Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain
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 wordsTripeptidylpeptidase aminopeptidase neuropeptides human brain cholecystokinin octapeptide neurokinin-A
Unable to display preview. Download preview PDF.
- 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
- 10.Dixon, M., and Webb, E.C. 1979. Enzymes (3rd Edition), Longman, Harlow, UK.Google Scholar
- 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
- 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
- 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
- 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