Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

1990 ◽  
Vol 68 (3) ◽  
pp. 616-621 ◽  
Author(s):  
Shinko Takagi ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Specific Activity ◽  
Binding Capacity ◽  
High Specific Activity ◽  
Relative Mass ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Therapeutic Effects ◽  
Inhibitory Effects

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass ≈ 50 000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 μCi/μg; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 × 109 to 1.74 × 109 M−1 for PTU and 1.79 × 109 M−1 for MMI), while they both decreased (p < 0.02) maximal binding capacity (from 0.36 ± 0.02 to 0.19 ± 0.02 pmol/mg protein for MMI and 0.17 ± 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with ≈25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats. Pretreatment of CNR for 1 h with 5 mM methyl methanethiosulfonate (an oxidant of thiol groups) abolished the inhibitory effects of PTU, MMI, or arsenite, but was not inhibitory in itself. From these studies it is concluded that (i) MMI and PTU could exert at least part of their therapeutic effects by inhibiting specific binding of L-T3 to its hepatic nuclear receptor; (ii) these inhibitory effects of MMI and PTU are likely due to an interaction with cysteine residues (some of which are not in a dithiol configuration) that are essential for T3 binding to its receptor; and (iii) binding of T3 is not inhibited by oxidation of receptor thiols to methyl dithiol groups.Key words: nuclear triiodothyronine (T3) receptor, methimazole, propylthiouracil, Scatchard analysis.

Thioredoxin and glutaredoxin enhance the binding of L-triiodothyronine to its hepatic nuclear receptors

1989 ◽  
Vol 67 (8) ◽  
pp. 477-480 ◽  
Author(s):  
Shinko Takagi ◽  
Ganesh B. Bhat ◽  
Brian C. W. Hummel ◽  
Paul G. Walfish
Keyword(s):  
Glutathione Reductase ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Thioredoxin Reductase ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Physiological Effects ◽  
Thioredoxin System

The influence of thioredoxin and glutaredoxin on binding of L-triiodothyronine (T3) to the rat hepatic nuclear T3 receptor was compared with that of the exogenous activator dithiothreitol. Specific [125I]T3 binding, the affinity constant, Ka, and the maximal binding capacity, MBC, were measured using whole nuclei, solubilized preparations of receptor, and chromatographed nuclear receptor. Both the thioredoxin system (thioredoxin, thioredoxin reductase, and NADPH) and the glutaredoxin system (glutaredoxin, glutathione reductase, glutathione, and NADPH) increased specific binding of T3 to nuclei, solubilized receptor, and chromatographed receptor significantly. Compared with the values obtained in the absence of added thiol (Ka = 1.6 ± 0.1 × 109 M−1 MBC = 1.7 ± 0.06 pM), the thioredoxin and glutaredoxin systems increased Ka by 147 and 112%, respectively, while decreasing MBC by 51 and 45%, respectively, when chromatographed receptor was used. The same tendency was observed with solubilized receptor. However, dithiothreitol increased Ka without affecting MBC when solubilized receptor was used. These results, the first demonstration of endogenous disulphide reductant systems enhancing binding of T3 to its receptor, suggest that the thioredoxin and (or) glutaredoxin systems may modulate the physiological effects of thyroid hormone.Key words: nuclear triiodothyronine (T3) receptor, thioredoxin, glutaredoxin, Scatchard analysis.

Hybridoma Screening by Antigen Capture: Capture or Sandwich ELISA in 96-Well Plates

2022 ◽  
Vol 2022 (1) ◽  
pp. pdb.prot103127
Author(s):  
Edward A. Greenfield
Keyword(s):  
Culture Supernatant ◽  
Detection Method ◽  
Specific Activity ◽  
Binding Capacity ◽  
Sandwich Elisa ◽  
High Specific Activity ◽  
Tissue Culture Supernatant ◽  
Capture Assay ◽  
Antigen Capture ◽  
Elisa Plate

In an antigen capture assay for hybridoma screening, the detection method identifies the presence of the antigen. Often this is achieved by labeling the antigen directly. In this assay, the polyvinyl chloride (PVC) wells of a high-binding-capacity ELISA plate are first coated with an affinity-purified rabbit anti-mouse immunoglobulin and then incubated with hybridoma tissue culture supernatant. Monoclonal antibodies in the supernatant are “captured” on the coated PVC surface and detected by screening with biotin- or histidine (His)–tagged antigen. The antigen can be labeled to a high specific activity and thus very little antigen is required for this procedure.

Changes in cytosolic 3,5,3′-tri-iodo-l-thyronine (T3) binding activity during administration of l-thyroxine to thyroidectomized rats: cytosolic T3-binding protein and its activator act as intracellular regulators for nuclear T3 binding

1989 ◽  
Vol 123 (1) ◽  
pp. 99-104 ◽  
Author(s):  
Y. Nishii ◽  
K. Hashizume ◽  
K. Ichikawa ◽  
T. Miyamoto ◽  
S. Suzuki ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Nuclear Receptor ◽  
Binding Capacity ◽  
Binding Protein ◽  
Binding Activity ◽  
Affinity Constant ◽  
Maximal Binding

ABSTRACT Changes in the amount of cytosolic 3,5,3′-tri-iodo-l-thyronine (T3)-binding protein (CTBP) and its activator during administration of l-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor. These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor. Journal of Endocrinology (1989) 123, 99–104

scholarly journals A new unextracted-sample radioimmunoassay method for hepatic endogenous nuclear l-tri-iodothyronine content. Validity of its use in determining nuclear receptor binding characteristics

1982 ◽  
Vol 208 (3) ◽  
pp. 641-649 ◽  
Author(s):  
Toshihiro Yagura ◽  
Paul G. Walfish
Keyword(s):  
Receptor Binding ◽  
Binding Capacity ◽  
Nuclear Extract ◽  
Affinity Constant ◽  
Lower Sensitivity ◽  
Scatchard Analysis ◽  
Binding Characteristics ◽  
Radioimmunoassay Method ◽  
True Values ◽  
Good Agreement

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-[125I]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.

Changes in serum somatomedin A and its binding to kidney membranes in growing rats

1981 ◽  
Vol 97 (3) ◽  
pp. 352-357 ◽  
Author(s):  
Naomi Hizuka ◽  
Kazue Takano ◽  
Kazuo Shizume ◽  
Yoko Hasumi
Keyword(s):  
Binding Capacity ◽  
Inverse Correlation ◽  
Close Correlation ◽  
Serum Levels ◽  
Affinity Constant ◽  
Scatchard Analysis ◽  
Receptor Sensitivity ◽  
Growing Rats ◽  
The Mean ◽  
Clear Change

Abstract. Changes in serum somatomedin A levels and [125I]somatomedin A binding to membrane fractions from kidney were studied in rats 1–80 days of age. The mean level of serum somatomedin A was 0.80 U/ml at birth and increased with age; at 80 days the mean level was 7.41 ± 0.67 U/ml. There was a close correlation between serum levels of somatomedin A and body weight. Labelled somatomedin A binding to membrane fractions from kidney was highest at birth and decreased with age up to 50 days. In Scatchard analysis of the data the affinity constant did not show a clear change with age, but the binding capacity decreased with age up to 30 days. An inverse correlation was observed between serum somatomedin A levels and labelled somatomedin A binding to membrane fractions from kidney. Compared to changes in circulating somatomedin A, the change in tissue binding was modest. This observation suggests that other circulating growth factors not measured by this radioreceptor assay or altered post-receptor sensitivity to somatomedins may be involved in growth.

scholarly journals Effects of Progesterone on Nuclear and Cytosol Steroid Receptor Levels in the Oestrogen-stimulated Uterus: Comparison of the Sheep and Mouse

1982 ◽  
Vol 35 (4) ◽  
pp. 403 ◽  
Author(s):  
GM Stone ◽  
C McCaffery ◽  
BG Miller
Keyword(s):  
Binding Site ◽  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Direct Effect ◽  
Dissociation Constants ◽  
Single Injection ◽  
Steroid Receptor ◽  
Scatchard Analysis ◽  
Mouse Uterus ◽  
Receptor Level

Methods for the measurement of nuclear receptors for oestradiol (Ez) and progesterone (P) in mouse uterus and sheep endometrium have been established. Scatchard analysis of nuclear receptors gave the following dissociation constants (nM) and binding site concentrations (pmol steroid/mg DNA): Ez receptor, mouse 3� 76 and 2� 68, sheep 2� 03 and 5� 37; P receptor, mouse 5� 77 and 2� 52, sheep 5 34 and 3� 58. The effects of a single injection of P on nuclear and cytosol levels of Ez and P receptors have been contrasted in these tissues from Ez-treated mice and sheep. In both species, P treatment resulted, in 30-120 min, in depletion of its own cytosol receptor and accumulation of its nuclear receptor. A significant reduction of cytosol E2 receptor was seen only in the mouse at 24 h. P decreased the nuclear Ez-receptor level in the mouse at 2-8 h, but had no such effect in the sheep. The results indicate that the anti-uterotrophic action of P in mouse uterus is caused by an early direct effect of P on nuclear Ez-receptor retention, and appear also to explain why P is not anti-uterotrophic in sheep uterus.

