scholarly journals Stimulation of the respiratory chain of rat liver mitochondria between cytochrome c1 and cytochrome c by glucagon treatment of rats

1978 ◽  
Vol 172 (3) ◽  
pp. 399-405 ◽  
Author(s):  
Andrew P. Halestrap
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Redox State ◽  
Electron Flow ◽  
Rat Liver Mitochondria ◽  
O2 Uptake ◽  
Pyruvate Metabolism ◽  
Cytochromes C ◽  
Cytochrome C1

Mitochondria from glucagon-treated rats oxidize succinate, but not ascorbate plus tetramethylphenylenediamine, faster in the uncoupled state than do control mitochondria. The rate of O2 uptake in the presence of both substrates is equal to the sum of the rates of the O2 uptake in the presence of either substrate alone. It is concluded that the mitochondrial respiratory chain is limited at some point between cytochromes b and c and that this step is regulated by glucagon. Measurement of the cytochrome spectra under uncoupled conditions in the presence of succinate and rotenone demonstrates a crossover between cytochromes c and c1 when control mitochondria are compared with those from glucagon-treated rats, cytochrome c being more oxidized and cytochrome c1 more reduced in control mitochondria. Under conditions where pyruvate metabolism is studied the control mitochondria are generally more oxidized than those from glucagon-treated rats, the redox state of cytochrome b-566 correlating with the rate of pyruvate metabolism in sucrose medium. However, when the redox state of the mitochondria is taken into account, a crossover between cytochromes c and c1 is again apparent. The spectra of the b cytochromes are complex, but cytochrome b-562 appears to become more reduced relative to cytochrome b-566 in mitochondria from glucagon-treated rats than in control mitochondria. This can be explained by the existence of a more alkaline matrix in glucagon-treated rats, the redox potential for cytochrome b being pH-sensitive. It is concluded that glucagon stimulates electron flow between cytochromes c1 and c. The physiological significance of these findings is discussed.

scholarly journals The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals

1982 ◽  
Vol 204 (1) ◽  
pp. 37-47 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Respiratory Chain ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Complex Iii ◽  
Secondary Site ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Q Cycle ◽  
Cytochromes C ◽  
Stimulation Of

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the ‘Q cycle’ [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.

scholarly journals The mechanism of transmembrane ΔμH+ generation in mitochondria by cytochrome c oxidase

1979 ◽  
Vol 182 (1) ◽  
pp. 133-147 ◽  
Author(s):  
M Lorusso ◽  
F Capuano ◽  
D Boffoli ◽  
R Stefanelli ◽  
S Papa
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Electron Flow ◽  
Aerobic Oxidation ◽  
Outer Side ◽  
Rat Liver Mitochondria ◽  
Proton Release ◽  
Ferrocytochrome C ◽  
Proton Production

In rat liver mitochondria treated with rotenone, N-ethylmaleimide or oligomycin the expected alkalinization caused by proton consumption for aerobic oxidation of ferrocyanide was delayed with respect to ferrocyanide oxidation, unless carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present. 2. When valinomycin or valinomycin plus antimycin were also present, ferricyanide, produced by oxidation of ferrocyanide, was re-reduced by hydrogenated endogenous reductants. Under these circumstances the expected net proton consumption caused by ferrocyanide oxidation was preceded by transient acidification. It is shown that re-reduction of formed ferricyanide and proton release derive from rotenone- and antimycin-resistant oxidation of endogenous reductants through the proton-translocating segments of the respiratory chain on the substrate side of cytochrome c. The number of protons released per electron flowing to ferricyanide varied, depending on the experimental conditions, from 3.6 to 1.5. 3. The antimycin-insensitive re-reduction of ferricyanide and proton release from mitochondria were strongly depressed by 2-n-heptyl-4-hydroxyquinoline N-oxide. This shows that the ferricyanide formed accepts electrons passing through the protonmotive segments of the respiratory chain at the level of cytochrome c and/or redox components of the cytochrome b-c1 complex situated on the oxygen side of the antimycin-inhibition site. Dibromothymoquinone depressed and duroquinol enhanced, in the presence of antimycin, the proton-release process induced by ferrocyanide respiration. Both quinones enhanced the rate of scalar proton production associated with ferrocyanide respiration, but lowered the number of protons released per electron flowing to the ferricyanide formed. 4. Net proton consumption caused by aerobic oxidation of exogenous ferrocytochrome c by antimycin-supplemented bovine heart mitochondria was preceded by scalar proton release, which was included in the stoicheiometry of 1 proton consumed per mol of ferrocytochrome c oxidized. This scalar proton production was associated with transition of cytochrome c from the reduced to the oxidized form and not to electron flow along cytochrome c oxidase. 5. It is concluded that cytochrome c oxidase only mediates vectorial electron flow from cytochrome c at the outer side to protons that enter the oxidase from the matrix side of the membrane. In addition to this consumption of protons the oxidase does not mediate vectorial proton translocation.

Kinetics of the cytochrome c oxidase and reductase reactions in energized and de-energized mitochondria

1977 ◽  
Vol 55 (7) ◽  
pp. 706-713 ◽  
Author(s):  
Lars Chr. Petersen ◽  
Hans Degn ◽  
Peter Nicholls
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxygen Concentration ◽  
Respiration Rate ◽  
Redox State ◽  
Rat Liver Mitochondria ◽  
Excess Oxygen ◽  
Succinate Oxidation ◽  
State Reduction

1. Coupled, cytochrome-c-depleted ('stripped') rat liver mitochondria reducing oxygen in the presence of exogenous cytochrome c, with succinate or ascorbate as substrates, show marked declines in the steady-state reduction of cytochrome c in excess oxygen on addition of uncouplers. Calculated ratios of maximal turnover in the uncoupled state and in the energized state for the cytochrome c oxidase (EC 1.9.3.1) reaction lie between 3 and 6, as obtained with reconstituted oxidase-containing vesicles. The succinate-cytochrome c reductase activity in such mitochondria shows a smaller response to uncoupler than that of the oxidase.2. The respiration rates of uncoupled mitochondria oxidizing ascorbate in the presence of added cytochrome c follow a Michaelis–Menten relationship with respect to oxygen concentration, in accordance with the pattern found previously with the solubilized oxidase. But succinate oxidation tends to give nonlinear concave-upward double-reciprocal plots of respiration rate against oxygen concentration, in accordance with the pattern found previously with intact uncoupled mitochondria.3. From simultaneous measurements of cytochrome c steady-state reduction, respiration rate, and oxygen concentration during succinate oxidation under uncoupled conditions it is found that at full reduction of cytochrome c, apparent Km for oxygen is 0.9 μM and the maximal oxidase (aa3) turnover is 400 s−1 (pH 7.4, 30 °C).4. The redox state of cytochrome c in uncoupled systems reflects a simple steady state; the redox state of cytochrome c in energized systems tends towards an equilibrium condition with the terminal cytochrome a3, whose apparent potential under these conditions is more negative than that of cytochrome c.

scholarly journals Multiple light-induced reactions of cytochromes b and c in Rhodopseudomonas spheroides

1969 ◽  
Vol 114 (4) ◽  
pp. 793-799 ◽  
Author(s):  
O. T. G. Jones
Keyword(s):  
Cytochrome C ◽  
Cytochrome B ◽  
Photochemical Reaction ◽  
Room Temperature ◽  
Close Association ◽  
Antimycin A ◽  
Reaction Centre ◽  
Difference Spectra ◽  
Cytochromes C ◽  
Rhodopseudomonas Spheroides

Illumination of chromatophore preparations from Rhodopseudomonas spheroides causes the oxidation of a cytochrome c and a slight oxidation of a cytochrome b with a maximum at 560nm. When illuminated in the presence of antimycin A the oxidation of cytochrome c was more pronounced and cytochrome b560 was reduced; the dark oxidation of cytochrome b560 was biphasic in the presence of succinate, but not in the presence of NADH, a less effective reductant. Split-beam spectroscopy showed that, in addition to the reduction of cytochrome b560, another pigment with maxima at 565 and 537nm. was reduced and was more rapidly oxidized in the dark than cytochrome b560. This pigment, tentatively identified as cytochrome b565, was also detected in spectra at 77°k, after brief illumination at room temperature; the maxima at 77°k were at 562 and 536nm. In the absence of antimycin A, light caused a transient reduction of cytochrome b565 and an oxidation of cytochrome b560. Dark oxidation of b565 was rapid, even in the presence of antimycin A and succinate. Difference spectra, at 77°k, of ascorbate-reduced minus succinate-reduced chromatophores or of anaerobic succinate-reduced minus aerobic succinate-reduced chromatophores suggested that two cytochromes c were present, with maxima at 547 and 549nm. When chromatophores frozen at 77°k were illuminated both these cytochromes c were oxidized, indicating a close association with the photochemical reaction centre. A scheme involving two reaction centres is proposed to explain these results.

The inhibition of mammalian mitochondrial NADU oxidation by chloramphenicol and its isomers and analogues

1968 ◽  
Vol 46 (9) ◽  
pp. 1003-1008 ◽  
Author(s):  
K. B. Freeman ◽  
D. Haldar
Keyword(s):  
Cytochrome B ◽  
Respiratory Chain ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Coenzyme Q ◽  
Liver Mitochondria ◽  
Heart Mitochondria ◽  
Rat Liver Mitochondria ◽  
Beef Heart ◽  
Beef Heart Mitochondria

Chloramphenicol and its isomers and analogues have been found to inhibit the oxidation of NADH, but not that of succinate, by beef heart mitochondria. They must therefore inhibit the NADH dehydrogenase segment of the respiratory chain. Chloramphenicol gave 50% inhibition at a concentration of 1 mM. The methylthio analogue of chloramphenicol inhibited NADH – coenzyme Q6 reductase but not NADH–ferricyanide reductase. Spectrophotometric observations suggest that these inhibitors act between NADH and flavin in coupled rat liver mitochondria and between flavin and cytochrome b in uncoupled beef heart mitochondria.

