Effects of pH and ionic strength on the binding of L-tri-iodothyronine to the solubilized nuclear receptor.
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ABSTRACT:
SUBMITTER: Wilson BD
PROVIDER: S-EPMC1152936 | biostudies-other | 1985 Dec
REPOSITORIES: biostudies-other
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Project description:Endogenous l-tri-iodothyronine content in an hepatic nuclear extract was measured by a new unextracted-sample radioimmunoassay method using 8-anilinonaphthalene-1-sulphonic acid to inhibit the l-[(125)I]tri-iodothyronine binding to the nuclear l-tri-iodothyronine receptor within the extract. For this method, the lower sensitivity limit was 3.125 pg/tube, the recovery of added l-tri-iodothyronine was 90-120%, and the between-assay coefficient of variation was 10%. The amount of endogenous l-tri-iodothyronine was 10-40 pg/0.2 ml of hepatic nuclear extract from euthyroid rats, compared with less than 3.125 pg/0.2 ml from thyroidectomized rats. The results obtained by this new method were compared with a Sephadex G-25 column extracted-sample radioimmunoassay method and showed a good agreement. The values for the endogenous l-tri-iodothyronine content were utilized to correct for the l-tri-iodothyronine concentration within the binding assay mixture in order to accurately determine by Scatchard analysis the binding characteristics of the nuclear l-tri-iodothyronine receptor. The validity of the correction for endogeneous l-tri-iodothyronine was demonstrated by using a nuclear extract from a thyroidectomized rat which was preincubated with a small known amount of l-tri-iodothyronine before determining the nuclear l-tri-iodothyronine receptor binding characteristics. When the Scatchard plots were corrected for the preincubated dose, the results obtained were similar to true values, but they were falsely lower when not corrected. It is concluded that the necessity and validity of using endogenous l-tri-iodothyronine corrections in the Scatchard analytical computations of the nuclear l-tri-iodothyronine receptor binding characteristics has been demonstrated, being particularly more important for affinity constant than maximum binding capacity.
| S-EPMC1154013 | biostudies-other
Project description:The dynamics of the induction of nuclear tri-iodothyronine receptors and mitochondrial alpha-glycerophosphate dehydrogenase were studied in rat liver after a single injection of tri-iodothyronine. The maximal binding capacity (C(max.)) and association constant (K(a)) of the nuclear receptors were determined by Scatchard analyses with and without correction for the endogenous tri-iodothyronine measured by radioimmunoassay. The administration of tri-iodothyronine induced sequential increases in the concentration of nuclear receptors and alpha-glycerophosphate dehydrogenase activity in the liver. The nuclear-receptor concentration was increased to 2.5 times that in the hypothyroid rat 1 day after the administration of hormone, and then decreased, with a half-life of about 2 days. alpha-Glycerophosphate dehydrogenase activity changed in parallel with the nuclear-receptor concentration, showing a delayed response. The total amount of non-histone protein in the liver was significantly increased 3 days after the administration. It seems likely therefore that the tri-iodothyronine-induced increase in nuclear-receptor concentration is responsible, at least in part, for the induction of this enzyme. The possibility is also suggested that nuclear receptors may be one of the non-histone proteins selectively synthesized at an early stage of the hormonal stimulation. Throughout the time course, the K(a) values of the nuclear receptors for tri-iodothyronine remained unchanged, when corrected for endogenous tri-iodothyronine bound to the non-histone proteins, although they were apparently changed when the correction was not made. The results obtained provide further evidence for hormonal modulation of the nuclear receptors which is closely linked with the hormonal effect.
