Characterization of Phytaspase Proteolytic Activity Using Fluorogenic Peptide Substrates.

Within the subtilase family of plant proteolytic enzymes, phytaspases are distinguished by their strict substrate cleavage specificity after an aspartate residue preceded by a characteristic tripeptide amino acid motif. This type of recognition resembles that of animal apoptotic proteases, caspases. Phytaspases attract attention not only because they are critically important for the accomplishment of stress-induced death of plant cells, but also due to their ability to specifically process precursor proteins, thus generating bioactive plant peptide hormones, systemin and phytosulfokine. As the activity of phytaspases appears to be essential for life and death decisions made by the plant cell, elaboration of an approach to characterize and quantitate phytaspase proteolytic activity is of importance. Here we provide a protocol for phytaspase activity determination and characterization using fluorogenic peptide substrates. This approach works well, both with purified phytaspase samples, and with crude extracts from plant tissues. We also discuss advantages of the assay, factors that may influence its sensitivity and specificity, as well as possible pitfalls.