Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: Characterization of receptor binding sites and receptor assay for benzodiazepine drugs

The epitopes of human thyroid-stimulating hormone (hTSH) recognized by two murine monoclonal antibodies (MAbs), designated MAb 279 and MAb 299, have been characterized. These MAbs are highly specific for the beta-subunit of TSH. The epitope recognized by MAb 279 appears to be completely conserved between bovine and human TSH and partially conserved in the porcine species. The TSH beta-subunit epitope recognized by MAb 299 is only partially conserved between the human, bovine and porcine species. Both MAbs are capable of inhibiting the binding of TSH to its receptor in a TSH radioreceptor assay, indicating that the epitopes either coincide or are located close to the TSH beta-subunit receptor-binding sites. The carbohydrate moieties of the TSH beta-subunit appear to play little or no role in the epitope recognition by MAb 279 or MAb 299 while the integrity of the disulphide bonds are essential. The epitopic recognition may also involve lysine residues, as determined by the immunoreactivity with both MAbs following citraconylation of TSH. In addition, the amino acid sequence region between residues bTSH beta 34-44 could be excised by trypsin digestion of bovine TSH beta (bTSH beta) without eliminating epitopic recognition by either MAb. These results provide further insight into the relationship between the structure of the TSH beta-subunit epitopes and location of the receptor-binding sites.

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Characterization of Insulin-Like Growth Factor (IGF)-I-Receptor Binding Sites during In Vitro Transformation of Rat Hepatic Stellate Cells to Myofibroblasts

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.1996.34.5.401 ◽

1996 ◽

Vol 34 (5) ◽

Cited By ~ 6

Author(s):

Arnfried Brenzel ◽

Axel M. Gressner

Keyword(s):

Growth Factor ◽

Receptor Binding ◽

Hepatic Stellate Cells ◽

Binding Sites ◽

Stellate Cells ◽

Insulin Like Growth Factor ◽

In Vitro Transformation ◽

Igf I

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Characterization of the Binding of Bovine Thrombin to Isolated Rat Hepatocytes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646983 ◽

1988 ◽

Vol 60 (03) ◽

pp. 419-427 ◽

Cited By ~ 5

Author(s):

Britta Weyer ◽

Torben E Petersen ◽

Ole Sonne

Keyword(s):

Binding Sites ◽

Time Course ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Rat Hepatocytes ◽

Isolated Rat Hepatocytes ◽

Prolonged Incubation ◽

Receptor Assay ◽

Isolated Rat

SummaryIsolated rat hepatocytes possess per cell 4,500 high-affinity binding sites for thrombin with a Kd of 30-40 pM, and 2.8 × 105 low-affinity sites with a Kd of 30 nM. These binding sites are highly specific for thrombin. Half-maximal binding of 125I-labelled thrombin is achieved after 3 min at 37¸ C and 7 min at 4¸ C. The reversibly bound fraction of the ligand dissociates according to a biexponential time course with the rate constants 1—2 × 10−2 s−1 and 3—4 × 10−4 s−1. Part of the tracer remains cell-associated even after prolonged incubation, but all cell-associated radioactivity migrates as intact thrombin upon sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bound thrombin is minimally endocytosed as judged by the resistance to pH 3-treatment. Cell-associated radioactivity dissociated from the cells binds just aswell in a receptor assay as tracer incubated in a conditioned medium under the same conditions, indicating the absence of a quantitatively important receptor-mediated degradation ofthe ligand.

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