METHIONINE-ENKEPHALIN METABOLISM BY MURINE MACROPHAGE ECTOPEPTIDASE(S)

Ectopeptidases which hydrolyze opioid and other neuropeptides have bee n identified in brain, kidney and intestine. In this study, identifica tion of the enzymes metabolizing the opioid peptide methionine enkepha lin (YGGFM) in murine macrophages was undertaken. Incubation of methio nine enkephalin with intact murine peritoneal macrophages results in f ive products identified as Y, F, FM, GFM and GGFM by amino acid analys is and peptide microsequencing after fractionation by HPLC. The spectr um of metabolites results from at least two distinct aminopeptidase ac tivities. The enzyme hydrolyzing YGGFM to GGFM is identified as the me mbrane-anchored aminopeptidase N (ApN; EC 3.4.11.2) based on its subst rate specificity and inhibitor profile. A distinct bestatin and amasta tin 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 glycosylati on differences rather than expression of a distinct protein. The ApN c atalytic activity and mRNA levels are increased in thioglycollate-elic ited as compared to resident peritoneal macrophages. RT-PCR analysis i dentified a 0.7 kb fragment of the ApN coding sequence which was ident ical in mouse kidney and thioglycollate-elicited peritoneal macrophage s and which has 89% identity with the corresponding rat kidney ApN cDN A sequence.