Cu dependence of HMA8 phosphorylation from [γ-32P]ATP
(A) Membrane preparations containing HMA8 (150 μg) or HMA6 (50 μg) were phosphorylated under reducing conditions (500 μM Na2SO3) without added metal (Ø, lanes 1–3, 7), with CuSO4 (1 μM lanes 4–6 and 5 μM lanes 8, 9). In each condition, BCA/BCS (lanes 2, 5, 9 and 10) or EGTA (lanes 3, 6) was added as indicated in the figure. (B) Lactococcus membranes containing HMA8 were phosphorylated under reducing conditions with various concentrations of CuSO4. Quantifications (lower panel) were made using the Optiquant software (PerkinElmer Life Sciences). Hundred percent corresponds to the maximum phosphorylation level of HMA8. Values correspond to the mean of four independent experiments. Ø corresponds to the values obtained without addition of Cu in the assay. Error bars indicate S.D.