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. 2013 Jun 21;288(31):22658–22669. doi: 10.1074/jbc.M113.485284

FIGURE 2.

FIGURE 2.

Purified mitochondrial ABCB6 binds ATP and shows intrinsic ATPase activity. A and B, solubilization and FLAG affinity purification of ABCB6-FLAG from mitochondria are shown. 2 and 5 μg of protein eluted from the affinity column was analyzed by SDS-PAGAE (4–15%) followed by Coomassie staining (A) and immunoblot (500 ng-purified protein) (B) using ABCB6-specific and FLAG-specific antibody. C--G, ATP binding and ATPase activity of detergent-solubilized FLAG-tagged ABCB6 are shown. C, shown is phosphor image analysis of purified ABCB6 (2 μm) labeled with 8-azido-[α-32P]ATP (top panel) followed by Coomassie Blue staining to verify equal protein loading (bottom panel). The effect of EDTA (1 mm), ATP (10 mm), and various nucleotides (0.3 mm each) on ABCB6 labeling with 8-azido-[α-32P]ATP is also shown. D, shown is a binding assay with increasing concentrations of 8-azido-[α-32P]ATP. The photo-cross-linking efficiencies were estimated from (D, top panel) phosphorimaging analysis and by (D, bottom panel) quantifying the spots and plotting the intensities against the concentration of 8-azido-[α-32P]ATP. a.u., arbitrary units. The apparent Kd(azidoATP) value for ABCB6 (1.39 μm) was obtained from the best fit of the binding data to a hyperbolic curve. E, shown is competition of 8-azido-[α-32P]ATP binding to ABCB6 (2 μm) by MgATP. E, photo-cross-linking was performed with 5 μm 8-azido-[α-32P]ATP with increasing concentrations of MgATP (top panel), and the IC50 for MgATP was derived by plotting labeling intensities corresponding to ABCB6 as a function of unlabeled MgATP concentrations (bottom panel). The sample without competitor was set to 100%. From the curve, an IC50 value of 0.82 μm for ABCB6 was estimated. F and G, ATPase activity of wild type and mutant ABCB6 is shown. The ATPase activity of ABCB6 (5 μm) was measured as a function of the ATP concentration at 37 °C. Purified ABCB6 is active in ATP hydrolysis. The data were fitted to Michaelis-Menten (F) and Lineweaver-Burk plots (G), resulting in a Km of 0.99 mm and a Vmax of 492.3 nmol/mg/min. The ABCB6-nonfunctional mutant (ABCB6-MT) showed only background ATPase activity. Results are representative of three independent experiments. TL, total lysate; ML, mitochondrial lysate; Flag-ab, FLAG-specific antibody; ABCB6-ab, ABCB6-specific antibody.