Skip to main content
NCBI home page
Cellular and Molecular Neurobiology logoLink to Cellular and Molecular Neurobiology
. 1989 Sep;9(3):379–400. doi: 10.1007/BF00711417

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

Elaine A DelBel 1, Afonso P Padovan 1, Gilberto J Padovan 1, Otto Z Sellinger 2, Antonio R Martins 1,✉
PMCID: PMC11567327  PMID: 2558804

Abstract

  1. Bradykinin (Bk; Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg8) inactivation by bulk isolated neurons from rat brain is described.

  2. Bk is rapidly inactivated by neuronal perikarya (4.2 ± 0.6 fmol/min/cell body).

  3. Sites of inactivating cleavages, determined by a kininase bioassay combined with a time-course Bk-product analysis, were the Phe5-Ser6, Pro7-Phe8, Gly4-Phe5, and Pro3-Gly4 peptide bonds. The cleavage of the Phe5-Ser6 bond inactivated Bk at least five fold faster than the other observed cleavages.

  4. Inactivating peptidases were identified by the effect of inhibitors on Bk-product formation. The Phe5-Ser6 bond cleavage is attributed mainly to a calcium-activated thiol-endopeptidase, a predominantly soluble enzyme which did not behave as a metalloenzyme upon dialysis and was strongly inhibited byN-[1(R,S)-carboyx-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate and endo-oligopeptidase A antiserum. Thus, neuronal perikarya thiol-endopeptidase seems to differ from endo-oligopeptidase A and endopeptidase 24.15.

  5. Endopeptidase 24.11 cleaves Bk at the Gly4-Phe5 and, to a larger extent, at the Pro7-Phe8 bond. The latter bond is also cleaved by angiotensin-converting enzyme (ACE) and prolyl endopeptidase (PE). PE also hydrolyzes Bk at the Pro3-Gly4 bond.

  6. Secondary processing of Bk inactivation products occurs by (1) a rapid cleavage of Ser6-Pro7-Phe8-Arg8 at the Pro7-Phe8 bond by endopeptidase 24.11, 3820ACE, and PE; (2) a bestatin-sensitive breakdown of Phe8-Arg9; and (3) conversion of Arg1-Pro7 to Arg1-Phe5, of Gly4-Arg9 to both Gly4-Pro7 and Ser6-Arg9, and of Phe5-Arg9 to Ser6-Arg9, Phe8-Arg9, and Ser6-Pro7, by unidentified peptidases.

  7. A model for the enzymatic inactivation of bradykinin by rat brain neuronal perikarya is proposed.

Key words: isolated nuerons, bradykinin inactivation, thiol-endopeptidase, endopeptidase 24.11, angiotensin-converting enzyme, prolyl endopeptidase

Abbreviations used

ACE

angiotensin-I converting enzyme

AMC

7-amino-4-methyl-coumarin

antiserum

rat brain endo-oligopeptidase A antiserum

Bk

bradykinin cF,N-[1(R,S)-carboxy-2-phenylethyl]

