DEMONSTRATION AND CHARACTERIZATION OF HIPPOCAMPAL CHOLINERGIC NEUROSTIMULATING PEPTIDE (HCNP) PROCESSING ENZYME-ACTIVITY IN RAT HIPPOCAMPUS

Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cho linergic activity of cultured medial septal nuclei explants. It consis ts of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and char acterization of an HCNP processing enzyme that cleaves the bioactive u ndecapeptide from the precursor. The enzyme was purified from the hipp ocampus of young Wistar rats. A synthetic deacetylated peptide (peptid e(1-26)) consisting of the N-terminal 26 amino acids of the HCNP precu rsor protein served as substrate. The product of the enzyme reaction w as identified and quantitated by HPLC using deacetylated HCNP as stand ard. The amount of undecapeptide generated was directly proportional t o the time of incubation of the enzyme reaction mixture. From molecula r sieving chromatography it was estimated that the molecular mass of t he enzyme is close to 68 kDa. The HCNP processing enzyme has a pH opti mum of 6.0 and a K-m of 0.59 mM for peptide(1-26). Preincubation at 56 degrees C causes rapid inactivation of the HCNP processing activity. Enzyme activity is enhanced by EDTA and 1,4-dithiothreitol, and inhibi ted by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzy me of the hippocampus may represent a valuable tool for further studie s on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.