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.
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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.
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.
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.
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.
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.
Bålöw, R.-M., Ragnarsson, U., and Zetterqvist, O. 1983. Tripeptidylaminopeptidase in the extralysosomal fraction of rat liver. J. Biol. Chem. 258:11622–11628.
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.
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.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.
Dixon, M., and Webb, E.C. 1979. Enzymes (3rd Edition), Longman, Harlow, UK.
Smith, A. I., and McDermott, J. R. 1984. High-performance liquid chromatography of neuropeptides using radially compressed polythene cartridges. J. Chromatog. 306:99–108.
Bidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. 1984. Rapid analysis of amino acids using precolumn derivitization. J. Chromatogr. 336:93–104.
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.
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.
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.
Bålöw, R.-M., and Eriksson, I. 1987. Tripeptidyl peptidase II in haemolysates and liver homogenates of various species. Biochem. J. 241:75–80.
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.
McDonald, J. K., and Barrett, A. J. 1986. Tripeptidylpeptidase I. Pages 147–149,in Mammalian Proteases vol. 2 Exopeptidases, Academic Press, London.
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.
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.
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.
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.
Rivett, A. J., 1989. High molecular mass intracellular proteases. Biochem. J. 263:625–633.
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.
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.
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Wilson, C., Gibson, A.M. & McDermott, J.R. Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain. Neurochem Res 18, 743–749 (1993). https://doi.org/10.1007/BF00966768
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DOI: https://doi.org/10.1007/BF00966768