The Oxidation of Malate by Isolated Plant Mitochondria

Coleman, J. O. D.; Palmer, John

doi:10.1111/j.1432-1033.1972.tb01792.x

Cited by 79 publications

(33 citation statements)

References 44 publications

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“…Other investigators claim that OAA is converted to pyruvate-not directly, but through malate dehydrogenase + NAD-malic enzyme activity (4,5,7,14). Data in this communication show that OAA added alone is not metabolized, indicating the absence of significant OAA decarboxylase activity in these mitochondrial preparations (Fig.…”

Section: Resultsmentioning

confidence: 59%

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Bowman

Ikuma²

1976

Plant Physiol.

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In order to investigate the relationship between malate oxidation and subsequent cycle reactions, the effects of oxaloacetate, pyruvate, and thiamine pyrophosphate on malate oxidation in mung bean (Phaseolus aureus var. Jumbo) hypocotyl mitochondria were quantitatively examined. Malate oxidation was optimally stimulated by addition of pyruvate and thiamine pyrophosphate, whose addition lowered the apparent Km for malate from 5 mm to 0.1 nM. Intermediate analysis showed that the stimulatory effect was correlated with removal of oxaloacetate to citrate.Oxaloacetate added alone was shown not to be metabolized until addition of pyruvate and thiamine pyrophosphate; then oxaloacetate was converted in part to pyruvate and also to citrate. These results establish that malate oxidation in mung bean mitochondria is subject to control by oxaloacetate levels, which are primarily detennrned by the resultant of the activities of malate dehydrogenase, citrate synthase, and pyruvate dehydrogenase.Malate oxidation by plant mitochondria becomes progressively inhibited with successive state 3 to 4 transitions (3). Oxaloacetate is believed to be the direct cause of inhibition (8,9,12,17,19). The inhibition can be reversed by agents which remove OAA5; agents used include pyruvate, TPP, and glutamate (6,8,12,17 MATERIALS AND METHODS Mitochondria were prepared from mung bean hypocotyls as described previously (3). Oxygen uptake and fluorescence changes in mitochondria were measured in standard reaction medium as before (3). Further additions are noted in the text, figure legends, and table legends. Citric acid cycle intermediates were extracted and assayed as described previously (3). The protein content of mitochondria was determined as before (3).Chemicals were obtained from the following sources: BSA from Nutritional Biochemicals Corporation; ATP, ADP, and TPP from Calbiochem; and NAD and NADH from Sigma Chemical Co. Enzymes were purchased from Boehringer-Mannheim. RESULTSThe requirement of pyruvate oxidation for a sparker is illustrated in Figure 1, which shows 02 electrode and fluorescence traces with pyruvate and/or malate as substrate in the presence of TPP. The oxidation of malate alone is rapid at high concentrations (30 mM) (Fig. 1A) and very slow at low levels (1 mM) (Fig. 1C). Pyruvate oxidation is not detectable in the absence of a sparker (Fig. 1B). Addition of 1 mm malate results in rapid pyruvate oxidation (Fig. 1iD)

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Section: Resultsmentioning

confidence: 59%

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Bowman

Ikuma²

1976

Plant Physiol.

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“…Traces I, I1 and I11 were obtained using wheat mitochondria; trace IV was obtained using rat liver mitochondria. The mitochondria (1 mg protein) were suspended in a total reaction volume of 0.5 ml, as described in Methods, containing 1 and 11) in contrast to rat liver mitochondria where no parallel inhibition was observed (Fig. 1, trace IV).…”

Section: Assay Of the Endogenous N A D F Poolmentioning

confidence: 99%

“…7). The external piericidin-A-resistant pathway is reported to be only linked to two phosphorylation sites [1,40]; it was therefore necessary to establish the number of phosphorylation sites associated with the internal resistant pathways. From the data shown in Table 1 it can be seen that the addition of rotenone to all NADf-linked substrates caused the ADP/O ratios to fall by the equivalent of one phosphorylation site, thus suggesting that the piericidin-A-and rotenone-resistant, internal electron-transport pathway by-passed the first phosphorylation site.…”

Section: Piericidin a Sensitivity And Location Of The Pyridine-nucleomentioning

confidence: 99%

Pathways for the Oxidation of Malate and Reduced Pyridine Nucleotide by Wheat Mitochondria

