Hydrolysis of α-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site

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Abstract
.alpha.-Human atrial natriuretic peptide (1-28) (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vitro 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 and 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.