The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver

Abstract

1. The concentrations of the oxidized and reduced substrates of the lactate-, beta-hydroxybutyrate- and glutamate-dehydrogenase systems were measured in rat livers freeze-clamped as soon as possible after death. The substrates of these dehydrogenases are likely to be in equilibrium with free NAD(+) and NADH, and the ratio of the free dinucleotides can be calculated from the measured concentrations of the substrates and the equilibrium constants (Holzer, Schultz & Lynen, 1956; Bücher & Klingenberg, 1958). The lactate-dehydrogenase system reflects the [NAD(+)]/[NADH] ratio in the cytoplasm, the beta-hydroxybutyrate dehydrogenase that in the mitochondrial cristae and the glutamate dehydrogenase that in the mitochondrial matrix. 2. The equilibrium constants of lactate dehydrogenase (EC 1.1.1.27), beta-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38 degrees ; I0.25). 3. The mean [NAD(+)]/[NADH] ratio of rat-liver cytoplasm was calculated as 725 (pH7.0) in well-fed rats, 528 in starved rats and 208 in alloxan-diabetic rats. 4. The [NAD(+)]/[NADH] ratio for the mitochondrial matrix and cristae gave virtually identical values in the same metabolic state. This indicates that beta-hydroxybutyrate dehydrogenase and glutamate dehydrogenase share a common pool of dinucleotide. 5. The mean [NAD(+)]/[NADH] ratio within the liver mitochondria of well-fed rats was about 8. It fell to about 5 in starvation and rose to about 10 in alloxan-diabetes. 6. The [NAD(+)]/[NADH] ratios of cytoplasm and mitochondria are thus greatly different and do not necessarily move in parallel when the metabolic state of the liver changes. 7. The ratios found for the free dinucleotides differ greatly from those recorded for the total dinucleotides because much more NADH than NAD(+) is protein-bound. 8. The bearing of these findings on various problems, including the following, is discussed: the number of NAD(+)-NADH pools in liver cells; the applicability of the method to tissues other than liver; the transhydrogenase activity of glutamate dehydrogenase; the physiological significance of the difference of the redox states of mitochondria and cytoplasm; aspects of the regulation of the redox state of cell compartments; the steady-state concentration of mitochondrial oxaloacetate; the relations between the redox state of cell compartments and ketosis.

Equilibrium protection studies of the interaction of bovine glutamate dehydrogenase with purine nucleotide effectors

“…Is GDH in mammalian liver so active that it equilibrates its reactants under all physiological conditions [36], or are there [37] conditions under which this reaction becomes rate-limiting and therefore perhaps subject to effective allosteric control? Another possibility that should be borne in mind is that the control properties of this enzyme may be relevant to its function in some tissue other than liver.…”

Mitochondrial metabolites extend lifespan

“…Increased amounts of the reduced, α‐hydroxyacid form of these α‐ketoacids are also observed. These pairs of compounds exist in a dehydrogenase‐catalyzed redox equilibrium with NAD (Williamson et al ., 1967). The α‐ketoacid dehydrogenase complexes are comprised of multiple E1, E2, and E3 subunits that assemble into a mini‐electron transport chain which normally operates sequentially in the order of α‐ketoacid oxidation (E1 subunit), lipoic acid reduction (E2 subunit), cystine reduction, FAD reduction, and NAD+ reduction (the three latter sites all reside within the E3 subunit).…”

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

“…10 If the function of a redox sensing ability is to form a link between changes to cellular metabolism and the control of transcription, one might expect that NmrA would be more likely to have its biological effects modulated by binding reduced dinucleotides. 27 The rationale for this argument is that it follows from the high (mM range) NAD(H) concentration and the high NAD þ /NADH ratio (500-700) 28,29 that conversion of NAD þ to NADH will have the greatest relative effect on the NADH level. 27 In this respect we note that the Human protein HSCARG, which belongs to the NmrA-like structural family, binds NADPH with a 360-fold lower K D than that for NADP þ .…”