FIGURE 4.
Liposome-reconstituted ABCB6 shows substrate-stimulated ATPase activity and ATP-dependent substrate transport. A, liposome-reconstituted ABCB6 interacts with its substrates. A, top panel, ABCB6 liposomes (4 μg protein) were subjected to hemin affinity chromatography in the presence or absence of either coproporphyrinogen III or porphobilinogen, and bound protein was subjected to 4–15% SDS-PAGE followed by Western blot using FLAG M2 antibody. Bottom panel, percent binding was estimated by plotting the band intensities corresponding to untreated ABCB6, which was set to 100%. B–D, liposome-reconstituted ABCB6 shows both basal and substrate-stimulated ATPase activity. ATPase activity of ABCB6-WT and ABCB6-MT proteoliposomes (25–50 μg protein) was measured as a function of the ATP concentration at 37 °C. The data were fitted to Michaelis-Menten (B) and Lineweaver-Burk plots (C), resulting in a Km of 0.97 ± 0.070 mm and a Vmax of 614.4 ± 11.53 nmol/mg/min. The ABCB6-nonfunctional mutant (ABCB6-MT) showed only background ATPase activity. D, liposome-reconstituted ABCB6 shows substrate-stimulated ATPase activity. -Fold change was calculated relative to basal ATPase activity. Values represent the mean ± S.D. *, significantly different from basal ATPase activity; p < 0.01. &, significantly different from CPIII ATPase activity; p < 0.01. E and F, shown is the effect of substrate concentration on ATP-dependent CPIII uptake by ABCB6 liposomes (25–50 μg of protein). Kinetic parameters were determined by fitting the data to Michaelis-Menten (E) and linear regression analysis of the Lineweaver-Burk transformation of the data points (F). Transport kinetics of liposome-reconstituted ABCB6 showed a Km of 11.97 μm and Vmax of 29.6 pmol/mg/min. Copro III, coproporphyrinogen; PBG, porphobilinogen; PPIX, protoporphyrin IX; PhA, pheophorbide A.