The Oxidation of Malate by Mitochondria Isolated from Cauliflower Buds

Macrae, Alasdair R.; Moorhouse, Ralph

doi:10.1111/j.1432-1033.1970.tb01058.x

Cited by 59 publications

(36 citation statements)

References 28 publications

(26 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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: 57%

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Bowman

Ikuma²

1976

Plant Physiol.

View full text Add to dashboard Cite

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)

show abstract

Section: Resultsmentioning

confidence: 57%

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Bowman

Ikuma²

1976

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…It is also possible that the inhibition could have occurred as a consequence of the direct inhibition of the malic enzyme by oxaloacetate. Macrae [20] has reported that 2 mM oxaloacetate can act as a competitive inhibitor of the solubilised malic enzyme. The relationship between the inhibition caused by 0.2 mM oxaloacetate in the presence of increasing concentrations of malate, as the respiratory substrate, is shown as a LineweaverBurke reciprocal plot in Fig.4.…”

Section: Assay Of the Endogenous N A D F Poolmentioning

confidence: 99%

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

Brunton

Palmer

1973

European Journal of Biochemistry

View full text Add to dashboard Cite

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...

show abstract

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Moreau

Romani²

1982

Plant Physiol.

View full text Add to dashboard Cite

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)

show abstract

The Oxidation of Malate by Mitochondria Isolated from Cauliflower Buds

Cited by 59 publications

References 28 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

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

Contact Info

Product

Resources

About