FIGURE 5.

Hydroxylamine treatment of SR membranes results in an increase of calcium ATPase activity. A, ATPase activity. Rabbit SR membranes were initially suspended at 4 mg·ml⁻¹ in the assay buffer and stored on ice. When employed, an equal volume of a freshly prepared 2 m hydroxylamine solution (NH₂OH, pH adjusted at 7.5 with saturated Tris) was added prior to incubation at 20 °C for 1 h. As a control, an equal volume of buffer was added before incubation at 20 °C for 1 h. ATPase activity was estimated by an enzyme-coupled assay as described under “Experimental Procedures.” The reaction was started by addition of 5 μg·ml⁻¹ ATPase (SR) to record the basal activity. It was followed by successive additions of 5 μg·ml⁻¹ of the A23187 ionophore (A23187) and 1 mg·ml⁻¹ C₁₂E₈ to reach maximum activity (in absence of any accumulation of calcium). The Ca²⁺-independent activity was estimated after addition of 1 mm EGTA. Note that this Ca²⁺-independent activity was systematically lower after treatment with hydroxylamine, suggesting that it had inactivated some contaminants. For presentation purpose, one of the traces was manually lowered by −0.5 units of absorbance. The initial “true” OD was almost the same for all the assays. When the SR membranes were treated with 1 m hydroxylamine, the final concentration of hydroxylamine in the cuvette was 5 mm. For comparison, 5 mm hydroxylamine was added to the cuvette prior to addition of the untreated SR. Note that we observed that 5 mm hydroxylamine slightly decreases the rate of regeneration of ATP (data not shown). B, effect of hydroxylamine on ATPase activity in rabbit and rat SR. Experiments depicted in panel A for rabbit SR were repeated 10 times for control samples and 6 times for hydroxylamine-treated samples, and are summarized in the left panel. The same series of experiments was done with rat SR and is presented in the right panel (n = 4 for control and n = 3 for hydroxylamine pre-treated membranes).