FIGURE 1.

Differing sensitivity of Cys substitutions in 1–2 loop and 3–4 loop to Ag⁺ and NEM inhibition. A 160-μl aliquot of membranes at 10 mg/ml in TMG-acetate were treated with 5 mm NEM for 15 min at room temperature and then diluted into 3.2 ml of HMK-nitrate buffer containing 0.3 μg/ml ACMA. Alternatively membranes were diluted into HMK-nitrate buffer, and AgNO₃ was added to 40 μm for 15 min at room temperature prior to addition of ACMA. ATP was added to 0.94 mm at 20 s, and the uncoupler nigericin was added to 0.5 μg/ml at 100 s. The traces indicate no treatment (blue), NEM treatment (green), or Ag⁺ treatment (red). The substitution tested is indicated in each panel.