FIG. 1.

ONOO⁻ impairs recombinant GTPCH1 activity but increases its ubiquitination associated with zinc release. A: Purification of recombinant FLAG-GTPCH1. The purity of FLAG-GTPCH1 was analyzed by SDS-PAGE using silver staining. B: Effect of ONOO⁻ on purified recombinant GTPCH1 activity. The enzyme activity of GTPCH1 was determined by HPLC. Data are shown as mean ± SD (n = 6). *P < 0.05 vs. control. C: Effect of NO on recombinant GTPCH1 activity. *P < 0.05 when compared to Vehicle. D: NO but not ONOO⁻ increases the S-nitrosylation of GTPCH1. E: ONOO⁻ stimulated zinc release from recombinant GTPCH1. Zinc was assayed as described in research design and methods and was expressed as percentage of maximal zinc release from GTPCH1 diluted in 7 mol/L guanidine HCl. n = 3. *P < 0.05 vs. control. F: The effect of the zinc chelator TPEN on GTPCH1 activity. n = 3. *P < 0.05 vs. control; #P < 0.05 vs. +TPEN 200 μmol/L. CTR (+), with biotin label; FT, flow through, after affinity purification; IB, immunoblot.