Rotenone-Insensitive Malate Oxidation by Isolated Plant Mitochondria

The inhibitory effect of rotenone, and the variability of theinhibition, on malate oxidation by isolated plant mitochondriahas been investigated. Under conditions where oxaloacetate removalis enhanced (by transamination with glutamate) the rotenoneinhibition is less severe. Where oxaloacetate is allowed toaccumulate, inhibition by rotenone is marked. Similarly, underconditions which would tend to increase the steady state levelof reduction of the NAD pool (restricting coupled electron flowwith oligomycin) rotenone inhibition is severe. It is concludedthat the degree of rotenone inhibition is related to factorsaffecting the equilibrium poise of malic dehydrogenase, anddoes not necessarily reflect the interaction between rotenoneand the respiratory chain. There appears to be no correlationbetween the effectiveness of rotenone inhibition and mitochondrialmalic enzyme activity. Although there may be separate pathwaysof intramitochondrial NADH oxidation, it appears to be prematureto postulate separation or compartmentation of intramitochondrialmalate oxidation pathways.     

The Effect of Carnitine on Palmitate Oxidation by Pea Cotyledon Mitochondria

The conditions for maximum O₂ uptake by pea cotyledon mitochondriaoxidizing palmitate were established. It was found that CoASH,Mg²⁺, ATP, malate, and carnitine were necessary additions tothe incubation medium. It is suggested that carnitine combineswith palmitoylCoA to produce palmitoylcarnitine with the releaseof CoASH. The palmitoylcarnitine thus formed may penetrate themembranes of the mitochondria with greater ease than palmitoylCoA.     

Characteristics of External NADH Oxidation by Beetroot Mitochondria

Mitochondria isolated from fresh red beetroot (Beta vulgaris L.) tissue do not oxidize external NADH with O₂ as the electron acceptor. These mitochondria have a rotenone- and antimycin-insensitive pathway of NADH oxidation associated with the outer membrane and are capable of reducing cytochrome c or potassium ferricyanide. They are also capable of oxidizing internal NADH via the inner membrane electron transport chain with normal rotenone and antimycin sensitivity and ADP/O ratios. They differ from other plant mitochondria in the apparent lack of the NADH dehydrogenase located on the outer surface of the inner membrane. It is shown that this activity develops during the aging of red beetroot slices in aerated dilute CaSO₄ solutions, and is present in the mitochondria isolated from aged tissue.     

Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)     

Cooperative Substrate Oxidation by Mitochondria from Castor Bean Hypocotyls

Cooperative oxidation of succinate and exogenous NADH was followed in the mitochondria from five- to six-day-old castor bean (Ricinus communisL.) seedlings. Although succinate was oxidized at a much higher rate than NADH, the former inconsiderably (less than 15%) inhibited the oxidation of the latter substrate in state 4, while, in state 3 (in the presence of ATP), the two substrates did not compete and were jointly oxidized. When two substrates were oxidized by the mitochondria with the alternative CN-resistant oxidase (AO) inhibited with salicylhydroxamic acid, the rate of NADH oxidation in state 4 dropped by over 40% as compared to the initial rate. Meanwhile, the rate of succinate oxidation was not considerably affected by AO inhibition. We believe that one of the AO functions in the mitochondria is to provide for noncompeting oxidation of two (or more) substrates by employing two (or several) dehydrogenases of the respiratory chain.     

NADH Oxidation by Mitochondria from the Thermogenic Plant Arum orientale

The enzyme content of the mitochondrial respiratory chain was investigated in the heat-producing plant Arum orientale. It is shown that mitochondria isolated from thermogenic tissues of this plant (with respect to non-thermogenic tissues of A. orientale or to Zea mays) demonstrate significantly elevated levels of activities of two non-coupled NADH dehydrogenases oxidizing intramitochondrial and cytoplasmic NADH pools. It is postulated that operation of a completely non-coupled respiratory chain consisting of non-coupled NADH:quinone oxidoreductases and cyanide-resistant alternative quinol-oxidase is the main mechanism of heat production in thermogenic plants.     

Tramsamination and the Oxidation of Some Amino-acids by Mung Bean Mitochondria

The transaminases for the reaction of -ketoglutarate with theamino-acids L-alanine, L-aspartic acid, glycine, L-leucine,L-ornithine, and L-valine have been shown to be associated withmung bean mitochondria. Aspartate-glutamate and alanine-glutamatetransaminases are two different enzymes both of which are reversibleand stimulated by pyridoxal-5'-phosphate. Aspartate is deaminated, with associated oxygen uptake, by mitochondriafrom 1-day 8eedlings, but there is no oxygen uptake with 2-daymitochondria and aspartate, nor with 1-or 2-day mitochondriaand L-alanine, glycine, L-leucine, or L-valine although theseamino-acids are all deaminated. The change in aspartate metabolismwith increased age of the seedlings is probably due to the reorganizationof the enzyme systems which resulted in DPNH being largely oxidizedby malic dehydrogenase and oxaloacetate, instead of mainly bya DPNH oxidase.     

