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.

scholarly journals Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

1985 ◽  
Vol 228 (1) ◽  
pp. 111-117 ◽  
Author(s):  
E Davies Jones ◽  
F A Hashim ◽  
Y Kajita ◽  
F M Creagh ◽  
P R Buckland ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyrotropin Receptor ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

scholarly journals Purification by affinity chromatography and immunological characterization of a 110kDa component of the chick oviduct progesterone receptor

1984 ◽  
Vol 217 (3) ◽  
pp. 685-692 ◽  
Author(s):  
J M Renoir ◽  
J Mester ◽  
T Buchou ◽  
M G Catelli ◽  
P Tuohimaa ◽  
...  
Keyword(s):  
Progesterone Receptor ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Sedimentation Coefficient ◽  
Binding Activity ◽  
B Subunit ◽  
Stokes Radius ◽  
A Subunit ◽  
Final Yield

A 110kDa component of the chick oviduct progesterone receptor (PR) has been purified to homogeneity according to electrophoretic criteria and specific activity (assuming one progestagen-binding site/110kDa). The procedure involved affinity chromatography of 0.3 M-KCl-prepared cytosol, followed by DEAE-Sephacel chromatography (elution at 0.2 M-KCl). The final yield was about 12% in terms of binding activity. Properties of the 110kDa component indicate that it is identical with the ‘B’ subunit described previously [Stokes radius approximately 6.1 nm; sedimentation coefficient, (S20, w) approximately 4S; frictional ratio approximately 1.77]. It reacted with the IgG-G3 polyclonal antibody, but not with BF4 monoclonal antibody raised against the 8S molybdate-stabilized chick oviduct PR and reacting with its 90kDa component. Another progesterone-binding component, corresponding to the ‘A’ subunit, also previously described, was eluted from the DEAE-Sephacel column at approximately 0.08 M-KCl, and contained a peptide of molecular mass approx. 75-80kDa, which had S20, w approximately 4S in a sucrose gradient. This component was also recognized by IgG-G3, but not by BF4; it was very unstable in terms of hormone-binding activity.

scholarly journals Analysis of thyrotropin receptors by photoaffinity labelling. Orientation of receptor subunits in the cell membrane

1985 ◽  
Vol 227 (2) ◽  
pp. 413-420 ◽  
Author(s):  
Y Kajita ◽  
C R Rickards ◽  
P R Buckland ◽  
R D Howells ◽  
B Rees Smith
Keyword(s):  
Cell Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Fat Cell ◽  
Disulphide Bridge ◽  
B Subunit ◽  
Hydrophilic Peptide ◽  
A Subunit

Porcine thyrotropin (TSH) receptors have been purified by Sepharose-TSH affinity chromatography and crosslinked to a 125I-labelled photoactive derivative (N-hydroxysuccinimidyl 4-azidobenzoate; HSAB) of TSH (125I-HSAB-TSH). Purification of the crosslinked complexes on Sephacryl S-300 followed by polyacrylamide-gel electrophoresis in sodium dodecyl sulphate showed that the receptor contained two subunits. One subunit (A) with Mr 45 000 was crosslinked to TSH and the other (B) subunit, Mr 25 000, was linked to the A subunit by a disulphide bridge(s). Other, as yet unidentified, subunits may have been non-covalently associated with the A and B subunits. Analysis of reduced and non-reduced crosslinked TSH receptor-125I-HSAB-TSH on Sephacryl S-300 in the presence and absence of detergent indicated that the A subunit was a hydrophilic peptide. This was confirmed in studies of the release into aqueous solution by reducing agent treatment of 125I-HSAB-TSH crosslinked to the TSH receptor A subunit in thyroid membranes. Similar results were obtained with TSH receptors in human thyroid and guinea pig fat cell membranes. These studies suggest that the hydrophilic A subunit of the receptor forms a binding site for TSH on the outside surface of the cell membrane and that the A subunit is linked to the cell membrane by way of a disulphide bridge to the receptor B subunit.

Isolation and characterization of distinct bioactive forms of LH from male buffalo pituitaries: differences localized to their α subunits

1994 ◽  
Vol 143 (2) ◽  
pp. 313-323 ◽  
Author(s):  
M R Sairam ◽  
A A H Zaky ◽  
A A Hassan
Keyword(s):  
Receptor Binding ◽  
Binding Activity ◽  
Α Subunit ◽  
Basic Fraction ◽  
Isolation And Characterization ◽  
Receptor Binding Activity ◽  
Acidic Fraction ◽  
Α Subunits

Abstract The isolation of highly purified forms of pituitary LH from Egyptian male (Nile) buffaloes is described. The total LH content (receptor binding activity) which was approximately 30 to 50 fold higher than FSH in the pituitary could be divided into three pools based upon fractionation patterns on a cation exchanger. The acidic fraction which also contained FSH was not purified to homogeneity. A basic fraction (bu-LH-2; 300 mg/kg anterior pituitary) and a very basic fraction (bu-LH-3; 80 mg/kg) were both highly purified and free of FSH activity as tested by specific FSH receptor and immunoassays. The basic buffalo LH fraction, bu-LH-2, was as active as highly purified ovine LH (oLH). The most basic form of buffalo LH, bu-LH-3, was, however, about twice as active as highly purified oLH in the in vitro bioassay using mouse Leydig tumour (MA-10) cells. In a receptor binding assay employing 125I-labelled buffalo LH (bu-LH-3) and porcine testicular membranes, the affinity of bu-LH-3 was about five times higher than purified oLH. The Mr of both forms of purified buffalo LH and subunits was similar to that of oLH. Amino acid composition of buffalo LH was also very similar to oLH except for small differences. Fractionation by fast protein liquid chromatography on Mono-Q columns revealed further evidence of microheterogeneity in each of the pools of buffalo LH with bu-LH-3 exhibiting a predominant single component. By reverse-phase high-pressure liquid chromotography analysis we have localized differences in the two purified isoforms of male buffalo LH to the α subunit. It is suggested that differences in biological potencies could be due to variations in terminal glycosylation and/or differences in branching of this subunit which is known to be important for signal transduction. Journal of Endocrinology (1994) 143, 313–323

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.

