Hydrolysis of intact and Cys-Phe-cleaved human atrial natriuretic peptide in vitro by human tissue kallikrein.
Atrial natriuretic peptide (ANP) is a 28-amino-acid hormone involved in the regulation of fluid balance. In circulation, the proteolytic inactivation of ANP has been demonstrated to involve both membrane metalloendopeptidase and an aprotonin-sensitive activity, probably corresponding to kallikrein [Vanneste, Y., Pauwels, S., Lambotte, L., Michel, A., Dimaline, R. & Deschodt-Lanckman, M. (1990) Biochem. J. 269, 801-806]. In the present study, we focused on the aprotinin-sensitive pathway of ANP metabolism. In order to identify the cleavage sites recognized by kallikrein within the sequence of the hormone, tissue kallikrein was purified to homogeneity from human urine and the degradation of human ANP by the enzyme preparation was studied. Our results demonstrate that both intact and Cys7-Phe8-cleaved ANP, the initial metabolite produced in circulation by the metallo-endopeptidase, are substrates in vitro for purified tissue kallikrein. However, the Cys-Phe-cleaved peptide was degraded approximately fourfold faster than the intact hormone by the purified enzyme. The first degradation step of ANP by tissue kallikrein involves two cleavages occurring at the bonds Arg3-Arg4 and Gly16-Ala17, generating an inactive, open-ring metabolite. Incubation of ANP for a longer period with the enzyme led to the generation of several additional degradation fragments. Ten peaks were separated by HPLC and characterized by amino acid analysis. The results allowed the identification of a total of eight peptide bonds susceptible to hydrolysis by tissue kallikrein in the sequence of ANP: Arg3-Arg4, Ser5-Ser6, Cys7-Phe8, Arg11-Met12, Gly16-Ala17, Gly20-Leu21, Ser25-Phe26 and Arg27-Tyr28. These results indicate that the aprotinin-sensitive activity involved in the metabolism of ANP in circulation could correspond to tissue kallikrein. However, clear identification of ANP as a novel physiological substrate of the enzyme will need further investigation.
Vanneste
Y
Y
Laboratoire Pluridisciplinaire de Recherche Expérimentale Biomédicale, Faculté de Médecine, Université Libre de Bruxelles, Belgium.
Michel
A
A
Deschodt-Lanckman
M
M
D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007610:Kallikreins / Q000302:isolation & purification / Q000378:metabolism* / Q000652:urine; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D010449:Peptide Mapping; D010649:Phenylalanine / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D010446:Peptide Fragments; D010649:Phenylalanine; D009320:Atrial Natriuretic Factor; D007610:Kallikreins; D003545:Cysteine