Citric Acid Cycle Activity in Mitochondria Isolated from Mung Bean Hypocotyls

Bowman, Emma Jean; Ikuma, Hiroshi; Stein, Howard J.

doi:10.1104/pp.58.3.426

Cited by 40 publications

(32 citation statements)

References 9 publications

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“…NADH-GDH is relatively easily solubilized from plant mitochondria and hence the enzyme is thought to be localized in the soluble matrix of mitochondria (4,28). However, the method of disrupting the mitochondria in those reports (4,28) involved a relatively vigorous treatment, such as freezing and thawing followed by sonication. To demonstrate the distribution ofNADH-GDH in corn mitochondria, we treated mitochondria, purified by centrifugation through a Percoll density gradient, with osmotic shock, a relatively gentle disruption method.…”

Section: Resultsmentioning

confidence: 99%

“…Most of the fumarase activity (a matrix marker) and succinate:Cyt c reductase (a membrane marker) were located in the expected fractions (29). NADH-GDH and malate dehydrogenase, which were thought to be in the mitochondrial matrix (4,28) were, however, found primarily in the membrane fraction (75%). When the mitochondria were disrupted by harsher treatments (freezing and thawing followed by sonication), about 85% of the NADH-GDH activity was Table I.…”

Section: Resultsmentioning

confidence: 99%

“…When intramitochondrial Ca distibution was determined, after submitochondrial fractionation following the gentle disruption with osmotic shock, about 35% of the total Ca was distributed in the mitochondrial matrix and the remaining 65% with the mitochondrial membrane. If one assumes a matrix water space of I ul/mg mitochondrial protein (4,15), and if the Ca detected in the matrix is all free, the Ca2" concentration in the matrix can be calculated to be 52 to 56 mm. This, together with the fact that the addition of Ca2e had little effect on the NADH-GDH in purified mitochondrial fractions (Table I), suggests that the mitochondrial enzyme is fully activated with respect to Ca2".…”

Section: Resultsmentioning

confidence: 99%

“…According to the work by Bowman et al (4), NAD is the only detectable pyridine nucleotide in mung bean mitochondria. Thus, concentrations of Ca2" and NADH in the mitochondria could be important in the regulation of NADH-GDH.…”

mentioning

confidence: 99%

“…NADH-GDH2 is easily solubilized from mitochondria from roots (30), shoots (4,25), and seeds (28) of various plants. Although it is the only mitochondrial enzyme with the potential for the assimilation of NH44, it has been suggested by Miflin and Lea (21) that the NH44 released by the conversion of glycine to serine in the mitochondria (3) is reassimilated via GS in the cytosol.…”

mentioning

confidence: 99%

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Characteristics of Glutamate Dehydrogenase in Mitochondria Prepared from Corn Shoots

Yamaya¹,

Oaks²,

Matsumoto³

1984

Plant Physiol.

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The amination of a-ketoglutarate (a-KG) by NADH-glutamate dehydrogenase (GDH) obtained from Sephadex G-75 treated crude extracts from shoots of 5-day-old seedlings was stimulated by the addition of Ca2+. The NADH-GDH purified 161-fold with ammonium sulfate, was also activated by Ca2" in the presence of 160 micromolar NADH. However, with 10 micromolar NADH, Ca2 had no effect on the NADH-GDH activity. The deamination reaction (NAD-GDH) was not influenced by the addition of Ca2".About 25% of the NADH-GDH activity was solubilized from purified mitochondria after a simple osmotic shock treatment, whereas the remaining 75% of the activity was associated with the mitochondrial membrane fraction. When the lysed mitochondria, mitochondrial matrix, or mitochondrial membrane fraction was used as the source of NADH-GDH, Ca2" had little effect on its activity. The mitochondrial fraction contained about 155 nanomoles Ca per milligram of mitochondrial protein, suggesting that the NADH-GDH in the mitochondria is already in an activated form with regard Ca2". In a simulated in vitro system using concentrations of 6A millimolar NAD, 0.21 millimolar NADH, 5 millimolar a-KG, and 5 millimolar glutamate thought to occur in the mitochondria, together with 1 millimolar Ca2", 10 and 50 millimolar NH4', and purified enzyme, the equilibrium of GDH was in the direction of glutamate formation.NADH-GDH2 is easily solubilized from mitochondria from roots (30), shoots (4, 25), and seeds (28) of various plants. Although it is the only mitochondrial enzyme with the potential for the assimilation of NH44, it has been suggested by Miflin and Lea (21) that the NH44 released by the conversion of glycine to serine in the mitochondria (3) is reassimilated via GS in the cytosol. They have also suggested that the principal function of mitochondrial GDH is the oxidation ofglutamate (21). However, a part of intramitochondrially generated NH44 from glycine is incorporated into glutamate by mitochondria isolated from pea shoots (I 1). Chou and Splittstoesser (7), Joy (14), and recently Furuhashi and Takahashi (10) Isolation of Mitochondria by Density Gradient Centrifugation. All reagents and buffer solutions were prepared with double glass-distilled H20 to minimize Ca24 contamination, and all operations were carried out at 4°C. Whole shoots of 5-d seedlings were harvested, washed in water, blotted dry with filter paper, and weighed. The shoots (40 to 60 g in fresh weight) were chopped with razor blades in extraction buffer consisting of 0.4 M mannitol, 0.1 M Hepes-KOH buffer (pH 7.5), 1 mM EDTA, 0.1% (w/v) BSA, and 0.6% (w/v) insoluble PVP, at a ratio of 1 g fresh weight per 3 ml of buffer. The chopped materials were ground with motar and pestle.The mitochondrial fraction was isolated by modification of the method described by Nishimura et al. (26) as described previously by us (32). The purified mitochondria after Percoll discontinuous density gradient were able to oxidize both malate and succinate with a high P/O and RCR ratios and appear to...

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