CNS

central nervous system

DFP

diisopropylfluorophosphate

DTT

dithiothreitol

MCA

4-methyl-coumarinyl-7-amide

MK 422

N-[(S)-1-carboxy-3-phenylpropyl]-l-Ala-l-Pro

Nsuc

N-succinyl

pAB

p-aminobenzoate

PCMB

p-mercuribenzoate

PE

prolyl endopeptidase

Z

N-benzyloxycarbonyl

References

  1. Acker, G. R., Molineaux, C., and Orlowski, M. (1987). Synaptosomal membrane-bound form of endopeptidase 24.15 generates Leuenkephalin from dynorphin 1-8,α- andβ-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8.J. Neurochem.18284–292. [DOI] [PubMed] [Google Scholar]
  2. Almenoff, J., and Orlowski, M. (1983). Membrane-bound kidney neutral metalloendopeptidase: Interaction with synthetic substrates, natural peptides and inhibitors.Biochemistry22590–599. [DOI] [PubMed] [Google Scholar]
  3. Andrews, P. C., Hines, C. M., and Dixon, J. E. (1983). Characterization of proline endopeptidase from rat brain.Biochemistry19590–599. [DOI] [PubMed] [Google Scholar]
  4. Bensadoun, A., and Weinstein, D. (1976). Assay of proteins in the presence of interfering materials.Anal. Biochem.70241–250. [DOI] [PubMed] [Google Scholar]
  5. Camargo, A. C. M., and Graeff, F. G. (1969). Subcellular distribution and properties of the bradykinin inactivation system in rabbit brain homogenates.Biochem. Pharmacol.18548–549. [DOI] [PubMed] [Google Scholar]
  6. Camargo, A. C. M., Ramalho-Pinto, F. J., and Greene, L. J. (1972). Brain peptidases: Conversion and inactivation of kinin hormones.J. Neurochem.1937–49. [DOI] [PubMed] [Google Scholar]
  7. Camargo, A. C. M., Shapanka, R., and Greene, L. J. (1973). Preparation, assay and partial characterization of a neutral endopeptidase from rabbit brain.Biochemistry121838–1844. [DOI] [PubMed] [Google Scholar]
  8. Camargo, A. C. M., Martins, A. R., and Greene, L. J. (1979). Steric constraints make polypeptides resistant to hydrolysis by tissue peptidases. InLimited Proteolysis in Micro-organisms (G. N. Cohen and H. Holzer, Eds.), DEW Publication No. (NIH) 79-1591, U.S. Government Printing Office, Washington, D.C., pp. 45–48. [Google Scholar]
  9. Camargo, A. C. M., Oliveira, E. B., Toffoletto, O., Metters, K. M., and Rossier, J. (1987). Brain endo-oligopeptidase A, a putative enkephalin converting enzyme.J. Neurochem.481234–1239. [DOI] [PubMed] [Google Scholar]
  10. Carvalho, K. M., and Camargo, A. C. M. (1981). Purification of rabbit brain endo-oligopeptidases and preparation of anti-enzyme antibodies.Biochemistry207082–7088. [DOI] [PubMed] [Google Scholar]
  11. Chao, J., Woodley, C., Chao, L., and Margolius, H. S. (1983). Identification of tissue kallikrein in brain and in the cell-free translation product encoded by brain mRNA.J. Biol. Chem.25815173–15178. [PubMed] [Google Scholar]
  12. Chu, T. G., and Orlowski, M. (1984). Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain.Biochemistry233598–3603. [DOI] [PubMed] [Google Scholar]
  13. Correa, F. M. A., Innis, R. B., Uhl, G. R., and Snyder, S. H. (1979). Bradykinin-like immunoreactive neuronal systems localized histochemically in rat brain.Proc. Natl. Acad. Sci. USA761489–1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Croft, D. N., and Luban, M. (1965). The estimation of deoxyribonucleic acid in the presence of sialic acid: Application to analysis of human gastric washings.Biochem. J.95612–620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. DelBel, E. A., Gambarini, A. G., and Martins, A. R. (1986). Neuropeptide-metabolizing peptidases in Neuro-2a neuroblastoma and C6 glioma cells.J. Neurochem.47938–944. [DOI] [PubMed] [Google Scholar]
