Metabolism of insect neuropeptides: properties of a membrane-bound endopeptidase from heads of Musca domestica.
A phosphoramidon-sensitive endopeptidase activity has been identified in membranes prepared from heads of Musca domestica. The enzyme hydrolyses the Gly3-Phe4 bond of the enkephalin analogue [D-Ala2,Leu5]enkephalin and the Asn3-Phe4 bond of AKH I. Phosporamidon (10 microM), a selective inhibitor of mammalian endopeptidase 24:11, was able to fully protect AKH I from degradation by head membranes. The breakdown of [D-Ala2,Leu5]enkephalin was only partially inhibited by phosphoramidon (10 microM), suggesting the presence of other enkephalin-degrading enzymes in this preparation. The endopeptidase activity was inhibited by 1 mM EDTA and 1 mM 1,10-phenanthroline and could be partially re-activated in the presence of ZnCl2 but not other divalent metal ions. The enzyme had a neutral pH optimum and behaved like an integral membrane protein when subjected to phase-separation with Triton X-114. Although they have a number of similar properties, the insect and mammalian enzymes could be distinguished by their sensitivity to site-directed inhibitors of endopeptidase 24:11. The fly endopeptidase was much less sensitive to phosphoramidon (IC50, 0.25 microM), thiorphan (IC50, 2.5 microM), SQ 28603 (IC50, 1.0 microM), SCH 39370 (IC50, 2.5 microM) and SCH 32615 (IC50, 30 microM). The fly enzyme is indistinguishable from the endopeptidase activity that is enriched in locust synaptic membranes and that found in membranes from heads of Drosophila melanogaster. In summary, we have identified a rich source of an insect neutral metallo-endopeptidase which is similar to endopeptidase 24:11, an enzyme known to play a key role in the metabolism and inactivation of neuropeptides in mammals.
Lamango
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Department of Pure and Applied Biology, University of Leeds, England.
Isaac
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D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D016308:Enkephalin, Leucine-2-Alanine / Q000737:chemistry / Q000378:metabolism; D006793:Houseflies / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007301:Insect Hormones / Q000737:chemistry / Q000378:metabolism; D008566:Membranes / Q000201:enzymology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013379:Substrate Specificity
D002413:Cations, Divalent; D007301:Insect Hormones; D009479:Neuropeptides; D009842:Oligopeptides; C044833:adipokinetic hormone; D016308:Enkephalin, Leucine-2-Alanine; D004492:Edetic Acid; D008666:Metalloendopeptidases; D011761:Pyrrolidonecarboxylic Acid