Fig. 3.
Labeling of aTMH2 and aTMH5 with 14C NEM. Membranes from various aTMH2 and aTMH5 mutants were treated at 10 mg/ml in TMG-acetate buffer (pH 7.5) with 1 mM 14C NEM for 1 h at room temperature. The membranes were then solubilized with SDS, and subunit a was purified with nickel-nitrilotriacetic acid-agarose. Samples were subjected to SDS/PAGE, and the radioactivity in the dried gel was quantitated with a PhosphorImager. (A) aTMH2 data. (B) aTMH5 data. (A and B Top) Coomassie stain of dried gel, used to normalize the labeling intensities. (A and B Middle) PhosphorImage of dried gel. (A and B Bottom) Bar graph of labeling intensity normalized to Coomassie staining intensity. In all experiments, aS206C membranes were used as a positive control for labeling, and the labeling intensity for aS206C represents 100% on the bar graph. The C bar on the graphs represent labeling results for the control membranes lacking Cys in F1Fo.