Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria

Marx, Ruth; Brinkmann, Klaus

doi:10.1007/bf00385124

Cited by 25 publications

(22 citation statements)

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“…In state 3, malate is oxidized by both MDH and ME, the Cyt pathway predominates, and the alternative pathway, if used, can be phosphorylating at the first site level. In the substrate state and state 4, malate is oxidized by a pathway involving ME, a rotenone-insensitive NADH dehydrogenase, which is presumably located on the inner face of the inner membrane (15,22,26), and the alternative oxidase (18,26). In this context, it seemed important to determine whether the state/3 state 4 transition in avocado mitochondria is accompanied by additional activation of the alternative path as proposed by Bahr and Bonner (2, 3).…”

Section: Methodsmentioning

confidence: 99%

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

Moreau

Romani²

1982

Plant Physiol.

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After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var be carried out by both MDH3 and ME (6,13,14,21), has recently been shown (25, 26) to exhibit a varying degree of cyanideinsensitivity, depending on the relative activity of either enzyme. It seemed promising, therefore, to re-examine malate oxidation by avocado mitochondria throughout the climacteric with special attention to the cyanide-insensitive alternative pathway. In a previous paper (20), we described the preparation of avocado mitochondria on self-generated Percoll gradients. The present study concerns the biochemical behavior of PGC avocado mitochondria with special attention to malate oxidation and cyanide-insensitive respiration. A preliminary report of this work has appeared (19). MATERIALS AND METHODSSource of Mitochondria. 'Fuerte' and 'Hass' avocado fruits (Persea americana, Mill) were obtained from the University of California, South Coast Field Station. The conditions for ripening, measurement of fruit respiration, and preparation ofmitochondria on self-generated Percoll gradients have been described (20). At intermediate ripening stages, the mitochondria separate into two bands on the gradients. In this work, the light and heavy mitochondria were combined for subsequent study.Mitochondrial 02 Uptake and Protein Determination. 02 uptake was measured at 25°C with a Clark-type polarographic electrode using a medium containing 0.3 M mannitol, 10 mm phosphate buffer, 10 mm KCI, 5 mIM MgC12, and 0.1% BSA. Depending on the experiment, pH was adjusted to 6.8, 7.2, or 7.5. Protein was measured by the Lowry procedure.Measurement of Products of Malate Oxidation. The products of malate oxidation (OAA and pyruvate) were determined spectrophotometrically. Mitochondria (about 1 mg protein/ml) were incubated at 25°C in 7 ml of the same medium as used for 02 uptake determinations plus 1 mm arsenite and 25 mm malate. This suspension was maintained aerobic by gentle stirring. After starting the reaction by the addition of 20 mM malate, 1-ml aliquots were taken every 2 min, mixed rapidly with 0.1 ml of cold 20o HC104 containing 1 mm EDTA, immediately neutralized with a few drops of 10 N KOH in the presence of 20 t,l methyl orange (0.05%), and then centrifuged at 2000g for 5 min. Aliquots of the supernatant fraction were added to 0.1 M phosphate buffer (pH 7.2) and 0.12 mm NADH in a final volume of 3 ml. The oxidation of NADH was determined at 30 nm after addition of 2 ,ul of MDH (Calbiochem, porcine heart, 11,370 IU/ml) or lactate dehydrogenase (Sigma Chemical Co., rabbit muscle, type 1, 3900 units/ml)

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Section: Methodsmentioning

confidence: 99%

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

Moreau

Romani²

1982

Plant Physiol.

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“…The curves represent oxygen consumption to drive the proton leak plus any extra oxygen consumption due to other processes such as the alternative oxidase. There is evidence that P/O ratios [43] and H + / O ratios [44] are not identical for oxidation of NADH and succinate in plant mitochondria. However, the differences are small, less than 20%, and for the purposes of assessing the alternative oxidase activities in this paper, we will ignore them and assume that the two substrates have similar H ' /O ratios [3,9].…”

Section: Activity Of Alternative Oxidase With Succinate As Substratementioning

confidence: 99%

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

Kesseler

Diolez²,

Brinkmann

et al. 1992

European Journal of Biochemistry

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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...

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“…The location of these two enzymes in the matrix space seems to be widely accepted now (3,19,22). Endogenous NADH produced by malate oxidation is also thought to be reoxidized by two distinct NADH dehydrogenases located on the inner face of the inner membrane (15,18,19,22,25). This occurs through a rotenone-sensitive NADH dehydrogenase connected to MDH while a rotenone-resistant NADH dehydrogenase would be linked to ME (22).…”

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confidence: 99%

Interaction of Benzylaminopurine with Electron Transport in Plant Mitochondria during Malate Oxidation

Chauveau¹,

Dizengremel²,

Roussaux³

1983

Plant Physiol.

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The effect of 6-benzylaminopurine (BA) was assayed on malate oxidation in mitochondria isolated from fresh and aged potato (Solanum tuberosum L.) slices. Depending on the experimental pH, two pathways for malate oxidation were selected. A pH of 7.7 favored the activity of malate dehydrogenase, which is connected with a rotenone-sensitive NADH dehydrogenase, whereas at pH 6.5 malic enzyme, linked to a rotenone-resistant NADH dehydrogenase, was more active.Experimental results indicate the existence of two sites of inhibition for BA. The first site is common with the site of inhibition of rotenone. The second site is on the classical cyanide-resistant alternative pathway, but is different from the site of salicylhydroxamic acid (SHAM) inhibition, as in succinate oxidation.Moreover, a distinct cyanide-resistant pathway, sensitive to SHAM but resistant to BA, is found to coexist with the well-known alternative pathway which is sensitive to SHAM and BA. This outlet of electrons can accommodate 10% of the total electron flow in mitochondria from fresh slices, and up to 30% in mitochondria from aged slices.

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Characteristics of rotenone-insensitive oxidation of matrix-NADH by broad bean mitochondria

Cited by 25 publications

References 22 publications

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

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

Interaction of Benzylaminopurine with Electron Transport in Plant Mitochondria during Malate Oxidation

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