L. V. Eggleston

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 degrees , pH7.0 and I 0.25 (required for the calculation of the [free NADP(+)]/[free NADPH] ratio), gave a value of 3.44x10(-2)m for the ;malic' enzyme (with CO(2) rather than HCO(3) (-) as the reactant) and a value of 1.98x10(-2)m(-1) for glutathione reductase.

1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

1. The changes in the metabolite content in freeze-clamped livers of fed rats occurring on perfusion with 10mm-d-fructose have been examined. 2. The most striking effects of fructose were an accumulation of fructose 1-phosphate, as already known, up to 8.7mumol/g of liver within 10min, a loss of total adenine nucleotides (up to 35% after 40min) with a decrease in the ATP content to 23% within 10min, a sevenfold rise in the concentration of IMP to 1.1mumol/g and an eightfold rise of alpha-glycerophosphate to 1.1mumol/g. 3. There was a transient decrease in P(i) from 4.2 to 1.7mumol/g. Within 40min the P(i) content recovered to the normal value, probably because of an uptake of P(i) from the perfusion medium. 4. The degradation of the adenine nucleotides beyond the stage of AMP can be accounted for by the decrease of ATP and P(i). As ATP inhibits 5-nucleotidase, and as P(i) inhibits AMP deaminase any AMP arising in the tissue is liable to undergo dephosphorylation or deamination under the conditions occurring after fructose loading. 5. The content of lactate increased to 4.3mumol/g at 80min; pyruvate also increased and the [lactate]/[pyruvate] ratio remained within physiological limits. 6. The concentration of free fructose within the liver remained much below that in the perfusion medium, indicating that the rate of penetration of fructose into the tissue was lower than the rate of utilization. 7. The fission of fructose 1-phosphate by liver aldolase is inhibited by several phosphorylated intermediates, especially by IMP. This inhibition is competitive with a K(i) of 0.1mm. 8. The maximal rates of the enzymes synthesizing and splitting fructose 1-phosphate are about equal. The accumulation of fructose 1-phosphate on fructose loading is due to the inhibition of the fission of fructose 1-phosphate by the IMP arising from the degradation of the adenine nucleotides.

Research Article| March 01 1940 The oxidation of pyruvate in pigeon breast muscle Hans Adolf Krebs; Hans Adolf Krebs 1The Department of Biochemistry, University of Sheffield Search for other works by this author on: This Site PubMed Google Scholar Leonard Victor Eggleston Leonard Victor Eggleston 1The Department of Biochemistry, University of Sheffield Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1940) 34 (3): 442–459. https://doi.org/10.1042/bj0340442 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Hans Adolf Krebs, Leonard Victor Eggleston; The oxidation of pyruvate in pigeon breast muscle. Biochem J 1 March 1940; 34 (3): 442–459. doi: https://doi.org/10.1042/bj0340442 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1940 CAMBRIDGE UNIVERSITY PRESS1940 Article PDF first page preview Close Modal You do not currently have access to this content.