P0294

The activity of both enzymes was determined by the production of ADP which was coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase^{58 (link)}. The assays were performed in a final volume of 100 µl 96-well deep-well plates and spectrophotometrically monitored (Omega multimode microplate reader) at 360 nm at 35 °C. KH₂PO₄ (100 mM) at pH 7.0, supplemented with 1.5 mM MgCl₂ and 100 mM KCl, was used as a buffer. For NADH, a molar extinction coefficient of 4,546.7 cm⁻¹ M⁻¹ was experimentally determined for the above-named conditions. To the buffer, 1 mM NADH, 2.5 mM Na₂SO₄, 1 mM phosphoenolpyruvate (PEP), 2 mM ATP, 2 U inorganic pyrophosphatase (Saccharomyces cerevisiae, 10108987001, Sigma-Aldrich), 1.1 U ml⁻¹ lactate dehydrogenase, 0.8 U ml⁻¹ pyruvate kinase (rabbit muscle, P0294, Sigma-Aldrich) and 0.5 mg ml⁻¹MtAPSK (all final concentrations) were added. The reaction was started by the addition of 0.5 mg ml⁻¹MtATPS. Addition of 0.02 mM Na₂MoO₄ did not affect activity (0.116 ± 0.027 µmol of oxidized NADH min⁻¹ mg⁻¹), but the addition of 2 mM Na₂MoO₄ resulted in a decrease (0.068 ± 0.019 µmol of oxidized NADH min⁻¹ mg⁻¹). All assays were performed in triplicates.

ATP was regenerated using ∼1 unit of rabbit muscle PK (P0294; Sigma-Aldrich) and PEP (concentration equimolar to Cy3-ATP; Fig. 1). PK, PEP, and Cy3-ATP were incubated for at least 60 min on ice.