The effect of the coupled oxidation of substrate on the permeability of blowfly flight-muscle mitochondria to potassium and other cations

Hansford, Richard G.; Lehninger, Albert L.

doi:10.1042/bj1260689

Cited by 25 publications

(17 citation statements)

References 40 publications

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“…The degree of condensation does, however, appear to be unusually extensive with brown-adipose-tissue mitochondria, which in 250 mM sucrose have a sucrose-impermeable space of under 0.4p.l/mg. This value approximates to the osmotic dead-space determined for blowfly flight muscle [26] and rat liver mitochondria [24]. These observations may be explained in terms of an exceptionally low matrix content of ions providing osmotic support in sucrose-stored brown-adiposetissue mitochondria.…”

Section: Discussionmentioning

confidence: 91%

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Mitochondria from Hamster Brown‐Adipose Tissue

Nicholls

Grav

Lindberg

1972

European Journal of Biochemistry

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1.The oxidation of palmitoyl-L-carnitine by mitochondria from the brown adipose tissue of cold-adapted hamsters is highly dependent on the nature of the incubation medium. No respiration occurs in media containing 200 mM sucrose or 100 mM choline chloride, while 100 mM NaCl allows rapid respiration.2. Media containing 100 mM KCl allow steadily increasing rates of palmitoyl-carnitine oxidation. High initial rates may be obtained by adding valinomycin, or by decreasing the concentration of KC1.3. Respiration can proceed in media containing sucrose or choline chloride if they are made sufficiently hypotonic. Pyruvate and 2-oxoglutarate oxidation is also sensitive to tonicity, whereas ~-glycerol-3-phosphate oxidation is unaffected.4. A close correlation was found between the sucrose-impermeable space and the rate of palmitoyl-carnitine oxidation under all conditions examined, a low space determination being associated with respiratory inhibition.5. Mitochondria stored in 250 mM sucrose have a very low sucrose impermeable space. Evidence is presented that this space must be increased by hypotonic conditions or by ion permeation before respiration can occur. Mitochondria stored in 100 mM KC1, 20 mM K-TES have a much larger sucrose-impermeable space and can immediately oxidize palmitoyl-carnitine. 6.The ion permeability properties of these mitochondria are discussed, together with the nature of the respiratory inhibition associated with matrix condensation.This work arose from observations that the oxidation of palmitoyl-carnitine by freshly prepared, uncoupled [l --51 hamster brown-adipose-tissue mitochondria was highly dependent on the nature of the major component of the incubation medium. The effects were so striking that a systematic study was undertaken, in the course of which it became apparent that all conditions which resulted in a high degree of condensation of the matrix compartment resulted in an inhibition of palmitoylcarnitine oxidation. This paper presents these results, together with evidence for a causal relationship between matrix condensation and respiratory inhibition. While such inhibition is unlikely to occur under physiological conditions, its importance as a factor complicating the interpretation of experimentation in vitro will be discussed. Abbreviation. K-TES, potassium N-tris(hydroxymethy1)-Enzyme. Adenylate kinase (EC 2.7.4.3).2-aminoethane sulphonate. MATERIALS AND METHODSSyrian hamsters were acclimatised for a t least 15 days a t 4 f 1 "C, with food and water ad libitum. The animals were decapitated, and pooled interscapular, cervical and axillary brown adipose tissue was rapidly excised into ice-cold 250 mM sucrose. Mitochondria were then prepared as described by Hittelman et al. [4] for rat brown adipose tissue and stored a t 0 "C in 250 mM sucrose. Alternatively, the mitochondria were taken before the final centrifugation, resuspended in 100 mM KCl, 20mM K-TES, pH 7.2, and resuspended h a l l y in this medium. Protein was determined by the biuret method.All experiments were performed ...

