PURIFICATION AND CHARACTERIZATION OF A TRIPEPTIDYL AMINOPEPTIDASE-I FROM RAT SPLEEN

A tripeptidyl aminopeptidase I with an M( r )of 47,000 Da has been pur ified from rat spleen. The N-terminal sequence of the enzyme and inter nal sequences did not resemble that of any known protein. The enzyme c leaves tripeptides from synthetic substrates provided that the N-termi nus is unsubstituted and the amino acid in the P, position is not char ged. The enzyme also cleaves small peptides (angiotensin II and glucag on) releasing tripeptides but does not appear to demonstrate any prefe rence 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 t ripeptidyl peptidase I was not inhibited by classical inhibitors of se rine, cysteine, aspartate or metalloproteinases, The peptidase was pot ently inhibited by a series of substrate-based tripeptidyl chloromethy l ketones (K-i's of 10(-6)-10(-8) M), Inhibition was rapid and reversi ble. This mode of inhibition is different to the interaction between c hloromethyl ketones and cysteine or serine peptidases. These tripeptid yl 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. (C) 1998 Elsevier Science B.V. All rights reserved,