IMMUNOLOGY & DISEASES OF THE GASTROINTESTINAL TRACT

这项研究计划是针对研究胃肠道 调节肽，特别强调与其 受体以及结构和功能表征 这些调节的细胞受体膜蛋白的特征 缩氨酸 与胃肠调节肽互补的肽 将通过基于mRNA的固相肽合成来合成 与人胃泌素I（G17）的密码子互补，羧基- 末端胃泌素四肽（G4）， 胆囊收缩素（CCK 8）、生长抑素-14（S14）和羧基末端 胃泌素释放肽（GRP 14）的十四肽。 配体结合 这些互补肽的性质将被评估， 通过孵育125 I-12 聚氯乙烯威尔斯孔中的胃肠调节肽 用各自的互补肽包被的板。 竞争 将进行研究以评估125 I- 调节肽 互补肽的抗体将是 通过用互补肽免疫兔子而产生 与作为载体蛋白的钥孔血蓝蛋白偶联。 潜在 这些抗体的结合，其将通过免疫亲和纯化 层析并用125 I放射性标记，与细胞膜受体结合 (on胃粘膜壁细胞、胃泌素细胞和生长抑素细胞） 将被审查。 将通过以下方法从犬胃底粘膜制备细胞： 胶原酶解离，然后淘洗。 抗体 互补肽，预期与膜结合位点结合 受体蛋白将用于免疫亲和性研究， 胃泌素、生长抑素和胃泌素释放的细胞膜受体 为了确定这些膜受体的结构。 初步研究将针对膜的表征 胃泌素受体 在拟议研究的后期阶段， 使用如研究犬胃底粘膜所述的技术 细胞，实验也将进行检查结构和 胃泌素瘤细胞膜受体的功能特征 胃泌素释放肽和生长抑素。

This research proposal is directed to the study of gastrointestinal regulatory peptides with special emphasis on interactions with their receptors as well as characterization of structure and functional characteristics of cell receptor membrane proteins for these regulatory peptides. Peptides complementary to gastrointestinal regulatory peptides will be synthesized by solid phase peptide synthesis based on mRNA complementarity to the codons of human gastrin I (G17), the carboxyl- terminal gastrin tetrapeptide (G4), the carboxyl-terminal octapeptide of cholecystokinin (CCK8), somatostatin-14 (S14) and the carboxyl-terminal tetradecapeptide of gastrin-releasing peptide (GRP14). Ligand binding properties of these complementary peptides will be assessed for the respective gastrointestinal regulatory peptides by incubation of 125 I- gastrointestinal regulatory peptides in wells of polyvinyl chloride plates coated with the respective complementary peptides. Competition studies will be performed to assess specificity of binding of 125 I- regulatory peptides. Antibodies to complementary peptides will be produced by immunization of rabbits with complementary peptides conjugated to keyhole limpet hemocyanin as carrier protein. Potential binding of these antibodies, which will be purified by immunoaffinity chromatography and radiolabelled with 125 I, to cell membrane receptors (on gastric mucosal parietal cells, gastrin cells and somatostatin cells) will be examined. Cells will be prepared from canine fundic mucosa by collagenase dissociation followed by elutriation. Antibodies to complementary peptides, anticipated to bind to binding sites of membrane receptor proteins will be utilized in immunoaffinity studies to purify cell membrane receptors for gastrin, somatostatin, and gastrin-releasing peptide in order to determine the structure of those membrane receptors. Initial studies will be directed to characterization of membrane receptors for gastrin. In the later stages of the proposed studies, using techniques as described for the study of canine fundic mucosal cells, experiments will also be performed to examine structural and functional characteristics of gastrinoma cell membrane receptors for gastrin-releasing peptide and somatostatin.