title: Ultrasensitive internally quenched substrates of human cathepsin L.
abstract: Internally quenched cathepsin L (Cat L) substrate ABZ-Bip-Arg-Ala-Gln-Tyr(3-NO2)-NH2 with high specificity constant (kcat/KM=2.6×10(7)M(-1)s(-1)) was synthesized. The resultant compound displayed high selectivity over other members of the cathepsin family (B, S, X, V, C, K, H, F, D, and A). Activity of Cat L at picomolar (pM) concentrations was found using this substrate. Moreover, it was established that the presence of the selective Cat L inhibitor suppressed the proteolysis of the substrate to a non-detectable level. Incubation of the synthesized compound with a cell lysate of healthy and cancer cell lines indicated significant differences in Cat L activity. Based on the obtained results, it is proposed that this substrate could be used for selective monitoring of Cat L activity in biological systems.
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.
authors.forename: Monika
authors.initials: M
authors.lastname: Lęgowska
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Wysocka
authors.affiliation: Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland.
authors.forename: Timo
authors.initials: T
authors.lastname: Burster
authors.affiliation: Department of Clinical Immunology and Transplantology, Medical University of Gdansk, 80-210 Gdansk, Poland.
authors.forename: Michał
authors.initials: M
authors.lastname: Pikuła
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Rolka
authors.affiliation: Department of Biochemistry, Faculty of Chemistry, University of Gdansk, 80-952 Gdansk, Poland. Electronic address: adam.lesner@ug.edu.pl.
authors.forename: Adam
authors.initials: A
authors.lastname: Lesner
mesh_terms: D001681:Biological Assay; D056668:Cathepsin L / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D062367:ortho-Aminobenzoates / Q000378:metabolism
chemical_list: D010455:Peptides; D062367:ortho-Aminobenzoates; C002744:3-nitrotyrosine; D014443:Tyrosine; D002403:Cathepsins; D056668:Cathepsin L
keywords: Cancer; Cathepsin L; FRET peptides; Fluorescent assay