Purification and characterisation of a tripeptidyl aminopeptidase I from rat spleen.
A tripeptidyl aminopeptidase I with an M(r) of 47,000 Da has been purified from rat spleen. The N-terminal sequence of the enzyme and internal sequences did not resemble that of any known protein. The enzyme cleaves tripeptides from synthetic substrates provided that the N-terminus is unsubstituted and the amino acid in the P1 position is not charged. The enzyme also cleaves small peptides (angiotensin II and glucagon) releasing tripeptides but does not appear to demonstrate any preference for amino acids on either side of the cleavage site. The enzyme had maximum activity at pH 4 but was unstable above pH 7. Rat spleen tripeptidyl peptidase I was not inhibited by classical inhibitors of serine, cysteine, aspartate or metalloproteinases. The peptidase was potently inhibited by a series of substrate-based tripeptidyl chloromethyl ketones (Ki's of 10(-6)-10(-8) M). Inhibition was rapid and reversible. This mode of inhibition is different to the interaction between chloromethyl ketones and cysteine or serine peptidases. These tripeptidyl chloromethyl ketones were also inhibitors of bone resorption using an in vitro assay suggesting that a tripeptidyl peptidase is involved in the degradation of bone matrix proteins.
Vines
D
D
Department of Histopathology, St George's Hospital Medical School, London, UK.
Warburton
M J
MJ
D000626:Aminopeptidases; D000818:Animals; D001862:Bone Resorption; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D010450:Endopeptidases / Q000302:isolation & purification*; D007531:Isoflurophate / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D057057:Serine Proteases; D013154:Spleen / Q000201:enzymology*; D000091346:Tripeptidyl-Peptidase 1
D007976:Leupeptins; D011480:Protease Inhibitors; C000717269:Tpp1 protein, rat; D000091346:Tripeptidyl-Peptidase 1; D007531:Isoflurophate; D010450:Endopeptidases; D057057:Serine Proteases; D000626:Aminopeptidases; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; C032854:leupeptin