title: Endothelin 1 hydrolysis by rat kidney membranes.
abstract: Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.
authors.affiliation: Department of Medicine, Kobe University School of Medicine, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: M
authors.initials: M
authors.lastname: Fukase
authors.forename: M
authors.initials: M
authors.lastname: Arao
authors.forename: T
authors.initials: T
authors.lastname: Sugimoto
authors.forename: K
authors.initials: K
authors.lastname: Chihara
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D016232:Endothelins / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D051381:Rats
chemical_list: D016232:Endothelins