Quantification of Plazomicin Acetyltransferase Activity

Total soluble proteins (enzymatic extracts) were prepared as before [42 (link)]. Briefly, cells were pelleted from cultures by centrifugation and resuspended in a 0.5 mM MgCl₂ solution. The cells were lysed by sonication with a Heat Systems Ultra-sonic, Inc., Model No. H-IA (Plainview, NY, USA) cell disrupter. The soluble protein fraction was then separated from unbroken cells, membranes, and cell debris by centrifugation in a microfuge for 10 min at 4 °C. The protein concentration of the extracts was measured using a commercial reagent (Bio-Rad Protein Assay). Acetyltransferase activity was assessed using the phosphocellulose paper binding assay [65 (link)]. Soluble extract (120 μg protein) obtained from E. coli TOP10(pUC57AAC2Ia) cells was added to the reaction mixture (200 mM Tris HCl pH 7.6 buffer, 0.25 mM MgCl₂, 330 μM plazomicin, the indicated concentrations of sodium acetate or silver acetate, and 0.05 μCi of [acetyl-1-¹⁴C]-acetyl-coenzyme A (specific activity 60 μCi/μmol). The reaction mixture final volume was 30 μL. Silver ions were added as silver acetate due to its adequate solubility in water. After incubating the reaction mixture at 37 °C for 30 min, 20 μL were spotted on phosphocellulose paper strips. The unreacted radioactive substrate [acetyl-1-¹⁴C]-acetyl-coenzyme A was removed from the phosphocellulose paper strips by submersion in 80 °C water followed by two washes by submersion in room temperature water. After this treatment, the only radioactive compound bound to the phosphocellulose paper strips was the acetylated plazomicin. The phosphocellulose paper strips were then dried and the radioactivity corresponding to enzymatic reaction product was determined in a scintillation counter.