Artigo
Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates
Fecha
2005-01-15Registro en:
Biochemical Journal. London: Portland Press Ltd, v. 385, n. 2, p. 389-397, 2005.
0264-6021
10.1042/BJ20040849
WOS:000226662500008
Autor
Molinaro, Giuseppe
Carmona, Adriana Karaoglanovic [UNIFESP]
Juliano, Maria Aparecida [UNIFESP]
Juliano, Luiz [UNIFESP]
Malitskaya, Elena
Yessine, Marie-Andrée
Chagnon, Miguel
Lepage, Yves
Simmons, William H.
Boileau, Guy
Adam, Albert
Institución
Resumen
APP (aminopeptidase P) has the unique ability to cleave the N-terminal amino acid residue from peptides exhibiting a proline at P-1'. Despite its putative involvement in the processing of bioactive peptides, among them the kinins, little is known about the physiological roles of both human forms of APP. The purpose of the present study is first to engineer and characterize a secreted form of hmAPP (human membrane-bound APP). Our biochemical analysis has shown that the expressed glycosylated protein is fully functional, and exhibits enzymic parameters similar to those described previously for mAPP purified from porcine or bovine lungs or expressed from a porcine clone. This soluble form of hmAPP cross-reacts with a polyclonal antiserum raised against a 469-amino-acid hmAPP fragment produced in Escherichia coli. Secondly, we synthesized three internally quenched fluorescent peptide substrates that exhibit a similar affinity for the enzyme than its natural substrates, the kinins, and a higher affinity compared with the tripeptide Arg-Pro-Pro used until now for the quantification of APP in biological samples. These new substrates represent a helpful analytical tool for rapid and reliable: screening of patients susceptible to adverse reactions associated with angiotensin-converting enzyme inhibitors or novel vasopeptidase (mixed angiotensin-converting enzyme/neprilysin) inhibitors.