Brunton

Palmer

1973

European Journal of Biochemistry

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piericidin A and oxaloacetate have been used as inhibitors to investigate the operation and localisation of NAD+-linked dehydrogenases and the possible compartmentation of these enzymes within the matrix of the mitochondrion. 1.Mitochondria isolated from etiolated shoots of wheat (Triticum vulgare) were shown to oxidise a variety of NAD+-linked substrates. Oxaloacetate, when added to rapidly respiring preparations (i.e. state 3) resulted in a large but transient inhibition ofthe oxidation of both pyruvate and citrate, but had little effect on the rate of oxidation of malate.2. The products of oxidation of malate, in the absence of added NAD+, were found to be a large quantity of pyruvate together with smaller quantities of oxaloacetate and citrate. Using potassium ferricyanide as a non-penetrating electron acceptor it was possible to show that the malate oxidation occurred in the matrix. This is consistent with the operation of both the malic enzyme and a low level of malate dehydrogenase within the matrix of the mitochondrion.3. The low level of inhibition of malate oxidation by oxaloacetate was a function of the concentration of malate and appeared to be of a competitive nature.4. Measurement of the redox state of the endogenous NAD+ pool showed that under phosphorylatig conditions both malate and citrate reduce approximately 15O/, of the total coenzyme. The addition of 0.2 mM oxaloacetate to phosphorylating aerobic mitochondria failed to oxidise any of the NAD+ reduced by malate but resulted in a partial oxidation of NAD+ reduced by citrate. The transition from the aerobic to anaerobic state in the presence of malate resulted in a biphasic reduction of the endogenous NAD+, whereas in the presence of citrate it was monophasic.5 . The oxidation of NAD+-linked substrates, including malate, was found to be only partially inhibited by piercidin A. The piericidin-A-resistant pathway was found to be located internally on the basis of experiments using ferricyanide as a non-penetrating electron acceptor. The first site of phosphorylation was not coupled to oxidation mediated by the piericidin-A-resistant pathway.6. The presence of piericidin A altered the shape of the malate saturation curve. I n the ahsence of the inhibitor it was found to be biphasic; saturating at 60 mM and in its presence to be monophasic saturing a t 15 mM. At high concentrations of malate (60 mM) piericidin A caused approximately a 30 O/, inhibition, similar to that found with other NAD+-linked substrates, while a t lower concentrations of malate (15 mM) piericidin A caused a much higher inhibition (80 O/,), which subsequently recovered to the much higher piericidin-A-resistant rate observed with higher concentrations of malate. 7.I n the presence of piericidin A oxaloacetate caused severe but transient inhibition of malate oxidation, similar in magnitude to that observed during pyruvate oxidation in the absence of piericidin A.8. It was concluded that the data were consistent with the spatial separation of both the internal malic enzyme and ma...

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“…In intact mitochondria, NADHcytochrome Previous studies with plant mitochondria (4,11,13,14) have indicated the presence of two NAD+-linked enzymes involved in the oxidation of malate; viz. malate dehydrogenase and "malic" enzyme.…”

Section: Introductionmentioning

confidence: 99%

“…Coleman and Palmer (4) suggested that the malic enzyme of plant mitochondria was situated in the intermembrane space and that the oxidation of malate by this enzyme was linked to the respiratory chain via the above mentioned NADH dehydrogenase. Douce et al.…”

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confidence: 99%

The Oxidation of Malate and Exogenous Reduced Nicotinamide Adenine Dinucleotide by Isolated Plant Mitochondria

Day¹,

Wiskich²

1974

Plant Physiol.

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Exogenous NADH oxidation by cauliflower (Brassica oleracea L.) bud mitochondria was sensitive to antimycin A and gave ADP/O ratios of 1.4 to 1.9. In intact mitochondria, NADHcytochrome Previous studies with plant mitochondria (4,11,13,14) have indicated the presence of two NAD+-linked enzymes involved in the oxidation of malate; viz. malate dehydrogenase and "malic" enzyme. Plant mitochondria also appear to possess an alternative system for the oxidation of exogenous NADH.The oxidation involves an NADH dehydrogenase, apparently situated on the outside of the inner membrane (8,19), which is linked to the respiratory chain and coupled to two sites of phosphorylation (5,9,15,19,24 were carried out at about 2 C, using chilled solutions and apparatus.

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The Oxidation of Malate by Isolated Plant Mitochondria

Cited by 79 publications

References 44 publications

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Pathways for the Oxidation of Malate and Reduced Pyridine Nucleotide by Wheat Mitochondria

The Oxidation of Malate and Exogenous Reduced Nicotinamide Adenine Dinucleotide by Isolated Plant Mitochondria

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