Fig. 2. S-Nitrosylation of COX-2 enhances enzyme activity. (A) COX-2 expressed in transfected HEK293T cells is S-nitrosylated in the presence of GSNO (100 µM) or glutathione (reduced form) (100 µM) as determined by biotin-switch assay. All the S-nitrosylated proteins were precipitated and COX-2 was detected by Western blot with COX-2–specific antibody. COX-2 was selectively S-nitrosylated by GSNO. (B) LPS–IFN- treatment of RAW264.7 cells elicits S-nitrosylation of COX-2, which is prevented by the iNOS inhibitor 1400W (100 µM). COX-2 was selectively S-nitrosylated by endogenously generated NO. (C) COX-2enzyme activity was measured from the cell lysate of transfected HEK293T cells expressing COX-2–Myc in the presence or absence of SNP and ascorbate. Bars represent the mean ± SEM of three independent cell cultures performed in triplicate (*statistically significant by Student's t test). (D) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated by various concentrations of GSNO. The dose-dependence of GSNO-mediated activation of PGE₂ was measured. Data were pooled from at least three independent determinations, each in triplicate. (E) COX-2–Myc expressed in transfected HEK293T cells is S-nitrosylated in the presence of SNP and reversed by the addition of ASC. All the S-nitrosylated proteins were precipitated, and COX-2 was detected by Western blot with COX-2–specific antibody. (F) Recombinant human COX-2 was treated with SNP, and COX-2 activity was measured (n = 3, control: V_max = 81.3 ± 4.8 nmol/min per mg, K_m = 16.2 ± 2.2 µM; SNP: V_max = 132 ± 6.5 nmol/min per mg, K_m = 17.0 ± 2.0 µM). (G) Recombinant human COX-2 was treated with SNP, and its turnover rate (k_cat) was measured in the presence of various concentrations of sucrose. Data were expressed as k_cat-control over k_cat in each viscosity versus viscosity ratio. [View Larger Version of this Image (125K JPEG file)]

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