Interference with thyrotropin receptor antibody determination by a spuriously occurring anti-bovine TSH antibody

1986 ◽  
Vol 112 (2) ◽  
pp. 197-203 ◽  
Author(s):  
Makoto Iitaka ◽  
Toshinori Tanikawa ◽  
Yoshiki Sakatsume ◽  
Morifumi Yanagisawa ◽  
Yoshihito Hara ◽  
...  
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Binding Activity ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pepsin Digestion ◽  
Thyrotropin Receptor Antibody ◽  
Maximum Binding Capacity ◽  
Antibody Determination ◽  
Bovine Tsh

Abstract. Abnormally negative values of thyrotropin binding inhibitor immunoglobulin (TBII) were found in the sera from a patient with Graves' disease. This was due to the presence of potent bovine TSH (bTSH) binding activity in the sera. This activity was demonstrated to be in immunoglobulin G (IgG) with a λ light chain isotype, which was shown to have an affinity for bTSH with a Ka value of 3.5 × 1010 m−1 and a maximum binding capacity of 1.1 × 10−14m/mg IgG. F(ab')2 fragments obtained through pepsin digestion from the patient's IgG retained bTSH binding activity. [125I] bTSH binding to this IgG was inhibited by the TSH receptor. The inhibition was not completely competitive, suggesting the presence of different binding sites for this IgG and the TSH receptor on the TSH molecule. This IgG, however, could not bind labelled human TSH (hTSH). Since neither TSH nor other pituitary derivatives had ever been given to the patient, this bTSH binding activity was considered to be due to a spuriously occurring anti-bTSH antibody.

Distribution of opioid receptors in canine small intestine: implications for function

1989 ◽  
Vol 256 (6) ◽  
pp. G966-G974 ◽  
Author(s):  
H. D. Allescher ◽  
S. Ahmad ◽  
P. Kostka ◽  
C. Y. Kwan ◽  
E. E. Daniel
Keyword(s):  
Small Intestine ◽  
Binding Sites ◽  
Myenteric Plexus ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Opiate Receptors ◽  
Circular Muscle ◽  
Maximum Binding Capacity ◽  
Opiate Binding ◽  
Subtype Selective

Distribution of the binding sites for [3H]diprenorphine, a non-selective opiate ligand, was studied in membrane fractions from longitudinal muscle/myenteric plexus and circular muscle containing deep muscular plexus. [3H]saxitoxin was used as a marker for neuronal plasma membranes and 5'-nucleotidase as a marker for smooth muscle plasma membranes. Saxitoxin binding correlated strongly with diprenorphine binding, but 5'-nucleotidase correlated poorly with diprenorphine or saxitoxin binding in these fractions. Opiate binding sites in membranes of myenteric and deep muscular plexus were of high affinity (Kd = 0.12 and 0.18 nM, respectively) with maximum binding capacity of 400 and 500 fmol/mg protein, respectively. Competition experiments using subtype-selective opiate ligands indicated that all three subtypes of opiate receptors were present in the same ratio of 40-45% mu-subtypes, 40-45% delta-subtypes, and 10-15% kappa-subtypes on both plexuses. Opiate receptors of canine small intestine, therefore, are located primarily or exclusively on nerves with similar distributions in nerve membranes containing only axonal varicosities (deep muscular plexus) as in those containing neurons, dendrites, and varicosities (myenteric plexus).

Uptake of 3,5,3′-l-tri-iodothyronine in human erythrocytes

1989 ◽  
Vol 121 (3) ◽  
pp. 585-591 ◽  
Author(s):  
K. Yamauchi ◽  
R. Horiuchi ◽  
H. Takikawa
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Human Erythrocytes ◽  
The Other ◽  
Transport Pathway ◽  
High Affinity ◽  
High Affinity Site ◽  
Maximum Binding Capacity ◽  
Energy Dependent ◽  
Dependent Pathway

ABSTRACT The mechanisms of 3,5,3′-l-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59·2±17·8 nmol/l; maximum binding capacity, 344·3 ± 95·5 fmol/μg protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3»thyroxine > 3,5,3′-d-tri-iodothyronine), energydependent and dominant at 15 °C; the other was not displaced by unlabelled T3 and was energyindependent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 °C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 °C and accounted for 83% of total T3 uptake of human erythrocytes. Journal of Endocrinology (1989) 121, 585–591

Bovine thyrotropin receptor cDNA is characterized by full-length and truncated transcripts

1997 ◽  
Vol 18 (2) ◽  
pp. 101-112 ◽  
Author(s):  
D W Silversides ◽  
A Houde ◽  
J-F Ethier ◽  
J G Lussier
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Transmembrane Domain ◽  
Full Length ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Pcr Cloning ◽  
Specific Expression ◽  
Terminal Domain ◽  
Bovine Tsh

