Methionine enkephalin metabolism by murine macrophage ectopeptidase(s).
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.
Miller
B C
BC
Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 75235-9038.
Ackroyd
A
A
Hersh
L B
LB
Cottam
G L
GL
D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018826:CD13 Antigens; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004744:Enkephalin, Methionine / Q000378:metabolism*; D006031:Glycosylation; D006868:Hydrolysis; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D018076:DNA, Complementary; D004744:Enkephalin, Methionine; D012313:RNA; D000626:Aminopeptidases; D018826:CD13 Antigens