title: Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates.
abstract: 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.
authors.affiliation: Faculté de Pharmacie, Université de Montréal, 2900, boul Edouard-Montpetit (bureau S338), C. P. 6128, succursale Centre-ville, Montréal, Québec, Canada H3C 3J7.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Molinaro
authors.forename: Adriana K
authors.initials: AK
authors.lastname: Carmona
authors.forename: Maria A
authors.initials: MA
authors.lastname: Juliano
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Elena
authors.initials: E
authors.lastname: Malitskaya
authors.forename: Marie-Andrée
authors.initials: MA
authors.lastname: Yessine
authors.forename: Miguel
authors.initials: M
authors.lastname: Chagnon
authors.forename: Yves
authors.initials: Y
authors.lastname: Lepage
authors.forename: William H
authors.initials: WH
authors.lastname: Simmons
authors.forename: Guy
authors.initials: G
authors.lastname: Boileau
authors.forename: Albert
authors.initials: A
authors.lastname: Adam
mesh_terms: D000626:Aminopeptidases / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012995:Solubility; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
chemical_list: D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000626:Aminopeptidases; C022750:X-Pro aminopeptidase