Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes.
A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.
D000818:Animals; D001921:Brain / Q000166:cytology* / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D009496:Neurotensin / Q000378:metabolism*; D017830:Octoxynol; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D010455:Peptides; D011092:Polyethylene Glycols; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013570:Synaptic Membranes / Q000201:enzymology*; D029867:Xenopus Proteins
D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011480:Protease Inhibitors; D029867:Xenopus Proteins; C042090:neuromedin N; D009496:Neurotensin; D011092:Polyethylene Glycols; C016832:xenopsin; D017830:Octoxynol; D010447:Peptide Hydrolases