Necessary Conditions for Isolation of Tightly Coupled Higher Plant Mitochondria

Ikuma, Hiroshi

doi:10.1104/pp.45.6.773

Cited by 28 publications

(15 citation statements)

References 39 publications

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“…Jumbo) seedlings, grown at 27 C in the dark. The mitochondria were prepared according to recommendations made by Ikuma (22). The grinding medium consisted of 500 mm mannitol, 1 mm EDTA, 5 mm cysteine-HCl, 0.2% BSA (w/v), and 10 mm K phosphate, pH 7.2; and the suspension medium contained 500 mm mannitol, 0.2% BSA, and 10 mM K phosphate, pH 7.2.…”

Section: Methodsmentioning

confidence: 99%

“…While ATPase has (22), NAD is the only detectable pyridine nucleotide in these mitochondria. In general, endogenous NAD levels observed in higher plant mitochondria are in the range of 3 to 6 nmol/mg protein (14,22,25 Figure 2 shows the results from a representative. In order to examine the relationship between respiratory chain phosphorylation and CAC activity, the experimental procedure was modified to emphasize the individual effects of ADP and ATP (Table V).…”

Section: Methodsmentioning

confidence: 99%

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Citric Acid Cycle Activity in Mitochondria Isolated from Mung Bean Hypocotyls

Bowman

Ikuma²,

Stein³

1976

Plant Physiol.

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Citric acid cycle activity in mitochondria from mung bean (Phaseolus aureus var. Jumbo) hypocotyls were examined by surveying (a) Higher plant mitochondria exhibit many respiratory activities different from those of mammalian mitochondria (14, cf. 23). For example (a) plant mitochondria readily oxidize malate and externally applied NADH, whereas animal mitochondria oxidize malate poorly and do not oxidize externally applied NADH; (b) respiratory patterns in the oxidation of succinate and other substrates by plant mitochondria differ from those demonstrated with animal mitochondria; and (c) rotenone, by far the most potent inhibitor of NADH oxidase in animal mitochondria, is a weak and incomplete inhibitor of plant mitochondrial respiration. In order to characterize the respiratory properties of plant mitochondria further, current research is concerned with elucidating metabolic regulation of CAC6 activity in mung bean mitochondria.In contrast with several extensive studies on metabolic control of CAC activity in mammalian mitochondria (e.g. 27, 28) attempts to examine the over-all functioning of higher plant mitochondria (3, 13) have been sparse in recent years. In this com-

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Citric Acid Cycle Activity in Mitochondria Isolated from Mung Bean Hypocotyls

Bowman

Ikuma²,

Stein³

1976

Plant Physiol.

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“…Mitochondria were prepared as described previously (6) from mung bean hypocotyls by the method of Ikuma (13). The standard reaction medium contained 500 mm mannitol, 10 mm KCI, 5 mM MgCl2, and 10 mm K phosphate buffer, pH 7.2. In studies aimed at evaluating the effect of adenylates on malate oxidation products, mitochondria were incubated with 10 mm malate as substrate, and 02 consumption was followed to a steady state level.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Malate Oxidation in Isolated Mung Bean Mitochondria

Bowman¹,

Ikuma²

1976

Plant Physiol.

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Effects of ADP and ATP on products of malate oxidation in the presence or absence of respiratory inhibitors and an uncoupler were investigated in mitochondria isolated from mung bean (Phaseolus aureus var. Jumbo) hypocotyls. Changes in levels of products from malate oxidation generally correlated directly with changes in oxygen uptake. Effects of ADP and ATP were indistinguishable from each other when respiratory chain activity was limited. We concluded that adenylates indirectly act on malate oxidation via the oxidation-reduction status of the pyridine nudeotides which are linked to the respiratory chain. The possibility of allosteric action of ADP and ATP on malate dehydrogenase activity was examined in both intact mitochondria and a partially purified enzyme preparation. Although small inhibition, 16% with 500 ,UM ATP and 8% with 500 jAM ADP, was observed at pH 9.5, this effect was abolished by the addition of magnesium ions or by lowering the pH to 7.2. We concluded that these adenylate effects are probably not a significant factor in regulation under physiological conditions. Furthermore, the equilibrium constant of malate dehydrogenase (to 1.5 x 10-5) in both mitochondria and the partially purified enzyme calculated from the steady state level of NADH formed suggested that the enzyme functions in an equilibrium manner in intact mitochondria.Studies of citric acid cycle activity in mitochondria isolated from mung bean hypocotyls led to the conclusion that malate oxidation plays an important role in cycle activity (6). Adenylates are implicated in the control of malate oxidation by the following observations in the literature: (a) ADP and ATP drastically altered the levels of OAA5 produced from oxidation of malate (6) purified from mung bean cotyledon mitochondria (1). On the other hand, some investigators contend that control of oxidation of cycle intermediates does not reside in the cycle itself but is confined to interaction of adenine nucleotides with the respiratory chain (25,27). The concept of respiratory control proposed by Chance and Williams (7) can also be viewed as supporting the latter possibility.This publichation is aimed at resolving how adenylates act in the regulation of malate oxidation in isolated mung bean mitochondria, whether they act directly as effectors of malate dehydrogenase, indirectly via their action on the oxidative-phosphorylation system, or both. MATERIALS AND METHODSPreparation and Incubation of Mitochondria. Mitochondria were prepared as described previously (6) from mung bean hypocotyls by the method of Ikuma (13). The standard reaction medium contained 500 mm mannitol, 10 mm KCI, 5 mM MgCl2, and 10 mm K phosphate buffer, pH 7.2. In studies aimed at evaluating the effect of adenylates on malate oxidation products, mitochondria were incubated with 10 mm malate as substrate, and 02 consumption was followed to a steady state level. During the period of linear 02 uptake, ADP or ATP was added, and the oxidation products were analyzed at two to three timed intervals ...

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“…Mitochondria were isolated from the mature part (10-40 mm from the tip) of 3-day-old seedling North-Holland Publishing Company -Amsterdam roots or from the seed embryonal axes after 1-2 h imbibition [4]. Mitochondria were disintegrated by freeze-thawing in hypotonic medium, and about 70-75% of mitochondrial protein were solubilized in 1% Triton X-100 (final protein concentration 6 mg/ml).…”

Section: Methodsmentioning

confidence: 99%

“…To prevent bacterial growth penicillin (20 #g/ml), streptomycin (20 #g/ml), and chloramphenicol (5/ag/ml) were added to incubation media. Mitochondria were isolated [4] from the combined all-and 14C-labelled samples. Proteins were precipitated by trichloroacetic acid (TCA) in cold, washed twice with 5% TCA containing nonlabelled leucine and further with acetone, dissolved in 0.01 M Na-phosphate buffer, pH 7.2, containing 8 M urea, 1% SDS, and 1% 2-mercaptoethanol during 3-5 min at 95°C, and separated by SDS-disc-electrophoresis in 13%-polyacrylamide gels [7].…”

Section: Methodsmentioning

confidence: 99%