CHARACTERIZATION AND LOCALIZATION OF MITOCHONDRIAL OLIGOPEPTIDASE (MOP) (EC-3.4.24.16) ACTIVITY IN THE HUMAN CERVICAL ADENOCARCINOMA CELL-LINE HELA

In this study we describe tile partial purification and characterizati on of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The H eLa enzyme was isolated initially by its ability to hydrolyse a nonape ptide substrate (P9) wh ich was cognate to the N-terminal cleavage sit e of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn2+-chelating agents and DTT and had an approxima te molecular weight of 55-63 kD determined by gel filtration. Neuroten sin, dynorphin A(1-17) and GnRH(1-9) were rapidly degraded by the enzy me while GnRH(1-10) and somatostatin were not. Neurotensin was cleaved at the Pro(10)-Tyr(11) bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K-m for neurotensin cleavage was 7 mu M and the K-i for the specific 24.16 dipeptide inhibitor (Pro-Ile) was 140 mu M which were similar to those observed from the human brai n enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be olig opeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is l ocated mainly in mitochondria though it was detected on the plasma mem brane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and r aise the issue of its function therein. (C) 1997 Wiley-Liss, Inc.