Figure 2.

Effect of Ag⁺ on AAC(2′)-Ia activity. Assays were performed in triplicate by the phosphocellulose paper binding method with soluble extracts obtained from E. coli TOP10(pUC57AAC2Ia) cells. The reaction mixture contained 200 mM Tris HCl pH 7.6 buffer, 0.25 mM MgCl₂, 330 μM plazomicin, the indicated concentrations of silver acetate (Ag⁺) or sodium acetate (Na⁺), and 0.05 μCi of [acetyl-1-¹⁴C]-acetyl-coenzyme A (specific activity 60 μCi/μmol) in a final volume of 30 μL. The enzymatic reactions were allowed to proceed at 37 °C. After 30 min incubation, 20 μL were spotted on phosphocellulose paper strips, which were immersed in water at 80 °C. Following this, the paper strips were washed twice by immersion in room temperature water. Finally, the phosphocellulose paper strips were left to dry and counted to determine the bound radioactivity, which corresponds to acetylated plazomicin.