ABSTRACT The complete coding sequence for the bovine thyrotropin (TSH) receptor was derived using a modified PCR cloning strategy. The bovine thyrotropin receptor conforms to the pattern of receptor interacting with membrane-bound G-protein already established in other species for TSH and gonadotropins receptors. The cDNA for the bovine TSH receptor consists of an open reading frame 2289 nucleotides in length, corresponding to a protein of 763 amino acids (estimated molecular mass of 86·4 kDa) which includes a 20 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 417 amino acids with 5 potential N-linked glycosylation sites, a transmembrane domain (265 amino acids) consisting of 7 putative membrane α-helix spanning segments, and an intracytoplasmic COOH-terminal domain (82 amino acids). The bovine TSH receptor is one amino acid less than the corresponding sequence in dog, human, rat and mouse. Cysteine residues (n=22) were conserved when compared with other TSH receptors. Three potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. As with other members of this receptor family, alternative splicing was observed. A transcribed but truncated TSH receptor of 1769 nucleotides was demonstrated, lacking half of the V segment of the transmembrane domain up to the COOH-terminal domain of the full length TSH receptor. Additionally, alternative transcriptional start sites were observed. Northern blot analysis using a probe (1170 bp) spanning part of the extracellular domain up to the first loop of the transmembrane domain showed specific expression in the bovine thyroid gland with major transcripts of 9·3 and 4·3 kb, and a minor transcript of 3·8 kb being detected.

PROGESTERONE-BINDING GLOBULIN AND TESTOSTERONE-BINDING ACTIVITY IN GUINEA PIG SERUM DURING PREGNANCY: RELATIONSHIP TO PROGESTERONE AND OESTROGENS

1976 ◽  
Vol 81 (2) ◽  
pp. 367-378 ◽  
Author(s):  
O. A. Lea ◽  
A. Bessesen ◽  
K. F. Støa
Keyword(s):  
Guinea Pig ◽  
Binding Sites ◽  
Binding Capacity ◽  
Post Partum ◽  
Equilibrium Dialysis ◽  
Binding Activity ◽  
Maximal Level ◽  
Sharp Drop ◽  
Endogenous Oestrogen ◽  
Competitive Protein Binding

ABSTRACT Progesterone levels in serum have been determined throughout pregnancy in guinea-pigs by a competitive protein-binding technique, using pregnant guinea pig plasma protein as binding agent. The concentrations of this protein (progesterone-binding globulin, PBG) as well as the testosteronebinding activity (TBA) have been quantitated by means of equilibrium dialysis. In order to correlate these parameters to the endogenous oestrogen production, the urinary oestrogen excretion was recorded. The concentration of progesterone, PBG and TBA showed a sharp rise on days 14–18 of gestation, reaching a maximal level on days 30–44. The progesterone level thereafter declined significantly towards parturition, followed by a sharp drop post partum. PBG and TBA followed a similar course, the decline towards parturition, however, being non significant. Neither progesterone, PBG nor TBA were significantly correlated to the urinary oestrogen excretion. The concentration of PGB was remarkably high, amounting to 1–2 × 10-5 m during most of the guinea pig pregnancy. This binding capacity generally exceeded the endogenous progesterone concentration by a factor of 20, as calculated on a molar basis. A mean of 64 % of the binding sites was available for testosterone binding, corresponding to a free binding capacity of 400 μg testosterone per 100 ml.

THYROID HORMONE-BINDING INTERACTIONS IN CYTOSOL OF RAT ANTERIOR PITUITARY

1977 ◽  
Vol 85 (2) ◽  
pp. 256-266 ◽  
Author(s):  
Valerie Anne Galton
Keyword(s):  
Thyroid Hormone ◽  
Binding Sites ◽  
Anterior Pituitary ◽  
Binding Capacity ◽  
Relative Effectiveness ◽  
Binding Activity ◽  
Rat Tissues ◽  
Hormone Binding ◽  
Cytosol Fraction ◽  
Binding Interactions

ABSTRACT Thyroxine (T4) and triiodothyronine (T3)-binding interactions in preparations of rat anterior pituitary gland have been studied. T4 is bound primarily to extranuclear binding sites located in the cytosol fraction of the cell. These sites have a medium affinity for T4: Ka = 2.5 × 108 1/mol and a maximum binding capacity (MBC) of 1.15 pmol/mg tissue (wet weight). Binding of T3 to these sites is minimal. The extent of binding of T4 is influenced by the pH of the system and the temperature of incubation. The relative effectiveness of T4 analogues in displacing bound T4 is tetrac > T4 > triac > D-T4 > T3. Similar T4-binding sites are present in other rat tissues, but in all except serum, binding activity is lower than in the pituitary. T4-binding by serum contaminating the pituitary preparations contributed only partially to the total activity observed. Concomitant assessment of T4-binding activity and T4 metabolism in pituitary homogenates prepared at different pH values indicated an inverse relationship between the two processes. The possible role of thyroid hormone binding in cytosol in influencing the intracellular distribution of thyroid hormones is discussed.

