Figure 3.

ATPase activity of ATP7B is stimulated by Atox1–Cu, but not free copper. A, expression of ATP7B in baculovirus-infected Sf9 cells detected by Western blot with antibodies against the ATP7B C terminus (left panel) and against the Strep tag (right panel). B, ATPase activity of microsomes prepared from noninfected Sf9 cells (1 μg of total protein/data point), cells infected with the virus encoding no exogenous protein (mock), D1027A-ATP7B, or wild-type ATP7B (n = 3). a.u., absorption at 600 nm. D1027A is a catalytically inactive variant of ATP7B used as a control. C, effect of free copper with various reducing agents (2 mm TCEP, 2 mm DTT, 10 mm cysteine, or 2 mm GSH) and of Atox1–Cu on the ATPase activity of Sf9 microsomes expressing wild-type ATP7B. Atox1 was added at 1:1 molar ratio with copper (Cu–Atox1). Equal amount of apo–Atox1 was used as a control for each experimental point (apo–Atox1).