FIGURE 3.
Tpr hydrolyzes Suc-Leu-Leu-Val-Tyr-AMC and Z-Phe-Arg-AMC with a lag-phase (A), which is apparently due to the enzyme inhibition by the N-terminal remaining non-covalently associated with Tpr33 (B and C). Recombinant Tpr (Tpr55/Tpr48) was preincubated in the assay buffer for 1 h before the substrate was added, and then fluorescence was recorded (λex = 355 nm and λem = 460 nm) for 3 h (A). Tpr processing was activated by calcium (2.5 mm final concentration), and at specific time points aliquots were withdrawn and subjected to the SDS-PAGE analysis (B). Simultaneously, the activity was measured employing Suc-Leu-Leu-Val-Tyr-AMC as the substrate (C). The inset shows substrates hydrolysis by Tpr preincubated in the presence of 2.5 mm CaCl2 for 4 h. RFU, relative fluorescence units.