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

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.     

A nicotinamide-adenine dinucleotide assay utilizing liver alcohol dehydrogenase

An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD⁺ and NADH are assayed by the same procedure, which is also used to measure NADP⁺ and NADPH after these nucleotides have been converted to NAD⁺ and NADH, respectively.     

The development of polyploidy in two classes of rat liver nuclei

Two classes of nuclei from livers of Sprague-Dawley rats were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.     

The regulation of rat liver tryptophan pyrrolase activity by reduced nicotinamide-adenine dinucleotide (phosphate). Experiments with glucose and nicotinamide.

1. Chronic administration of glucose or nicotinamide in drinking water inhibits the activity of rat liver tryptophan pyrrolase, and subsequent withdrawal causes an enhancement. The enzyme activity is also inhibited by administration in drinking water of sucrose, but not fructose, which is capable of preventing the glucose effect. 2. The inhibition by glucose or nictinamide is not due to a defective apoenzyme synthesis nor a decreased cofactor availability. 3. The inhibition by nicotinamide is reversed by regeneration of liver NAD+ and NADP+ in vivo by administration of fructose, pyruvate or phenazine methosulphate. Inhibition by glucose is also reversed by the above agents and by NH4Cl. Reversal of inhibition by glucose or nicotinamide is also achieved in vitro by addition of NAD+ or NADP+. 4. Glucose or nicotinamide increases liver [NADPH]. [NADP+] is also increased by nicotinamide. [NADPH] is also increased by sucrose, but not by fructose, which prevents the glucose effect. Phenazine methosulphate prevents the increase in [NADPH] caused by both glucose and nicotinamide. 5. It is suggested that the inhibition of tryptophan pyrrolase activity by glucose or nicotinamide is mediated by both NADPH and NADH.