Figure 1

Tricarboxylic acid cycle enzyme assays. A, The first segment of the tricarboxylic acid cycle can be conveniently evaluated using a single assay to measure five enzymes by spectrophotometrically recording the reduction of DCPIP. The sequential assay begins by measurement of succinyl-CoA ligase activity based on oxidation of the produced succinate by succinate dehydrogenase, which forwards the electrons to the electron acceptors (DQ, DCPIP, and PMS). Coupling of these two activities to estimate succinyl-CoA ligase activity is permitted by the much higher activity of SDH than of succinyl-CoA ligase. After SDH inhibition by malonate, simultaneous addition of glutamate and aspartate aminotransferase ensures elimination of any oxaloacetate in the assay medium, thereby allowing further measurement of MDH activity. Incidentally, the required presence of NAD+ permits the measurement of glutamate dehydrogenase activity. Adding more DCPIP allows subsequent measurement of fumarase and MDH activity. Again, the coupling assay to estimate fumarase activity using MDH activity is permitted by the much higher activity of MDH. B, A second spectrophotometric assay subsequently measures pyridine dinucleotide reduction by three additional enzymes starting with α-ketoglutarate dehydrogenase. The next enzyme to be measured is aconitase, whose product, isocitrate, is readily oxidized by isocitrate dehydrogenase, producing NADPH. A saturating isocitrate concentration is finally added to enable measurement of isocitrate dehydrogenase activity. C, respective proportions of TCAC enzyme activities in mouse heart. The inset shows the TCAC depicted as two interacting enzyme cycles, A and B. Cycle A: α-ketoglutarate dehydrogenase (6), succinyl CoA ligase (1), succinate dehydrogenase (2), fumarase (4), and malate dehydrogenase (5); Cycle B: citrate synthase (9), aconitase (7), and isocitrate dehydrogenase (8). The two cycles interact via the activity of aspartate aminotransferase (10).