Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1968 Jul;108(4):513–520. doi: 10.1042/bj1080513

The redox state of the nicotinamide–adenine dinucleotides in rat liver homogenates

H A Krebs 1,*, T Gascoyne 1
PMCID: PMC1198844  PMID: 4299127

Abstract

1. The redox state of the NAD couple of rat liver mitochondria, as measured by the [β-hydroxybutyrate]/[acetoacetate] ratio, rapidly changed in the direction of oxidation during the preparation of homogenates in a saline medium. The value of the [β-hydroxybutyrate]/[acetoacetate] ratio fell from 2·3 to 0·15 in 10min. EDTA diminished the fall and succinate prevented it. 2. The redox state of the rat liver cytoplasm, as measured by the [lactate]/[pyruvate] ratio, changed slightly in the direction of reduction during the preparation of homogenate. This was prevented by succinate. 3. In unsupplemented homogenates the differences in the redox states of mitochondria and cytoplasm decreased. Succinate and EDTA together maintained the differences within the physiological range. A measure of the ability of the mitochondria to maintain different redox states in mitochondria and cytoplasm is the value of the expression [lactate][acetoacetate]/[pyruvate][β-hydroxybutyrate]. If there are no differences in the redox states of the NAD in the two cell compartments the value of the expression is 444 at 37°. The value in the intact rat liver is between 4·7 and 21. 4. α-Oxoglutarate or glutamate were still more effective than succinate in maintaining high [β-hydroxybutyrate]/[acetoacetate] ratios in the homogenates because these substrates supply a reducing agent of NAD+ and, through succinate, an inhibitor of the oxidation of NADH. 5. When supplemented with α-oxoglutarate and EDTA, homogenates readily adjust the redox state of the β-hydroxybutyrate dehydrogenase system after it has been upset by the addition of either acetoacetate or β-hydroxybutyrate. 6. Amytal and rotenone raised the value of the [β-hydroxybutyrate]/[acetoacetate] ratio. This is taken to indicate that the reduction of acetoacetate in the homogenates was not an energy-linked process. 7. 2,4-Dinitrophenol shifted the [β-hydroxybutyrate]/[acetoacetate] ratio in the presence of succinate in favour of oxidation because it inhibited the oxidation of succinate and accelerated the oxidation of NADH. 8. Rotenone increased the rate of ketone-body formation of liver homogenates, though it decreased the rate of oxygen uptake.

Full text

PDF
516

Selected References

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

  1. AZZONE G. F., ERNSTER L., WEINBACH E. C. Succinate-linked acetoacetate reduction. I. Endergonic reduction of acetoacetate by succinate in liver mitochondria. J Biol Chem. 1963 May;238:1825–1833. [PubMed] [Google Scholar]
  2. BORST P., SLATER E. C. The oxidation of glutamate by rat-heart sarcosomes. Biochim Biophys Acta. 1960 Jun 17;41:170–171. doi: 10.1016/0006-3002(60)90391-7. [DOI] [PubMed] [Google Scholar]
  3. GOLDMAN D. S. Studies on the fatty acid oxidizing system of animal tissues. VII. The beta-ketoacyl coenzyme A cleavage enzyme. J Biol Chem. 1954 May;208(1):345–357. [PubMed] [Google Scholar]
  4. Good N. E., Winget G. D., Winter W., Connolly T. N., Izawa S., Singh R. M. Hydrogen ion buffers for biological research. Biochemistry. 1966 Feb;5(2):467–477. doi: 10.1021/bi00866a011. [DOI] [PubMed] [Google Scholar]
  5. Griffiths D. E., Roberton A. M. Energy-linked reactions in mitochondria: studies on the mechanism of the energy-linked transhydrogenase reaction. Biochim Biophys Acta. 1966 Jun 15;118(3):453–464. doi: 10.1016/s0926-6593(66)80089-9. [DOI] [PubMed] [Google Scholar]
  6. HASLAM R. J., KREBS H. A. THE METABOLISM OF GLUTAMATE IN HOMOGENATES AND SLICES OF BRAIN CORTEX. Biochem J. 1963 Sep;88:566–578. doi: 10.1042/bj0880566. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. HOLZER H., LYNEN F., SCHULTZ G. Bestimmung des Quotienten DPNH/DPN in lebenden Hefezellen durch Analyse stationärer-Alkohol- und Acetaldehyd-Konzentrationen. Biochem Z. 1956;328(4):252–263. [PubMed] [Google Scholar]
  8. Horgan D. J., Singer T. P. Reaction sites of rotenone in the respiratory chain and in soluble DPNH-coenzyme Q reductase. Biochem Biophys Res Commun. 1967 May 5;27(3):356–360. doi: 10.1016/s0006-291x(67)80106-2. [DOI] [PubMed] [Google Scholar]
  9. KAUFMAN B. T., KAPLAN N. O. Mechanism of depletion of mitochondrial pyridine nucleotides. Biochim Biophys Acta. 1960 Apr 8;39:332–342. doi: 10.1016/0006-3002(60)90171-2. [DOI] [PubMed] [Google Scholar]
  10. KLINGENBERG M., HAFEN H. V. [Hydrogen pathway in mitochondria. I. Hydrogen transfer from succinate to acetoacetate]. Biochem Z. 1963;337:120–145. [PubMed] [Google Scholar]
  11. KREBS H. A., BELLAMY D. The interconversion of glutamic acid and aspartic acid in respiring tissues. Biochem J. 1960 Jun;75:523–529. doi: 10.1042/bj0750523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. KREBS H. A., EGGLESTON L. V., D'ALESSANDRO A. The effect of succinate and amytal on the reduction of acetoacetate in animal tissues. Biochem J. 1961 Jun;79:537–549. doi: 10.1042/bj0790537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. KREBS H. A., EGGLESTON L. V. The effect of dinitrophenol and amytal on the reduction of acetoacetate in the presence of succinate. Biochem J. 1962 Jan;82:134–143. doi: 10.1042/bj0820134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. KULKA R. G., KREBS H. A., EGGLESTON L. V. The reduction of acetoacetate to beta-hydroxybutyrate in animal tissues. Biochem J. 1961 Jan;78:95–106. doi: 10.1042/bj0780095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Krebs H. A., Eggleston L. V. Metabolism of acetoacetate in animal tissues. 1. Biochem J. 1945;39(5):408–419. [PMC free article] [PubMed] [Google Scholar]
  16. Krebs H. A., Johnson W. A. Metabolism of ketonic acids in animal tissues. Biochem J. 1937 Apr;31(4):645–660. doi: 10.1042/bj0310645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Krebs H. A. The redox state of nicotinamide adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Adv Enzyme Regul. 1967;5:409–434. doi: 10.1016/0065-2571(67)90029-5. [DOI] [PubMed] [Google Scholar]
  18. NOSSAL P. M. Estimation of L-malate and fumarate by malic decarboxylase of Lactobacillus arabinosus. Biochem J. 1952 Jan;50(3):349–355. doi: 10.1042/bj0500349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. SCHIMASSEK H. [Metabolites of carbohydrate metabolism in the isolated perfused rat liver]. Biochem Z. 1963;336:460–467. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Williamson D. H., Lund P., Krebs H. A. The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem J. 1967 May;103(2):514–527. doi: 10.1042/bj1030514. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES