The ATPase assay was carried out using an enzyme coupled reaction in which the regeneration of ATP is coupled to the oxidation of NADH, as previously described (64). Activity was detected as a decrease in NADH absorbance at 340 nm, which was measured every 1 min for 120 min using a microplate reader (Synergy, BioTek) and the path-length correction function. All reactions were carried out in triplicate in a final volume of 100 μl using a 96-well plate. The decrease in NADH absorbance at 340 nm was converted to micromoles of ATP using Beer’s law and then expressed as a function of time. The final conditions for all the reactions were 25 mM Hepes (pH 7.2), 125 mM NaCl, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM DTT, 0.6 mM NADH, 2 mM ATP, 1 mM phosphoenol pyruvate, 2.5 μl of pyruvate kinase/lactate dehydrogenase (Sigma-Aldrich), and 0.02% dimethyl sulfoxide. Identical reactions were treated with 10 μM thapsigargin and subtracted from untreated reactions to identify specific SERCA activity. Protein concentration of light membrane samples was quantified using the Pierce BCA Protein Assay Kit and 1 μg of protein was added to each well. Reactions were started by adding the regenerating system consisting of DTT, NADH, ATP, phosphoenol pyruvate, and pyruvate kinase/lactate dehydrogenase to the resuspended light membranes. Fit lines were calculated with the following equation: Y = ((Bmax * X)/(Kapp + X)) + X0. SERCA ATPase activity is shown as the percentage of the ATPase activity inhibited by thapsigargin in the light membranes and as ATPase activity per microgram of protein in the light membranes.

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