IMMUNOPRECIPITATION AND CHARACTERIZATION OF A BINDING-PROTEIN SPECIFIC FOR THE PEPTIDE, INTESTINAL TREFOIL FACTOR
IMMUNOPRECIPITATION AND CHARACTERIZATION OF A BINDING-PROTEIN SPECIFIC FOR THE PEPTIDE, INTESTINAL TREFOIL FACTOR
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DOI：
10.1016/0196-9781(95)00045-l
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发表时间：
1995-01-01
期刊：
影响因子：
3
通讯作者：
COX, HM
中科院分区：
文献类型：
--
作者：
CHINERY, R;COX, HM
重组大鼠肠三叶因子（rITF）和人解痉挛多肽（hSP）与溶解大鼠肠上皮膜和人腺癌细胞的特异性结合位点不可逆交联。通过免疫印迹法对免疫沉淀物进行分析，发现了一个类似于45 kDa的交联蛋白复合物，在还原条件下，该复合物与28 kDa的条带相似，后者在结合复合物中显示出配体刺激的酪氨酸磷酸化，而不是苏氨酸或丝氨酸残基。[I-125]rITF通过大鼠胃肠道组织冷冻切片放射自显影来定位结合位点。高密度的特异性[I-125]rITF结合位点存在于胃、结肠和空肠粘膜腺中。与相同浓度的未标记的rITF相比，未标记的hSP在1 μ M浓度下部分抑制[I-125]rITF的结合。这些研究支持了早期关于胃肠道中存在三叶结合位点的观察结果，并进一步表明hSP对粘膜rITF结合位点具有亲和力。
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 similar to 45 kDa, which under reducing conditions appeared as a similar to 28-kDa band and the latter displayed ligand-stimulated phosphorylation of a tyrosine, but not a threonine or serine, residue in the binding complex. [I-125]rITF was used to localize binding sites by autoradiography of frozen sections from rat gastrointestinal tissues. A high density of specific [I-125]rITF binding sites was present within gastric, colonic, and jejunal mucosal glands. Unlabeled hSP partially inhibited [I-125]rITF binding at a concentration of 1 mu M 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.