  16. Dresdner, K., Barker, L. A., Orlowski, M., and Wilk, S. (1982). Subcellular distribution of prolyl endopeptidase and cation-sensitive neutral endopeptidase in rabbit brain.J. Neurochem.381151–1154. [DOI] [PubMed] [Google Scholar]
  17. Folk, J. E., Piez, K. A., Carroll, W. R., and Gladner, J. A. (1960). Carboxypeptidase B. IV. Purification and characterization of the porcine enzyme.J. Biol. Chem.2352272–2277. [PubMed] [Google Scholar]
  18. Greene, L. J., Spadaro, A. C. C., Martins, A. R., Perussi de Jesus, W. D., and Camargo, A. C. M. (1982). Brain endo-oligopeptidase B: A post-proline cleaving enzyme that inactivates angiotensin I and II.Hypertension4178–184. [DOI] [PubMed] [Google Scholar]
  19. Hersh, L. B. (1981). Immunological, physical and chemical evidence for the identity of brain and kidney post-proline cleaving enzyme.J. Neurochem.37172–178. [DOI] [PubMed] [Google Scholar]
  20. Kariya, K., Yamauchi, A., Hattori, S., Tsuda, Y., and Okada, Y. (1982). The disappearance rate of intraventricular bradykinin in the brain of the conscious rat.Biochem. Biophys. Res. Commun.1071461–1466. [DOI] [PubMed] [Google Scholar]
  21. Kariya, K., Yamauchi, A., and Sasaki, T. (1985). Regional distribution and characterization of kinin in the CNS of the rat.J. Neurochem.441892–1897. [DOI] [PubMed] [Google Scholar]
  22. Kato, T., Nakano, T., Kojima, K., Nagatsu, T., and Sakakibara, S. (1980). Changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance P by the purified enzyme.J. Neurochem.35527–535. [DOI] [PubMed] [Google Scholar]
  23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem.193265–275. [PubMed] [Google Scholar]
  24. Lynch, D. R., and Snyder, S. H. (1986). Neuropeptides: Multiple molecular forms, metabolic pathways, and receptors.Annu. Rev. Biochem.55773–799. [DOI] [PubMed] [Google Scholar]
  25. Martins, A. R., and De Mello, F. G. (1985). Screening for neuropeptide metabolizing peptidases during the differentiation of chick embryo retina.Dev. Brain Res.21147–151. [DOI] [PubMed] [Google Scholar]
  26. Martins, A. R., Caldo, H., Coelho, H. L. L., Moreira, A. C., Antunes-Rodrigues, J., Greene, L. J., and Camargo, A. C. M. (1980). Screening for rabbit brain neuropeptide-metabolizing peptidases. Inhibition of endopeptidase B by bradykinin potentiating peptide 9a (SQ 20881).J.Neurochem.34100–107. [DOI] [PubMed] [Google Scholar]
  27. Martins, A. R., Izumi, C., Pretel, H. S., and De Mello, F. G. (1987). Ontogenesis of prolyl endopeptidase in the chick retina.Neurosci. Lett.8889–94. [DOI] [PubMed] [Google Scholar]
  28. Matsas, R., Kenny, A. J., and Turner, A. J. (1984). The metabolism of neuropeptides.Biochem. J.223433–440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McDermott, J. R., Gibson, A. M., and Turner, J. D. (1987). Involvement of endopeptidase 24.15 in the inactivation of bradykinin by rat brain slices.Biochem. Biophys. Res. Commun.146154–158. [DOI] [PubMed] [Google Scholar]
  30. McPhie, P. (1971). Dialysis. InMethods in Enzymology (S. P. Colowick and N. O. Kaplan, Eds), Academic Press, New York, Vol. 22, pp. 23–26. [Google Scholar]
  31. Oliveira, E. B., Martins, A. R., and Camargo, A. C. M. (1976). Isolation of brain endopeptidases: Influence of size and sequence of substrates structurally related to bradykinin.Biochemistry151967–1974. [DOI] [PubMed] [Google Scholar]
  32. Orlowski, M. (1983). Pituitary endopeptidases.Mol. Cell. Biochem.5249–74. [DOI] [PubMed] [Google Scholar]
