scholarly journals The proton-translocating nicotinamide–adenine dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1973 ◽  
Vol 132 (3) ◽  
pp. 571-585 ◽  
Author(s):  
Jennifer Moyle ◽  
Peter Mitchell
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Isocitrate Dehydrogenase ◽  
Proton Translocation ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Catalytic Amount ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Ph Change ◽  
Carbonyl Cyanide

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an →H+/2e− quotient of 1.94±0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K+-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of −450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.

scholarly journals Respiration-driven proton translocation in rat liver mitochondria

1967 ◽  
Vol 105 (3) ◽  
pp. 1147-1162 ◽  
Author(s):  
Peter Mitchell ◽  
Jennifer Moyle
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Ionic Composition ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Proton Conductance ◽  
Carbonyl Cyanide ◽  
M Phase ◽  
Rate Of Decay

1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H+ (or OH−) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the →H+/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H+/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the →H+/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na+. These apparent changes of →H+/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H+/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H+/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.

scholarly journals Proton translocation by the mitochondrial cytochrome b-c1 complex is inhibited by NN′-dicyclohexylcarbodi-imide

1982 ◽  
Vol 206 (2) ◽  
pp. 419-421 ◽  
Author(s):  
B D Price ◽  
M D Brand
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Rat Liver ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Cytochrome ◽  
Low Concentrations ◽  
Mitochondrial Cytochrome B

NN'-Dicyclohexylcarbodi-imide at low concentrations decreases the H+/2e ratio for rat liver mitochondria over the span succinate to oxygen from 5.9 +/- 0.3 (mean +/- S.E.M.) to 4.0 +/- 0.1 and for the cytochrome b-c1 complex from 3.8 +/- 0.2 to 1.9 +/- 0.1, but has little effect on the H+/2e ratio of cytochrome oxidase. The decrease in stoicheiometry is due, not to uncoupling or inhibition of electron transport, but to inhibition of proton translocation. NN'-Dicyclohexylcarbodi-imide thus ‘decouples’ proton translocation in the cytochrome b-c1 complex.

scholarly journals The control of isocitrate oxidation by rat liver mitochondria

1969 ◽  
Vol 114 (2) ◽  
pp. 215-225 ◽  
Author(s):  
D. G. Nicholls ◽  
P. B. Garland
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Isocitrate Dehydrogenase ◽  
Adenine Nucleotides ◽  
Kinetic Studies ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Dependent Inhibition ◽  
Nad 4

1. The factors capable of affecting the rate of isocitrate oxidation in intact mitochondria include the rate of isocitrate penetration, the activity of the NAD-specific and NADP-specific isocitrate dehydrogenases, the activity of the transhydrogenase acting from NADPH to NAD+, the rate of NADPH oxidation by the reductive synthesis of glutamate and the activity of the respiratory chain. A quantitative assessment of these factors was made in intact mitochondria. 2. The kinetic properties of the NAD-specific and NADP-specific isocitrate dehydrogenases extracted from rat liver mitochondria were examined. 3. The rate of isocitrate oxidation through the respiratory chain in mitochondria with coupled phosphorylation is approximately equal to the maximal of the NAD-specific isocitrate dehydrogenase but at least ten times as great as the transhydrogenase activity from NADPH to NAD+. 4. It is concluded that the energy-dependent inhibition of isocitrate oxidation by palmitoylcarnitine oxidation is due to an inhibition of the NAD-specific isocitrate dehydrogenase. 5. Kinetic studies of NAD-specific isocitrate dehydrogenase demonstrated that its activity could be inhibited by one or more of the following: an increased reduction of mitochondrial NAD, an increased phosphorylation of mitochondrial adenine nucleotides or a fall in the mitochondrial isocitrate concentration. 6. Uncoupling agents stimulate isocitrate oxidation by an extent equal to the associated stimulation of transhydrogenation from NADPH to NAD+. 7. A technique is described for continuously measuring with a carbon dioxide electrode the synthesis of glutamate from isocitrate and ammonia.

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 Effect of 2-n-heptyl-4-hydroxyquinoline N-oxide on proton permeability of the mitochondrial membrane

1980 ◽  
Vol 186 (2) ◽  
pp. 637-639 ◽  
Author(s):  
K Krab ◽  
M Wikström
Keyword(s):  
Respiratory Chain ◽  
Rat Liver ◽  
Proton Transport ◽  
Mitochondrial Membrane ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Proton Permeability ◽  
Isolated Rat Liver ◽  
Isolated Rat

The respiratory-chain inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide catalyses transmembrane proton transport driven by a pH gradient in isolated rat liver mitochondria. This effect explains the apparent blockade of net proton translocation by this compound in mitochondria respiring with ferrocyanide as described by Papa, Lorusso, Guerrieri, Boffoli, Izzo & Capuano [(1977) in Bioenergetics of Membranes (Packer, Papageorgiu & Trebst, eds.), pp. 377-388, Elsevier/North-Holland, Amsterdam] and by Lorusso, Capuano, Boffoli, Stefanelli & Papa [(1979) Biochem. J. 182, 133-147].

scholarly journals Characterization of the glucose 6-phosphate dehydrogenase activity in rat liver mitochondria

1976 ◽  
Vol 159 (3) ◽  
pp. 683-687 ◽  
Author(s):  
M Grunwald ◽  
H Z Hill
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
The Other ◽  
Rat Liver Mitochondria ◽  
Kinetic Behaviour ◽  
Two Peaks ◽  
Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase activity in rat liver mitochondria can be released by detergent. The released activity is separated by chromatography into two peaks. One peak has the kinetic behaviour and mobility similar to the soluble sex-linked enzyme, whereas the other peak is similar to the microsomal hexose 6-phosphate dehydrogenase. There is no evidence for the existence of a new glucose 6-phosphate dehydrogenase activity in rat liver mitochondria.

Variations of D(—)-β-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during post-natal development

Biochimie ◽  
1978 ◽  
Vol 60 (1) ◽  
pp. 71-76 ◽  
Author(s):  
Norbert Latruffe ◽  
Marie-Noëlle Feuvrier ◽  
Nicole Bichet ◽  
Yves Gaudemer
Keyword(s):  
Dehydrogenase Activity ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria
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