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. 1994 May 1;299(Pt 3):651–657. doi: 10.1042/bj2990651

Identification and properties of a peptidyl dipeptidase in the housefly, Musca domestica, that resembles mammalian angiotensin-converting enzyme.

N S Lamango 1, R E Isaac 1
PMCID: PMC1138070  PMID: 8192653

Abstract

[D-Ala2,Leu5]Enkephalin was readily metabolized by membranes (40,000 g pellet) prepared from heads of the housefly, Musca domestica, with Gly3-Phe4 being the major site of cleavage. This hydrolysis was only partially inhibited (40%) by 10 microM phosphoramidon, an inhibitor of endopeptidase-24.11, but was almost totally abolished in the presence of a mixture of 10 microM phosphoramidon and 10 microM captopril, a potent inhibitor of mammalian angiotensin-converting enzyme (ACE). An assay for ACE employing Bz-Gly-His-Leu as the substrate was used to confirm the presence of an ACE-like peptidyl dipeptidase activity in fly head membranes. The peptidase had a Km of 1.91 mM for Bz-Gly-His-Leu and a pH optimum of 8.2. The activity was inhibited by 100 microM EDTA and was greatly activated by ZnCl2 but not other bivalent metal ions. Captopril, lisinopril, fosinoprilat and enalaprilat, all selective inhibitors of mammalian ACE, were also good inhibitors of the insect enzyme with IC50 values of 400 nM, 130 nM, 16 nM and 290 nM respectively. An M(r) value of around 87,000 was obtained for this enzyme from gel-filtration chromatography, indicating that the insect enzyme is similar in size to mammalian testicular ACE (M(r) = 90,000-110,000) and not the larger form of the enzyme (M(r) = 150,000-180,000) found in mammalian somatic tissues. The fly peptidyl dipeptidase was released from membranes into a soluble fraction by incubating the head membranes at 37 degrees C but not at 0 degree C, suggesting that the insect ACE-like enzyme can be solubilized from cell surfaces through the activity of a membrane-bound enzyme activity. In conclusion, we have shown the existence of a peptidyl dipeptidase in membranes from the heads of M. domestica, which has similar properties to those of mammalian ACE.

