Identification and properties of a neuropeptide-degrading endopeptidase (neprilysin) of Ascaris suum muscle.
We have previously identified in membranes of the locomotory muscle of Ascaris suum a phosphoramidon-sensitive endopeptidase which hydrolyses the neuropeptide AF1 (Lys-Asn-Glu-Phe-Ile-Arg-Phe-NH2) by cleavage of the Glu3-Phe4 bond (Sajid & Isaac, 1994). We have determined the properties of this neuropeptide-degrading enzyme of A. suum muscle using AKH-1 (pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2) and [D-Ala2, Leu5]enkephalin as convenient endopeptidase substrates. Phosphoramidon, thiorphan and SQ 28603, potent inhibitors of mammalian neprilysin (neutral endopeptidase, endopeptidase 24.11), inhibited the endopeptidase activity towards AKH-I with IC50 values of 0.13 microM, 22 microM and 6.3 microM, respectively. Two other neprilysin inhibitors (SCH 32615 and SCH 39370) and the bivalent metal ion chelators, EDTA (1 mM) and 1, 10 bis-phenanthroline (1 mM) failed to inhibit the nematode enzyme. The endopeptidase had a neutral pH optimum and a significant proportion (45%) of the enzyme activity partitioned into the detergent-rich phase of Triton X-114, indicating that the enzyme is an integral membrane protein. The muscle enzyme also attacked [D-Ala2, Leu5]enkephalin cleaving the Gly3-Phe4 bond and this hydrolytic activity was inhibited by phosphoramidon and thiorphan (IC50, 0.28 microM and 15.8 microM, respectively) but not by EDTA and 1, 10 bis-phenanthroline. The phosphoramidon-sensitive endopeptidase activity was detected on intact muscle cells prepared by collagenase treatment of the body wall musculature, indicating that endopeptidase is accessible to peptide molecules that interact with the cell surface.
Sajid
M
M
Department of Pure and Applied Biology, University of Leeds.
Isaac
R E
RE
D000595:Amino Acid Sequence; D000818:Animals; D017165:Ascaris suum / Q000201:enzymology*; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine / Q000378:metabolism; D015801:Helminth Proteins / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009479:Neuropeptides / Q000378:metabolism*
D015801:Helminth Proteins; D009479:Neuropeptides; D016308:Enkephalin, Leucine-2-Alanine; D010450:Endopeptidases