Study of the role of histidyl, tyrosyl, α- or ε-amino residues in the specific binding of 3,5,3'-triiodothyronine to rat liver nuclear receptors

1988 ◽  
Vol 117 (2) ◽  
pp. 159-165 ◽  
Author(s):  
Julius Brtko ◽  
Ján Knopp
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Biologically Active ◽  
Amino Groups ◽  
Trinitrobenzenesulfonic Acid ◽  
Receptor Molecule

Abstract. The role of histidyl, tyrosyl, α-or ε-amino residues of rat liver nuclear receptors for the specific binding of T3 was studied by chemically modifying the receptor molecule. The kinetics of the formation of N-carbethoxyhistidyl derivative from histidyl groups of nuclear receptors by diethylpyrocarbonate was examined. The modified nuclear receptor fraction was separated from diethylpyrocarbonate by gel filtration and the T3 binding parameters (Ka and MBC) at pH 8.0 were tested by Scatchard plot analysis. At 0.1 mmol/l diethylpyrocarbonate, the value of Ka was significantly (P < 0.01) decreased without any change in maximal binding capacity (MBC). The modification of α- or ε-amino groups of nuclear receptors by excess of trinitrobenzenesulfonic acid, 6.3 mmol/l at pH 8.5, resulted in a 4-fold increase in MBC of T3 specific binding without any change in Ka. In addition, acetylation of tyrosyl residues of nuclear receptors at pH 7.5 with an excess of 24 mmol/l N-acetylimidazole was performed. No changes in nuclear receptor Ka or MBC were observed after N-acetylimidazole treatment. Histidine and/or amino groups of the receptor molecule seem to hold a key position in the generation of the biologically active T3-nuclear receptor complex in the rat liver.

scholarly journals ANTIBODY PRODUCTION IN VITRO

1968 ◽  
Vol 127 (4) ◽  
pp. 661-674 ◽  
Author(s):  
Gilmour Harris
Keyword(s):  
Antibody Production ◽  
Small Proportion ◽  
Specific Activity ◽  
Specific Antigen ◽  
High Specific Activity ◽  
Inhibitory Effects ◽  
Spleen Cells ◽  
Dividing Cells ◽  
Stimulation Of

The effect of high specific activity thymidme-3H on proliferation and antibody production, using the hemolytic plaque-forming technique, by spleen cell suspensions in vitro from rabbits killed after a boost of SRC's has been studied. High specific activity thymidine-3H inhibited the proliferative ay well as the antibody response to antigen, and it was conduded that this was the result of the incorporation of radioactive 3H into the nuclei of dividing cells which were synthesizing antibody in these cultures. The stimulation of the rate of DNA synthesis by specific antigen could be correlated with the ability of antigen to maintain antibody production, as measured by the specific hemolytic plaque-forming technique, above levels found in control cultures, incubated without antigen. Radioautographic studies of PFC's in vitro showed that the majority of the cells arose from the DNA-synthesizing population of cells in these cultures, confirming the conclusions from the results of the inhibitory effects of high specific-activity thymidine-3H on PFC's. It was found that these PFC's, labeling with thymidine-14C, formed only a small proportion of all the cells labeled in this way in these cultures. The postulation was made that antigen, in vitro, provided a stimulation for cell proliferation in the responsive population of rabbit spleen cells, but that only a small proportion of this population could be induced by antigen to synthesize antibody.

scholarly journals Studies on the biosynthesis of quinones in fungi. Incorporation of 6-methylsalicylic acid into fumigatin and related compounds in Aspergillus fumigatus I.M.I. 89353

1965 ◽  
Vol 97 (2) ◽  
pp. 321-332 ◽  
Author(s):  
NM Packter
Keyword(s):  
Aspergillus Fumigatus ◽  
Specific Activity ◽  
High Specific Activity ◽  
The Other ◽  
Inhibitory Effects ◽  
Orsellinic Acid ◽  
Aromatic Components ◽  
Related Compounds

