FIGURE 2.

Tpr55 is a zymogen requiring Ca²⁺ for sequential autoproteolytic processing to generate active forms of the enzyme: first Tpr48, then Tpr37, and finally stable Tpr33. A, purified Tpr55/Tpr48 (in buffer containing EDTA) was incubated in assay buffer without calcium (EDTA +/Ca²⁺ −) or with CaCl₂ (EDTA −/Ca²⁺ +), and samples were treated with DCG-04. After a 15-min incubation at 37 °C, the reaction was terminated by boiling in denaturing sample buffer. Proteins were resolved by SDS-PAGE and subjected to Western blot analysis. Lanes 1 and 2, protein staining; lanes 3 and 4, detection of probe labeled active Tpr. B and C, purified Tpr55/Tpr48 was supplemented with CaCl₂ and treated with DCG-04 (at time 0 or after incubation at 37 °C for specific time intervals). A sample incubated in buffer with EDTA (Tpr-ctrl) served as a control. Proteins were resolved by SDS-PAGE, and gels were stained for protein (B) or subjected to Western blot analysis to detect probe labeled proteins (C). SDS-PAGE-separated proteins were subjected to N-terminal sequencing analysis. D, purified Tpr55/Tpr48 was incubated in the assay buffer with Ca²⁺ at 21 °C for the indicated periods of time, and samples were analyzed by SDS-PAGE.