Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor.
Recombinant rat intestinal trefoil factor (rITF) and human spasmolytic polypeptide (hSP) were irreversibly cross-linked to specific binding sites in solubilized rat intestinal epithelial membranes and human adenocarcinoma cells. Analysis of the immunoprecipitates by immunoblotting identified a cross-linked protein complex of approximately 45 kDa, which under reducing conditions appeared as a approximately 28-kDa band and the latter displayed ligand-stimulated phosphorylation of a tyrosine, but not a threonine or serine, residue in the binding complex. [125I]rITF was used to localize binding sites by autoradiography of frozen sections from rat gastrointestinal tissues. A high density of specific [125I]rITF binding sites was present within gastric, colonic, and jejunal mucosal glands. Unlabeled hSP partially inhibited [125I]rITF binding at a concentration of 1 microM when compared with the same concentration of unlabeled rITF. These studies support earlier observations for the existence of trefoil binding sites in the gastrointestinal tract and further suggest that hSP has affinity for the mucosal rITF binding site.
D000818:Animals; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008297:Male; D009077:Mucins; D009124:Muscle Proteins; D009363:Neoplasm Proteins / Q000378:metabolism*; D009479:Neuropeptides; D010455:Peptides / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism
D003432:Cross-Linking Reagents; D006133:Growth Substances; D007457:Iodine Radioisotopes; D009077:Mucins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D009479:Neuropeptides; D010455:Peptides; D011994:Recombinant Proteins; C099037:TFF3 protein, rat; C000604008:Tff2 protein, rat; D000071164:Trefoil Factor-2; D000071165:Trefoil Factor-3; D014443:Tyrosine