FIG. 4.
Quantification and characterization of exported radioactively labeled nucleotides by E. coli cells expressing the ATP/ADP translocase NTT1Li from L. intracellularis. Preloading with radioactivity was achieved by incubation of induced E. coli cells in phosphate buffer containing 50 μM [α-32P]ATP for 5 min at 30°C. Washed cells were incubated in 500 μM nonlabeled adenine nucleotides or phosphate buffer (control) for 2.5 min at 30°C. Back-exchange substrates are indicated on the x axis, i.e., potassium phosphate buffer (KPi; white bars), ATP (light gray bars), ADP (dark gray bars), and AMP (black bars). (A) Fraction of exported label (white, gray, and black bars) and remaining radioactivity in the cells (hatched bars). (B) Nature of the exported label (indicated above the bars) as determined by thin-layer chromatography and quantification of the radioactive spots by scintillation counting. Data are the mean of three independent replicates.