Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1967 May;103(2):514–527. doi: 10.1042/bj1030514

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

D H Williamson 1, Patricia Lund 1, H A Krebs 1
PMCID: PMC1270436  PMID: 4291787

Abstract

1. The concentrations of the oxidized and reduced substrates of the lactate-, β-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 β-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), β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and malate dehydrogenase (EC 1.1.1.37) were redetermined for near-physiological conditions (38°; 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 β-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.

Full text

PDF
514

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. AMOORE J. E., BARTLEY W. The permeability of isolated rat-liver mitochondria to sucrose, sodium chloride and potassium chloride at 0 degrees. Biochem J. 1958 Jun;69(2):223–236. doi: 10.1042/bj0690223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. AMOORE J. E. The permeability of isolated rat-liver mitochondria at 0 degree to the metabolites pyruvate, succinate, citrate, phosphate, adenosine 5'-phosphate and adenosine triphosphate. Biochem J. 1958 Dec;70(4):718–726. doi: 10.1042/bj0700718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. BROWN R. H., DUDA G. D., KORKES S., HANDLER P. A colorimetric micromethod for determination of ammonia; the ammonia content of rat tissues and human plasma. Arch Biochem Biophys. 1957 Feb;66(2):301–309. doi: 10.1016/s0003-9861(57)80005-8. [DOI] [PubMed] [Google Scholar]
  4. BURTON K., WILSON T. H. The free-energy changes for the reduction of diphosphopyridine nucleotide and the dehydrogenation of L-malate and L-glycerol 1-phosphate. Biochem J. 1953 Apr;54(1):86–94. doi: 10.1042/bj0540086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergmeyer H. U., Gawehn K., Klotzsch H., Krebs H. A., Williamson D. H. Purification and properties of crystalline 3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Biochem J. 1967 Feb;102(2):423–431. doi: 10.1042/bj1020423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. CHAPPELL J. B., CROFTS A. R. CALCIUM ION ACCUMULATION AND VOLUME CHANGES OF ISOLATED LIVER MITOCHONDRIA. CALCIUM ION-INDUCED SWELLING. Biochem J. 1965 May;95:378–386. doi: 10.1042/bj0950378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dixon M., Zerfas L. G. The role of coenzymes in dehydrogenase systems. Biochem J. 1940 Mar;34(3):371–391. doi: 10.1042/bj0340371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Edson N. L. Ketogenesis-antiketogenesis: The influence of ammonium chloride on ketone-body formation in liver. Biochem J. 1935 Sep;29(9):2082–2094. doi: 10.1042/bj0292082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. FRIEDEN C. Coenzyme binding, observed by fluorescence enhancement, apparently unrelated to the enzymic activity of glutamic dehydrogenase. Biochim Biophys Acta. 1961 Feb 18;47:428–430. doi: 10.1016/0006-3002(61)90316-x. [DOI] [PubMed] [Google Scholar]
  10. GAMBLE J. L., Jr ACCUMULATION OF CITRATE AND MALATE BY MITOCHONDRIA. J Biol Chem. 1965 Jun;240:2668–2672. [PubMed] [Google Scholar]
  11. GLOCK G. E., MCLEAN P. The intracellular distribution of pyridine nucleotides in rat liver. Exp Cell Res. 1956 Aug;11(1):234–236. doi: 10.1016/0014-4827(56)90214-2. [DOI] [PubMed] [Google Scholar]
  12. HAKALA M. T., GLAID A. J., SCHWERT G. W. Lactic dehydrogenase. II. Variation of kinetic and equilibrium constants with temperature. J Biol Chem. 1956 Jul;221(1):191–209. [PubMed] [Google Scholar]
  13. HOHORST H. J. Enzymatische Bestimmung von L(+)-Milchsäure. Biochem Z. 1957;328(7):509–521. [PubMed] [Google Scholar]
