The redox state of free nicotinamide–adenine dinucleotide phosphate in the cytoplasm of rat liver

Abstract

1. The concentrations of the oxidized and reduced substrates of the ‘malic’ enzyme (EC 1.1.1.40) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in freeze-clamped rat livers. By assuming that the reactants of these dehydrogenase systems are at equilibrium in the cytoplasm the [free NADP+]/[free NADPH] ratio was calculated. The justification of the assumption is discussed. 2. The values of this ratio obtained under different nutritional conditions (well-fed, 48hr.-starved, fed with a low-carbohydrate diet, fed with a high-sucrose diet) were all of the same order of magnitude although characteristic changes occurred on varying the diet. The value of the ratio fell on starvation and on feeding with the low-carbohydrate diet and rose slightly on feeding with the high-sucrose diet. 3. The mean values of the ratio were calculated to be between 0·001 and 0·015, which is about 100000 times lower than the values of the cytoplasmic [free NAD+]/[free NADH] ratio. 4. The differences in the redox state of the two nicotinamide–adenine dinucleotide couples can be explained on a simple physicochemical basis. The differences are the result of equilibria that are determined by the equilibrium constants of a number of highly active readily reversible dehydrogenases and transaminases and the concentrations of the substrates and products of these enzymes. 5. The decisive feature is the fact that the NAD and NADP couples share substrates. This sharing provides a link between the redox states of the two couples. 6. The application of the method of calculation to data published by Kraupp, Adler-Kastner, Niessner & Plank (1967), Goldberg, Passonneau & Lowry (1966) and Kauffman, Brown, Passonneau & Lowry (1968) shows that the redox states of the NAD and NADP couples in cardiac-muscle cytoplasm and in mouse-brain cytoplasm are of the same order as those in rat liver. 7. The determination of the equilibrium constant at 38°, pH7·0 and I 0·25 (required for the calculation of the [free NADP+]/[free NADPH] ratio), gave a value of 3·44×10−2m for the ‘malic’ enzyme (with CO2 rather than HCO3− as the reactant) and a value of 1·98×10−2m−1 for glutathione reductase.