Characterization of large neuromedin-N using antisera towards regions of the neurotensin/neuromedin-N precursor

Processing of the precursor to neurotensin/neuromedin-N was studied in brain and intestine from four mammalian species (dog, cat, guinea pig and rat) using previously characterized immunoassays for neurotensin and neuromedin-N, as well as newly developed assays towards the 35-44 sequence (P1) and the 70-85 sequence (P2) of the canine precursor. While neurotensin was the major product (approximately 98%) with neurotensin immunoreactivity in brain and ileum, a large molecular form of neuromedin-N was found to comprise 55-91% of the neuromedin-N activity in the ileum of these species and only 2-8% that in brain. Large neuromedin-N, which behaved as a single substance during multiple chromatographic steps, was found to cross-react in the assays for P1 and P2, indicating that this molecule extended at least from residues 40-148, neuromedin-N being located at its C-terminus. Western blots confirmed the results obtained by immunoassay. Partially purified preparations of large neuromedin-N from dog, cat and rat were also found to contract the isolated guinea pig ileum, exhibiting potencies near to that of neuromedin-N. These results indicate that tissue-specific storage of large neuromedin-N, a biologically active molecule with greater than 100 amino acids, occurs in these four mammals.