(A) Lactococcus membranes expressing HMA8 (HMA8) or the inactive enzyme HMA8-AKT (AKT) were phosphorylated from ATP in the presence of 1 μM CuSO4 (Cu) or 1 mM BCA/100 μM BCS (BCA/BCS) at pH 6 or pH 7. Quantifications 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 five independent experiments. Error bars indicate S.D. (B) Lactococcus membranes containing HMA8 (150 μg) and HMA8-AKT (150 μg) were phosphorylated from ATP at pH 6 in the presence of 1 μM CuSO4. Following 1 min reaction, the sample was acid-quenched (lanes 1 and 5) or incubated 1 min more either without any addition (lanes 2 and 5) or with BCA/BCS (single arrow, lanes 3 and 7) or with BCA/BCS plus 1 mM ADP (double arrow, lanes 4 and 8) prior to acid-quenching. Samples were submitted to an acidic SDS/PAGE and phosphorylation was detected using a phosphoimager. Quantifications (C) were made using the Optiquant software. 100% corresponds to the phosphorylation level of HMA8 in presence of Cu following 2 min reaction (lane 2). Values correspond to the mean of three independent experiments. Error bars indicate S.D.