To test AtSBT1.1-myc for activity against pAtPSK4, we generated a fluorogenic peptide called fpAtPSK4 with the sequence Abz-RRSLVLHTDY(NO2)D-OH [where Abz is a 2-aminobenzoyl fluorescent group and Y(NO2)D-OH is a 3-nitrotyrosine quencher], representing a probable subtilase recognition site in pAtPSK4. The site was chosen because di-basic amino acid residues (in this case, RR) are characteristic signatures of subtilase recognition sites (Barr, 1991) . When bead-bound protein from transgenic plants expressing AtSBT1.1-myc was incubated with fluorescence-quenched fpAtPSK4, a time-dependent increase in fluorescence was observed (Figure 4a ). Because the enzyme was produced in a homologous system, we were mindful of potential contamination by endogenous Arabidopsis subtilases in our preparations. To preclude that possibility, we thoroughly washed the beads and routinely ran controls in which the same pull-down procedure was conducted with extracts from non-transgenic seedlings and from transgenic seedlings expressing a tagged subtilase (S552A) that was mutated at the active site ( Figure 4a ). The kinetics of the reaction with the fluorogenic peptide substrate followed Michaelis-Menten kinetics, and the K m for the reaction was determined to be approximately 18 lM (Figure 4b ). The K m value lies within the range of affinity constants for fluorogenic peptide substrates with comparable subtilases, such as mammalian prohormone convertases (Basak et al., 2004 (Basak et al., , 2007 . The activity was inhibited by PMSF (Figure 4a ), an inhibitor of serine proteases. The pH optimum for the reaction was in the acidic range, centered on pH 6 ( Figure 4c) .