scholarly journals Biochemical heterogeneity of rat liver mitochondria separated by rate zonal centrifugation

1972 ◽  
Vol 129 (1) ◽  
pp. 209-218 ◽  
Author(s):  
M. A. Wilson ◽  
J. Cascarano
Keyword(s):  
Cytochrome C ◽  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Cytochrome B ◽  
Rat Liver ◽  
Nadh Dehydrogenase ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Osmotic Gradient ◽  
Glycerophosphate Dehydrogenase

1. Rat liver mitochondria were separated on the basis of their sedimentation coefficients in an iso-osmotic gradient of Ficoll–sucrose by rate zonal centrifugation. The fractions (33, each of 40ml) were collected in order of decreasing density. Fractions were analysed by spectral analysis to determine any differences in the concentrations of the cytochromes and by enzyme analyses to ascertain any differences in the activities of NADH dehydrogenase, succinate dehydrogenase and α-glycerophosphate dehydrogenase. 2. When plotted as% of the highest specific concentration, the contents of cytochrome a+a3 and cytochrome c+c1 were constant in all fractions but cytochrome b was only 65% of its maximal concentration in fraction 7 and increased with subsequent fractions. As a result, the cytochrome b/cytochrome a+a3 ratio almost doubled between fractions 7 and 25 whereas the cytochrome c+c1/cytochrome a+a3 ratio was unchanged. 3. Expression of the dehydrogenase activities as% of highest specific activity showed the following for fractions 6–26: NADH dehydrogenase activity remained fairly constant in all fractions; succinate dehydrogenase activity was 62% in fraction 6 and increased steadily to its maximum in fraction 18 and then decreased; the activity of α-glycerophosphate dehydrogenase was only 53% in fraction 6 and increased slowly to its peak in fractions 22 and 24. 4. These differences did not result from damaged or fragmented mitochondria or from microsomal contamination. 5. These results demonstrate that isolated liver mitochondria are biochemically heterogeneous. The importance of using a system for separating biochemically different mitochondria in studies of mitochondrial biogenesis is discussed.

scholarly journals Steady-state H+/O stoichiometry of liver mitochondria

1981 ◽  
Vol 200 (3) ◽  
pp. 539-546 ◽  
Author(s):  
M K Al-Shawi ◽  
M D Brand
Keyword(s):  
Steady State ◽  
Cytochrome C ◽  
Consumption Rate ◽  
Initial Rate ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
O2 Consumption ◽  
Ejection Rate ◽  
Novel Method

We have measured the H+/O stoichiometry of rat liver mitochondria respiring in a steady-state, using a novel method. This involves measuring the initial rate of H+ back-flow into mitochondria after respiratory inhibition, with the assumption that this is equal to the steady-state H+-ejection rate. Division by the steady-state O2-consumption rate yields the H+/O ratio. The H+/O values obtained were: 8.3 +/- 1.0 (mean +/- S.E.M.) for 3-hydroxybutyrate: 8.2 +/- 0.7 for glutamate plus malate; 6.0 +/- 0.2 for succinate; 4.1 +/- 0.3 for ascorbate/tetramethylphenylenediamine and 3.0 +/- 0.1 for ascorbate/ferrocyanide. These values correspond to H+/O stoichiometries for electron flow to oxygen from NAD+-linked substrates, succinate and cytochrome c of 8, 6 and 2 (charge/O ratio = 4) respectively.

scholarly journals Oxygen affinity of the respiratory chain of Acanthamoeba castellanii

1983 ◽  
Vol 214 (1) ◽  
pp. 47-51 ◽  
Author(s):  
D Lloyd ◽  
H Mellor ◽  
J L Williams
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Cytochrome B ◽  
Respiratory Chain ◽  
Oxygen Affinity ◽  
Acanthamoeba Castellanii ◽  
Intact Cells ◽  
Cytochrome Aa3 ◽  
Isolated Mitochondria ◽  
Soil Amoeba

Apparent Km values for O2 for the soil amoeba Acanthamoeba castellanii determined polarographically and by bioluminescence gave similar values (0.37 and 0.41 microM respectively). Mitochondria oxidizing succinate or NADH in the presence or absence of ADP gave values in the range 0.21-0.36 microM-O2. Oxidation of respiratory-chain components to 50% of the aerobic steady states in intact cells was observed at the following O2 concentrations: cytochrome aa3, 0.1-0.25 microM; cytochrome c, 0.3-0.6 microM; cytochrome b, 0.35-0.45 microM; flavoprotein, 2 microM. In isolated mitochondria corresponding values for a-, c- and b-type cytochromes were 0.007, 0.035-0.05 and 0.06-0.09 microM-O2. It is concluded that an O2 gradient exists between plasma membrane and mitochondria in A. castellanii.

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