Figure 8.

Ca²⁺-ATPase assays. COS7 (A) and HEK293 (C) cells were transfected with the indicated cDNA construct(s) using a [C]_final of 1 μg/ml. 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 from both cell lines show that coexpression of sAnk1 with SERCA1 leads to a reduction of SERCA1's apparent affinity for Ca²⁺, but the effect of sAnk1 is less than that of SLN, as shown previously (1). B and D, K_Ca ([Ca²⁺]_free required for half-maximal activation) values were determined from each curve and are summarized in Tables 1 and 2. COS7 mean K_Ca: SERCA1 pCa = 6.33 (468 nm); SERCA1 + sAnk1 pCa = 6.15 (708 nm); SERCA1 + SLN pCa = 5.95 (1122 nm); and SERCA1 + sAnk1 + SLN pCa = 6.12 (759 nm). HEK293 mean K_Ca: SERCA1 pCa = 6.38 (415 nm); SERCA1 + sAnk1 pCa = 6.17 (680 nm); SERCA1 + SLN pCa = 6.08 (830 nm); and SERCA1 + sAnk1 + SLN pCa = 6.26 (560 nm). Statistics used one-way ANOVA: *, p < .05 versus SERCA1; **, p < .01 versus SERCA1; and #, p < .05 versus SERCA1 + SLN. ^† indicates data presented previously (1) and performed at same time as SERCA1 + sAnk1 + SLN.