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Selected References

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  1. Barnes K., Turner A. J., Kenny A. J. An immunoelectron microscopic study of pig substantia nigra shows co-localization of endopeptidase-24.11 with substance P. Neuroscience. 1993 Apr;53(4):1073–1082. doi: 10.1016/0306-4522(93)90490-7. [DOI] [PubMed] [Google Scholar]
  2. Barnes K., Turner A. J., Kenny A. J. Membrane localization of endopeptidase-24.11 and peptidyl dipeptidase A (angiotensin converting enzyme) in the pig brain: a study using subcellular fractionation and electron microscopic immunocytochemistry. J Neurochem. 1992 Jun;58(6):2088–2096. doi: 10.1111/j.1471-4159.1992.tb10950.x. [DOI] [PubMed] [Google Scholar]
  3. Bernstein K. E., Martin B. M., Edwards A. S., Bernstein E. A. Mouse angiotensin-converting enzyme is a protein composed of two homologous domains. J Biol Chem. 1989 Jul 15;264(20):11945–11951. [PubMed] [Google Scholar]
  4. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed] [Google Scholar]
  5. Cheung H. S., Wang F. L., Ondetti M. A., Sabo E. F., Cushman D. W. Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. J Biol Chem. 1980 Jan 25;255(2):401–407. [PubMed] [Google Scholar]
  6. Cushman D. W., Cheung H. S. Concentrations of angiotensin-converting enzyme in tissues of the rat. Biochim Biophys Acta. 1971 Oct;250(1):261–265. doi: 10.1016/0005-2744(71)90142-2. [DOI] [PubMed] [Google Scholar]
  7. De Loof A., Schoofs L. Homologies between the amino acid sequences of some vertebrate peptide hormones and peptides isolated from invertebrate sources. Comp Biochem Physiol B. 1990;95(3):459–468. doi: 10.1016/0305-0491(90)90003-c. [DOI] [PubMed] [Google Scholar]
  8. Duve H., Thorpe A. Immunocytochemical mapping of gastrin/CCK-like peptides in the neuroendocrine system of the blowfly Calliphora vomitoria (Diptera). Cell Tissue Res. 1984;237(2):309–320. doi: 10.1007/BF00217150. [DOI] [PubMed] [Google Scholar]
  9. Duve H., Thorpe A. Mapping of enkephalin-related peptides in the nervous system of the blowfly, Calliphora vomitoria, and their co-localization with cholecystokinin (CCK)- and pancreatic polypeptide (PP)-like peptides. Cell Tissue Res. 1988 Feb;251(2):399–415. doi: 10.1007/BF00215849. [DOI] [PubMed] [Google Scholar]
  10. Ehlers M. R., Fox E. A., Strydom D. J., Riordan J. F. Molecular cloning of human testicular angiotensin-converting enzyme: the testis isozyme is identical to the C-terminal half of endothelial angiotensin-converting enzyme. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7741–7745. doi: 10.1073/pnas.86.20.7741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ehlers M. R., Riordan J. F. Angiotensin-converting enzyme: new concepts concerning its biological role. Biochemistry. 1989 Jun 27;28(13):5311–5318. doi: 10.1021/bi00439a001. [DOI] [PubMed] [Google Scholar]
  12. El-Dorry H. A., Bull H. G., Iwata K., Thornberry N. A., Cordes E. H., Soffer R. L. Molecular and catalytic properties of rabbit testicular dipeptidyl carboxypeptidase. J Biol Chem. 1982 Dec 10;257(23):14128–14133. [PubMed] [Google Scholar]
  13. Erdös E. G., Skidgel R. A. Neutral endopeptidase 24.11 (enkephalinase) and related regulators of peptide hormones. FASEB J. 1989 Feb;3(2):145–151. [PubMed] [Google Scholar]
  14. Holman G. M., Nachman R. J., Wright M. S. Insect neuropeptides. Annu Rev Entomol. 1990;35:201–217. doi: 10.1146/annurev.en.35.010190.001221. [DOI] [PubMed] [Google Scholar]
  15. Hooper N. M. Angiotensin converting enzyme: implications from molecular biology for its physiological functions. Int J Biochem. 1991;23(7-8):641–647. doi: 10.1016/0020-711x(91)90032-i. [DOI] [PubMed] [Google Scholar]
  16. Hooper N. M., Keen J., Pappin D. J., Turner A. J. Pig kidney angiotensin converting enzyme. Purification and characterization of amphipathic and hydrophilic forms of the enzyme establishes C-terminal anchorage to the plasma membrane. Biochem J. 1987 Oct 1;247(1):85–93. doi: 10.1042/bj2470085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hooper N. M., Turner A. J. Isolation of two differentially glycosylated forms of peptidyl-dipeptidase A (angiotensin converting enzyme) from pig brain: a re-evaluation of their role in neuropeptide metabolism. Biochem J. 1987 Feb 1;241(3):625–633. doi: 10.1042/bj2410625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Howard T. E., Shai S. Y., Langford K. G., Martin B. M., Bernstein K. E. Transcription of testicular angiotensin-converting enzyme (ACE) is initiated within the 12th intron of the somatic ACE gene. Mol Cell Biol. 1990 Aug;10(8):4294–4302. doi: 10.1128/mcb.10.8.4294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Isaac R. E. Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes. Biochem J. 1988 Nov 1;255(3):843–847. doi: 10.1042/bj2550843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Isaac R. E. Proctolin degradation by membrane peptidases from nervous tissues of the desert locust (Schistocerca gregaria). Biochem J. 1987 Jul 15;245(2):365–370. doi: 10.1042/bj2450365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Konopińska D., Rosiński G., Sobótka W. Insect peptide hormones, an overview of the present literature. Int J Pept Protein Res. 1992 Jan;39(1):1–11. doi: 10.1111/j.1399-3011.1992.tb01548.x. [DOI] [PubMed] [Google Scholar]
  22. Kumar R. S., Kusari J., Roy S. N., Soffer R. L., Sen G. C. Structure of testicular angiotensin-converting enzyme. A segmental mosaic isozyme. J Biol Chem. 1989 Oct 5;264(28):16754–16758. [PubMed] [Google Scholar]
