Enzyme activity assay

6PGD activity was determined on the basis of the rate of NADPH production in assay buffer containing 0.1 mM NADP⁺, 1 mM MgCl₂ and 50 mM Tris (pH 8.1) with 0.2 mM 6-phosphogluconate as a substrate. The increase of absorbance at 340 nm was measured by a spectrophotometer.

G6PD activity was determined by the NADPH production rate from G6PD and 6PGD, and then subtracting that of 6PGD, because a product of G6PD, 6-phosphogluconolactone, is rapidly hydrolysed to a substrate of 6PGD, 6-phosphogluconate, in cells.

IDH1 activity was measured by using cytosolic extracts.The reaction mixture contains 20 mM Gly-Gly (pH 7.5), 0.6 mM MnCl₂, 1 mM NADP⁺ and 0.44 mM D-(+)-threo-isocitrate. Increase in 340 nm absorbance as a measure of NADPH production was detected every 20 s for 10 min on a DU800 Spectrophotometer (Beckman Coulter).

The reaction buffer of ME1 activity assay contained 67 mM triethanolamine, 3.3 mM l-malic acid, 0.3 mM NADP⁺ and 5.0 mM manganese chloride.The reactions were started by adding cytosolic extracts and were monitored by absorbance at 340 nm every 5 s for up to 10 min. Background control was run without l-malic acid as substrate.