Fig. 6: ATPase activities and transport measurements.
From: Rhodamine6G and Hœchst33342 narrow BmrA conformational spectrum for a more efficient use of ATP
A Schematic ATPase activity of the transporter B ATPase activity can be measured by following disappearance of ATP via a coupled enzymatic assay; decrease of absorbance at 340 nm is directly correlated to ATP hydrolysis. Blue curve for BmrA apo, and red in complex with R6G. The slope is greater with R6G showing a faster ATP hydrolysis. This example is for the point 300 µM ATP for BmrA in detergent as shown in (D). C same as (B) for 1 mM ATP showing no stimulation. D ATPase activity measured in detergent, liposomes or in membranes. Each point represents a kinetic experiment as in (B) for a range of ATP concentration, acquired with or without R6G. The slope of each curve was measured at initial speeds, and the normalized ratio is presented on the graph to show the activation. E Scheme of Hœchst33342 transport in membranes occurring with ATP hydrolysis. F Transport efficiency of Hœchst33342 or Doxorubicin transported per ATP hydrolyzed along the range of ATP-Mg2+ sampled in (D). Individual measures are indicated on the graph (quadruplicates) and the line links the average of each concentration of ATP-Mg2+ sampled. Details of this experiment are shown in Supplementary Figs. 29–30.