  23. Lamango N. S., Isaac R. E. Metabolism of insect neuropeptides: properties of a membrane-bound endopeptidase from heads of Musca domestica. Insect Biochem Mol Biol. 1993 Oct;23(7):801–808. doi: 10.1016/0965-1748(93)90068-4. [DOI] [PubMed] [Google Scholar]
  24. Langford K. G., Shai S. Y., Howard T. E., Kovac M. J., Overbeek P. A., Bernstein K. E. Transgenic mice demonstrate a testis-specific promoter for angiotensin-converting enzyme. J Biol Chem. 1991 Aug 25;266(24):15559–15562. [PubMed] [Google Scholar]
  25. Lattion A. L., Soubrier F., Allegrini J., Hubert C., Corvol P., Alhenc-Gelas F. The testicular transcript of the angiotensin I-converting enzyme encodes for the ancestral, non-duplicated form of the enzyme. FEBS Lett. 1989 Jul 31;252(1-2):99–104. doi: 10.1016/0014-5793(89)80897-x. [DOI] [PubMed] [Google Scholar]
  26. McKelvy J. F., Blumberg S. Inactivation and metabolism of neuropeptides. Annu Rev Neurosci. 1986;9:415–434. doi: 10.1146/annurev.ne.09.030186.002215. [DOI] [PubMed] [Google Scholar]
  27. Nässel D. R., Lundquist C. T. Insect tachykinin-like peptide: distribution of leucokinin immunoreactive neurons in the cockroach and blowfly brains. Neurosci Lett. 1991 Sep 16;130(2):225–228. doi: 10.1016/0304-3940(91)90402-f. [DOI] [PubMed] [Google Scholar]
  28. Nässel D. R., O'shea M. Proctolin-like immunoreactive neurons in the blowfly central nervous system. J Comp Neurol. 1987 Nov 15;265(3):437–454. doi: 10.1002/cne.902650311. [DOI] [PubMed] [Google Scholar]
  29. Nässel D. R., Ohlsson L. G., Cantera R. Metamorphosis of identified neurons innervating thoracic neurohemal organs in the blowfly: transformation of cholecystokininlike immunoreactive neurons. J Comp Neurol. 1988 Jan 15;267(3):343–356. doi: 10.1002/cne.902670305. [DOI] [PubMed] [Google Scholar]
  30. O'Shea M., Schaffer M. Neuropeptide function: the invertebrate contribution. Annu Rev Neurosci. 1985;8:171–198. doi: 10.1146/annurev.ne.08.030185.001131. [DOI] [PubMed] [Google Scholar]
  31. Ondetti M. A., Cushman D. W. Enzymes of the renin-angiotensin system and their inhibitors. Annu Rev Biochem. 1982;51:283–308. doi: 10.1146/annurev.bi.51.070182.001435. [DOI] [PubMed] [Google Scholar]
  32. Oppong S. Y., Hooper N. M. Characterization of a secretase activity which releases angiotensin-converting enzyme from the membrane. Biochem J. 1993 Jun 1;292(Pt 2):597–603. doi: 10.1042/bj2920597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Scharrer B. Insects as models in neuroendocrine research. Annu Rev Entomol. 1987;32:1–16. doi: 10.1146/annurev.en.32.010187.000245. [DOI] [PubMed] [Google Scholar]
  34. Schoofs L., Tips A., Holman G. M., Nachman R. J., De Loof A. Distribution of locustamyotropin-like immunoreactivity in the nervous system of Locusta migratoria. Regul Pept. 1992 Feb 18;37(3):237–254. doi: 10.1016/0167-0115(92)90618-5. [DOI] [PubMed] [Google Scholar]
  35. Schooneveld H., Tesser G. I., Veenstra J. A., Romberg-Privee H. M. Adipokinetic hormone and AKH-like peptide demonstrated in the corpora cardiaca and nervous system of Locusta migratoria by immunocytochemistry. Cell Tissue Res. 1983;230(1):67–76. doi: 10.1007/BF00216028. [DOI] [PubMed] [Google Scholar]
  36. Shai S. Y., Fishel R. S., Martin B. M., Berk B. C., Bernstein K. E. Bovine angiotensin converting enzyme cDNA cloning and regulation. Increased expression during endothelial cell growth arrest. Circ Res. 1992 Jun;70(6):1274–1281. doi: 10.1161/01.res.70.6.1274. [DOI] [PubMed] [Google Scholar]
  37. Soubrier F., Alhenc-Gelas F., Hubert C., Allegrini J., John M., Tregear G., Corvol P. Two putative active centers in human angiotensin I-converting enzyme revealed by molecular cloning. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9386–9390. doi: 10.1073/pnas.85.24.9386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stephenson S. L., Kenny A. J. Metabolism of neuropeptides. Hydrolysis of the angiotensins, bradykinin, substance P and oxytocin by pig kidney microvillar membranes. Biochem J. 1987 Jan 1;241(1):237–247. doi: 10.1042/bj2410237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Strittmatter S. M., Thiele E. A., Kapiloff M. S., Snyder S. H. A rat brain isozyme of angiotensin-converting enzyme. Unique specificity for amidated peptide substrates. J Biol Chem. 1985 Aug 15;260(17):9825–9832. [PubMed] [Google Scholar]
  40. Thekkumkara T. J., Livingston W., 3rd, Kumar R. S., Sen G. C. Use of alternative polyadenylation sites for tissue-specific transcription of two angiotensin-converting enzyme mRNAs. Nucleic Acids Res. 1992 Feb 25;20(4):683–687. doi: 10.1093/nar/20.4.683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Turner A. J., Dowdall M. J. The metabolism of neuropeptides. Both phosphoramidon-sensitive and captopril-sensitive metallopeptidases are present in the electric organ of Torpedo marmorata. Biochem J. 1984 Aug 15;222(1):255–259. doi: 10.1042/bj2220255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Turner A. J., Hryszko J., Hooper N. M., Dowdall M. J. Purification and characterization of a peptidyl dipeptidase resembling angiotensin converting enzyme from the electric organ of Torpedo marmorata. J Neurochem. 1987 Mar;48(3):910–916. doi: 10.1111/j.1471-4159.1987.tb05603.x. [DOI] [PubMed] [Google Scholar]
  43. Turner A. J., Matsas R., Kenny A. J. Are there neuropeptide-specific peptidases? Biochem Pharmacol. 1985 May 1;34(9):1347–1356. doi: 10.1016/0006-2952(85)90669-0. [DOI] [PubMed] [Google Scholar]

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