title: Substrate specificity differences between recombinant rat testes endopeptidase EC 3.4.24.15 and the native brain enzyme.
abstract: We have characterized and compared the substrate specificity of affinity-purified recombinant rat testes endopeptidase EC 3.4.24.15 (EP 24.15) with that reported for the isolated brain enzyme. Of the peptides tested, only bradykinin, dynorphin A1-8, and neurotensin were efficiently cleaved by the recombinant enzyme (kcat/Km = 3.0, 2.8 and 0.5 x 10(5) M-1sec-1, respectively); other peptides considered substrates of EP 24.15 (gonadotropin-releasing hormone, substance P, somatostatin and angiotensin) were not metabolized. The enzyme was inhibited by metal ion chelators and thiol-reactive agents, as well as a specific EP 24.15 inhibitor (N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate), thus confirming the enzyme as a thiol-dependent metalloendopeptidase. The observed discrepancies in substrate specificity of the recombinant testicular and the isolated brain enzymes may result from tissue-specific forms and/or post-translational modifications of EP 24.15.
authors.affiliation: Peptide Biology Laboratory, Baker Medical Research Institute, Prahran, Victoria, Australia.
authors.forename: R A
authors.initials: RA
authors.lastname: Lew
authors.forename: N J
authors.initials: NJ
authors.lastname: Hey
authors.forename: T J
authors.initials: TJ
authors.lastname: Tetaz
authors.forename: M J
authors.initials: MJ
authors.lastname: Glucksman
authors.forename: J L
authors.initials: JL
authors.lastname: Roberts
authors.forename: A I
authors.initials: AI
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013737:Testis / Q000201:enzymology*
chemical_list: D007527:Isoenzymes; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase