Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria

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Abstract
The contributions of NAD-specific isocitrate dehydrogenase [EC 1.1.1.41] and NADP-specific isocitrate dehydrogenases [EC 1.1.1.42] to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-.alpha.-methylisocitrate (3-hydroxy-1,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase. Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20-40% by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite .alpha.-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of .alpha.-methylisocitrate (400 .mu.M) sufficient for 99.99% inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 .+-. 6% and 19 .+-. 11% (mean .+-. SEM [standard error of the mean]), respectively. With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3- to 4-fold, decreased NADPH levels by 30-40% and 2-oxoglutarate accumulation by about 40%. The further addition of 600 .mu.M .alpha.-methylisocitrate decreased the NH4Cl-stimulated isocitrate oxidation by about 40% and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and .alpha.-methylisocitrate as in their absence. Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the 2 isocitrate dehydrogenases. In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase [EC 1.1.1.30], 85-100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400-900 .mu.M .alpha.-methylisocitrate. In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase [EC 1.6.4.2], the rate of glutathione reduction extrapolated to infinite .alpha.-methylisocitrate concentration was from 20-40% of the uninhibited rate. In the coupled synthesis of glutamate from isocitrate and NH4Cl, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase [EC 1.4.1.3], the rate of glutamate production extrapolated to infinite .alpha.-methylisocitrate concentration was about 60% of the uninhibited rate.