Demonstration and characterization of hippocampal cholinergic neurostimulating peptide (HCNP) processing enzyme activity in rat hippocampus.
Hippocampal cholinergic neurostimulating peptide (HCNP) stimulates cholinergic activity of cultured medial septal nuclei explants. It consists of eleven amino acids that are located at the N-terminal region of its precursor protein. This report concerns the demonstration and characterization of an HCNP processing enzyme that cleaves the bioactive undecapeptide from the precursor. The enzyme was purified from the hippocampus of young Wistar rats. A synthetic deacetylated peptide (peptide(1-26)) consisting of the N-terminal 26 amino acids of the HCNP precursor protein served as substrate. The product of the enzyme reaction was identified and quantitated by HPLC using deacetylated HCNP as standard. The amount of undecapeptide generated was directly proportional to the time of incubation of the enzyme reaction mixture. From molecular sieving chromatography it was estimated that the molecular mass of the enzyme is close to 68 kDa. The HCNP processing enzyme has a pH optimum of 6.0 and a K m of 0.50 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 inhibited by antipain, chymostatin and E-64. These findings suggest that the enzyme probably has a thiol group in its active site. This novel enzyme of the hippocampus may represent a valuable tool for further studies on the general protein metabolism in the central nervous system, as well as for elucidating the neurochemical aspects of neurodegenerative disorders.
Otsuka
Y
Y
Second Department of Internal Medicine, Medical School, Nagoya City University, Kawasumi, Japan.
Ojika
K
K
D000595:Amino Acid Sequence; D000818:Animals; D018678:Cholinergic Agents / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000302:isolation & purification; D004795:Enzyme Stability; D006624:Hippocampus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D018678:Cholinergic Agents; D009479:Neuropeptides; D010455:Peptides; D011480:Protease Inhibitors; C075274:hippocampal cholinergic neurostimulating peptide; D003546:Cysteine Endopeptidases; C105434:HCNP-processing enzyme