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Section: Discussionmentioning

confidence: 91%

“…This appears to be a distinctive property of brown-adipose-tissue mitochondria. Rat liver mitochondria [34,35] or beef heart mitochondria [36] become permeable to chloride in the absence of respiration at high pH, while beef heart [36-391 and blowfly mitochondria [26] appear to allow penetration of chloride under energised conditions.…”

Section: Discussionmentioning

confidence: 99%

Mitochondria from Hamster Brown‐Adipose Tissue

Nicholls

Grav

Lindberg

1972

European Journal of Biochemistry

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“…The values of m used were 2.0#1/mg of protein and 1.01/mg of protein for glycerol 3-phosphate-energized and de-energized mitochondria respectively (Hansford & Lehninger, 1972), and 1.3ju1/mg of protein for pyruvateenergized mitochondria (R. G. Hansford, unpublished observations). Each value was corrected for the osmotic dead-space of 0.4ju1/mg of protein (Hansford & Lehninger, 1972), giving final values of 1.6,u1/mg for glycerol 3-phosphate-energized, 0.9,ul/ mg for pyruvate-energized and 0.6p1/mg for deenergized mitochondria in the absence of substrate. The study of Hansford & Lehninger (1972) is not strictly comparable in that none of the space determination was made in an experimental system containing valinomycin.…”

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confidence: 99%

“…Each value was corrected for the osmotic dead-space of 0.4ju1/mg of protein (Hansford & Lehninger, 1972), giving final values of 1.6,u1/mg for glycerol 3-phosphate-energized, 0.9,ul/ mg for pyruvate-energized and 0.6p1/mg for deenergized mitochondria in the absence of substrate. The study of Hansford & Lehninger (1972) is not strictly comparable in that none of the space determination was made in an experimental system containing valinomycin. However, it is noted that blowfly mitochondria show an active K+ uniport when energized in the absence of valinomycin (Hansford & Lehninger, 1972) and that the concentration of valinomycin-transportable cation used in the present work was limited to 0.21 mm, so that volume changes of large amplitude were avoided.…”

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confidence: 99%

The nature of controlled respiration and its relationship to protonmotive force and proton conductance in blowfly flight-muscle mitochondria

Johnson

Hansford

1977

Biochemical Journal

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1. To determine whether controlled (State 4) pyruvate oxidation can support a high energy state, measurements of the redox span NAD-cytochrome c, phosphorylation potential and protonmotive force (the gradient in electrochemical activity of protons across the mitochondrial inner membrane) were made as indices of energy status. For comparison, these three measurements were also made with glycerol 3-phosphate, an alternative substrate. The two substrates gave essentially identical values for the redox span NAD-cytochrome c in State 4, and the phosphorylation potential was of sufficient magnitude to be considered in equilibrium with the redox span over the first two phosphorylation sites. The magnitude of the protonmotive force in State 4 was much less and the implications of this finding are discussed. 2. Measurements made during the controlled (State 4) to active (State 3) transition indicated that with glycerol 3-phosphate as substrate, both the redox span NAD-cytochrome c and the protonmotive force were diminished; the State 4 --> State 3 transition with pyruvate as substrate was accompanied by an increase in the redox span but a decrease in protonmotive force. The contrary behaviour of these two energetic parameters in the presence of pyruvate was ascribed to a transient excess in the flux of protons through the adenosine triphosphatase relative to the protonpumping respiratory chain, in spite of the increased dehydrogenase activity. 3. The lower protonmotive force seen in State 3 relative to State 4 with pyruvate as substrate was due to a diminution of both the electrical (DeltaPsi) and the chemical (DeltapH) components; with glycerol 3-phosphate, the magnitude of the decrease in protonmotive force during the State 4 --> State 3 transition was similar to that seen with pyruvate, but was due to a large decrease in the electrical component (DeltaPsi) and a small rise in the chemical component (DeltapH). The reason for the difference seen in the behaviour of the components of the protonmotive force was investigated but not established. 4. In the presence of oligomycin and ADP, oxidation of pyruvate, but not of glycerol 3-phosphate, supported a greater protonmotive force than in State 4, in keeping with the dehydrogenase activation and increased redox span NAD-cytochrome c found under these conditions. 5. Experiments involving the use of uncoupling agent to stimulate respiration are compared with those in which limiting concentrations of ADP were used. Estimates of the proton conductance of the inner membrane indicate a similar non-linear dependence on uncoupler concentration with the two substrates. 6. A model is proposed as an explanation of the high rates of controlled glycerol 3-phosphate oxidation. The model relies on a high permeability of the inner membrane to protons and other ions being induced by glycerol 3-phosphate oxidation in State 4.

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“…The matrix volume of these mitochondria is approx. 2.5p1/mg of protein during state 4 respiration (Hansford & Lehninger, 1972), indicating a total concentration of only 0.2mM. It is possible that some loss has occurred during isolation, as a comparison with the content of the thorax indicates that only 30% of the total CoA and thioester is recovered in the mitochondrial fraction.…”

Section: Discussionmentioning

confidence: 99%