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Methionine enkephalin metabolism by murine macrophage ectopeptidase(s)
B C Miller1, A Ackroyd, L B Hersh
1Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038.
Abstract:
Ectopeptidases which hydrolyze opioid and other neuropeptides have been identified in brain, kidney and intestine. In this study, identification of the enzymes metabolizing the opioid peptide methionine enkephalin (YGGFM) in murine macrophages was undertaken. Incubation of methionine enkephalin with intact murine peritoneal macrophages results in five products identified as Y, F, FM, GFM and GGFM by amino acid analysis and peptide microsequencing after fractionation by HPLC. The spectrum of metabolites results from at least two distinct aminopeptidase activities. The enzyme hydrolyzing YGGFM to GGFM is identified as the membrane-anchored aminopeptidase N (ApN; EC 3.4.11.2) based on its substrate specificity and inhibitor profile. A distinct bestatin and amastatin sensitive aminopeptidase catalyzes hydrolysis of GGFM to GFM. The macrophage ApN protein has a larger mass and is antigenically distinct from murine kidney ApN, which is suggested to result from glycosylation differences rather than expression of a distinct protein. The ApN catalytic activity and mRNA levels are increased in thioglycollate-elicited as compared to resident peritoneal macrophages. RT-PCR analysis identified a 0.7 kb fragment of the ApN coding sequence which was identical in mouse kidney and thioglycollate-elicited peritoneal macrophages and which has 89% identity with the corresponding rat kidney ApN cDNA sequence.
Insights
Murine macrophages metabolize methionine enkephalin via aminopeptidase N (ApN) and another enzyme. ApN activity and mRNA increase in elicited macrophages, suggesting a role in immune response.
Area of Science:
- Biochemistry
- Immunology
- Neuroscience
Background:
- Ectopeptidases in the brain, kidney, and intestine hydrolyze neuropeptides.
- Methionine enkephalin is an opioid peptide with potential roles in various physiological processes.
Purpose of the Study:
- To identify enzymes metabolizing methionine enkephalin in murine macrophages.
- To characterize the properties and regulation of these enzymes.
Main Methods:
- Incubation of methionine enkephalin with murine peritoneal macrophages.
- High-performance liquid chromatography (HPLC) for peptide fractionation.
- Amino acid analysis and peptide microsequencing for metabolite identification.
- Enzyme inhibition assays and Western blotting for enzyme characterization.
- RT-PCR for mRNA analysis.
Main Results:
- Methionine enkephalin was metabolized into Y, F, FM, GFM, and GGFM.
- Membrane-anchored aminopeptidase N (ApN) was identified as the enzyme hydrolyzing YGGFM to GGFM.
- A distinct aminopeptidase sensitive to bestatin and amastatin catalyzed GGFM to GFM hydrolysis.
- Macrophage ApN exhibited distinct antigenic properties and higher mass compared to kidney ApN, likely due to glycosylation.
- ApN catalytic activity and mRNA levels were elevated in thioglycollate-elicited macrophages versus resident macrophages.
- RT-PCR confirmed identical ApN coding sequences in kidney and elicited macrophages, with high identity to rat ApN.
Conclusions:
- Murine macrophages possess distinct aminopeptidase activities capable of metabolizing methionine enkephalin.
- Membrane-anchored aminopeptidase N (ApN) is a key enzyme in this process.
- ApN expression and activity are upregulated in activated macrophages, suggesting a role in macrophage function during inflammation or immune response.