Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain.
A peptidase inactivating neurotensin at the Pro(10)-Tyr(11) peptidyl bond, leading to the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o-phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin (Km = 2.3 itM). Studies on its specificity indicated that: (i) neurotensin(9-13) was the shortest neurotensin fragment with full inhibitory potency of [(3)H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [(3)H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [(3)H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive "neurotensinase" activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1-8 and 1-13, atriopeptin III and bradykinin potently inhibited [(3)H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.
Barelli
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Centre de Biochimie du CNRS, Faculté des Sciences de Nice, Parc Valrose, 06034 Nice Cédex, France.
Girard
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F
St Pierrr
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S
Kitabgi
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Vincent
J P
JP
Checler
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F