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 hydrol yses the Gly3-Phe4 bond of the enkephalin analogue [D-Ala2,Leu5]enkeph alin and the Asn3-Phe4 bond of AKH I. Phosphoramidon (10 muM), a selec tive inhibitor of mammalian endopeptidase 24:11, was able to fully pro tect AKH I from degradation by head membranes. The breakdown of [D-Ala 2,Leu5]enkephalin was only partially inhibited by phosphoramidon (10 m uM), suggesting the presence of other enkephalin-degrading enzymes in this preparation. The endopeptidase activity was inhibited by 1 mM EDT A 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 ha d a neutral pH optimum and behaved like an integral membrane protein w hen subjected to phase-separation with Triton X-114. Although they hav e a number of similar properties, the insect and mammalian enzymes cou ld be distinguished by their sensitivity to site-directed inhibitors o f endopeptidase 24:11. The fly endopeptidase was much less sensitive t o phosphoramidon (IC50, 0.25 muM), thiorphan (IC50, 2.5 muM), SQ 28603 (IC50, 1.0 muM), SCH 39370 (IC50, 2.5 muM) and SCH 32615 (IC50, 30 mu M). The fly enzyme is indistinguishable from the endopeptidase activit y that is enriched in locust synaptic membranes and that found in memb ranes from heads of Drosophila melanogaster. In summary, we have ident ified a rich source of an insect neutral metallo-endopeptidase which i s similar to endopeptidase 24:11, an enzyme known to play a key role i n the metabolism and inactivation of neuropeptides in mammals.