Figure 4.

Effect of exogeneous polysulfides on CSE β-lyase activity toward cystine. (A) The immobilized CSE was pre-incubated with 10 µM Na₂S₄ for 10 min at 30 °C. Na₂Sn (n = 2–4) were removed by centrifugation, and CSE activity was assayed in the presence of 50 µM PLP and 1 mM cystine. The synthesized Cys-SSH levels in each reaction mixture were measured by the SSP4 probe. For the DTT sample, an additional incubation with 20 mM DTT (20 min at 30 °C) was performed prior to assessment. (B) The immobilized CSE was pre-incubated with indicated concentrations of Na₂Sn (n = 2–4) for 10 min at 30 °C. CSE activity in each reaction mixture was assayed in the presence of 50 µM PLP and 1 mM cystine, as described in panel A. (C) Cys-SSH was generated by purified CSE in the buffer containing 50 µM PLP and 1 mM cystine, either in the presence (CSE/Cystine) or absence (Cystine) of CSE. An aliquot of reaction mixture was subjected to the SSP4 assay. (D) Proportional amounts of either each reaction mixture in panel C or buffer alone (Control) were initially incubated with the immobilized CSE for 10 min at 30 °C. CSE activity in each reaction mixture was assayed in the presence of 50 µM PLP and 1 mM cystine, as described in panel A. Results are the mean ± SE of three-four independent experiments. **** p < 0.0001 when compared with control or indicated column. n.s.; not significant.