PURIFICATION AND CHARACTERIZATION OF TRIPEPTIDYLPEPTIDASE-II FROM POSTMORTEM 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 > 10(6)) and smalle r forms. The enzyme hydrolyses Ala-Ala-Phe-7-amido-4-methylcoumarin wi th a pH optimum of around 7.5 and K(m) of 148 muM. It did not hydrolys e N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, aminoacyl- or dipep tidyl-7-amido-methylcoumarins and was not inhibited by bestatin. The e nzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroiso coumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide s howing that its activity is serine and cysteine dependent. The purifie d enzyme released tripeptides from several naturally occurring neurope ptides with quite broad specificity. Cholecystokinin octapeptide, angi otensin III and neurokinin A were the most rapidly hydrolysed. Peptide s with Pro residues arount the point of cleavage were not hydrolysed.