title: Isolation of a neuropeptide-degrading endopeptidase from the leech Theromyzon tessulatum.
abstract: Extracts of head parts prepared from the leech Theromyzon tessulatum hydrolyse the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Gly-His bond of benzoyl-Gly-His-Leu. The metabolism of benzoyl-Gly-His-Leu was completely inhibited by captopril, consistent with an angiotensin-converting enzyme activity. Such an enzyme has recently been isolated from T. tessulatum. However, the enkephalin hydrolysis by captopril (100 microM) was inhibited to a maximum of 70%. The residual activity hydrolyzing enkephalin was inhibited by phosphoramidon, consistent with the presence of endopeptidase-24.11, a mammalian enzyme implicated in the metabolism of neuropeptides. This enzyme was isolated using four steps of purification including gel-permeation and anion-exchange chromatographies followed by reverse-phase HPLC. This neuropeptide endopeptidase (of approximate molecular mass 45 kDa) hydrolyses, at pH 7 and 37 degrees C, both the Gly3-Phe4 bond of synthetic [D-Ala2, Leu5]enkephalin and the Phe8-His9 bond of angiotensin I. Cleavage of [D-Ala2, Leu5]enkephalin yields, respectively, the Tyr-D-Ala-Gly and Phe-Leu peptides with a specific activity of 29 nmol Tyr-D-Ala-Gly.min-1.mg protein-1 (Km 95 microM). The hydrolysis of angiotensin I yields angiotensin II and the dipeptide His-Leu with a specific activity of 1.2 nmol angiotensin min-1.mg protein-1 (Km 330 microM). The metabolism of these peptides was totally inhibited by phosphoramidon. This study therefore provides biochemical evidence for neuropeptide-degrading endopeptidases in leeches.
authors.affiliation: Laboratoire de Phylogénie moléculaire des Annélides, Université des Sciences et Technologies de Lille, France.
authors.forename: V
authors.initials: V
authors.lastname: Laurent
authors.forename: M
authors.initials: M
authors.lastname: Salzet
mesh_terms: D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000737:chemistry / Q000378:metabolism; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002216:Captopril / Q000494:pharmacology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism*; D016308:Enkephalin, Leucine-2-Alanine / Q000737:chemistry / Q000378:metabolism; D006020:Glycopeptides / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D007865:Leeches / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D006020:Glycopeptides; D009479:Neuropeptides; D009842:Oligopeptides; D011480:Protease Inhibitors; C010980:hippuryl-histidyl-leucine; D016308:Enkephalin, Leucine-2-Alanine; D000803:Angiotensin I; D002216:Captopril; D010450:Endopeptidases; C008890:phosphoramidon