Figure 5

Relationship between ATPase activity and the ACOT activity of ABCD1 (A) Effect of ATP on ABCD1-catalyzed ACOT activity. ABCD1-liposomes were incubated with NBD-C16-CoA in the presence of various concentration of ATP for 30 min at 37 °C. The aliquots were subjected to TLC as shown in Fig. S7. The relative ACOT activities were evaluated by quantifying the intensities of NBD-C16 using the image analysis software Image J. Error bars indicate the standard error (n = 3). (B) His-ABCD1(a.a.1–431) and His-ABCD1(K513A) were prepared using the same procedure as the purification of His-ABCD1 and then reconstituted into liposomes. Proteoliposomes containing ABCD1wild type, ABCD1(K513A) or ABCD1(1–431) (2.68 μg, 3.21 μg or 3.71 μg, respectively) were incubated with NBD-C16-CoA for 30 min at 37 °C. The aliquots were subjected to TLC to separate hydrolyzed NBD-C16 and NBD-C16-CoA. (C) Relative ACOT activities of ABCD1(a.a.1–431) and ABCD1(K513A) were evaluated. The intensities of NBD-C16 were normalized by the signal intensities of each of the proteins obtained by immunoblot analysis using the image analysis software Image J. Error bars indicate the standard error (n = 3). Wild type activity (0.33 nmol/min/mg) has been normalized to 1. (D) Aliquots were also subjected to SDS-PAGE. The gel was fixed with methanol and stained with CBB (left panel) after the detection of NBD fluorescence (right panel). The arrow heads indicate the wild type, a.a.1–431 or K513A.