The principal aim of this proposal is to isolate novel neuro- and hormonal peptides from the gut and pancreas using a variety of the detection methods and subsequently characterize the chemical, biological and immunochemical properties of the isolated peptides. Large-scale extractions of the tissue materials will be followed by purification using gel permeation, ion exchange and high performance liquid chromatography (HPLC). Specifically, the following gut peptides will be screened: 1) novel peptides which possess the C-terminal amide structure using a chemical assay method; 2) novel peptide which contract or relax smooth muscles using smooth muscle preparations; 3) endogenous gut factors which alter the release of the hormones from pituitary, hypothalamic and intestinal cell cultures. The primary structure of the isolated peptides will be determined using automatic gas-phase sequencing, amino acid analysis and fast- atom-bombardment (FAB) mass spectrometry. Synthetic replicates of the peptides will be prepared using solid-phase synthesizers and will be utilized for generating antibodies, and for pharmacological studies. Further, peptide analogues of peptide YY and neuropeptide Y that have agonist and antagonist activities will be synthesized using a multiple solid-phase peptide synthesis technique. Antibodies raised against the peptides will be used for cellular localization studies using radioimmunoassay and immunohistochemical methods. The structure of precursors of the peptides will be determined by gene cloning techniques and oligonucleotide probes will be utilized for studies of mRNA levels in tissues and for in situ hybridization studies. This study is likely to find a host of new neuropeptides and hormones which have important biological significance and stimulate various physiological, pharmacological, biochemical, and clinical studies. We anticipate that such studies will shed further light on the regulatory mechanisms of the digestive system and will have implications toward understanding the pathogenesis of some digestive diseases.
