Advertisement

Detection and Assessment of Endothelin-Converting Enzyme Activity

  • Carolyn D. Jackson
  • Anthony J. Turner
Protocol
  • 448 Downloads
Part of the Methods in Molecular Biology™ book series (MIMB, volume 206)

Abstract

The biologically active vasoconstrictor peptide endothelin (ET-1) is produced from its inactive precursor big endothelin (big ET-1) in a process catalyzed by an endothelin-converting enzyme (ECE). Two potential human endothelin-converting enzymes, ECE-1 and ECE-2, have been cloned (1, 2, 3) and a third ECE activity, designated ECE-3, has been described in bovine iris (4). ECE-1 is a member of the M13 family of zinc metalloproteases that includes the well-characterized neprilysin, or NEP, to which ECE-1 has 37% identity (5). Like NEP, ECE-1 is a type II integral membrane protein with a short N-terminal cytoplasmic region, a single transmembrane domain and a large C-terminal region containing the active site, the zinc-binding domain and a number of potential glycosylation sites (Fig. 1). Significant differences exist between NEP and ECE-1. ECE-1 is a disulfide-linked dimer whereas NEP is a monomer, and they also exhibit different inhibitor profiles. The zinc metallopeptidase inhibitors phosphoramidon and thiorphan both inhibit NEP at nanomolar concentrations whereas micromolar levels of phosphoramidon are required to inhibit ECE-1, which is also relatively insensitive to thiorphan (I50 > 200 μM). There are also marked differences in substrate specificity, NEP having a much broader profile of substrates. ECE-1 cleaves big ET-1 and big ET-2 at the Trp21-Val22 bond and big ET-3 at the Trp21-Ile22 to produce ET- 1, -2 and -3 respectively (Fig. 2). Originally thought to be specific for these big ET substrates, it has since been shown that ECE-1 efficiently cleaves some other substrates including bradykinin and substance P, and even hydrolyzes insulin B chain at multiple sites (6,7). ECE-1 is expressed predominantly in endothelial cells but it has also been shown to be expressed in smooth muscle cells and neurones and glia in the brain (8). The subcellular localization of the enzyme has been the subject of much debate, with both intracellular and cell surface localization being reported, and is further complicated by the presence of four known isoforms of ECE-1, designated ECE-1a, -1b, -1c and -1d in the human, produced from distinct promoters within the same gene (9,10). These isoforms are identical in their C-terminal and transmembrane domains but differ significantly in their N-terminal domains (see Table 1). Potential tyrosine and dileucine based localization sequences in these short regions have been suggested to be responsible for different subcellular localizations of the isoforms (11, 12, 13, 14).
Fig. 1.

The topology of ECE-1 as a dimeric type II integral membrane protein. The subunits are linked by disulfide bonds as illustrated. Each subunit has a zinc ion coordinated by three zinc ligands that form the catalytic centre. The hatched areas at the N-termini represent the regions that differ between the different isoforms of ECE-1.

Fig. 2.

Schematic representation of the cleavage of some substrates of ECE-1. (A) Cleavage of big ET-1 to produce ET-1 and the C-terminal fragment, (B) the synthetic peptide [Phe22] big ET-1, and (C) bradykinin. Cleavage sites are indicated by arrows.

Table 1

Summary of the Characteristics of the Human ECE-1 Isoforms

ECE-1 Isoform

Number of residues

N-terminal sequence

Localization

Reference

a(β)

758

MPLQGLGLQRNPFLQGKR GPGLTSSPPLLPPSLQ-

Predominantly plasma membrane

Shimada et al. (2)

b

770

MRGVWPPPVSALLSALGM STYKRATLDEEDLVDSLS EGDAYPNGLQ-

Exclusively intracellular

Schweizer et al.(9)

c(α)

754

MMSTYKRATLDEEDLVDS LSEGDAYPNGLQ-

Plasma membrane and intracellular

Schmidt et al.(1)

d

767

MEALRESVLHLALQMTYK RATLDEEDLVDSLSEGDA YPMGLQ-

Intermediate between a and c

Valdenaire et al.(10)

Two isoforms of ECE-1 were originally identified in the rat and designated a and b. Subsequently, the human forms were identified and given Roman rather than Greek symbols for identification. It is now established that the rat isoforms a and b correspond to human ECE-1 c and ECE-1 a, respectively.

Keywords

Booster Injection Phenylhydrazine Hydrochloride Bovine Pulmonary Artery Smooth Muscle DTNB Assay Bovine Iris 
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.

