![]() Substrate specificity of cathepsins D and E determined by N-terminal and C-terminal sequencing of peptide pools. ![]() A rapid method for determination of endoproteinase substrate specificity: Specificity of the 3C proteinase from hepatitis A virus. Petithory, J.R., Masiarz, F.R., Kirsch, J.F., Santi, D.V. Determination of enzyme specificity in a complex mixture of peptide substrates by N-terminal sequence analysis. ![]() Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin. A combinatorial approach for determining protease specificities: application to interleukin-1β converting enzyme (ICE). Rapid identification of highly active and selective substrates for stromelysin and matrilysin using bacteriophage display libraries. Substrate phage: selection of protease substrates by monovalent phage display. Our results indicate that a small set of libraries can be used to quickly profile an expanding protease family, providing information applicable to the design of inhibitors and to the identification of protein substrates. The library data led us to identify the proteoglycan neurocan as a novel MMP-2 substrate. The results were validated by comparison with previous literature and by analyzing the cleavage of individually synthesized peptide substrates. The method was used to determine cleavage site motifs for six enzymes in the matrix metalloprotease (MMP) family. ![]() We describe a method for determining the cleavage site specificity of proteolytic enzymes that involves pooled sequencing of peptide library mixtures. Although one can guess at the functions of these novel enzymes by considering sequence homology to known proteases, there is a need for new tools to rapidly provide functional information on large numbers of proteins. The number of known proteases is increasing at a tremendous rate as a consequence of genome sequencing projects. ![]()
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