Fig. 3.

Inhibition of Cys reduction is a mechanism of NO cytoprotection. (A) Effect of Cys and NO on Fenton-mediated DNA damage in vitro. As indicated, the supercoiled pBR322 plasmid (0.5 μg) was treated with 30 μM FeCl₃, 10 mM Cys, 45 μM NO, or 10 mM H₂O₂ in 20 mM Tris·HCl buffer (pH 7.9). After a 10-min incubation at room temperature, the reaction was stopped and separated in a 1% agarose gel. RF, relaxed form; SF, supercoiled form. (B) NO-mediated suppression of cellular RE rereduction. The graph shows the negative effect of NO on the rate of formazan dye formation in the culture of wt cells grown in LB. Where indicated, cells were treated with 30 μM NO for 5 sec before the addition of 0.1 mg/ml MTS. Values shown are the means ± SE from three experiments. (C) Effects of NO, diamide, or TepAu on the rate of formazan dye accumulation. Conditions are as in B. TepAu and diamide were added 30 sec before MTS. Values shown are the means ± SD from three experiments. (D) Effect of carbon availability on oxidative stress survival and NO protection. Cells in mid-log phase were resuspended in M9 minimal medium containing 200 μg/ml Cm and incubated with or without glucose (50 mM for 15 min at 37°C). NO (30 μM) was added for 5 sec, followed by H₂O₂ (10 mM). Values shown are the means ± SD from three experiments.