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Published on: July 17, 2018
Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical
D R Krause1, T J Piva, S B Brown
1QCF Cancer Research Unit, Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Australia.
Abstract:
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.
Insights
This study identifies and characterizes oligopeptidase M (endopeptidase 3.4.24.16) in HeLa cells, revealing its metalloprotease activity and substrate specificity. Its presence in human cultured cells suggests novel cellular functions.
Area of Science:
- Biochemistry
- Enzymology
- Cell Biology
Background:
- Oligopeptidase M (endopeptidase 3.4.24.16) is a metalloprotease.
- Its presence and function in human cultured cells remain largely uncharacterized.
Purpose of the Study:
- To partially purify and characterize oligopeptidase M from HeLa cells.
- To investigate its enzymatic activity, substrate specificity, and cellular localization.
Main Methods:
- Enzyme purification using a nonapeptide substrate (P9) cognate to prepro-TGF alpha.
- Characterization of metalloprotease activity using inhibitors and gel filtration.
- Substrate degradation assays with neurotensin, dynorphin A1-17, GnRH1-9, GnRH1-10, and somatostatin.
- Enzyme localization studies using antibodies and subcellular fractionation.
Main Results:
- HeLa cell oligopeptidase M is a metalloprotease (55-63 kD) that degrades neurotensin, dynorphin A1-17, and GnRH1-9.
- Cleavage of neurotensin occurs at the Pro10-Tyr11 bond.
- The enzyme exhibits kinetic parameters similar to the human brain enzyme.
- Oligopeptidase M was found mainly in mitochondria, with some inactive presence on the plasma membrane.
- It was successfully separated from thimet oligopeptidase.
Conclusions:
- This study reports the first instance of oligopeptidase M in human cultured cells.
- The characterization provides insights into its enzymatic properties and localization.
- Further research is warranted to elucidate its specific functions within human cells.
