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Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria.

ABSTRACT: 1. High rates of state 3 pyruvate oxidation are dependent on high concentrations of inorganic phosphate and a predominance of ADP in the intramitochondrial pool of adenine nucleotides. The latter requirement is most marked at alkaline pH values, where ATP is profoundly inhibitory. 2. Addition of CaCl(2) during state 4, state 3 (Chance & Williams, 1955) or uncoupled pyruvate oxidation causes a marked inhibition in the rate of oxygen uptake when low concentrations of mitochondria are employed, but may lead to an enhancement of state 4 oxygen uptake when very high concentrations of mitochondria are used. 3. These properties are consistent with the kinetics of the NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) from this tissue, which is activated by isocitrate, citrate, ADP, phosphate and H(+) ions, and inhibited by ATP, NADH and Ca(2+). 4. Studies of the redox state of NAD and cytochrome c show that addition of ADP during pyruvate oxidation causes a slight reduction, whereas addition during glycerol phosphate oxidation causes a ;classical' oxidation. Nevertheless, it is concluded that pyruvate oxidation is probably limited by the respiratory chain in state 4 and by the NAD-linked isocitrate dehydrogenase in state 3. 5. The oxidation of 2-oxoglutarate by swollen mitochondria is also stimulated by high concentrations of ADP and phosphate, and is not uncoupled by arsenate.

SUBMITTER: Hansford RG

PROVIDER: S-EPMC1178582 | biostudies-other | 1972 Mar

REPOSITORIES: biostudies-other

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Project description: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.

Project description:1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.

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Some properties of pyruvate and 2-oxoglutarate oxidation by blowfly flight-muscle mitochondria.

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