Figure 4.

Substitution of S1334 with an amino acid that eliminates the hydroxyl group at this position impaired ATP binding/hydrolysis at the mutated NBD2. Photolabeling experiments were carried out in 10 μL of solution containing 10 μg of membrane vesicle proteins (the amount of MRP1 protein determined in Figure 2A was adjusted to a similar amount by adding varying amounts of membrane vesicles prepared from parental BHK cell), 10 mM MgCl₂, 800 μM vanadate, and 10 μM [α-³²P]-8-N₃ATP (α) or 10 μM [γ-³²P]-8-N₃ATP (γ) at 37 °C for 2 min. The reaction mixture was subjected to UV irradiation (+) or without UV irradiation (−) on ice for 2 min, separated by SDS–PAGE (7%), and electroblotted to a nitrocellulose membrane. Molecular mass markers (kDa) are indicated on the left. MRP1 on the right of the gel indicates the ³²P-nucleotide-labeled MRP1 protein which was confirmed in Western blot by employing the NBD1-specific monoclonal antibody 42.4 (8).