Potential precursors to neuromedin N in the cat and their processing by pepsin

Acidic (0.1 N HCl) extracts of feline brain and ileal mucosa were found to contain a number of basic proteins which yielded immunoreactive neuromedin N (NMN) when treated with hog pepsin. These proteins substrates were separated using Sephadex G-25, Sephadex G-75, and reverse phase HPLC. In a calibrated sodium dodecyl sulfate-polyacrylamide gel electrophoresis system, the major substrates from cat ileum gave mol wt of about 15K and 20K. Minor bands of substrates, possible aggregates, were observed under both reducing and nonreducing conditions at 40K and 75K. Substrate-derived immunoreactive NMN was indistinguishable from synthetic or native NMN during reverse phase HPLC and gave equal measurements with two antisera with different specificities. During equilibrium centrifugation of isotonic homogenates of cat brain and ileum in a gradient of sucrose, NMN and the substrates banded together, apparently associated with particles resembling synaptosomes and vesicles. These results suggest that these substrates represent precursors to NMN and that pepsin can mimic the physiological processing enzyme(s) involved in the biosynthesis of this regulatory peptide.