or a critical mixture of these two steroids, with the oxidized form predominating.

Sign in / Sign up

Export Citation Format

Share Document