Figure 6

Transport of the NBD-C16 derived from NBD-C16-CoA into ABCD1- liposomes. (A) Proteoliposomes containing ABCD1 (4.81 μg) or non-specific protein were incubated with NBD-C16-CoA at 37 °C for the indicated periods. After incubation, the remaining NBD-C16-CoA in the outside portion of the liposomes was quenched with sodium dithionite. In some cases, the liposomes were treated with sodium dithionite after the addition of 0.01% Triton X-100. Subsequently, ABCD1-liposomes were precipitated by centrifugation and then resuspended with 80% acetone. NBD-C16 and NBD-C16-CoA were separated by TLC. (B) Transport of NBD-C16 into ABCD1-liposomes was tested under various conditions. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of pCMB (1 mM) or palmitoyl-CoA (20 μM), or in the absence of ATP. NBD-C16-3′-dephosphoCoA (NBD-C16-dePiCoA) and NBD-C6-CoA were also examined as the substrate. Relative NBD-C16 transport activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J (right graph). Error bars indicate the standard error (n = 3). (C) NBD-C16 transport activity for ABCD1(K513A) was tested by the same procedure as used for the wild type. The relative transport activity ABCD1(K513A) was evaluated as follows. The intensities of NBD-C16 were normalized by the signal intensities of the wild type and K513A obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3).