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ATPase activity assays

HS Hendrik R. Sikkema
BP Bert Poolman
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As described in detail in29, we used a coupled enzyme assay with pyruvate kinase (PK) and lactate dehydrogenase (LDH) to determine the ATPase activity of OpuA, which is stoichiometrically coupled to the NADH absorbance decrease at 340 nm. The enzymes PK and LDH were present in excess over OpuA in terms of activity. The NADH absorbance was followed in 96-well plates using a Tecan Spark 10 m plate reader. Each well contains 50 mM KPi pH 7, 300 mM KCl, 4 mM phosphoenolpyruvate, 62 μM glycine betaine, 300 μM NADH, 2.1–3.5 units of pyruvate kinase and 3.2–4.9 units of lactate dehydrogenase. The wells were then supplemented with 100 μL of the elution fraction after labelling, corresponding to roughly 1–5 μM of OpuA. The reaction was started by the addition of 10 mM Mg ATP.

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