| S-EPMC1161318 | biostudies-other
Project description:The effect of long-term administration of small amounts of tri-iodothyronine was examined on the nuclear tri-iodothyronine receptors in rat liver. The maximal binding capacity (C(max.)) and association constant (K(a)) of the receptors were determined in thyroidectomized rats given vehicle alone (group A), 2mug of tri-iodothyronine/100g body wt. (group B) or 7mug of tri-iodothyronine/100g body wt. (group C) for 2 weeks. Scatchard analyses with correction for the amount of endogenous tri-iodothyronine revealed that C(max.) values per g of liver were increased to 1.5 and 2.7 times the control value in groups B and C respectively. Since concentrations of DNA per g of liver were significantly increased in the two groups of hormone-treated rats, C(max.) values per mg of DNA were nearly the same in group B, but still increased significantly in group C compared with group A. K(a) values remained unchanged in all three groups of animals. Mitochondrial alpha-glycerophosphate dehydrogenase activity was 9.6 and 28.7 times as high in groups B and C, respectively, as in group A. Concentrations of endogenous tri-iodothyronine bound to non-histone protein were substantially increased in groups B and C, although concentrations of serum tri-iodothyronine remained rather low. The results obtained indicate that the long-term administration of tri-iodothyronine in small doses induces an increase in the nuclear receptors associated with increased DNA with and without accompanying a relative increase in the receptor concentration in thyroidectomized rats. Also the hormonal effect is closely related to the total number of the nuclear receptors and the concentrations of nuclear tri-iodothyronine bound to the receptors rather than the serum tri-iodothyronine concentrations.
| S-EPMC1161684 | biostudies-other
Project description:In order to quantify the interactions between molecules of biological interest, the determination of the dissociation constant (K d) is essential. Estimation of the binding affinity in this way is routinely performed in "favorable" conditions for macromolecules. Crucial data for ligand-protein binding elucidation is mainly derived from techniques (e.g., macromolecular crystallography) that require the addition of high concentration of salts and/or other additives. In this study we have evaluated the effect of temperature, ionic strength, viscosity, and hydrophobicity on the K d of three previously characterized protein-ligand systems, based on variation in their binding sites, in order to provide insight into how these often overlooked unconventional circumstances impact binding affinity. Our conclusions are as follows: (1) increasing solvent viscosity in general is detrimental to ligand binding, (2) moderate increases in temperature have marginal effects on the dissociation constant, and (3) the degree of hydrophobicity of the ligand and the binding site determines the extent of the influence of cosolvents and salt concentration on ligand binding affinity.
| S-EPMC4137368 | biostudies-literature
Project description:Reversible interactions between DNA and silica are utilized in the solid phase extraction and purification of DNA from complex samples. Chaotropic salts commonly drive DNA binding to silica but inhibit DNA polymerase amplification. We studied DNA adsorption to silica using conditions with or without chaotropic salts through bulk depletion and quartz crystal microbalance (QCM) experiments. While more DNA adsorbed to silica using chaotropic salts, certain buffer conditions without chaotropic salts yielded a similar amount of eluted DNA. QCM results indicate that under stronger adsorbing conditions the adsorbed DNA layer is initially rigid but becomes viscoelastic within minutes. These results qualitatively agreed with a mathematical model for a multiphasic adsorption process. Buffer conditions that do not require chaotropic salts can simplify protocols for nucleic acid sample preparation. Understanding how DNA adsorbs to silica can help optimize nucleic acid sample preparation for clinical diagnostic and research applications.
| S-EPMC3766398 | biostudies-literature
Project description:Although, glyphosate (N-(phosphonomethyl) glycine) is one of the most widely used herbicides in the world, its interaction with poorly crystalline iron oxides, such as ferrihydrite, is not well studied. In this research, we examined the adsorption of glyphosate onto ferrihydrite using infrared spectroscopy (FT-IR), electron paramagnetic resonance spectroscopy (EPR), adsorption kinetic models and adsorption isotherm models. The effect of pH and sodium chloride concentration on the adsorption of glyphosate onto ferrihydrite as well as the effect of extractors (CaCl2 0.010 mol L-1 and Mehlich) on the desorption of glyphosate were also evaluated. There are two important findings described in this work. First, 84% of adsorbed glyphosate strongly interacted to ferrihydrite as an inner-sphere complex and phosphate and amine groups are involved in this interaction. Second, an increase of sodium chloride salt concentration increased the adsorption of glyphosate onto ferrihydrite. The non-linear Langmuir model and pseudo second order model showed a good agreement of theoretical limit of glyphosate adsorbed onto ferrihydrite, 54.88 µg mg-1 and 48.8 µg mg-1, respectively. The adsorption of glyphosate onto ferrihydrite decreased when the pH increased. Under the conditions used in this work, EPR spectra did not show dissolution of ferrihydrite. Surface area, pore volume and pHpzc of ferrihydrite decreased after adsorption of glyphosate.
