Determination of 6PG Consumption and KDPG Formation in a Discontinuous Assay System

The KDPG formation was assayed in a discontinuous assay (0.5 ml) containing 200 mM HEPES buffer pH 8, 5 mM MnCl₂, 2.5 mM 6PG and 8.1 μg purified EDD at 37°C. 100 μl samples were removed in regular intervals, and the reaction was stopped by addition of 10 μl 20% (w/v) TCA and incubation on ice for 10 min. After a 15 min centrifugation at 21,000 × g and 4°C, 50 μl of the supernatant were mixed with 2 μl 2 M NaOH for neutralization. For KDPG determination 11.4 μl of these samples were added to 100 mM HEPES buffer pH 8, containing 0.2 mM NADH, 3 U L-lactate dehydrogenase (rabbit muscle, Sigma-Aldrich) and 50 μg KD(P)GA-aldolase from S. acidocaldarius (500 μl final volume) and preincubated at 37°C for 2 min. The oxidation of NADH was followed spectrophotometrically [Analytik Jena, ε(NADH) = 6.22 mM^–1 cm^–1]. When the reaction ran to completion 5 μl of the purified EDD (corresponding to 0.22 U and 3.6 μg of protein) was added to determine the residual 6PG. All measurements were performed in triplicates.