RECEPTOR HETEROGENEITY IN THE HUMAN THYROID: DIFFERENCES BETWEEN THYROTROPHIN BINDING SITES IN MEMBRANE AND NUCLEAR FRACTIONS

1981 ◽  
Vol 91 (1) ◽  
pp. 163-173 ◽  
Author(s):  
S. W. MANLEY ◽  
J. R. BOURKE
Keyword(s):  
Binding Sites ◽  
Thyroid Tissue ◽  
Binding Activity ◽  
Human Thyroid ◽  
Affinity Binding ◽  
Binding Properties ◽  
Human Thyroid Tissue ◽  
Triton X ◽  
Scatchard Plots ◽  
Bovine Tsh

Binding of 125I-labelled bovine TSH to crude membrane fractions of human thyroid tissue was a saturable, hormonally specific process which yielded non-linear Scatchard plots with limiting affinities of approximately 109 and 107l/mol. Binding activity in membranes was soluble in Triton X-100, was inhibited specifically by immunoglobulins from patients with Graves's disease, and was increased by the beta-blocking drug, propranolol. In contrast, purified nuclear preparations showed a predominance of lower affinity binding, and their binding activity was insoluble in Triton and insensitive to immunoglobulins from patients with Graves's disease and to propranolol. Tryptic digestion liberated only low affinity binding activity from membranes or nuclei. It was concluded that human thyroid tissue contains independent classes of TSH-binding sites, which differ in their chemical, immunological and hormone-binding properties.

Erythropoietin binding sites in human foetal tissues

1987 ◽  
Vol 116 (4) ◽  
pp. 561-567 ◽  
Author(s):  
F. Pekonen ◽  
K. Rosenlöf ◽  
E.-M. Rutanen ◽  
F. Fyhrquist
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Recombinant Dna ◽  
Binding Capacity ◽  
Specific Binding ◽  
First Trimester ◽  
Binding Activity ◽  
Renin Substrate ◽  
Human Erythropoietin ◽  
Foetal Liver

Abstract. Using 125I labelled recombinant DNA human erythropoietin (EP), we have explored the presence and properties of EP binding sites in foetal human tissues. The EP binding site is present in the foetal liver already during the first trimester of pregnancy. The binding site has an equilibrium association constant of 4.1–6.2 × 109 1/mol and is specific for EP. The cross-reactivities of FSH, TSH, hCG, insulin and renin substrate were less than 0.01%. The EP binding capacity of foetal liver was 5.4–16 fmol/mg membrane protein. In foetal lung tissue, a slight EP binding activity was observed, whereas foetal spleen, muscle, brain, thyroid and placental tissues were virtually devoid of EP binding capacity. The same level of binding was reached at 37°C in 1 h and at 4°C in 24 h. The binding was pH-dependent with maximal specific binding at pH 7.7. SDS-PAGE gel electrophoresis analysis of covalently cross-linked 125I-EP to foetal liver membranes suggested that the EP binding site was composed of two subunits with an apparent mol wt of 41000 and 86000 dalton, respectively.

scholarly journals Affinity-labelling of the thyrotropin receptor. Characterization of the photoactive ligand

1985 ◽  
Vol 225 (3) ◽  
pp. 753-760 ◽  
Author(s):  
P R Buckland ◽  
R D Howells ◽  
C R Rickards ◽  
B Rees Smith
Keyword(s):  
Binding Activity ◽  
Tsh Receptor ◽  
Thyrotropin Receptor ◽  
Disulphide Bridge ◽  
Receptor Complexes ◽  
Receptor Binding Activity ◽  
Affinity Labelling ◽  
A Subunit ◽  
B Subunits

Thyrotropin (TSH) has been coupled to the photoactive heterobifunctional reagent N-hydroxysuccinimidyl 4-azidobenzoate (HSAB) and the properties of the product (HSAB-TSH) investigated. Preparations of HSAB-TSH containing two molecules of HSAB per molecule of TSH were used in most experiments and these preparations retained about 40% of the original receptor-binding activity of the TSH. HSAB-TSH could be labelled with 125I and cross-linked to porcine and human TSH receptors. Analysis of the cross-linked complexes indicated that the receptors consisted of two subunits (designated A and B) linked by a disulphide bridge. In the case of the human TSH receptor, the A- and B-subunits had approximate Mr values of 50 000 and 30 000 respectively, whereas the Mr values for porcine TSH-receptor A- and B-subunits were approx. 45 000 and 25 000 respectively. Only the A subunit was cross-linked to TSH. Comparison of the effects of trypsin and mercaptoethanol on the TSH-TSH-receptor complexes suggested that the trypsin cleavage point on the A-subunit was at a point close to the disulphide bridge.

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