References

  1. 1.
    Schmidt M. Kroger B. Jacob E. Seulberger H. Subkowski T. Otter R., et al. (1994) Molecular characterization of human and bovine endothelin converting enzyme (ECE-1). FEBS Lett. 356, 238–243.PubMedCrossRefGoogle Scholar
  2. 2.
    Shimada K. Matsushita Y. Wakabayashi K. Takahashi M. Matsubara A. Iijima Y., and Tanzawa K. (1995) Cloning and functional expression of human endothelin-converting enzyme cDNA. Biochem. Biophys. Res. Comm. 207, 807–812.PubMedCrossRefGoogle Scholar
  3. 3.
    Emoto N. and Yanagisawa M. (1995) Endothelin-converting enzyme-2 is a membrane-bound, phosphoramidon-sensitive metalloprotease with acidic pH optimum. J. Biol. Chem. 270, 15,262–15,268.PubMedCrossRefGoogle Scholar
  4. 4.
    Hasegawa H. Hiki K. Sawamura T. Aoyama T. Okamoto Y. Miwa S., et al. (1998) Purification of a novel endothelin-converting enzyme specific for big endothelin-3. FEBS Lett. 428, 304–308.PubMedCrossRefGoogle Scholar
  5. 5.
    Turner A. J. Isaac R. E., and Coates D. (2001) The neprilysin (NEP) family of zinc metalloendopeptidases: genomics and function. Bioessays 23, 261–269.PubMedCrossRefGoogle Scholar
  6. 6.
    Hoang M. V. and Turner A. J. (1997) Novel activity of endothelin-converting enzyme: hydrolysis of bradykinin. Biochem. J. 327, 23–26.PubMedGoogle Scholar
  7. 7.
    Johnson G. D. Stevenson T., and Ahn K. (1999) Hydrolysis of peptide hormones by endothelin-converting enzyme-1. J. Biol. Chem. 274, 4053–4058.PubMedCrossRefGoogle Scholar
  8. 8.
    Barnes K. Walkden B. J. Wilkinson T. C., and Turner A. J. (1997) Expression of endothelin-converting enzyme in both neuroblastoma and glial cell lines and its localization in rat hippocampus. J. Neurochem. 68, 570–577.PubMedCrossRefGoogle Scholar
  9. 9.
    Schweizer A. Valdenaire O. Nelbock P. Deuschle U. Dumas Milne Edwards J. B. Stumpf J. G., and Loffler B. M. (1997) Human endothelin-converting enzyme (ECE-1): three isoforms with distinct subcellular localizations. Biochem. J. 328, 871–877.PubMedGoogle Scholar
  10. 10.
    Valdenaire O. Lepailleur-Enouf D. Egidy G. Thouard A. Barret A. Vranckx R., et al. (1999) A fourth isoform of endothelin-converting enzyme (ECE-1) is generated from an additional promoter. Eur. J. Biochem. 264, 341–349.PubMedCrossRefGoogle Scholar
  11. 11.
    Barnes K. Brown C., and Turner A. J. (1998) Endothelin-converting enzyme: ultrastructural localization and its recycling from the cell surface. Hypertension 31, 3–9.PubMedGoogle Scholar
  12. 12.
    Emoto N. Nurhantari Y. Alimsardjono H. Xie J. Yamada T. Yanagisawa M., and Matsuo M. (1999) Constitutive lysosomal targeting and degradation of bovine endothelin-converting enzyme-1a mediated by novel signals in its alternatively spliced cytoplasmic tail. J. Biol. Chem. 274, 1509–1518.PubMedCrossRefGoogle Scholar
  13. 13.
    Valdenaire O. Barret A. Schweizer A. Rohrbacher E. Mongiat F. Pinet F., et al. (1999b) Two di-leucine-based motifs account for the different subcellular localizations of the human endothelin-converting enzyme (ECE-1) isoforms. J. Cell Sci. 112, 3115–3125.PubMedGoogle Scholar
  14. 14.
    Cailler F. Zappulla J. P. Boileau G., and Crine P. (1999) The N-terminal segment of endothelin-converting enzyme (ECE)-1b contains a di-leucine motif that can redirect neprilysin to an intracellular compartment in Madin-Darby canine kidney (MDCK) cells. Biochem J. 341, 119–126.PubMedCrossRefGoogle Scholar
  15. 15.
    Yanagisawa H. Yanagisawa M. Kapur R. P. Richardson J. A. Williams S. C. Clouthier D. E., et al. (1998) Dual genetic pathways of endothelin-mediated intercellular signalling revealed by targeted disruption of endothelin-converting enzyme-1 gene. Development 125, 825–836.PubMedGoogle Scholar
  16. 16.
    Barker S. Khan N. Q. Wood E. G. and Corder R. (2001) Effect of an antisense oligodeoxynucleotide to endothelin-converting enzyme-1c (ECE-1c) on ECE-1c mRNA, ECE-1 protein and endothelin-1 synthesis in bovine pulmonary artery smooth muscle cells. Mol. Pharmacol. 59, 163–169.PubMedGoogle Scholar