1. Orsellinic acid has been detected as a metabolite of Aspergillus fumigatus. 2. The other principal aromatic components of the medium are fumigatin and the quinol, fumigatol. Fumigatol has been shown to be dihydrofumigatin after oxidation to the quinone followed by acetylation. 3. (14)C-labelled 6-methylsalicylic acid can be hydroxylated in A. fumigatus to form orsellinic acid and decarboxylated to give m-cresol. 4. (14)C-labelled 6-methylsalicylic acid is incorporated into fumigatin and fumigatol (1.0-1.5%), but the conversion does not occur until about 2-3 days after supplementation of the medium. At this stage of growth, the organism has already synthesized approx. 20 times as much fumigatol as fumigatin and this ratio is reflected in the much lower specific activity of the quinol. 5. Supplementation of the medium with either orsellinic acid or orcinol, in addition to (14)C-labelled 6-methylsalicylic acid, greatly decreases the latter's incorporation into fumigatin. At the same time, the cultures containing these substances are stimulated to produce another quinone with relatively high specific activity. 6. 6-Methylsalicylic acid has not been detected in the medium of normal cultures. The results indicate that 6-methylsalicylic acid itself is not a direct precursor of fumigatin and fumigatol but that it is converted into a true intermediate, probably after hydroxylation to orsellinic acid. 7. Supplementation of the medium with 6-methylsalicylic acid (15-25mg./200ml.) greatly affects the metabolism of A. fumigatus. Growth is inhibited and the synthesis of fumigatol is markedly depressed in these cultures. The inhibitory effects may possibly be related in some way to the production of m-cresol.

Water retention after oral chlorpropamide is associated with an increase in renal papillary arginine vasopressin receptors

1995 ◽  
Vol 132 (4) ◽  
pp. 459-464 ◽  
Author(s):  
J Hensen ◽  
M Haenelt ◽  
P Gross
Keyword(s):  
Arginine Vasopressin ◽  
Water Retention ◽  
Specific Activity ◽  
High Specific Activity ◽  
Plasma Osmolality ◽  
Dependent Diabetes Mellitus ◽  
Scatchard Analysis ◽  
Sprague Dawley ◽  
Water Excretion ◽  
Vasopressin Receptors

Hensen J, Haenelt M, Gross P. Water retention after oral chlorpropamide is associated with an increase in renal papillary arginine vasopressin receptors. Eur J Endocrinol 1995;132:459–64. ISSN 0804–4643 Chlorpropamide (CP), a sulfonylurea used for treatment of non-insulin dependent diabetes mellitus, is known to potentiate the antidiuretic action of arginine vasopressin (AVP), predisposing to hyponatremia. It has been suggested that CP acts directly on the antidiuretic vasopressin receptor. Detailed studies on the influence of CP on the AVP receptor, however, have been hampered by lack of a suitable radioligand. Using a newly developed radioiodinated derivative of AVP with high specific activity and high affinity for the AVP V2-receptor (125I-[8-(p-(OH)-phenylpropionyl)]-LVP), we studied the role of AVP V2-receptors in CP-induced water retention. Male-Sprague-Dawley rats were treated orally with 40 mg CP/day or placebo for 7 days, after which Scatchard analysis was performed using membranes prepared from homogenized renal papilla. After oral water load, CP-treated rats but not control rats showed a significant decrease in plasma osmolality (289 ±2.2 to 284±0.8 mosmol/kg, p < 0.05). The Kd was 0.69 ± 0.16 nmol/l in controls and 0.70 ± 0.12 nmol/l after CP treatment (NS); Bmax was 129 ± 5.3 nmol/kg protein in controls (N = 8). Chlorpropamide significantly increased receptor density (Bmax) to 167±8.4 nmol/kg protein (N = 8) (p<0.05). Plasma AVP did not change significantly during CP treatment. These data show for the first time that CP in vivo increases the density of AVP V2 receptors without altering plasma AVP. This is associated with an impairment in water excretion. Our experiments and recent reports on CP-induced inhibition of AVP binding suggest that the AVP augmentation effect of CP is related to interference of CP with the AVP V2-receptor. Johannes Hensen, Department of Medicine I, Division of Endocrinology and Metabolism, Krankenhausstr. 12, 91054 Erlangen, Germany

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