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. 1989 Aug 1;261(3):951–958. doi: 10.1042/bj2610951

Endopeptidase 24.15 from rat testes. Isolation of the enzyme and its specificity toward synthetic and natural peptides, including enkephalin-containing peptides.

M Orlowski 1, S Reznik 1, J Ayala 1, A R Pierotti 1
PMCID: PMC1138921  PMID: 2803255

Abstract

Endopeptidase 24.15, a metalloendopeptidase (EC 3.4.24.15) with an Mr of about 70,000, was purified to homogeneity from rat testes. The enzyme cleaves preferentially bonds on the carboxyl side of hydrophobic amino acids. Secondary enzyme-substrate interactions at sites removed from the scissile bond are indicated by the finding that a hydrophobic or bulky residue in the P3' position greatly contributes to substrate binding and catalytic efficiency. The isolated enzyme is inhibited by metal chelators and by thiols. Loss of enzymic activity after dialysis against EDTA can be restored by low concentrations of Zn2+ and Co2+ ions. The rate of reaction of the Co2+ enzyme with a synthetic substrate was higher than that of the Zn2+ enzyme. These results are consistent with the classification of the enzyme as a metalloendopeptidase. N-Carboxymethyl peptides that fulfil the binding requirements of the substrate recognition site of the enzyme act as potent competitive inhibitors. Biologically active peptides such as luteinizing hormone-releasing hormone, bradykinin and neurotensin are cleaved at sites consistent with the specificity of the enzyme deduced from studies with synthetic peptides. Dynorphin A (1-8)-peptide, beta-neoendorphin, metorphamide, and Metenkephalin-Arg6-Gly7-Leu8 are rapidly converted to the corresponding enkephalins. The testis enzyme is catalytically and immunologically closely related to the previously identified brain enzyme.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Acker G. R., Molineaux C., Orlowski M. Synaptosomal membrane-bound form of endopeptidase-24.15 generates Leu-enkephalin from dynorphin1-8, alpha- and beta-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8. J Neurochem. 1987 Jan;48(1):284–292. doi: 10.1111/j.1471-4159.1987.tb13160.x. [DOI] [PubMed] [Google Scholar]
  2. Almenoff J., Orlowski M. Membrane-bound kidney neutral metalloendopeptidase: interaction with synthetic substrates, natural peptides, and inhibitors. Biochemistry. 1983 Feb 1;22(3):590–599. doi: 10.1021/bi00272a011. [DOI] [PubMed] [Google Scholar]
  3. Almenoff J., Wilk S., Orlowski M. Membrane bound pituitary metalloendopeptidase: apparent identity to enkephalinase. Biochem Biophys Res Commun. 1981 Sep 16;102(1):206–214. doi: 10.1016/0006-291x(81)91508-4. [DOI] [PubMed] [Google Scholar]
  4. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  5. Camargo A. C., Shapanka R., Greene L. J. Preparation, assay, and partial characterization of a neutral endopeptidase from rabbit brain. Biochemistry. 1973 Apr 24;12(9):1838–1844. doi: 10.1021/bi00733a028. [DOI] [PubMed] [Google Scholar]
  6. Carvalho K. M., Camargo A. C. Purification of rabbit brain endooligopeptidases and preparation of anti-enzyme antibodies. Biochemistry. 1981 Dec 8;20(25):7082–7088. doi: 10.1021/bi00528a005. [DOI] [PubMed] [Google Scholar]
  7. Checler F., Vincent J. P., Kitabgi P. Inactivation of neurotensin by rat brain synaptic membranes partly occurs through cleavage at the Arg8-Arg9 peptide bond by a metalloendopeptidase. J Neurochem. 1985 Nov;45(5):1509–1513. doi: 10.1111/j.1471-4159.1985.tb07220.x. [DOI] [PubMed] [Google Scholar]
  8. Chu T. G., Orlowski M. Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain. Biochemistry. 1984 Jul 31;23(16):3598–3603. doi: 10.1021/bi00311a005. [DOI] [PubMed] [Google Scholar]
  9. Chu T. G., Orlowski M. Soluble metalloendopeptidase from rat brain: action on enkephalin-containing peptides and other bioactive peptides. Endocrinology. 1985 Apr;116(4):1418–1425. doi: 10.1210/endo-116-4-1418. [DOI] [PubMed] [Google Scholar]
  10. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Camargo A. C., Da Fonseca M. J., Caldo H., De Morais Carvalho K. Influence of the carboxyl terminus of luteinizing hormone-releasing hormone and bradykinin on hydrolysis by brain endo-oligopeptidases. J Biol Chem. 1982 Aug 25;257(16):9265–9267. [PubMed] [Google Scholar]
  12. Kerr M. A., Kenny A. J. The purification and specificity of a neutral endopeptidase from rabbit kidney brush border. Biochem J. 1974 Mar;137(3):477–488. doi: 10.1042/bj1370477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Malfroy B., Swerts J. P., Guyon A., Roques B. P., Schwartz J. C. High-affinity enkephalin-degrading peptidase in brain is increased after morphine. Nature. 1978 Nov 30;276(5687):523–526. doi: 10.1038/276523a0. [DOI] [PubMed] [Google Scholar]
  16. Matsas R., Kenny A. J., Turner A. J. The metabolism of neuropeptides. The hydrolysis of peptides, including enkephalins, tachykinins and their analogues, by endopeptidase-24.11. Biochem J. 1984 Oct 15;223(2):433–440. doi: 10.1042/bj2230433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Molineaux C. J., Lasdun A., Michaud C., Orlowski M. Endopeptidase-24.15 is the primary enzyme that degrades luteinizing hormone releasing hormone both in vitro and in vivo. J Neurochem. 1988 Aug;51(2):624–633. doi: 10.1111/j.1471-4159.1988.tb01084.x. [DOI] [PubMed] [Google Scholar]
  18. OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1–78. [PubMed] [Google Scholar]
  19. Oliveira E. B., Martins A. R., Camargo A. C. Isolation of brain endopeptidases: influence of size and sequence of substrates structurally related to bradykinin. Biochemistry. 1976 May 4;15(9):1967–1974. doi: 10.1021/bi00654a026. [DOI] [PubMed] [Google Scholar]
  20. Orlowski M., Michaud C., Chu T. G. A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides. Eur J Biochem. 1983 Sep 1;135(1):81–88. doi: 10.1111/j.1432-1033.1983.tb07620.x. [DOI] [PubMed] [Google Scholar]
  21. Orlowski M., Michaud C., Molineaux C. J. Substrate-related potent inhibitors of brain metalloendopeptidase. Biochemistry. 1988 Jan 26;27(2):597–602. doi: 10.1021/bi00402a015. [DOI] [PubMed] [Google Scholar]
  22. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  23. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  24. Toffoletto O., Metters K. M., Oliveira E. B., Camargo A. C., Rossier J. Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A, but not by metalloendopeptidase EC 3.4.24.15. Biochem J. 1988 May 15;252(1):35–38. doi: 10.1042/bj2520035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]

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