کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1947941 1054663 2010 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Selective detection of Cathepsin E proteolytic activity
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Selective detection of Cathepsin E proteolytic activity
چکیده انگلیسی

BackgroundAspartic proteases Cathepsin (Cath) E and D are two different proteases, but they share many common characteristics, including molecular weight, catalytic mechanism, substrate preferences, proteolytic conditions and inhibition susceptibility. To define the biological roles of these proteases, it is necessary to elucidate their substrate specificity. In the present study, we report a new peptide–substrate that is only sensitive to Cath E but not Cath D.MethodsSubstrate e, Mca-Ala-Gly-Phe-Ser-Leu-Pro-Ala-Lys(Dnp)-DArg-CONH2, designed in such a way that due to the close proximity of a Mca-donor and a Dnp-acceptor, near complete intramolecular quenching effect was achieved in its intact state. After the proteolytic cleavage of the hydrophobic motif of peptide substrate, both Mca and Dnp would be further apart, resulting in bright fluorescence.ResultsSubstrate e showed a 265 fold difference in the net fluorescence signals between Cath E and D. This Cath E selectivity was established by having -Leu**Pro- residues at the scissile peptide bond. The confined cleavage site of substrate e was confirmed by LC-MS. The catalytic efficiency (Kcat/KM) of Cath E for substrate e was 16.7 μM−1 S−1. No measurable catalytic efficiency was observed using Cath D and no detectable fluorescent changes when incubated with Cath S and Cath B.ConclusionsThis study demonstrated the promise of using the developed fluorogenic substrate e as a selective probe for Cath E proteolytic activity measurement.General significanceThis study forms the foundation of Cath E specific inhibitor development in further studies.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - General Subjects - Volume 1800, Issue 9, September 2010, Pages 1002–1008
نویسندگان
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