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Neurochemical Research

, Volume 18, Issue 7, pp 743–749 | Cite as

Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain

  • C. Wilson
  • A. M. Gibson
  • J. R. McDermott
Original Articles

Abstract

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (Mr>106) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amino-4-methylcoumarin with a pH optimum of around 7.5 and Km of 148 μM. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amino-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues arount the point of cleavage were not hydrolysed.

Key words

Tripeptidylpeptidase aminopeptidase neuropeptides human brain cholecystokinin octapeptide neurokinin-A 

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References

  1. 1.
    McDermott, J. r., Mantle, D., Lauffart, B., and Kidd, A. M. 1985. Purification and characterization of a neuropeptide degrading aminopeptidase from human brain. J. Neurochem. 45:752–759.PubMedGoogle Scholar
  2. 2.
    McDermott, J. R., Mantle, D., Lauffart, B., Gibson, A. M., and Biggins, J. A. 1988. Purification and characterization of two soluble Cl-activated arginyl aminopeptidases from human brain and their endopeptidase action on neuropeptides. J. Neurochem. 50:176–182.PubMedGoogle Scholar
  3. 3.
    Gibson, A. M., Biggins, J. A., Lauffart, B., Mantle, D., and McDermott, J. R. 1991. Human brain leucyl aminopeptidase: isolation, characterization and specificity against some neuropeptides. Neuropeptides 19:163–169.PubMedGoogle Scholar
  4. 4.
    Lauffart, B., McDermott, J. R., Biggins, J. A., Gibson, A. M., and Mantle, D. 1988. Purification and characterization of pyroglutamylaminopeptidase from human cerebral cortex. Biochem. Soc. Trans. 17:207–208.Google Scholar
  5. 5.
    Lees, T., Lauffart, B., McDermott, J. R., Gibson, A. M., and Mantle, D. 1990. Purification and characterization of tripeptidylaminopeptidase from human cerebral cortex. Biochem. Soc. Trans. 18:667.PubMedGoogle Scholar
  6. 6.
    Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. 1983. Tripeptidylaminopeptidase in the extralysosomal fraction of rat liver. J. Biol. Chem. 258:11622–11628.PubMedGoogle Scholar
  7. 7.
    Bålöw, R.-M., Tomkinson, B., Ragnarsson, U., and Zetterqvist, O. 1986. Purification, substrate specificity and classification of tripeptidylpeptidase II. J. Biol. Chem. 261:2409–2417.PubMedGoogle Scholar
  8. 8.
    Lowry, D. H., Rosebrough, N. J., Farr A. L., and Randall R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275.PubMedGoogle Scholar
  9. 9.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.PubMedGoogle Scholar
  10. 10.
    Dixon, M., and Webb, E.C. 1979. Enzymes (3rd Edition), Longman, Harlow, UK.Google Scholar
  11. 11.
    Smith, A. I., and McDermott, J. R. 1984. High-performance liquid chromatography of neuropeptides using radially compressed polythene cartridges. J. Chromatog. 306:99–108.Google Scholar
  12. 12.
    Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. 1984. Rapid analysis of amino acids using precolumn derivitization. J. Chromatogr. 336:93–104.PubMedGoogle Scholar
  13. 13.
    Dodd, P. R., Hardy, J. A., Oakley, A. E., and Strong, A. J. 1981. Synaptosomes prepared from fresh human cerebral cortex; morphology, respiration and release of transmitter amino acids. Brain Res. 224:419–425.PubMedGoogle Scholar
  14. 14.
    McDermott, J. R., Gibson, A. M., and Biggins, J. A. 1988. Peptidases involved in the degradation of neurotensin by human brain synaptic membranes. Biochem. Soc. Trans. 16:851–852.Google Scholar
  15. 15.
    McDermott, J. R., Gibson, A. M., Oakley, A. E., and Biggins, J. A. 1991. Multicatalytic, high-M, endopeptidase from post-mortem human brain. J. Neurochem. 56:1509–1517.PubMedGoogle Scholar
  16. 16.
    Bålöw, R.-M., and Eriksson, I. 1987. Tripeptidyl peptidase II in haemolysates and liver homogenates of various species. Biochem. J. 241:75–80.PubMedGoogle Scholar
  17. 17.
    Tomkinson, B., Wernstedt, C., Hellman, U., and Zetterqvist, O. 1987. Active site of tripeptidyl peptidase II from human erthyrocytes is of the subtilisin type. Proc. Natl. Acad. Sci. USA 84:7508–7512.PubMedGoogle Scholar
  18. 18.
    McDonald, J. K., and Barrett, A. J. 1986. Tripeptidylpeptidase I. Pages 147–149,in Mammalian Proteases vol. 2 Exopeptidases, Academic Press, London.Google Scholar
  19. 19.
    MacPherson, E., Tomkinson, B., Bålöw, R.-M., Hoglund, S., and Zetterqvist, O. 1987. Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity. Biochem. J. 248:259–263.PubMedGoogle Scholar
  20. 20.
    Tomkinson, B., and Jonsson, A. K. 1991. Characterization of cDNA for human tripeptidylpeptidase II: the N-terminal part of the enzyme is similar to subtilisin. Biochemistry 30:168–174.PubMedGoogle Scholar
  21. 21.
    Bresnahan, P. A., Leduc, R., Thomas, L., Thorner, J., Gibson, H. L., Brake, A. J., Barr, P. J., and Thomas, G. 1990. Humanfur gene encodes a yeast KEX2-like endoprotease that cleaves pro-β-NGFin vivo. J. Cell. Biol. 111:2851–2859.PubMedGoogle Scholar
  22. 22.
    Smeekens, S. P., and Steiner, D. F. 1990. Identification of a human insulinoma cDNA encoding a novel mammalian protein structurally related to the yeast dibasic processing protease Kex2. J. Biol. Chem. 265:2997–3000.PubMedGoogle Scholar
  23. 23.
    Rivett, A. J., 1989. High molecular mass intracellular proteases. Biochem. J. 263:625–633.PubMedGoogle Scholar
  24. 24.
    McDermott, J. R., Dodd, P. R., Edwardson, J. A., Hardy, J. A., and Smith, A. I. 1983. Pathway of inactivation of cholecystokinin octapeptide (CCK-8) by synaptosomal fractions. Neurochem. Int. 5:641–647.Google Scholar
  25. 25.
    Rose, C., Camus, A., and Schwartz, J. C. 1988. A serine peptidase responsible for the inactivation of endogenous cholecystokinin in brain. Proc. Natl. Acad. Sci. USA 85:8326–8330.PubMedGoogle Scholar

Copyright information

© Plenum Publishing Corporation 1993

Authors and Affiliations

  • C. Wilson
    • 1
  • A. M. Gibson
    • 1
  • J. R. McDermott
    • 1
  1. 1.MRC Neurochemical Pathology UnitNewcastle General HospitalNewcastle upon TyneUK

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