Figure 3.

CD2068 mediated drug transport. (a) Ethidium transport in CD2068-expressing and control E. coli cells. The de-energized cells (OD₆₀₀ = 0.5) of the CD2068-expressing and control cells were pre-equilibrated with 20 μM ethidium bromide, and 25 mM glucose was added (indicated by the first arrow) to initiate ethidium bromide efflux. CCCP at 10 μM was then added to the reaction, as indicated by the second arrow. (b) CD2068-mediated transport of Hoechst 33342 in inside-out membrane vesicles. After the steady state was reached, active transport of Hoechst 33342 was initiated by the addition of 5 mM ATP or nonhydrolysable ATP analogue AMP-PNP (indicated by the arrow). The IMV prepared from the E. coli cells with the empty plasmid were included as a control. (c and d) Competitive transport by ciprofloxacin. The ATP-dependent transport of Hoechst 33342 in IMV containing CD2068 (c) and control vesicles (d) was measured. Ciprofloxacin was added to the system at the final concentration of 5 μM prior to the addition of Hoechst 33342. (e) Ciprofloxacin stimulated the CD2068-associated ATPase activity in inside-out membrane vesicles. The vanadate-sensitive ATPase activity of inside-out membrane vesicles with CD2068 (○) and control membrane vesicles (●) in the presence of various concentrations of ciprofloxacin was measured as described in Methods. The error bars and the asterisks represent the standard errors and the statistical significance with the p values < 0.05, respectively.