HPLC analysis of the conversion of NADH into NADPH. Cells exposed to control and menadione-stressed conditions were isolated and the soluble and membrane fractions were utilized to assess the conversion of NADH into NADPH.
Panel A) The soluble CFE from P. fluorescens grown in control and menadione-stressed media were was incubated for 60 min in a reaction mixture containing 10 mM oxaloacetate, 1 mM ATP, and 1 mM NADH. The levels of NADH, NADPH, oxaloacetate, and pyruvate were monitored at various time points by HPLC. Peaks were quantified using EMPOWER software. n = 3, p≤0.05, mean±S.D. Panel B) ME inhibition promotes malate accumulation in menadione-treated cells. The soluble CFE from P. fluorescens grown in control and menadione-stressed conditions were incubated in a reaction mixture containing 10 mM oxaloacetate, 1 mM ATP, 1 mM NADH, and 1 mM 3-bromopyruvate. Following a 60 min incubation, the levels of malate were quantified using EMPOWER software. n = 3, p≤0.05, mean±S.D. Panel C) The membrane CFE from control and menadione-stressed cells was incubated for 60 min in a reaction buffer containing 1 mM GTP, and 1 mM HCO3−. The levels of pyruvate and oxaloacetate were monitored at various time points by HPLC. Oxaloacetate and pyruvate were identified by injecting known standards. Peaks were quantified using EMPOWER software. n = 3, p≤0.05, mean±S.D. Cells were were isolated at 25 h for control and 30 h for menadione-stressed conditions to afford a proper comparison.
