Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site.
alpha-Human atrial natriuretic peptide (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vivo it has been shown to be catabolized partly by the kidney. Crude microvillar membranes of human kidney degrade 125I-ANP at several internal bonds generating metabolites among which the C-terminal fragments were identified. Formation of the C-terminal tripeptide was blocked by phosphoramidon, indicating the involvement of endopeptidase-24.11 in this cleavage. Subsequent cleavages by aminopeptidase(s) yielded the C-terminal dipeptide and free tyrosine. Using purified endopeptidase 24.11, we identified seven sites of hydrolysis in unlabelled alpha-hANP: the bonds Arg-4-Ser-5, Cys-7-Phe-8, Arg-11-Met-12, Arg-14-Ile-15, Gly-16-Ala-17, Gly-20-Leu-21 and Ser-25-Phe-26. However, the bonds Gly-16-Ala-17 and Arg-4-Ser-5 did not fulfil the known specificity requirements of the enzyme. Cleavage at the Gly-16-Ala-17 bond was previously observed by Stephenson & Kenny [(1987) Biochem. J. 243, 183-187], but this is the first report of an Arg-Ser bond cleavage by this enzyme. Initial attack of alpha-hANP by endopeptidase-24.11 took place at a bond within the disulphide-linked loop and produced a peptide having the same amino acid composition as intact ANP. The bond cleaved in this metabolite was determined as the Cys-7-Phe-8 bond. Determination of all the bonds cleaved in alpha-hANP by endopeptidase-24.11 should prove useful for the design of more stable analogues, which could have therapeutic uses in hypertension.
Vanneste
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Y
Laboratoire Pluridisciplinaire de Recherche Expérimentale Biomédicale, Faculté de Médecine, Campus Erasme, Université Libre de Bruxelles, Belgium.
Michel
A
A
Dimaline
R
R
Najdovski
T
T
Deschodt-Lanckman
M
M
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D007672:Kidney Cortex / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D006020:Glycopeptides; D010446:Peptide Fragments; D009320:Atrial Natriuretic Factor; D015260:Neprilysin; C008890:phosphoramidon