Neuropeptide-degrading endopeptidase activity of locust (Schistocerca gregaria) synaptic membranes.
Locust adipokinetic hormone (AKH, pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) was used as the substrate to measure neuropeptide-degrading endopeptidase activity in neutral membranes from ganglia of the locust Schistocerca gregaria. Initial hydrolysis of AKH at neural pH by peptidases of washed neural membranes generated pGlu-Leu-Asn and Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2 as primary metabolites, demonstrating that degradation was initiated by cleavage of the Asn-Phe bond. Amastatin protected the C-terminal fragment from further metabolism by aminopeptidase activity without inhibiting AKH degradation. The same fragments were generated on incubation of AKH with purified pig kidney endopeptidase 24.11, and enzyme known to cleave peptide bonds that involve the amino group of hydrophobic amino acids. Phosphoramidon (10 microM), a selective inhibitor of mammalian endopeptidase 24.11, partially inhibited the endopeptidase activity of locust neural membranes. This phosphoramidon-sensitive activity was shown to enriched in a synaptic membrane preparation with around 80% of the activity being inhibited by 10 microM-phosphoramidon (IC50 = 0.2 microM). The synaptic endopeptidase was also inhibited by 1 mM-EDTA, 1 mM-1,10-phenanthroline and 1 microM-thiorphan, and the activity was maximal between pH 7.3 and 8.0. Localization of the phosphoramidon-sensitive enzyme in synaptic membranes is consistent with a physiological role for this endopeptidase in the metabolism of insect peptides at the synapse.
Isaac
R E
RE
Department of Pure and Applied Biology, University of Leeds, U.K.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology; D006110:Grasshoppers / Q000201:enzymology*; D007301:Insect Hormones / Q000378:metabolism; D015260:Neprilysin / Q000378:metabolism; D009479:Neuropeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013570:Synaptic Membranes / Q000187:drug effects / Q000201:enzymology*
D000596:Amino Acids; D006020:Glycopeptides; D007301:Insect Hormones; D009479:Neuropeptides; D011480:Protease Inhibitors; C014956:adipokinetic hormone (locust); D010450:Endopeptidases; D015260:Neprilysin; C008890:phosphoramidon