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.
Yamaguchi
T
T
Division of Enzyme Chemistry, University of Tokushima, Japan.
Kido
H
H
Katunuma
N
N
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