FIG. 6.

Nitration and inhibition of PGHS-2. (A) Purified PGHS-2 (10 U/reaction) was treated with 0.1 μM 12-HpETE for activation and variable concentrations of NO₂⁻ in the presence of different levels of unlabeled substrate AA for 30 min. Before ¹⁴C-AA was added for activity detection, levels of unlabeled AA were supplemented to add up to the maximal concentration of 10 μM AA in all samples. The activity of the enzyme was then assessed by the addition of ¹⁴C-AA for 3 min. (B) Samples preincubated with 0.1 μM unlabeled AA and increasing concentrations of NO₂⁻ were subjected to SDS-PAGE. Consecutive staining with anti-PGHS-2 and anti-3-NT antibodies demonstrated the appearance of a nitrated band with increasing concentrations of NO₂⁻ at the same molecular weight as PGHS-2. (C) Purified human PGHS-2 (10 U/reaction) in the presence of 0.1 μM 12-HpETE was incubated with variable concentrations of either NO₂⁻ or authentic peroxynitrite. Following an incubation period of 2 h, the activity was the assessed by the addition of ¹⁴C-AA. (D) For the detection of radical formation, PGHS-2 (10 U/reaction), together with 0.1 μM 12-HpETE was treated with increasing concentrations of NO₂⁻ in the presence of DHR 123 (1 μM). DHR 123 is a relatively selective dye for the detection of peroxynitrite, respectively, the ^•NO₂ radical. Rhodamine fluorescence (λ_ex 485 nm; λ_em 538 nm) was then detected after 2 h. In a second experiment, PGHS-2 was treated with 0.1 μM 12-HpETE, 100 μM NO₂⁻, and 1 μM DHR 123 and varying concentrations of the peroxynitrite/^•NO₂ radical scavenger uric acid for 2 h. *p<0.05 versus conc.=0. 12-HpETE, 12-hydroperoxy-5Z,8Z,10E,14Z-eicosatetraenoic acid; DHR, dihydrorhodamine; SDS-PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis.