A3B3 or A3B(L65Y)3 (10 mg/ml protein) was reconstituted with the DF complex (5 mg/ml protein) in buffer G [100 mM MES, 5 mM MgSO4, 100 mM NaCl, and 10% glycerol (pH 6.0)] by incubating the mixture on ice for 1 hour (18, 19). The ATPase activity of A3B3 or the reconstituted A3B3DF was measured using an ATP-regenerating system (18, 19). The reaction mixture contained 1 mM ATP/MgSO4, 2.5 mM phosphoenolpyruvate, pyruvate kinase (50 μg/ml), lactate dehydrogenase (50 μg/ml), 0.2 mM β-NADH (nicotinamide adenine dinucleotide) (dipotassium salt), and 2 to 20 μg of protein in 1 ml of reaction mixture (buffer G). The reaction was initiated by adding ATP/MgSO4. The rate of ATP hydrolysis was monitored at 23°C by measuring the rate of oxidation of NADH, as determined by a decrease in absorbance at 340 nm.

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