title: Activity profiling of aminopeptidases in cell lysates using a fluorogenic substrate library.
abstract: Aminopeptidases are exopeptidases that process peptide bonds at the N-terminus of protein substrates, and they are involved in controlling several metabolic pathways. Due to their involvement in diseases such as cancer or rheumatoid arthritis, their presence can also be used as a predictive biomarker. Here, we used a library of fluorogenic substrates containing natural and unnatural amino acids to reliably measure the aminopeptidase N (APN) activity in cell lysates obtained from human, pig and rat kidneys. We compared our results to the substrate specificity profile of isolated APN. Our data strongly support the observation that fluorogenic substrates can be successfully used to identify aminopeptidases and to measure their activity in cell lysates. Moreover, in contrast to assays using single substrates, which can result in overlapping specificity due to cleavage by several aminopeptidases, our library fingerprint can provide information about single enzymes.
authors.affiliation: Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Byzia
authors.affiliation: Department of Medical Laboratory Diagnostics and Bank of Frozen Tissues and Genetic Specimens, Medical University of Gdansk, Debinki 7, 80-211 Gdansk, Poland.
authors.forename: Agata
authors.initials: A
authors.lastname: Szeffler
authors.affiliation: Department of Medical Laboratory Diagnostics and Bank of Frozen Tissues and Genetic Specimens, Medical University of Gdansk, Debinki 7, 80-211 Gdansk, Poland.
authors.forename: Leszek
authors.initials: L
authors.lastname: Kalinowski
authors.affiliation: Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, 50-370 Wroclaw, Poland. Electronic address: marcin.drag@pwr.edu.pl.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drag
mesh_terms: D000818:Animals; D018826:CD13 Antigens / Q000378:metabolism*; D002457:Cell Extracts / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology; D057075:Enzyme Assays / Q000295:instrumentation / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology*; D015394:Molecular Structure; D051381:Rats; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
chemical_list: D002457:Cell Extracts; D005456:Fluorescent Dyes; D054852:Small Molecule Libraries; D018826:CD13 Antigens
keywords: Aminopeptidase; CD13; Exopeptidase; Fluorogenic substrate; Protease