  17. 17.
    DeLombaert S. Ghai R. D. Jeng A. Y. Trapani A. J., and Webb R. L. (1994) Biochem. Biophys. Res. Comm. 204, 407–412.CrossRefGoogle Scholar
  18. 18.
    Wallace E. M. Moliterni J. A. Moskal M. A. Neubert A. D. Marcopulos N. Stamford L. B., et al. (1998) Design and synthesis of potent, selective inhibitors of endothelin-converting enzyme. J. Med. Chem. 41, 1513–1523.PubMedCrossRefGoogle Scholar
  19. 19.
    Ahn K. Sisneros A. M. Herman S. B. Pan S. M. Hupe D. Lee C., et al. (1998) Novel quinazoline inhibitors of endothelin-converting enyme-1. Biochem. Biophys. Res. Comm. 243, 184–190.PubMedCrossRefGoogle Scholar
  20. 20.
    Russell F. D. and Davenport A. P. (1999) Evidence for intracellular endothelinconverting enzyme-2 expression in cultured human vascular endothelial cells. Circ. Res. 84, 891–896.PubMedGoogle Scholar
  21. 21.
    Wada A. Tsutamoto T. Ohnishi M. Sawaki M. Fukai D. Maeda Y., and Kinoshita M. (1999) Effects of a specific endothelin-converting enzyme inhibitor on cardiac, renal, and neurohumoral functions in congestive heart failure: comparison of effects with those of endothelin A receptor antagonism. Circulation 99, 570–577.PubMedGoogle Scholar
  22. 22.
    Yanagisawa M. Kurihara H. Kimura S. Tomobe Y. Kobayashi M. Mitsui Y., et al. (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411–415.PubMedCrossRefGoogle Scholar
  23. 23.
    Baldwin S. A. (1994) Methods in Molecular Biology, vol 27: Biomembrane Protocols: II, Architecture and Function. (Graham J. M., ed.) Humana, Totowa, NJ.Google Scholar
  24. 24.
    Smith P. K. Krohn R. I. Hermanson G. T. Mallia A. K. Gartner F. H. Provenzano M. D., et al. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 76–85.PubMedCrossRefGoogle Scholar
  25. 25.
    Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  26. 26.
    Luciani N. De Rocquigny H. Turcaud S. Romieu A., and Roques B. P. (2001) Highly sensitive and selective fluorescence assays for rapid screening of endothelin-converting enzyme inhibitors. Biochem. J. 356, 813–819.PubMedCrossRefGoogle Scholar
  27. 27.
    Johnson G. D. and Ahn K. (2000) Development of an internally quenched fluorescent substrate selective for endothelin-converting enzyme-1. Anal. Biochem. 286, 112–118.PubMedCrossRefGoogle Scholar
  28. 28.
    Matsumura Y. Umekawa T. Kawamura H. Takaoka M. Robinson P. S. Cook N. D., and Morimoto S. (1992) A simple method for measurement of phosphoramidon-sensitive endothelin-converting enzyme activity. Life Sci. 51, 1603–1611.PubMedCrossRefGoogle Scholar
  29. 29.
    Warner T. D. Budzik G. P. Mitchell J. A. Huang Z. J., and Murad F. (1992) Detection by bioassay and specific enzyme-linked-immunosorbent assay of phosphoramidon-inhibitable endothelin-converting enzyme activity in brain and endothelium. J. Cardiovasc. Pharmacol. 20(Suppl. 12), S19–S21.PubMedGoogle Scholar
  30. 30.
    Fawzi A. B. Cleven R. M., and Wright D. L. (1994) A rapid and selective endothelin-converting enzyme assay-characterization of a phosphoramidon-sensitive enzyme from guinea-pig membrane. Anal. Biochem. 222, 342–350.PubMedCrossRefGoogle Scholar
  31. 31.
    Plumpton C. Haynes W. G. Webb D. J., and Davenport A. P. (1995) Measurement of C-terminal fragment of big endothelin-1 in humans. J. Cardiovasc. Pharmacol. 26(Suppl. 3), S34–S36.PubMedGoogle Scholar
  32. 32.
    Parnot C. LeMoullec J. M. Cousin M. A. Guedin D. Corvol P., and Pinet F. (1997) A live cell assay for studying extracellular and intracellular endothelinconverting enzyme activity. Hypertension 30, 837–844.PubMedGoogle Scholar
  33. 33.
    Turner A. J. and Murphy L. J. (1996) Molecular pharmacology of endothelinconverting enzymes. Biochem. Pharmacol. 51, 91–102.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  • Carolyn D. Jackson
    • 1
  • Anthony J. Turner
    • 1
  1. 1.School of Biochemistry and Molecular BiologyUniversity of LeedsLeedsUK

Personalised recommendations