The TSH receptor: Structure and interaction with autoantibodies in thyroid disease

1987 ◽  
Vol 116 (1_Suppl) ◽  
pp. S157-S165 ◽  
Author(s):  
J. Furmaniak ◽  
Y. Nakajima ◽  
F. A. Hashim ◽  
F. M. Creagh ◽  
E. Davies Jones ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Thyroid Tissue ◽  
Tsh Receptor ◽  
Sds Page ◽  
Affinity Labelling ◽  
Stokes Radius ◽  
A Subunit ◽  
Receptor Antibodies ◽  
Derivatives Of ◽  
Tsh Receptor Antibodies

Abstract. Studies of the TSH receptor using affinity labelling with photoactive derivatives of TSH and analysis by SDS-PAGE have shown that the receptor contains 2 subunits (A and B), linked by a disulphide bridge. Similar results are obtained with TSH receptors from human, porcine and guinea pig thyroid tissue and from guinea pig fat. Analysis of affinity labelled receptors under non-denaturing conditions suggest that subunits additional to the A and B subunits are not present. Hydrodynamic measurements indicate that the receptor A subunit has an approximately spherical structure (Stokes' radius 70Å) and when this interacts with TSH (an elongated structure with Stokes' radius 56Å) a very elongated complex (Stokes' radius 104Å) is formed. Isoelectric focusing studies of the TSH receptor A subunit, TSH and TSH receptor antibodies indicate that charge-charge interactions are of considerable importance in the binding of hormone and antibody to the receptor.

Interaction of sodium molybdate with the thyroid hormone receptor

1990 ◽  
Vol 68 (3) ◽  
pp. 630-634 ◽  
Author(s):  
Robert Faure ◽  
Jean H. Dussault
Keyword(s):  
Thyroid Hormone ◽  
Hormone Receptor ◽  
Thyroid Hormone Receptor ◽  
Sodium Molybdate ◽  
Binding Activity ◽  
Exchange Chromatography ◽  
Receptor Interaction ◽  
Affinity Constant ◽  
Receptor Complexes ◽  
Receptor Binding Activity

The 3,5,3′-triiodothyronine (T3) binding activity of solubilized nuclear proteins from rat liver was decreased when molybdate (10 mM) was present in the incubation medium in the absence of thiol reagents. The equilibrium affinity constant was reduced by 40%. The rate of degradation of T3-receptor complexes at 37 °C remained unchanged, but when the extracts were further reincubated in the presence of β-mercaptoethanol, molybdate had a protective effect after 5 h incubation at 37 °C. In contrast, the thyroxine (T4) binding activity was not affected by heating at 37 °C or by molybdate. Ion-exchange chromatography confirmed the existence of a molybdate–receptor interaction: the T3–receptor complexes shifted from elution at 0.22 to 0.20 M NaCl with the progressive appearance of a small leader peak, whereas the T4-receptor complexes eluted in a large and split peak (0.22–0.4 M NaCl). The destabilizing effect on T3 binding induced by exogenous dephosphorylation is more efficiently reversed by β-mercaptoethanol when the extracts were pretreated by molybdate. In controls, the loss of saturable T3 binding activity was recovered by 50% at a 10 mM concentration of β-mercaptoethanol, but in the presence of molybdate, the loss of T3 binding activity was recovered by 50% at a 5 mM concentration of β-mercaptoethanol. This molybdate–receptor interaction is similar to that with nuclear receptor models in term of (i) stabilization of hormone binding, (ii) dependency on a thiol, and (iii) reversibility of the destabilizing effect by exogenous dephosphorylation.Key words: thyroid hormone receptor, binding activity, sodium molybdate, alkaline phosphatase.

Anti-thyrotrophin receptor antibodies in Graves' disease as demonstrated directly by immunoprecipitation assay

1983 ◽  
Vol 102 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Tjerk W. A de Bruin ◽  
Daan van der Heide ◽  
Maria C. Krol
Keyword(s):  
Binding Site ◽  
Tsh Receptor ◽  
Graves Disease ◽  
Immunoprecipitation Assay ◽  
Receptor Complexes ◽  
Auto Antibody ◽  
Receptor Antibodies ◽  
Normal Controls

Abstract. An immunoprecipitation assay was developed to determine the presence of antibodies against human TSH1 receptors. With this assay we were able to demonstrate that in comparison with sera from normal controls, 24 out of 30 (80%) sera from patients with untreated Graves' disease could immunoprecipitate more [125I]TSH-TSH receptor complexes. In 9 assays, an average of 14.1 ± 3.7% (sd) of the [125I]TSH-TSH receptor complexes was immunoprecipitated by the 30 Graves' sera vs 9.8 ± 3.0% by the normal pool serum (n = 23) (P < 0.001) and 7.7 ± 2.8% by the 22 normal sera (P < 0.001). One serum of the 24 positive Graves' sera was studied in detail. The results suggest that this serum contained an anti-TSH receptor auto-antibody directed towards a different determinant on the TSH receptor than the TSH binding site.

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