| S-EPMC6743134 | biostudies-literature
Project description:The binding performance of dissolved organic matters (DOM) plays a critical role in the migration, diffusion and removal of various residual pollutants in the natural water environment. In the current study, four typical DOMs (including bovine serum proteins BSA (proteins), sodium alginate SAA (polysaccharides), humic acid HA and fulvic acid FA (humus)) are selected to investigate the binding roles in zwitterionic tetracycline (TET) antibiotic under various ionic strength (IS = 0.001-0.1 M) and pH (5.0-9.0). The dialysis equilibration technique was employed to determine the binding concentrations of TET, and the influence of IS and pH on binding performance was evaluated via UV-vis spectroscopy, total organic carbon (TOC), and Excitation-Emission-Matrix spectra (EEM), zeta potentials and molecule size distribution analysis. Our results suggested that carboxyl and phenolic hydroxyl were identified as the main contributors to TET binding based on the fourier transform infrared spectroscopy (FTIR) analysis, and the binding capability of four DOMs followed as HA > FA » BSA > SAA. The biggest binding concentrations of TET by 10 mg C/L HA, FA, BSA and SAA were 0.863 μM, 0.487 μM, 0.084 μM and 0.086 μM, respectively. The higher binding capability of HA and FA is mainly attributed to their richer functional groups, lower zeta potential (HA/FA = -15.92/-13.54 mV) and the bigger molecular size (HA/FA = 24668/27750 nm). IS significantly inhibits the binding interaction by compressing the molecular structure and the surface electric double layer, while pH had a weak effect. By combining the Donnan model and the multiple linear regression analysis, a modified Karickhoff model was established to effectively predict the binding performance of DOM under different IS (0.001-0.1 M) and pH (5.0-9.0) conditions, and the R2 of linear fitting between experiment-measured logKDOC and model-calculated logKOC were 0.94 for HA and 0.91 for FA. This finding provides a theoretical basis for characterizing and predicting the binding performance of various DOMs to residual micropollutants in the natural water environment.
| S-EPMC9488040 | biostudies-literature
Project description:Iodothyronine deiodinases (Dios) are important selenoproteins that control the concentration of the active thyroid hormone (TH) triiodothyronine through regioselective deiodination. The X-ray structure of a truncated monomer of Type III Dio (Dio3), which deiodinates TH inner rings through a selenocysteine (Sec) residue, revealed a thioredoxin-fold catalytic domain supplemented with an unstructured ?-loop. Loop dynamics are driven by interactions of the conserved Trp207 with solvent in multi-microsecond molecular dynamics simulations of the Dio3 thioredoxin(Trx)-fold domain. Hydrogen bonding interactions of Glu200 with residues conserved across the Dio family anchor the loop's N-terminus to the active site Ser-Cys-Thr-Sec sequence. A key long-lived loop conformation coincides with the opening of a cryptic pocket that accommodates thyroxine (T4) through an I?Se halogen bond to Sec170 and the amino acid group with a polar cleft. The Dio3-T4 complex is stabilized by an I?O halogen bond between an outer ring iodine and Asp211, consistent with Dio3 selectivity for inner ring deiodination. Non-conservation of residues, such as Asp211, in other Dio types in the flexible portion of the loop sequence suggests a mechanism for regioselectivity through Dio type-specific loop conformations. Cys168 is proposed to attack the selenenyl iodide intermediate to regenerate Dio3 based upon structural comparison with related Trx-fold proteins.
