A membrane-bound metallo-endopeptidase from rat kidney. Characteristics of its hydrolysis of peptide hormones and neuropeptides.
A membrane-bound metallo-endopeptidase that hydrolyzes human parathyroid hormone (1-84) and reduced hen egg lysozyme between hydrophilic amino acid residues was isolated from rat kidney [Yamaguchi et al. (1991) Eur. J. Biochem. 200, 563-571]. In this study, the hydrolyses of various peptide hormones and neuropeptides by the metallo-endopeptidase were examined using an automated gas-phase protein sequencer. The purified enzyme hydrolyzed the oxidized insulin B chain and substance P most rapidly, followed by big endothelin 1, neurotensin, angiotensin 1, endothelin 1, rat alpha-atrial natriuretic peptide and bradykinin, in this order. The enzyme mainly cleaved these peptides at bonds involving a hydrophilic amino acid residue. However, it cleaved bonds between less hydrophilic amino acid pairs in several short peptides, e.g. at the His5-Leu6 bond in oxidized insulin B chain, the Ile28-Val29 bond in big endothelin-1 and the Ile5-His6 and Phe8-His9 bonds in angiotensin 1. The enzyme cleavage sites of oxidized insulin B chain and angiotensin 1 were different from the reported sites cleaved by meprin and by endopeptidase 2, respectively. Kinetic determination of bradykinin hydrolysis by the purified enzyme yielded values of Km = 18.1 microM and kcat = 0.473 s-1, giving a ratio of kcat/Km = 2.62 x 10(4) s-1.M-1. The Km value was about 20-fold lower than that reported for meprin and endopeptidase 2. These results indicate that the membrane-bound metallo-endopeptidase from rat kidney is distinguished from meprin and endopeptidase 2 in its substrate specificity and is not parathyroid hormone specific, but has potential capacities to inactivate various biologically active peptide hormones and neuropeptides in vivo.
Division of Enzyme Chemistry, University of Tokushima, Japan.
T
T
Yamaguchi
H
H
Kido
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N
Katunuma
D000595:Amino Acid Sequence; D000803:Angiotensin I / Q000378:metabolism; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D016232:Endothelins / Q000378:metabolism; D006728:Hormones / Q000378:metabolism*; D006868:Hydrolysis; D007668:Kidney / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000378:metabolism*; D009496:Neurotensin / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation; D051381:Rats; D013373:Substance P / Q000378:metabolism
D016232:Endothelins; D006728:Hormones; D009479:Neuropeptides; D010455:Peptides; D013373:Substance P; D009496:Neurotensin; D009320:Atrial Natriuretic Factor; D000803:Angiotensin I; D008666:Metalloendopeptidases; D001920:Bradykinin