This experiment was performed at 4°C, at pH 6. Lactococcus membranes containing HMA8, HMA6, HMA8-AKT and HMA6-AKT were incubated under reducing conditions (500 μM Na₂SO₃) with Cu and 1 μM [γ-³²P]ATP. After 2 min (for HMA6) or 4 min (for HMA8) an aliquot was taken and acid-quenched (lane 1). On the remaining sample, a mix of BCA/BCS was added and the reaction was stopped at the indicated times (single arrow, lanes 2–5 and 7). After 10 min of incubation with BCA/BCS, 1 mM ADP was added to the reaction for 2 min more (double arrow, lanes 6 and 8). All the experiments described in this figure were performed with 1 μM CuSO₄ for the phosphorylation of Lactococcus membranes containing HMA8/HMA8-AKT (150 μg) or with 5 μM CuSO₄ for the phosphorylation of Lactococcus membranes containing HMA6/HMA6-AKT (50 μg). Quantifications (B) were made using the Optiquant software. Hundred percent dephosphorylation correspond to the intensity of the background level as measured on the HMA8-AKT mutant. Values correspond to the mean of three independent experiments. Error bars indicate S.D.