Characterization of thiol-, aspartyl-, and thiol-metallo-peptidase activities in Madin-Darby canine kidney cells

We combined fluorogenic substrates or internally quenched fluorescent pepti des with specific inhibitors in the pH profile of proleolytic activity expe riments in order to detect proteolytic activities in lysates of MDCK cells. Hydrolytic activities related to cathepsin B, L, and D were observed. Seri ne-proteinase was not detected; however, we clearly demonstrated the presen ce of a thiol-metallo-endo-oligopeptidase, also called thimet-oligopeptidas e (TOP). This peptidase from MDCK cells has substrate and inhibitor specifi cities as well as an activation profile with mercaptoethanol that are indis tinguishable from the recombinant rat testis TOP (EC 3.4.24.15). In additio n, polyclonal purified antibodies to this enzyme depleted the TOP activity of MDCK cells in whole homogenate. Although we present only preliminary dat a, TOP is secreted by MDCK cells. The presence of TOP in a phenotype polari zed MDCK cells can have special significance in the cytoplasmic selection, transport, or clearance of short peptides due to restriction of the enzyme to sequences from 6 to 17 amino acids. Therefore, the MDCK cell could be a very useful cellular model with which to study some of the suggested TOP bi ological functions as processing of biological active peptides and antigen presentation. (C) 2000 Wiley-Liss, Inc.