FIGURE 6.

Ca²⁺-ATPase assay in COS7 microsomes. A, COS7 cells were transfected with the indicated cDNA construct(s). ATPase activity was determined at each [Ca²⁺]_free compared with the V_max measured for SERCA1 alone, following normalization of the levels of SERCA1 expression as determined by immunoblotting (see “Experimental Procedures”). Data were fitted to the equation for a general cooperative model for substrate binding. Results show that co-expression of sAnk1 with SERCA leads to a reduction of the apparent affinity of SERCA for Ca²⁺ but that the effect of sAnk1 is less than that of SLN. B, the K_Ca2+ ([Ca²⁺]_free required for half-maximal activation) values were determined from each curve. Mean K_Ca2+ values were as follows: SERCA1, pCa = 6.33 (468 nm); SERCA1 + sAnk1, pCa = 6.15 (708 nm); SERCA1 + SLN, pCa = 5.95 (1122 nm). C, unlike sAnk1 (WT), sAnk1 (all-L) did not significantly alter the affinity of SERCA1 for Ca²⁺. D, mean K_Ca2+ values were as follows: SERCA1, pCa = 6.31 (488 nm); SERCA1 + sAnk1, pCa = 6.10 (794 nm); SERCA1 + sAnk1 (all-L), pCa = 6.30 (502 nm). Statistics used one-way analysis of variance. *, p < 0.05 versus SERCA1; **, p < 0.01 versus SERCA1; #, p < 0.05 versus SERCA1 + sAnk1 (all-L). Error bars, S.E.