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A prolyl endopeptidase from murine macrophages, its assay and specific inactivation
Abstract:
The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.
Insights
Murine macrophages contain a prolyl endopeptidase enzyme specific for proline residues. This enzyme can be selectively inhibited and resynthesized by macrophages, offering insights into cellular protein processing.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- Prolyl endopeptidase activity has been identified in various mammalian tissues.
- Understanding the role and characteristics of this enzyme in immune cells like macrophages is crucial.
Purpose of the Study:
- To report the presence and characterize the prolyl endopeptidase in murine peritoneal macrophages.
- To investigate the enzyme's specificity, inhibition, and resynthesis in macrophages.
Main Methods:
- Development of a sensitive fluorogenic assay substrate (benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide).
- Enzyme inhibition studies using benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone and diisopropyl fluorophosphate.
- Macrophage culture experiments to assess selective inhibition and resynthesis, with cycloheximide used to block resynthesis.
Main Results:
- A prolyl endopeptidase was detected in the soluble fraction of murine peritoneal macrophages, highly specific for cleaving after proline residues.
- The enzyme was rapidly inactivated by specific cysteine and serine proteinase inhibitors.
- Selective inhibition of the cytoplasmic enzyme was achieved in cultured macrophages, which then resynthesized the enzyme over several days.
- Similar enzyme activity was observed in both normal and elicited macrophages.
Conclusions:
- Murine peritoneal macrophages possess a specific prolyl endopeptidase.
- The enzyme's activity can be modulated through selective inhibition and subsequent resynthesis, indicating dynamic regulation within macrophages.
- The macrophage enzyme shares similarities with prolyl endopeptidases found in other tissues, suggesting conserved functions.