title: Digestion of invertebrate neuropeptides by preparations from the free-living nematode Panagrellus redivivus.
abstract: Proteases in the soluble fraction of homogenates prepared from the free-living nematode Panagrellus redivivus hydrolysed the amidated invertebrate neuropeptides FMRFa and FLRFa, and nematode FMRFa-like peptides (FLPs) KPNFLRFa (FLP-1-H), APKPKFIRFa (FLP-5-A), KNEFIRFa (FLP-8), KPSFVRFa (FLP-9), RNKFEFIRFa (FLP-12) and KHEYLRFa (FLP-14) in vitro. Results were assessed by analysing reaction components with RP-HPLC, UV detection at 210 nm and peak integration. Based upon substrate peak size, more than 90% of most of the peptide substrates was consumed after 1 h at 27 degrees C, but digestion was not complete even with a crude protease mixture. Two peptides, FLP-12 and FLP-14, were significantly less susceptible to digestion than the others. FLP-12 was the least susceptible of all sequences (71% loss; P < 0.0001), while FLP-14 was digested less (84% loss; P < 0.0004) than all but FLP-12. Product peak digestion patterns of FLP-12, a second nonapeptide (FLP-5-A), and FMRFa, incubated with aminopeptidase (amastatin) and serine endoprotease (AEBSF) inhibitors, demonstrated highly specific behaviours of each sequence to protease cleavage. Amastatin significantly (P < 0.03) reduced digestion of FLP-12 (54% loss) and FMRFa (61% loss; P < 0.0005), but had no effect on FLP-5-A. AEBSF had no protective effect on FMRFa but significantly decreased hydrolysis of FLP-5-A (77% loss; P < 0.0001) and FLP-12 (59% loss; P < 0.03). The combination of both inhibitors had additive effects only for FMRFa (34% loss; P < 0.0005). Further analysis of FMRFa digestion using peptides with D-amino acid substitutions demonstrated nearly complete protection of FdMRFa (2% loss; P < 0.0001) from all proteolytic digestion, whereas digestion of FMRdFa was complete. Results suggest that in addition to aminopeptidase and serine proteases, both deamidase and aminopeptidase P participate in neuropeptide metabolism in P. redivivus.
authors.affiliation: Nematology Laboratory, United States Department of Agriculture, Agricultural Research Service, 10300 Baltimore Avenue, BARC-West, Beltsville, MD 20705-2350, USA. edward.masler@ars.usda.gov
authors.forename: E P
authors.initials: EP
authors.lastname: Masler
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000737:chemistry; D004063:Digestion / Q000502:physiology; D019835:FMRFamide / Q000737:chemistry / Q000302:isolation & purification*; D007448:Invertebrates / Q000378:metabolism / Q000502:physiology*; D009348:Nematoda / Q000378:metabolism / Q000502:physiology*; D011480:Protease Inhibitors / Q000302:isolation & purification*; D018010:Receptors, Invertebrate Peptide / Q000302:isolation & purification*; D015222:Sodium Channels / Q000737:chemistry
chemical_list: D015415:Biomarkers; D011480:Protease Inhibitors; D018010:Receptors, Invertebrate Peptide; D015222:Sodium Channels; D019835:FMRFamide