  14. HOHORST H. J., KREUTZ F. H., REIM M., HUEBENER H. J. The oxidation/reduction state of the extramitochondrial DPN/DPNH system in rat liver and the hormonal control of substrate levels in vivo. Biochem Biophys Res Commun. 1961 Mar 10;4:163–168. doi: 10.1016/0006-291x(61)90263-7. [DOI] [PubMed] [Google Scholar]
  15. HUCKABEE W. E. Relationships of pyruvate and lactate during anaerobic metabolism. I. Effects of infusion of pyruvate or glucose and of hyperventilation. J Clin Invest. 1958 Feb;37(2):244–254. doi: 10.1172/JCI103603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. KIRSTEN E., GEREZ C., KIRSTEN R. [An enzymatic microdetermination method for ammonia, specifically for extracts of animal tissues and fluids. Determination of NH4 ions in blood]. Biochem Z. 1963;337:312–319. [PubMed] [Google Scholar]
  17. KLINGENBERG M., PETTE D. Proportions of mitochondrial enzymes and pyridine nucleotides. Biochem Biophys Res Commun. 1962 Jun 4;7:430–432. doi: 10.1016/0006-291x(62)90329-7. [DOI] [PubMed] [Google Scholar]
  18. Krebs H. A. Bovine ketosis. Vet Rec. 1966 Feb 5;78(6):187–192. doi: 10.1136/vr.78.6.187. [DOI] [PubMed] [Google Scholar]
  19. Krebs H. A., Gascoyne T., Notton B. M. Generation of extramitochondrial reducing power in gluconeogenesis. Biochem J. 1967 Jan;102(1):275–282. doi: 10.1042/bj1020275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LEHNINGER A. L., SUDDUTH H. C., WISE J. B. D-beta-Hydroxybutyric dehydrogenase of muitochondria. J Biol Chem. 1960 Aug;235:2450–2455. [PubMed] [Google Scholar]
  21. LOEFFLER G., WIELAND O. [An isotope method for the enzymatic determination of oxalacetate]. Biochem Z. 1963;336:447–454. [PubMed] [Google Scholar]
  22. LUSTY C. J., SINGER T. P. LIPOYL DEHYDROGENASE. FREE AND COMPLEXED FORMS IN MAMMALIAN MITOCHONDRIA. J Biol Chem. 1964 Nov;239:3733–3742. [PubMed] [Google Scholar]
  23. Löffler G., Matschinsky F., Wieland O. Uber den Mechanismus der gesteigerten Ketonköperbildung. II. Redox-Status des DPN der isolierten Rattenleber bei Durchströmung mit Fettsäuren. Biochem Z. 1965 Jun 3;342(1):76–84. [PubMed] [Google Scholar]
  24. MASSEY V. The identity of diaphorase and lipoyl dehydrogenase. Biochim Biophys Acta. 1960 Jan 15;37:314–322. doi: 10.1016/0006-3002(60)90239-0. [DOI] [PubMed] [Google Scholar]
  25. OLSON J. A., ANFINSEN C. B. Kinetic and equilibrium studies on crystalline 1-glutamic acid dehydrogenase. J Biol Chem. 1953 Jun;202(2):841–856. [PubMed] [Google Scholar]
  26. RAVAL D. N., WOLFE R. G. Malic dehydrogenase. II. Kinetic studies of the reaction mechanism. Biochemistry. 1962 Mar;1:263–269. doi: 10.1021/bi00908a012. [DOI] [PubMed] [Google Scholar]
  27. RECKNAGEL R. O., POTTER V. R. Mechanism of the ketogenic effect of ammonium chloride. J Biol Chem. 1951 Jul;191(1):263–275. [PubMed] [Google Scholar]
  28. SHUSTER C. W., DOUDOROFF M. A cold-sensitive D(-) beta-hydroxybutyric acid dehydrogenase from Rhodospirillum rubrum. J Biol Chem. 1962 Feb;237:603–607. [PubMed] [Google Scholar]
  29. WILLIAMSON D. H., MELLANBY J., KREBS H. A. Enzymic determination of D(-)-beta-hydroxybutyric acid and acetoacetic acid in blood. Biochem J. 1962 Jan;82:90–96. doi: 10.1042/bj0820090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. WILLIAMSON D. H., MELLANBY J., KREBS H. A. Enzymic determination of D(-)-beta-hydroxybutyric acid and acetoacetic acid in blood. Biochem J. 1962 Jan;82:90–96. doi: 10.1042/bj0820090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. WINER A. D., SCHWERT G. W., MILLAR D. B. Lactic dehydrogenase. VI. Fluorimetric measurements of the complex of enzyme and reduced diphosphopyridine nucleotide. J Biol Chem. 1959 May;234(5):1149–1154. [PubMed] [Google Scholar]
  32. WOLLENBERGER A., RISTAU O., SCHOFFA G. [A simple technic for extremely rapid freezing of large pieces of tissue]. Pflugers Arch Gesamte Physiol Menschen Tiere. 1960;270:399–412. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES