PEPCK enzyme activity was measured through the oxaloacetate formation from PEP, with NaHCO3 serving as the source of CO2 and was determined by measuring the oxidation of NADH in the presence of malate dehydrogenase (MDH) at 340 nm.
Respiration state 3 and 4 (LEAK) and ETS capacity were measured with pyruvate, malate and succinate (5, 5 and 10 mM, respectively) or palmitoylcarnitine and malate (40 μM and 5 mM, respectively).
In contrast, expression of human DIC was very low (data not shown) and transport activity for its canonical substrate malate, measured as malate/[C]-malate exchange, was negligible (Fig. S1).
All attempts to measure malate activation with different nucleoside triphosphates (GTP, UTP) or CoA donors (succinyl-CoA) were unsuccessful (data not shown).
In panel T, citrate and malate were measured using the citrate and malate analysis kits (R-Biopharm, Marshall, MI), according to the manufacturer's instructions60.
Therefore, glutamate developed into lactate through malate overflow (Fig. 5c).
These amino acids can be catabolised to pyruvate through malate and fumarate [ 35].
While citrate synthase (CS) was measured with the DTNB assay, malate dehydrogenase (MDH) was monitored via the production of NADH in the presence of malate [14].
Malate and citrate were chosen as representatives for unspecific leakage of organic acids, and their concentration was measured following the protocol of the malate and citrate enzymatic bioanalysis (r-biopharma, Roche).
Citrate and malate were measured by gas chromatography-mass spectrometry (GC-MS 8. GC-MS 8
