Evaluation of recombinant caspase specificity by competitive substrates.
The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.
Benkova
Bistra
B
Department of Medical Chemistry and Biochemistry, Medical University of Sofia, Sofia 1431, Bulgaria.
Lozanov
Valentin
V
Ivanov
Ivaylo P
IP
Mitev
Vanio
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000609:Aminoacridines / Q000737:chemistry; D001667:Binding, Competitive; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D000609:Aminoacridines; D010455:Peptides; D011994:Recombinant Proteins; C072515:2-aminoacridone; D020169:Caspases