FIGURE 3.

Expression profile and ATPase activity of D164C/D805C mutant P-gp expressed in insect cells. Crude membranes were prepared from cysless-WT and mutant P-gp-expressing High-Five insect cells. A, expression of cysless-WT and D164C/D805C were checked by separating crude membrane samples on 7% Tris-acetate gel (30 μg of protein/lane) followed by detection with Colloidal blue staining and by Western blot analysis (1 μg of protein/lane) using anti-P-gp antibody C219 at 1:2000 dilution. The arrow shows the position of the P-gp band, and the position of pre-stained molecular weight (111 kDa) is also shown. B, effect of selected compounds on the ATPase activity of cysless-WT and D164C/D805C mutant P-gp. Crude membranes (100 μg/ml) were incubated with DMSO (for basal activity), 10 μm CsA, 5 μm nilotinib, 10 μm valinomycin, or 30 μm verapamil, and ATPase activity was measured as described under “Experimental Procedures.” Values represent the mean ± S.D. (cysless-WT: n = 3; DD: n = 3). C, ATPase activity of cysless-WT and double mutant P-gp measured as a function of varying concentrations (0–10 mm) of ATP. Due to low basal activity of D164C/D805C, the measurements were performed in the presence of 30 μm verapamil. Each data point is the mean ± S.E.M. of three independent experiments. The data were fitted to the Michaelis-Menten curve using GraphPad Prism 5.0.