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 (M(r) > 10(6) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amido-4-methylcoumarin with a pH optimum of around 7.5 and Km of 148 microM. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amido-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amido-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 around the point of cleavage were not hydrolysed.
MRC Neurochemical Pathology Unit, Newcastle General Hospital, United Kingdom.
C
C
Wilson
A M
AM
Gibson
J R
JR
McDermott
D000595:Amino Acid Sequence; D000626:Aminopeptidases; D000805:Angiotensin III / Q000378:metabolism; D002540:Cerebral Cortex / Q000201:enzymology*; D002845:Chromatography; D002850:Chromatography, Gel; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015288:Neurokinin A / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism; D013379:Substrate Specificity
D009479:Neuropeptides; D000805:Angiotensin III; D015288:Neurokinin A; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C048001:tripeptidyl-peptidase 2; D012697:Serine Endopeptidases; D007930:Leucine; C012211:ubenimex; D012844:Sincalide