  33. Orlowski, M., Wilk, E., Pearce, S., and Wilk, S. (1979). Purification and properties of a prolyl endopeptidase from rabbit brain.J. Neurochem.33461–469. [DOI] [PubMed] [Google Scholar]
  34. Orlowski, M., Michaud, C., and Chu, T. G. (1983). A soluble metalloendopeptidase from rat brain. Purification of the enzyme and determination of specificity with synthetic and natural peptides.Eur. J. Biochem.13581–88. [DOI] [PubMed] [Google Scholar]
  35. Patchett, A. A., Harris, E., Tristam, E. W., Wyvrat, M. J., Wu, M. T., Taub, D., Peterson, E. R., Ikeler, T. J., tenBroeke, J., Payne, L.-G., Ondeyka, D. L., Thorsett, E. D., Greenlee, W. J., Lohr, N. S., Hoffsommer, R. D., Joshua, H., Ruyle, W. V., Rothrock, J. W., Aster, S. D., Maycock, A. L., Robinson, F. M., Hirschmman, R., Sweet, C. S., Ulm, E. H., Gross, D. M., Vassil, T. C., and Stone, C. A. (1980). A new class of angiotensin-converting enzyme inhibitors.Nature288280–283. [DOI] [PubMed] [Google Scholar]
  36. Perry, D. C., and Snyder, S. H. (1984). Identification of bradykinin in mammalian brain.J. Neurochem.431072–1080. [DOI] [PubMed] [Google Scholar]
  37. Sellinger, O. Z., Azcurra, J. M., Johnson, E., Ohlsson, W. G., and Lodin, Z. (1971). Independence of protein synthesis and drug uptake in nerve cell bodies and glial cells isolated by a new technique.Nature (New Biol.)130253–256. [DOI] [PubMed] [Google Scholar]
  38. Shikimi, T., Kema, R., Matsumoto, M., Yamahata, Y., and Miyata, S. (1973). Studies on kinin like-substances in the brain.Biochem. Pharmacol.22567–573. [DOI] [PubMed] [Google Scholar]
  39. Snyder, S. H. (1980). Brain peptides as neurotransmitters.Science209976–983. [DOI] [PubMed] [Google Scholar]
  40. Soffer, R. L. (1981). Angiotensin-converting enzyme. InBiochemical Regulation of Blood Pressure (R. L. Soffer, Ed.), John Wiley & Sons, New York, pp. 123–164. [Google Scholar]
  41. Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids.Anal. Chem.301190–1206. [PubMed] [Google Scholar]
  42. Suzuki, K., Abiko, T., Endo, N., Kameyama, T., Sasaki, K., and Nabeshima, J. (1969). Biologically active synthetic fragments of bradykinin.Jpn. J. Pharmacol.19325–327. [DOI] [PubMed] [Google Scholar]
  43. Toffoletto, O., Metters, K. M., Oliveira, E. B., Camargo, A. C. M., and Rossier, J. (1988). Enkephalin is liberated from metorphamide and dynorphin A1-8 by endo-oligopeptidase A but not by metalloendopeptidase E. C. 3.4.24.15.Biochem. J.25335–38. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Turner, A. J., Matsas, R., and Kenny, A. J. (1985). Are there neuropeptide-specific peptidases?Biochem. Pharmacol.341347–1356. [DOI] [PubMed] [Google Scholar]
  45. Vallee, B. L., Rupley, J. A., Coombs, T. L., and Neurath, H. (1960). The role of zinc in carboxipeptidase.J. Biol. Chem.23564–69. [Google Scholar]
  46. White, J. D., Stewart, K. D., Krause, J. E., and Mckelvy, J. F. (1985). Biochemistry of peptide-secreting neurons.Physiol. Rev.65553–605. [DOI] [PubMed] [Google Scholar]
  47. Wilk, S. (1983). Prolyl endopeptidase.Life Sci.332149–2157. [DOI] [PubMed] [Google Scholar]
  48. Wilk, S., and Orlowski, M. (1980). Cation-sensitive neutral endopeptidase: Isolation and specificity of the bovine pituitary enzyme.J. Neurochem.351172–1182. [DOI] [PubMed] [Google Scholar]
  49. Wilk, S., and Orlowski, M. (1983). Inhibition of rabbit brain prolyl endopeptidase by N-benzyloxycarbonyl-prolyl-prolinal, a transition state aldehyde inhibitor.J. Neurochem.4169–75. [DOI] [PubMed] [Google Scholar]

Articles from Cellular and Molecular Neurobiology are provided here courtesy of Springer

RESOURCES