Gastrointestinal peptides play an important role in the physiology of the gut and brain. GI peptides act as hormones, neurotransmitters, or trophic factors. They are biosynthesized as prohormones and produced by extensive proteolytic processing. The prohormones often contain two or more GI peptides, some of which have been shown to have receptors, but whose functions are unknown. In many cases, several mRNA transcripts can be produced from one gene, thus increasing the diversity and number of GI peptides derived from a single gene. The proteolytically processed peptides are often subjected to further posttranslational modifications such as conversion of glutamine to pyroglutamic acid at the N-terminus, C-terminal amidation, sulfation, phosphorylation, and glycosylation. The majority of structural studies in the past have focused on the more readily available GI peptides from pig. In our previous work we have demonstrated the ability to extend these studies to human GI, brain, and neuroendocrine tumor peptides. In this proposal we plan to isolate and characterize of gastrin and GRP (gastrin-related peptide), gene-related peptides which have been predicted from the molecular cloning work. We will isolate the peptides from tumor tissue and cell lines by reverse phase HPLC and characterize the peptides by a combination of Fast Atom Bombardment/Mass Spectrometry (FAB/MS) and microsequence analysis. FAB/MS will allow us to determine exact molecular weights of the peptides, including posttranslational modifications. We will also biosynthetically label neuroactive peptides produced in small cell lung carcinomal cell lines, and establish their product-precursor relationships. As part of the structure verification and isolation methodology we will synthesize peptides corresponding to the peptide hormones, raise antibodies to the peptides, and develop sensitive immunoassays. These studies will be performed in close collaboration with Drs. Walsh, Reeve, and Vigna at the Wadsworth VA in Los Angeles. Our collaborators will perform physiological and membrane receptor studies with the newly identified peptide hormones.
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