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

Abstract

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.

Determination of the Proton Electrochemical Gradient across Biological Membranes

“…ATP synthesis in aerobic mitochondria and bacteria, or vesicles obtained from these organelles, is coupled to Δ/Ι Η depression (e.g., Nicholls, 1974;Sorgato et aL, 1978;Kell et aL, 1978a;Leiser and Gromet-Elhanan, 1977;Johnson and Hansford, 1977), and titrations confirm the close relationship between the rate of ATP synthesis and the extent of Δ/Ι Η depression . ATP synthesis in aerobic mitochondria and bacteria, or vesicles obtained from these organelles, is coupled to Δ/Ι Η depression (e.g., Nicholls, 1974;Sorgato et aL, 1978;Kell et aL, 1978a;Leiser and Gromet-Elhanan, 1977;Johnson and Hansford, 1977), and titrations confirm the close relationship between the rate of ATP synthesis and the extent of Δ/Ι Η depression .…”

“…Similar conclusions have been drawn from double-inhibitor titration studies (Baum et al, 1971;Hitchens and Kell, 1982a;Westerhoff et al, 1982). Several reports contradict this (Padan and Rottenberg, 1973;Azzone et al, 1978c;Zoratti et al, 1981;Johnson and Hansford, 1977;Branca et al, 1981;Wilson and Forman, 1982;Sorgato et al, 1978;Holian and Wilson, 1980;see, however, Küster et al, 1981;Nicholls and Bernson, 1977;Nicholls, 1979), suggesting that ATP synthesis drains energy from the redox H + pump through a pathway which is not equivalent to Δ/Ι Η utilization for cation transport. The model proposes a stepwise activation of the ATPase depending on the number of H + ions interacting with the enzyme.…”

Relation between mitochondrial calcium transport and control of energy metabolism

“…It seems to the reviewer that these experiments instead establish the role of the mitochondrion as providing ATP for some other membrane Ca 2÷ transport system, quite possibly in the endoplasmic reticulum. Oligomycin would not be expected to inhibit mitochondrial Ca 2+ uptake per se, as it does not diminish the driving force, ~0 , and indeed may even increase this quantity (Johnson and Hansford 1977). Oligomycin does, however, prevent mitochondrial synthesis of ATP.…”

The uptake of silicic acid by rat liver mitochondria

“…Spaces were calculated for each radioactive isotope (see above), giving the values c for methylamine, h for acetate and m for the matrix spaces respectively. By analogy with a previously described approach (Johnson & Hansford, 1977), an absolute value (N) was calculated for (h-c)/m. The magnitude of ApH was then given exactly by log [(N+V\N2+4)/2], and the sign of ApH depended on the sign of (h-c)/m: defining ApH as pHmedium-pHmatrjx, for (h-c)/m >0, ApH <0, and vice versa.…”

“…The effect of mitochondrial energy status on Si(OH)4 uptake was investigated by using a silicone-oil-centrifugation technique, since such a method has been shown to preserve mitochondrial energy states until complete separation of organelles from the suspending medium has been accomplished (Davis & Lumeng, 1975;Johnson & Hansford, 1977). Control experiments with rotenone-treated mitochondria showed that neither the silicone oil nor the dextran added to the medium (LaNoue et al, 1973) affected the results appreciably.…”