scholarly journals Equilibrium relations between the oxidation—reduction reactions and the adenosine triphosphate synthesis in suspensions of isolated liver cells

1974 ◽  
Vol 140 (1) ◽  
pp. 57-64 ◽  
Author(s):  
David F. Wilson ◽  
Marion Stubbs ◽  
Richard L. Veech ◽  
Maria Erecińska ◽  
Hans A. Krebs
Keyword(s):  
Cytochrome C ◽  
Adenine Nucleotides ◽  
Atp Synthesis ◽  
Mitochondrial Respiratory Chain ◽  
Liver Cells ◽  
Inner Mitochondrial Membrane ◽  
Phosphorylation State ◽  
Oxidation Reduction ◽  
Rat Liver Cells ◽  
The Individual

1. The redox state of cytochrome c, cytochrome a and the mitochondrial NAD couple, and the phosphorylation state of the adenine nucleotides, were measured in suspensions of isolated rat liver cells. 2. The ΔG for the transfer of two electrons from the mitochondrial NAD to the cytochrome c couple is calculated to be 104kJ (24.8kcal). 3. The ΔG associated with the synthesis of ATP at the measured phosphorylation state is calculated to be 95kJ (22.7kcal)/2mol of ATP. 4. The near equality of ΔG of the electron-transport process and ΔG required for ATP synthesis indicates near-equilibrium between the mitochondrial respiratory chain and the extramitochondrial phosphorylation state. 5. The existence of near-equilibrium in the coupled reactions implies that the respiratory activity depends on the ratio [ATP]/[ADP][Pi] and not on the concentrations of the individual reactants. 6. If the overall system of oxidative phosphorylation is at near-equilibrium, all intermediary reactions must also be at equilibrium. Hence if the intramitochondrial and extramitochondrial phosphorylation states are indeed different, it follows that any differences in the activities of ATP, ADP and Pi must be coupled to ion gradients and/or potentials across the inner mitochondrial membrane in such a way that translocation occurs without loss of free energy. 7. The metabolic state of the mitochondria in the cell can be defined by the turnover number of the cytochromes, the cytoplasmic phosphorylation state, and the oxidation–reduction potential of the NAD couple, rather than by the availability of ADP, substrate and O2.

scholarly journals Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells

1976 ◽  
Vol 156 (1) ◽  
pp. 91-102 ◽  
Author(s):  
E A Siess ◽  
O H Wieland
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Active Form ◽  
Inverse Correlation ◽  
Liver Cells ◽  
Diabetic Rats ◽  
Pyruvate Dehydrogenase Kinase ◽  
Intact Cells ◽  
Phosphorylation State

1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio.

scholarly journals The metabolism of l-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism

1980 ◽  
Vol 192 (2) ◽  
pp. 673-686 ◽  
Author(s):  
S A Smith ◽  
F P A Carr ◽  
C I Pogson
Keyword(s):  
Benzene Ring ◽  
Kynurenine Pathway ◽  
Liver Cells ◽  
Relative Importance ◽  
Isolated Rat Liver ◽  
Rat Liver Cells ◽  
The Individual ◽  
Ylacetic Acid ◽  
Isolated Rat

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5–6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.

CYTOCHROME OXIDASE ACTIVITY OF CELL NUCLEI

1954 ◽  
Vol 32 (1) ◽  
pp. 548-552 ◽  
Author(s):  
David Rubinstein ◽  
Orville F. Denstedt
Keyword(s):  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Nuclear Material ◽  
Liver Cells ◽  
Nuclear Fraction ◽  
Cell Nuclei ◽  
Nuclear Membranes ◽  
Rat Liver Cells ◽  
Chicken Erythrocytes

The presence of cytochrome oxidase in the nuclear material from chicken erythrocytes and rat-liver cells has been demonstrated with the aid of p-phenylenediamine as the agent for reducing cytochrome c. The three reagents p-phenylenediamine, ascorbate, and hydroquinone are effective in the estimation of cytochrome oxidase in mitochondrial preparations, but only the first mentioned agent can effect the reduction of endogenous cytochrome c. The addition of cytochrome c will increase the oxidase activity of the liver-cell mitochondria but not of the nuclear fraction from the erythrocytes or the liver cells, presumably because of the impermeability of the nuclear membranes to added cytochrome c.

Ultrastructural Cytochemistry of Some Oxidative Enzymes

1974 ◽  
Vol 32 ◽  
pp. 322-323
Author(s):  
Arnold M. Seligman
Keyword(s):  
Electron Transport ◽  
Cytochrome C ◽  
Succinic Dehydrogenase ◽  
Atp Synthesis ◽  
Oxidative Enzymes ◽  
Reaction Products ◽  
Inner Mitochondrial Membrane ◽  
Design And Synthesis ◽  
Membrane Bound ◽  
Membrane Bound Enzymes

The membrane-bound enzymes of the succinic oxidase chain of electron transport on the cristae of mitochondria have been the target of ultrastructural cytochemical research for a number of years. Methods for succinic dehydrogenase have been improved by the continuous design and synthesis of better tetrazolium salts. The most recent is BSPT, which is not osmiophilic, but yields an osmiophilic, lipophobic, insoluble formazan. The terminal triplet of the chain of electron transport or cytochrome oxidase, consisting of cytochrome c, a and a3 has been demonstrated very well via cytochrome c with diaminobenzidine (DAB). The localization of these two reaction products specifically on the outer surface of the inner mitochondrial membrane, lends some support to speculation concerning the mechanism of transfer of oxidative energy for ATP synthesis.

CYTOCHROME OXIDASE ACTIVITY OF CELL NUCLEI

1954 ◽  
Vol 32 (5) ◽  
pp. 548-552 ◽  
Author(s):  
David Rubinstein ◽  
Orville F. Denstedt
Keyword(s):  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Oxidase Activity ◽  
Nuclear Material ◽  
Liver Cells ◽  
Nuclear Fraction ◽  
Cell Nuclei ◽  
Nuclear Membranes ◽  
Rat Liver Cells ◽  
Chicken Erythrocytes

The presence of cytochrome oxidase in the nuclear material from chicken erythrocytes and rat-liver cells has been demonstrated with the aid of p-phenylenediamine as the agent for reducing cytochrome c. The three reagents p-phenylenediamine, ascorbate, and hydroquinone are effective in the estimation of cytochrome oxidase in mitochondrial preparations, but only the first mentioned agent can effect the reduction of endogenous cytochrome c. The addition of cytochrome c will increase the oxidase activity of the liver-cell mitochondria but not of the nuclear fraction from the erythrocytes or the liver cells, presumably because of the impermeability of the nuclear membranes to added cytochrome c.

scholarly journals Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex.

1980 ◽  
Vol 85 (3) ◽  
pp. 501-515 ◽  
Author(s):  
N Borgese ◽  
J Meldolesi
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Golgi Complex ◽  
Cytochrome B5 ◽  
Liver Cells ◽  
Cytochrome C Reductase ◽  
Cytochrome B5 Reductase ◽  
Galactosyl Transferase ◽  
Rat Liver Cells ◽  
Nadh Cytochrome

The subcellular distribution of NADH-cytochrome b5 reductase in rat liver cells was reinvestigated. In fresh heavy and light Golgi fractions (GF3 and GF1 + 2) and in mitochondria, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was approximately 100, 60, and 30%, respectively, of the value found in microsomes. However, the Golgi enzyme was unstable inasmuch as pelleting and resuspending the fresh fractions resulted in a considerable inactivation (40--60%), which was further increased with subsequent storage at 4 degrees C. A similar inactivation was observed using cytochrome b5 but not ferricyanide as electron acceptor. The inactivation of Golgi NADH-cytochrome c reductase activity was independent of the protein concentration of the fractions during storage, was unaffected by the addition of the antioxidant butylated hydroxytoluene, but was partly prevented by buffering the fractions at neutral pH and by storage at--20 degrees C. A total Golgi fraction was analyzed by density equilibration on continuous sucrose gradients after exposure to digitonin. As expected, the distribution of both protein and galactosyl transferase were shifted to higher densities by this treatment. However, not all galactosyl transferase-bearing elements were shifted to the same extent by exposure to the detergent, suggesting a biochemical heterogeneity of the Golgi complex. In contrast to their behavior in microsomes, the distribution of NADH-cytochrome c reductase and cytochrome b5 of Golgi fractions was shifted by digitonin, although to a lesser extent than that of galactosyl transferase. These results indicate that NADH-cytochrome b5 reductase is an authentic component of Golgi membranes, as well as of microsomes and of mitochondria. The conflicting results reported in the past on the Golgi localization of the enzyme could be due, on the one hand, to the differential lability of the activity in its various subcellular locations and, on the other, to the heterogeneity of the Golgi complex in terms of both cholesterol and enzyme distribution.

