Molecular Structure of Neutral Endopeptidase 24.11 (Enkephalinase)

  • Philippe Crine
  • Guy Boileau
  • Alain Devault
  • Max Zollinger
  • Muriel Aubry
Part of the Biochemical Endocrinology book series (BIOEND)


Whereas termination of the action of classical neurotransmitters is known to occur either by reuptake or degradation, inactivation has been proposed to be a major mechanism for terminating peptidergic signals. When incubated in vitro with crude brain extracts, the neuropeptides Leu- and Met-enkephalins are rapidly hydrolysed to inactive metabolites2,3. In the perfused brain in vivo, the tripeptide Tyr-Gly-Gly has been found to be a major metabolite of the enkephalins arising by hydrolysis of the Gly3-Phe4 bond. Preparations from the striatum, a brain region rich in peptidergic nerve terminals, contain a membrane-bound enzyme that is able to hydrolyse the enkephalins at the same position4. This enzyme termed “enkephalinase” was detected in other tissues and was shown to be particularly enriched in the kidney cortex. Its identity with Neutral Endopeptidase (NEP: E.C., has been established using specificity, sensitivity to inhibitors and immunological criteria5. Both renal and brain enzymes hydrolyse peptides on the amino-terminal side of a hydrophobic residue. The enzyme has therefore the potential to hydrolyse a wide range of regulatory peptides6. Although the enkephalins may be important physiological substrates for the enzyme, evidence is accumulating that the tachykinin peptides such as substance P may also be inactivated by this enzyme in the brain7,8. There is little to support the concept of specific peptide hydrolases tailored for individual neuropeptides9.


