FIGURE 4.

Characterization of PA1b binding. A, left, SDS-PAGE analysis of purified M. sexta V-ATPase with staining with silver (lane 1) or Coomassie Brilliant Blue (lane 2) and M indicating molecular mass markers. Right, identification of the staining polypeptides by immunoblot analysis. B, photoaffinity labeling of M. sexta V-ATPase with the ¹²⁵I-PA1b-benzophenone. For labeling, V₁V_o holoenzyme (V₁V_o), V_o complex (V_o), or V₁ complex (V₁) was incubated with ¹²⁵I-PA1b-benzophenone and exposed to UV light or kept in the dark. After separation by SDS-PAGE, the stained and dried gel was exposed to a phosphorimaging screen. Left, SDS-PAGE of the V-ATPase complexes with Coomassie Blue staining. Right, readout of the phosphorimaging screen for labeled holoenzyme (lanes 1–3), V_o (lanes 4–6), and V₁ (lanes 7–9) after either UV irradiation (U) or maintenance in the dark (D). Bands L1–L3 (arrows) indicate UV-dependent labeled species. C, slices of the dried gel from samples of the V₁V_O holoenzyme (open circles) and V_o (filled circles) subjected to scintillation counting. The column to the left of B indicates positions of gel slices subjected to counting. The majority of the radioactivity was found in slice 3 near the dye front. D, molecular mass determination of ¹²⁵I-labeled V-ATPase subunits. A calibration curve was prepared by plotting log molecular masses of standard proteins (triangles, dashed line) and V-ATPase subunits (deduced from their primary structure) against their relative migration on the SDS-polyacrylamide gels shown in A (open circles, dotted line) and B (filled circles, solid line). In each case, linear regression gave r²>0.99.