scholarly journals Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm

1972 ◽  
Vol 126 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Marion Stubbs ◽  
R. L. Veech ◽  
H. A. Krebs
Keyword(s):  
Rat Liver ◽  
Redox State ◽  
Adenine Nucleotide ◽  
Adenine Nucleotides ◽  
Phosphoglycerate Kinase ◽  
Phosphorylation State ◽  
Crotyl Alcohol ◽  
Order Of Magnitude ◽  
The Individual

1. A study has been made of the ability of rat liver in vivo to maintain equilibrium in the combined glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase and lactate dehydrogenase reactions, i.e. in the system: [Formula: see text] Attempts were made to upset equilibrium. The [lactate]/[pyruvate] ratio was rapidly changed by injection of ethanol or crotyl alcohol, and the value of [ATP]/[ADP][HPO42-] was rapidly changed by injection of ethionine or carbonyl cyanide p-trifluoromethoxy-phenylhydrazone. 2. The concentrations of the metabolites occurring in the above equation were measured in freeze-clamped liver. 3. Although the injected agents caused large changes in the concentrations of the individual components, near-equilibrium in the system was maintained, as indicated by the fact that the value of [ATP]/[ADP][HPO42-], referred to as the phosphorylation state of the adenine nucleotides, measured directly agreed with the value calculated for equilibrium conditions from the above equation. 4. The results are discussed and taken to confirm that the order of magnitude of the value of the redox state of the cytoplasmic NAD couple in rat liver is controlled by the phosphorylation state of the adenine nucleotide system.

scholarly journals Influence of the Phosphorylation State on Ethanol Oxidation and NADH Translocation in Isolated Rat Liver Cells.

1978 ◽  
Vol 32b ◽  
pp. 125-130 ◽  
Author(s):  
A. R. Pösö ◽  
Kåre Olsson ◽  
Sören Karlsson ◽  
Jan Sandström ◽  
Curt R. Enzell ◽  
...  
Keyword(s):  
Rat Liver ◽  
Ethanol Oxidation ◽  
Liver Cells ◽  
Isolated Rat Liver ◽  
Phosphorylation State ◽  
Rat Liver Cells ◽  
Isolated Rat

scholarly journals Thermodynamic efficiency, reversibility, and degree of coupling in energy conservation by the mitochondrial respiratory chain

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Mårten Wikström ◽  
Roger Springett
Keyword(s):  
Respiratory Chain ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Respiratory Chain ◽  
Point Of View ◽  
Proton Gradient ◽  
Thermodynamic Efficiency ◽  
Inner Mitochondrial Membrane ◽  
Degree Of Coupling ◽  
Mitochondrial Respiratory

AbstractThe protonmotive mitochondrial respiratory chain, comprising complexes I, III and IV, transduces free energy of the electron transfer reactions to an electrochemical proton gradient across the inner mitochondrial membrane. This gradient is used to drive synthesis of ATP and ion and metabolite transport. The efficiency of energy conversion is of interest from a physiological point of view, since the energy transduction mechanisms differ fundamentally between the three complexes. Here, we have chosen actively phosphorylating mitochondria as the focus of analysis. For all three complexes we find that the thermodynamic efficiency is about 80–90% and that the degree of coupling between the redox and proton translocation reactions is very high during active ATP synthesis. However, when net ATP synthesis stops at a high ATP/ADP.Pi ratio, and mitochondria reach “State 4” with an elevated proton gradient, the degree of coupling drops substantially. The mechanistic cause and the physiological implications of this effect are discussed.

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