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