Whole-cell lysate preparation for enzyme assays.

Bacterial strains containing the pdef plasmid (AS192 or AS194) or mutant pdef derivatives (AS348 or AS349) were grown overnight in 5 ml LB medium and then diluted 1:100 in 50 ml fresh LB medium. Cells were induced for 1 h with 0.5 mM IPTG at an OD₆₀₀ of 0.5 to 0.6. Cells were harvested, washed once with assay buffer (50 mM HEPES [pH 7.4] and 25 mM NaCl), and resuspended in 0.5 ml assay buffer containing 0.1 mM diethylenetriamine penta-acetic acid (DTPA). Cells were lysed mechanically using 0.1-mm silica beads, and cell debris was removed by centrifugation. The clear cell lysate was immediately used for enzyme assays. The protein content of cell lysates was estimated using the Coomassie protein assay reagent kit (Thermo Fisher Scientific). To measure the effects of zinc or NO· on enzyme activity, 125 μM ZnSO₄ or 25 to 75 μM spermine NO· were added during the IPTG induction phase.