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.
| Wolfe, M M; Bougoulias, M; McGuigan, J E (1990) Cholecystokinin binding and degradation by isolated rat liver cells. Regul Pept 27:27-36 |
