Figure 3.

The ‘dynamic catalytic loop' of human SSADH senses ROS both in vitro and in vivo. (A, B) SSADH senses ROS in vitro. To investigate how SSADH senses ROS through the disulphide bond formation, wild type (WT) and the C342A mutant were treated with various concentrations of H₂O₂ in vitro and SSADH activities were measured. We then added 10 mM DTT to switch the environment to reduced state, and compared the activity recovery between the two different forms of SSADH. Over 80% of activity was recovered from wild-type SSADH that was treated with even 500 μM H₂O₂ (A), whereas almost no activities were recovered from the C342A mutant that was treated with more than 400 μM H₂O₂ (B). (C) SSADH senses ROS in vivo. Signal sequence-containing wild-type (WT) and C342A mutant SSADH were expressed in HEK293 cell. Cells were treated with 500 μM of H₂O₂ for 1 h to provide oxidative stress, and lysed anaerobically. In addition, SSADH activities were measured with samples with and without H₂O₂ treatment. To investigate the activity recovery, 10 mM of DTT was added to the H₂O₂-treated cell lysates, and SSADH activities were measured.