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 the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase 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 located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein.
Krause
D R
DR
QCF Cancer Research Unit, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Australia.
Piva
T J
TJ
Brown
S B
SB
Ellem
K A
KA
D002462:Cell Membrane / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D010949:Plasma / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology
D007527:Isoenzymes; D011480:Protease Inhibitors; D004492:Edetic Acid; D008666:Metalloendopeptidases; C062316:neurolysin