Skip to main content
SpringerLink
Log in

Enzymatic inactivation of bradykinin by rat brain neuronal perikarya

  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Summary

  1. 1.

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

  2. 2.

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

  3. 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. 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. 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. 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. 7.

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

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

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

  • 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.

    Google Scholar 

  • Almenoff, J., and Orlowski, M. (1983). Membrane-bound kidney neutral metalloendopeptidase: Interaction with synthetic substrates, natural peptides and inhibitors.Biochemistry 22590–599.

    Google Scholar 

  • Andrews, P. C., Hines, C. M., and Dixon, J. E. (1983). Characterization of proline endopeptidase from rat brain.Biochemistry 19590–599.

    Google Scholar 

  • Bensadoun, A., and Weinstein, D. (1976). Assay of proteins in the presence of interfering materials.Anal. Biochem. 70241–250.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Camargo, A. C. M., Shapanka, R., and Greene, L. J. (1973). Preparation, assay and partial characterization of a neutral endopeptidase from rabbit brain.Biochemistry 121838–1844.

    Google Scholar 

  • 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 

  • 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.

    Google Scholar 

  • Carvalho, K. M., and Camargo, A. C. M. (1981). Purification of rabbit brain endo-oligopeptidases and preparation of anti-enzyme antibodies.Biochemistry 207082–7088.

    Google Scholar 

  • 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.

    Google Scholar 

  • Chu, T. G., and Orlowski, M. (1984). Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain.Biochemistry 233598–3603.

    Google Scholar 

  • 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. USA 761489–1493.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.Hypertension 4178–184.

    Google Scholar 

  • Hersh, L. B. (1981). Immunological, physical and chemical evidence for the identity of brain and kidney post-proline cleaving enzyme.J. Neurochem. 37172–178.

    Google Scholar 

  • 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.

    Google Scholar 

  • Kariya, K., Yamauchi, A., and Sasaki, T. (1985). Regional distribution and characterization of kinin in the CNS of the rat.J. Neurochem. 441892–1897.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Lynch, D. R., and Snyder, S. H. (1986). Neuropeptides: Multiple molecular forms, metabolic pathways, and receptors.Annu. Rev. Biochem. 55773–799.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Matsas, R., Kenny, A. J., and Turner, A. J. (1984). The metabolism of neuropeptides.Biochem. J. 223433–440.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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 

  • 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.Biochemistry 151967–1974.

    Google Scholar 

  • Orlowski, M. (1983). Pituitary endopeptidases.Mol. Cell. Biochem. 5249–74.

    Google Scholar 

  • Orlowski, M., Wilk, E., Pearce, S., and Wilk, S. (1979). Purification and properties of a prolyl endopeptidase from rabbit brain.J. Neurochem. 33461–469.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.Nature 288280–283.

    Google Scholar 

  • Perry, D. C., and Snyder, S. H. (1984). Identification of bradykinin in mammalian brain.J. Neurochem. 431072–1080.

    Google Scholar 

  • 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.

    Google Scholar 

  • Shikimi, T., Kema, R., Matsumoto, M., Yamahata, Y., and Miyata, S. (1973). Studies on kinin like-substances in the brain.Biochem. Pharmacol. 22567–573.

    Google Scholar 

  • Snyder, S. H. (1980). Brain peptides as neurotransmitters.Science 209976–983.

    Google Scholar 

  • 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 

  • Spackman, D. H., Stein, W. H., and Moore, S. (1958). Automatic recording apparatus for use in chromatography of amino acids.Anal. Chem. 301190–1206.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Turner, A. J., Matsas, R., and Kenny, A. J. (1985). Are there neuropeptide-specific peptidases?Biochem. Pharmacol. 341347–1356.

    Google Scholar 

  • 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 

  • White, J. D., Stewart, K. D., Krause, J. E., and Mckelvy, J. F. (1985). Biochemistry of peptide-secreting neurons.Physiol. Rev. 65553–605.

    Google Scholar 

  • Wilk, S. (1983). Prolyl endopeptidase.Life Sci. 332149–2157.

    Google Scholar 

  • Wilk, S., and Orlowski, M. (1980). Cation-sensitive neutral endopeptidase: Isolation and specificity of the bovine pituitary enzyme.J. Neurochem. 351172–1182.

    Google Scholar 

  • 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.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Departamento de Farmacologia e Laboratório de Química de Proteinas, Faculdade de Medicina de Ribeirão Prêto, Universidade de São Paulo, Ribeirão Prêto, São Paulo, Brasil

    Elaine A. DelBel, Afonso P. Padovan, Gilberto J. Padovan & Antonio R. Martins

  2. Mental Health Research Institute, University of Michigan, 48109, Ann Arbor, Michigan, USA

    Otto Z. Sellinger

Authors
  1. Elaine A. DelBel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Afonso P. Padovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gilberto J. Padovan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Otto Z. Sellinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Antonio R. Martins
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

DelBel, E.A., Padovan, A.P., Padovan, G.J. et al. Enzymatic inactivation of bradykinin by rat brain neuronal perikarya. Cell Mol Neurobiol 9, 379–400 (1989). https://doi.org/10.1007/BF00711417

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00711417

Key words

Search

Navigation