| S-EPMC7506546 | biostudies-literature
Project description:Gestational Diabetes Mellitus (GDM) is characterized by abnormal maternal D-glucose metabolism and altered insulin signaling. Dysregulation of thyroid hormones (TH) tri-iodethyronine (T3) and L-thyroxine (T4) Hormones had been associated with GDM, but the physiopathological meaning of these alterations is still unclear. Maternal TH cross the placenta through TH Transporters and their Deiodinases metabolize them to regulate fetal TH levels. Currently, the metabolism of TH in placentas with GDM is unknown, and there are no other studies that evaluate the fetal TH from pregnancies with GDM. Therefore, we evaluated the levels of maternal TH during pregnancy, and fetal TH at delivery, and the expression and activity of placental deiodinases from GDM pregnancies. Pregnant women were followed through pregnancy until delivery. We collected blood samples during 10-14, 24-28, and 36-40 weeks of gestation for measure Thyroid-stimulating hormone (TSH), Free T4 (FT4), Total T4 (TT4), and Total T3 (TT3) concentrations from Normal Glucose Tolerance (NGT) and GDM mothers. Moreover, we measure fetal TSH, FT4, TT4, and TT3 in total blood cord at the delivery. Also, we measured the placental expression of Deiodinases by RT-PCR, western-blotting, and immunohistochemistry. The activity of Deiodinases was estimated quantified rT3 and T3 using T4 as a substrate. Mothers with GDM showed higher levels of TT3 during all pregnancy, and an increased in TSH during second and third trimester, while lower concentrations of neonatal TT4, FT4, and TT3; and an increased TSH level in umbilical cord blood from GDM. Placentae from GDM mothers have a higher expression and activity of Deiodinase 3, but lower Deiodinase 2, than NGT mothers. In conclusion, GDM favors high levels of TT3 during all gestation in the mother, low levels in TT4, FT4 and TT3 at the delivery in neonates, and increases deiodinase 3, but reduce deiodinase 2 expression and activity in the placenta.
| S-EPMC7685482 | biostudies-literature
Project description:Repair of G·T mismatches arising from deamination of 5-methylcytosine (m(5)C) involves excision of thymine and restoration of a G·C pair via base excision repair (BER). Thymine DNA glycosylase (TDG) is one of two mammalian enzymes that can specifically remove thymine from G·T mispairs. While TDG can excise other bases, it maintains stringent specificity for a CpG context, suggesting deaminated m(5)C is an important biological substrate. Recent studies reveal TDG is essential for embryogenesis; it helps to maintain an active chromatin complex and initiates BER to counter aberrant de novo CpG methylation, which may involve excision of actively deaminated m(5)C. The relatively weak G·T activity of TDG has been implicated in the hypermutability of CpG sites, which largely involves C?T transitions arising from m(5)C deamination. Thus, it is important to understand how TDG recognizes and process substrates, particularly G·T mispairs. Here, we extend our detailed studies of TDG by examining the dependence of substrate binding and catalysis on pH, ionic strength, and temperature. Catalytic activity is relatively constant for pH 5.5-9, but falls sharply for pH>9 due to severely weakened substrate binding, and, potentially, ionization of the target base. Substrate binding and catalysis diminish sharply with increasing ionic strength, particularly for G·T substrates, due partly to effects on nucleotide flipping. TDG aggregates rapidly and irreversibly at 37°C, but can be stabilized by specific and nonspecific DNA. The temperature dependence of catalysis reveals large and unexpected differences for G·U and G·T substrates, where G·T activity exhibits much steeper temperature dependence. The results suggest that reversible nucleotide flipping is much more rapid for G·T substrates, consistent with our previous findings that steric effects limit the active-site lifetime of thymine, which may account for the relatively weak G·T activity. Our findings provide important insight into catalysis by TDG, particularly for mutagenic G·T mispairs.
| S-EPMC3084331 | biostudies-literature