Neutral Endopeptidase Cyanogen Bromide Fragment CNBr Fragment Kidney Brush Border Membrane Crude Brain Extract 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    D. R. Lynch and S. H. Snyder, Neuropeptides: multiple molecular forms, metabolic pathways and receptors, Ann. Rev. Biochem. 55:773 (1986).PubMedCrossRefGoogle Scholar
  2. 2.
    J. M. Hambrooke, B. A. Morgan, M. J. Rance, and C. F.Smith, Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates, Nature 262:782 (1976).CrossRefGoogle Scholar
  3. 3.
    J. L. Meek, H. Y. Yang, and E. Costa, Enkephalin catabolism in vitro and in vivo, Neuropharmacology 16:151 (1977).PubMedCrossRefGoogle Scholar
  4. 4.
    B. Malfroy, J. P. Swerts, A. Guyon, B. P. Roques, and J. C. Schwartz, High-affinity enkephalin-degrading peptidase in mouse brain and its enhanced activity following morphine, Nature 276:523 (1978).PubMedCrossRefGoogle Scholar
  5. 5.
    A. J. Turner, A. J. Kenny, and R. Matsas, Pharmacology of enkephalinase inhibitors, Trends Pharmacol Sci. 7:88 (1986).CrossRefGoogle Scholar
  6. 6.
    A. J. Turner, R. Matsas, and A. J. Kenny, Are there neuropeptide-specific peptidases?, Biochem. Pharmacol. 34:1347 (1985).Google Scholar
  7. 7.
    N. M. Hooper, A. J. Kenny, and A. J. Turner, Neurokinin A (substance K) is a substrate for endopeptidase-24.11 but not for dipeptidase A (angiotensine-converting-enzyme), Biochem. J. 231:357 (1985).PubMedGoogle Scholar
  8. 8.
    N. M. Hooper, and A. J. Turner, Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (enkephalinase) but not by angiotensin converting enzyme, FEBS Lett. 190:133 (1985).PubMedCrossRefGoogle Scholar
  9. 9.
    J. D. White, K. D. Stewart, J. E. Krause, and J. F. McKelvy, Biochemistry of peptide-secreting neurons, Physiol. Rev. 65:553 (1985).PubMedGoogle Scholar
  10. 10.
    B. P. Roques, M. C. Fournié-Zaluski, E. Soroca, J. M. Lecomte, B. Malfroy, C. Llorens, and J. C. Schwartz, The enkephalinase inhibitor thiorphan shows antinociceptive activity in mice, Nature 288:286 (1980).PubMedCrossRefGoogle Scholar
  11. 11.
    R. Matsas, A. J. Kenny, and A. J. Turner, An immunohistochemical study of endopeptidase -24.11 (“enkephalinase”) in the pig nervous system, Neuroscience 15:991 (1986).CrossRefGoogle Scholar
  12. 12.
    G. Waksman, E. Hamel, P. Delay-Goyet, and B. P. Roques, Neuronal localization of the neutral endopeptidase “enkephalinase” in rat brain revealed by lesions and autoradiography, EMBO J. 5:3163 (1986).PubMedGoogle Scholar
  13. 13.
    S. F. Atweh, and M. J. Kuhar, Autoradiographic localization of opiate receptors in rat brain: III the telencephalon, Brain Res. 134:393 (1977).PubMedCrossRefGoogle Scholar
  14. 14.
    G. Waksman, E. Hamel, P. Delay-Goyet, and B. P. Roques, Neutral endopeptidase-24.11, μ and δ opioid receptors after selective brain lesions: an autoradiographic study, Brain Res. 436:205 (1987).PubMedCrossRefGoogle Scholar
  15. 15.
    M. Del Faccio, G. Paxinos, and A. C. Cuello, Neostriatal enkephalin-immunoreactive neurons project to the globus pallidus,Brain Res. 231:1 (1982).Google Scholar
  16. 16.
    P. Crine, C. LeGrimellec, E. Lemieux, L. Labonté, S. Fortin, A. Blachier, and M. Aubry, The production and characterization of a monoclonal antibody specific for the 94 000 dalton enkephalin-degrading peptidase from rabbit kidney brush border, Biochem. Biophys. Res. Commun. 131:255 (1985).CrossRefGoogle Scholar
  17. 17.
    T. Ikemura, Codon usage and tRNA content in unicellular and multicellular organisms, Mol. Biol. Evol. 2:13 (1985).Google Scholar
  18. 18.
    R. J. Lathe, Synthetic oligonucleotide probes deduced from amino acid sequence data. Theorical and practical considerations, J. Mol. Biol. 183:1 (1985).PubMedCrossRefGoogle Scholar
  19. 19.
    A. Devault, C. Lazure, C. Nault, H. Le Moual, N. G. Seidah, M. Chretien, P. Kahn, J. Powell, J. Mallet, A. Beaumont, B. P. Roques, P. Crine, and G. Boileau, Amino acid sequence of rabbit kidney neutral endopeptidase 24.11 (enkephalinase) deduced from a complementary DNA, EMBO J. 6:13177 (1987).Google Scholar
  20. 20.
    F. Sanger, S. Nicklen, and A. R. Coulson, DNA sequencing with chain terminating inhibitors, Proc. Natl. Acad. Sci. USA 74:5463 (1977).PubMedCrossRefGoogle Scholar
  21. 21.
    M. Tomita, H. Furthmayr, and V. T. Marchesi, Primary structure of human glycophorin A. Isolation and characterization of peptides and complete amino acid sequence, Biochemistry 17:4756 (1978).PubMedCrossRefGoogle Scholar
  22. 22.
    T. Yamamoto, C. G. Davis, M. S. Brown, W. J. Schneider, M. L. Casey, J. L. Goldstein, and D. W. Russel, The human LDL receptor: a cysteine-rich protein with multiple ALU sequences in its mRNA, Cell 39:27 (1984).PubMedCrossRefGoogle Scholar
  23. 23.
    M. Hunziker, M. Speiss, G. Semenza, and H. F. Lodish, The sucrase-isomaltase complex: primary structure, membrane-orientation, and evolution of a stalked, intrinsic brush border protein, Cell 46:227 (1986).PubMedCrossRefGoogle Scholar
  24. 24.
    Y. Laperche, F. Bulle, T. Aissani, M. N. Chobert, M. Aggerbeck, J. Hanoune, and G. Guelaen, Molecular cloning and nucleotide sequence of the rat kidney lutamyl transpeptidase cDNA, Proc. Natl. Acad. Sci. USA 83:937 (1986).PubMedCrossRefGoogle Scholar
  25. 25.
