Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures
Jazyk angličtina Země Spojené státy americké Médium print-electronic
Typ dokumentu časopisecké články, práce podpořená grantem
PubMed
24388866
DOI
10.1016/j.ab.2013.12.032
PII: S0003-2697(13)00618-0
Knihovny.cz E-zdroje
- Klíčová slova
- Cysteine peptidases, Enzymatic peptide synthesis, Multicomponent enzyme mixtures, Selective peptide substrates, Substrates of peptidases,
- MeSH
- cysteinové proteasy izolace a purifikace metabolismus MeSH
- enzymatické testy metody MeSH
- fluorescenční barviva analýza metabolismus MeSH
- hydrolýza MeSH
- molekulární modely MeSH
- peptidy chemie metabolismus MeSH
- substrátová specifita MeSH
- Tenebrio enzymologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- cysteinové proteasy MeSH
- fluorescenční barviva MeSH
- peptidy MeSH
This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes.
A N Belozersky Institute of Physico Chemical Biology Moscow State University Moscow 119992 Russia
Department of Chemistry Moscow State University Moscow 119992 Russia
Entomological Institute Biology Centre AV ĈR Ĉeské Budĕjovice CZ 37005 Czech Republic
Faculty of Bioengineering and Bioinformatics Moscow State University Moscow 119992 Russia
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