Characterisation of the control of respiration in potato tuber mitochondria using the top‐down approach of metabolic control analysis

Abstract

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8 -1 .O, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were I .0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and + 0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.A unique property of photosynthetic plant cells is that they possess two energy-conserving organelles whose regulation may be closely interconnected [l, 21. Reducing Abbreviations. C, overall flux control coefficient; s, overall elasticity coefficient; Jc, rate of oxygen consumption by cytochrome oxidase; Jp, rate of oxygen consumption required to pump protons out at a rate equal to their rate of return through the phosphorylating system; JL, rate of oxygen consumption required to pump protons out at a rate equal to their rate of...

Localisation of the Sites of Action of Cadmium on Oxidative Phosphorylation in Potato Tuber Mitochondria Using Top‐Down Elasticity Analysis

“…tuber mitochondria [20]. The differences in ApH were insignificant for different cadmium concentrations, though Fig.…”

“…Mitochondria were prepared from potato tubers and purified by density-gradient centrifugation exactly as described in [20]. Mitochondria1 protein was determined as described in [23].…”

“…The steady-state rate after this transition is defined as the state 4 rate. The experimental run was completed by the addition of 25 nM valinomycin, which completely uncouples potato mitochondria due to the presence of an active H+/K+ antiporter [20] and therefore brings protonmotive force ( d p ) to zero. Respiration rates were calculated based on the calibration of the oxygen electrode with air-saturated medium at 25"C, containing 506 nmol O/ml [26].…”

“…The accumulation ratio of radioactive labelled ['H]Ph,MeP+ was calibrated against the accumulation ratio of 86Rb+ over the range of respiration rates and various cadmium concentrations used. The binding correction factor a is given by the accumulation ratio of 86Rb+ divided by that of [3H]Ph,MeP+ [20]. It has been demonstrated that a is independent of the respiratory state [31].…”

Tissue specificity of the regulation of ATP hydrolysis by isolated plant mitochondria

“…as it has been shown, in the case of membranebound chloroplast ATPase, that the maximum rate of catalytic turnover was 4 times higher for ATP synthesis than for ATP hydrolysis [22]. However, one should recall that in plant mitochondria, ATP synthesis is controlled mainly by the respiratory chain, and not by the ATPase [23]. in contrast to what has been described for animal mitochondria [24].…”