De l'utilisation des copolymères d'acide méthacrylique dans le transport intracellulaire des oligonucléotides antisens

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

Human recombinant membrane-bound aminopeptidase P: production of a soluble form and characterization using novel, internally quenched fluorescent substrates

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.Univ Montreal, Fac Pharm, Montreal, PQ H3C 3J7, CanadaUNIFESP, Escola Paulista Med, Dept Biophys, Sao Paulo, BrazilUniv Montreal, Fac Med, Dept Biochim, Montreal, PQ H3C 3J7, CanadaUniv Montreal, Fac Arts & Sci, Dept Math & Stat, Montreal, PQ H3C 3J7, CanadaLoyola Univ, Stritch Sch Med, Dept Biochem, Dept Cell Biol Neurobiol & Anat, Maywood, IL 60153 USAUNIFESP, Escola Paulista Med, Dept Biophys, Sao Paulo, BrazilWeb of Scienc