    L. T. Tarn, S. Engelbrecht, J. M. Talent, R. W. Gracy, and E. G. Erdos, The importance of disulfide bridges in human endopeptidase (enkephalinase) after proteolytic cleavage, Biochem. Biophys. Res. Commun. 133:1187 (1985).CrossRefGoogle Scholar
  26. 26.
    J. Kyte, and F. Doolittle, A simple method for displaying the hydropathic character of a protein, J. Mol. Biol. 157:105 (1982).PubMedCrossRefGoogle Scholar
  27. 27.
    T. J. Bos, A. R. Davis, and D. P. Nayak, NH2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation, Proc. Natl. Acad. Sci. USA 81:2327 (1984).PubMedCrossRefGoogle Scholar
  28. 28.
    M. Speiss, A. L. Schwartz, and H. F. Lodish, Sequence of human asialoglycoprotein cDNA. An internal signal sequence for membrane insertion, J. Biol. Chem. 260:1979 (1985).Google Scholar
  29. 29.
    P. Y. Chou, and G. D. Fasman, Empirical predictions of protein conformation, Annu. Rev. Biochem. 47:251 (1978).CrossRefGoogle Scholar
  30. 30.
    A. J. Kenny, and I. S. Fulcher, Microvillar endopeptidase, an enzyme with special topological features and a wide distribution, Ciba Found. Symp. 95:12 (1983).Google Scholar
  31. 31.
    G. I. Evan, A simple and rapid solid phase enzyme-linked immunoadsorbence assay for screening monoclonal antibodies to poorly soluble proteins, J. Immunol. Methods 73:427 (1984).PubMedCrossRefGoogle Scholar
  32. 32.
    W. Haase, A. Schafer, H. Murer, and R. Kinne, Studies on the orientation of brush-border membrane vesicles, Biochem. J. 172:57 (1978).PubMedGoogle Scholar
  33. 33.
    A. G. Booth, and A. J. Kenny, Assymetric labelling of the membrane by lactoperoxydase-catalysed radioiodination and by photolysis of 3,5-di[125I]iodo-4-azidobenzenesulphonate, Biochem. J. 187:31 (1980).PubMedGoogle Scholar
  34. 34.
    L. D. Fricker, C. J. Evans, F. S. Esch, and E. Herbert, Cloning and sequence analysis of cDNA for bovine carboxypeptidase E, Nature 323:461 (1986).PubMedCrossRefGoogle Scholar
  35. 35.
    W. R. Kester, and B. W. Matthews, Crystallographic study of the binding of dipeptide inhibitors to thermolysin: implication for the mechanism of catalysis, Biochemistry 16:2505 (1977).CrossRefGoogle Scholar
  36. 36.
    B. P. Roques, and M. C. Fournié-Zaluski, Opioid peptides: molecular pharmacology, biosynthesis and analysis, NIDA Research Monograph Series 70:128 (1986).Google Scholar
  37. 37.
    M. Pozsgay, C. Michaud, M. Liebman, and M. Orlowski, Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin., Biochemistry 25:1292 (1986).PubMedCrossRefGoogle Scholar
  38. 38.
    L. B. Hersh, and K. Morihara, Comparison of the subsite specificity of the mammalian neutral endopeptidase 24.11 (enkephalinase) to the bacterial neutral endopeptidase thermolysin, J. Biol. Chem. 261:6433 (1986).PubMedGoogle Scholar
  39. 39.
    A. Beaumont, and B. P. Roques, Presence of a histidine at the active site of neutral endopeptidase-24.11, Biochem. Biophys. Res. Commun. 139:733 (1986).PubMedCrossRefGoogle Scholar
  40. 40.
    F. A. Quiocho, and W. N. Lipscomp, Carboxypeptidase A: a protein and an enzyme, Adv. Prot. Chem. 25:1 (1971).Google Scholar
  41. 41.
    M. A. Kerr, and A. J. Kenny, The molecular weight and properties of a neutral metallo-endopeptidase from rabbit kidney brush borders, Biochem. J. 137:489 (1974).PubMedGoogle Scholar
  42. 42.
    A. Guyon, B. P. Roques, F. Guyon, A. Foucault, R. Perdrisot, J. P. Swerts, and J. C. Schwartz, Enkephalin degradation in mouse brain studied by a new H.P.L.C. method: further evidence for the involvement of carboxypeptidase, Life Sc. 25:1605 (1979).CrossRefGoogle Scholar
  43. 43.
    W. L. Scott, L. Mendelsohn, M. L. Cohen, D. A. Evans, and R. C. A. Frederickson, Enantiomers of (R-S)thiorphan: dissociation of analgesia from enkephalinase inhibition, Life Sci. 36:1307 (1985).PubMedCrossRefGoogle Scholar
  44. 44.
    M. C. Fournié-Zaluski, E. Lucassoroca, J. Devin, and B. P. Roques, 1H NMR configurational correlation for retro-inverso dipeptides; application to the determination of the absolute configuration of “enkephalinase” inhibitors. Relationship between stereochemistry and enzyme recognition, J. Med. Chem. 29:751 (1986).PubMedCrossRefGoogle Scholar
  45. 45.
    G. Waksman, R. Bouboutou, J. Devin, R. Besselievre, M. C. Fournié-Zaluski, and B. P. Roques, Binding of the bidentate inhibitor [3H]HACBO-Gly to the rat brain neutral endopeptidase “enkephalinase”, Biochem. Biophys. Res. Commun. 131:262 (1985).CrossRefGoogle Scholar
  46. 46.
    D. G. Hangauer, A. F. Monzingo, and B. W. Matthews, An interactive computer graphics study of thermolysin-catalysed peptide cleavage and inhibition by N-carboxymethyl dipeptides, Biochemistry 23:5730 (1984).PubMedCrossRefGoogle Scholar
  47. 47.
    M. C. Fournié-Zaluski, A. Coulaud, R. Bouboutou, P. Chaillet, J. Devin, G. Waksman, J. Costentin, and B. P. Roques, New bidentates as full inhibitors of enkephalin degrading enzymes: synthesis and analgesic properties, J. Med. Chem. 28:1158 (1985).PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1988

Authors and Affiliations

  • Philippe Crine
    • 1
  • Guy Boileau
    • 1
  • Alain Devault
    • 1
  • Max Zollinger
    • 1
  • Muriel Aubry
    • 1
  1. 1.Département de BiochimieUniversité de MontréalMontréalCanada

Personalised recommendations