The Respiratory Chain of Plant Mitochondria: VII. Kinetics of Flavoprotein Oxidation in Skunk Cabbage Mitochondria

The oxidation kinetics of the two high potential flavo-proteins, one (Fp_hf) fluorescent and the other (Fp_ha) nonfluorescent, in mitochondria from skunk cabbage (Symplocarpus foetidus) spadices have been measured by combined spectrophotometry and fluorimetry. In the absence of respiratory inhibitors, both flavoproteins are oxidized at nearly the same rate with half-times between 120 and 160 milliseconds at 24 C. When slight differences in rate are observed, it is Fp_ha which consistently has the shorter half-time. The presence of 0.3 millimolar KCN has no perceptible effect on the oxidation rate of either component. Antimycin A (2 nanomoles per milligram of protein) increases the oxidation half-time of Fp_ha about 3-fold, but it has no effect on the oxidation half-time of Fp_hf. In contrast to these two inhibitors, m-chlorobenzhydroxamic acid—an inhibitor specific to the cyanide insensitive, alternate oxidase pathway in these mitochondria—increases the oxidation half-time of Fp_hf 10-fold to about 2 seconds, while increasing that of Fp_ha only some 20%. This result implies that the flavoprotein Fp_hf mediates electron transport to the alternate oxidase from the region of the mitochondrial respiratory chain encompassing Fp_ha, ubiquinone, and the cytochromes b. The oxidation rate of cytochrome b₅₅₇ is unaffected by either m-chlorobenzhydroxamic acid or cyanide but is strongly inhibited by antimycin A. This result implies that cytochrome b₅₅₇ plays no direct role in the respiratory pathway to the alternate oxidase and is different from cytochrome b₇ found in mitochondria from the spadices of Arum maculatum.     

The Influence of Exogenous Nicotinamide Adenine Dinucleotide on the Oxidation of Malate by Jerusalem Artichoke Mitochondria

Mitochond$$$a isolated from Jerusalem artichoke tubers oxidizedendogenous NADH by both a piericidin A-sensitive and -resistantdehydrogenase if the level of oxaloacetate was kept low. Inwashed mitochondria the addition of NAD⁺ stimulated respirationin the presence of a variety of NAD⁺ -linked substrates. Inmitochondria purified through a sucrose density gradient exogenousNAD⁺ caused a substantial stimulation of respiration only inthe presence of malate. The apparent K_m for malate was 20 mMin the absence of NAD⁺ and 2 mM in the presence of exogenousNAD⁺ The products of malate oxidation were altered by the additionof exogenous NAD⁺. Oxaloacetate and pyruvate were produced inequal amounts in the absence of added NAD⁺, but in the presenceof exogenous NAD⁺ more pyruvate was formed. The rapid oxidationof malate in the absence of added NAD⁺ required phosphate whilethe NAD⁺-stimulated component was not affected by the absenceof phosphate. Phenylsuccinate inhibited the reduction of exogenousNAD⁺ by malate; this compound was found to inhibit isolatedma!ate dehydrogenase and mahe enzyme. Several lines of evidencesuggest that electron flux through one of the NADH dehydrogenasesystems may directly affect the flow through the other dehydrogenases.     

Metmyoglobin Oxidation during Electron Transport Reactions in Mitochondria

Studies of the intracellular role of myoglobin were carried out by recording spectrophotometric changes in acid metmyoglobin and oxymyoglobin during electron transport reactions with mitochondria prepared from pigeon heart muscle by the method of Chance and Hagihara. The absorption peak of metmyoglobin at 409 mµ disappeared when substrate was added to normal or antimycin-inhibited preparations, and was replaced by a new maximum at 423 to 424 mµ, identified as due to the oxidation to ferrylmyoglobin. Further investigation revealed that the oxidation of metmyoglobin took place with the simultaneous oxidation of reduced flavoprotein. Hydrogen peroxide, formed by the reaction of reduced flavoprotein with oxygen, was considered to be the probable intermediate for the oxidation of metmyoglobin in experiments in which catalase was added as a competitor for the oxidant. When DPNH was added to the reaction mixture, the reductant acted to resynthesize the ferri-derivative by reaction with ferrylmyoglobin. Oxymyoglobin could not be used in place of metmyoglobin in these systems. Under the experimental conditions, oxymyoglobin dissociated when dissolved oxygen was depleted from the medium by enzyme oxidations; the resultant ferromyoglobin underwent oxidation to metmyoglobin.     

豌豆叶片线粒体内甘氨酸氧化对苹果酸和异柠檬酸氧化的影响

豌豆幼苗叶片线粒体中,Gly,Mal和Isocit的氧化速率均受光促进。Gly的氧化抑制Mal和Isocit的氧化,而其本身不受影响。用INH抑制Gly氧化或提高NAD~+浓度均会降低其抑制程度。线粒体氧化Gly,Mal和Isocit的K_m(NAD~+)分别为66.67,119.1μmol/L和152.2μmol/L。Gly抑制Mal和Isocit氧化是由于Gly氧化在竞争NAD~+中占优势。     

Enhancement of Oxidation and Phosphorylation of Maize Scutellum Mitochondria by Physiological Concentration of Indoleacetic Acid

Mitochondria isolated from inbred and hybrid maize scutella were found to exhibil enhancd oxidative and phosphorylative activity in many cases when treated with physiological concentrations of IAA. It was found that the genotype being tested the substrate utilized, and the concentration of IAA applied had significant effects on the degree of enhancement or inhibition observed in each case. These results suggest that IAA is related in some manner to theactivity of the respiratory enzymes of the mitochondria.     

Effect of NAD on Malate Oxidation in Intact Plant Mitochondria

Potato tuber mitochondria oxidizing malate respond to NAD⁺ addition with increased oxidation rates, whereas mung bean hypocotyl mitochondria do not. This is traced to a low endogenous content of NAD⁺ in potato mitochondria, which prove to take up added NAD⁺. This mechanism concentrates NAD⁺ in the matrix space. Analyses for oxaloacetate and pyruvate (with pyruvate dehydrogenase blocked) are consistent with regulation of malate oxidation by the internal NAD⁺/NADH ratio.