Figure 2.

TrwD binding to ¹⁴C-labeled palmitic acid and palmitoyl-CoA. A, TrwD (80 μm) was incubated at increasing ¹⁴C-labeled palmitic acid ratios (1:0.25, 1:0.5, 1:0.75, and 1:1 protein:palmitic acid molar ratios, respectively). Lane C1 corresponds to TrwD (80 μm) in the absence of fatty acids. Lane C2 is also a control experiment, in which an alien protein, TrwA (80 μm), was incubated with ¹⁴C-labeled palmitic acid at a 1:1 protein:palmitic acid molar ratio. B, TrwD (80 μm) was incubated at increasing ¹⁴C-labeled palmitoyl-CoA ratios (1:0.5, 1:0.75, and 1:1 protein:palmitoyl-CoA molar ratios, respectively). Lane C1 corresponds to ¹⁴C-labeled palmitoyl-CoA (80 μm) in the absence of any protein. Lane C2, as in A, corresponds to TrwA (80 μm) incubated with ¹⁴C-labeled palmitoyl CoA (80 μm). Lane C1 and C2 radioactivity signals at the front of the gel correspond to free ¹⁴C-labeled palmitoyl-CoA. In both cases, protein–fatty acid complexes were analyzed by native PAGE, and radiolabeled images were obtained after overnight exposure, as described under “Experimental procedures.” C, TrwD (80 μm, lane D1) and TrwA (80 μm, lane D2) were analyzed by Coomassie Brilliant Blue–stained native PAGE, as described under “Experimental procedures.”