Table 3.
Kinetic parameters of ATP hydrolysis by wild-type and mutant BCRP
| Wild-type BCRP | N387A | Q398A | T402A | N629A | T642A | Y645F | |
|---|---|---|---|---|---|---|---|
| Vmax, nmol Pi·min−1·mg protein−1 | 14.5 ± 1.9 | 16.9 ± 1.5 | 14.9 ± 0.93 | 17.4 ± 1.7 | 11.5 ± 1.1 | 17.9 ± 1.9 | 14.5 ± 1.9 |
| Vmax normalized to BCRP protein level, nmol Pi·min−1·mg protein−1 | 14.5 ± 1.9 | 7.0 ± 0.63 | 6.5 ± 0.41 | 8.3 ± 0.81 | 28.0 ± 2.7 | 16.3 ± 1.7 | 12.0 ± 1.6 |
| Km for ATP, mM | 0.89 ± 0.40 | 0.90 ± 0.27 | 0.48 ± 0.12 | 1.1 ± 0.35 | 0.40 ± 0.15 | 0.93 ± 0.34 | 0.89 ± 0.43 |
| Vmax/Km, nmol Pi·min−1·mg protein−1·mM−1 | 16.3 | 7.8 | 13.5 | 7.5 | 70.0 | 17.5 | 13.5 |
Values are means ± SD of 3 independent determinations. Kinetic parameters for ATP hydrolysis were determined using plasma membrane preparations of HEK-293 cells expressing wild-type and mutant BCRP and were calculated by fitting the Michaelis-Menten equation to the data points by nonlinear regression using the GraphPad software. The Vmax values were normalized to the BCRP protein levels determined by immunoblotting of plasma membranes shown in Fig. 2.