Activities Of TCA Cycle Enzymes Research Articles

Tricarboxylic Acid Cycle Activity in Mitochondria from Soybean Nodules and Cotyledons

Infected cells of soybean (Glycine max) nodules require NADH, ATP, and 2-oxoglutarate for ammonia assimilation. The role of mitochondria in nodule metabolism was investigated by determining their respiratory properties and comparing them with cotyledon mitochondria. Nodule mitochondria oxidized malate at a rate twice that of any other NAD-linked substrate although their malic enzyme activity was very low, accounting for only 12% of malate oxidation at pH 6.4 compared to 56% for cotyledon mitochondria. The reduction of NAD+ in mitochondria of nodules on adding malate (determined by fluorescence) was rapid and reached a stable level, whereas in cotyledon mitochondria the NADH level declined rapidly as oxaloacetate accumulated. An oxaloacetate scavenging system in the mitochondrial reaction medium increased malate oxidation by cotyledon mitochondria 4-fold, but increased that of nodule mitochondria by less than 50%. This demonstrates that the efflux of oxaloacetate by the oxaloacetate carrier is highly regulated by the extra-mitochondrial oxaloacetate concentration in cotyledon mitochondria compared to nodule mitochondria. The activity of TCA cycle enzymes, except malate and succinate dehydrogenases, was low in nodule mitochondria. Their oxaloacetate export during malate oxidation was rapid. The aspartate amino transferase activity associated with nodule mitochondria was sufficient to account for significant formation of 2-oxoglutarate from oxaloacetate and glutamate. These results suggest that nodule mitochondria operate a truncated form of the TCA cycle and primarily oxidize malate to provide oxaloacetate and ATP for NH3 assimilation.

Carbon catabolism in continuous cultures and bacteroids of Rhizobium leguminosarum MNF 3841

Bacteroids of R. leguminosarum MNF3841 isolated from pea nodules using Percoll gradients had activities of TCA cycle enzymes up to 6-fold higher than those measured in free-living cells grown on fumarate or sucrose. Activities of sugar catabolic enzymes on the other hand were 2–14-fold lower in isolated bacteroids than in sucrose-grown free-living cells. In continuous culture, cells of strain MNF3841 grown on sucrose under Pi limitation had 2–3-fold higher activities of invertase, glucose-6-phosphate dehydrogenase, the Entner-Doudoroff enzymes and 6-phosphogluconate dehydrogenase, than cells grown on fumarate. With one exception O2 limited cultures had similar activities of the carbon catabolic enzymes to Pi-limited cultures grown in the same substrate. Glucose-6-phosphate dehydrogenase in O2-limited cells grown of fumarate was 50% lower than in Pi-limited cells. Co-utilization of fumarate and sucrose occurred with chemostat cultures supplied with both under a variety of conditions.

Effects of an anabolic hormone on aerobic capacity of rat striated muscle.

The effect of chronic administration for 6 weeks of an anabolic steroid, nandrolone phenylpropionate (Durabolin), was studied in three predominantly glycolytic muscles, and three oxidative muscles of sedentary female rats. Mean blood pressure and resting heart rate (HR) were lower in the anabolic-treated group, while the increase in HR during stimulation of EDL was reduced. No change was noted in the aerobic capacity of ventricular myocardium, although there was an increase in skeletal muscles due to a combination of increased capillary supply and/or TCA cycle enzyme activity. Capillary:fibre ratio (C:F) increased around 10% in glycolytic muscle with little effect on resting blood flow (BF). In EDL C:F was 1.1 +/- 0.02 vs. 1.2 +/- 0.01 and BF was 7.0 +/- 1.45 vs. 6.2 +/- 1.82 ml min-1 100 g-1 for control and Durabolin treated animals, respectively (means +/- SEM, n = 7). No increase in citrate synthase (CS) activity was evident. In soleus, where C:F was not significantly different between groups, CS activity increased from 3.9 +/- 0.34 to 5.9 +/- 0.40 microM g-1 min-1 (means +/- SEM, n = 7). Glycolytic capacity, indicated by pyruvate kinase activity, increased only in diaphragm. These data demonstrate that total oxidative metabolism of striated muscle does not necessarily increase with greater proportion of FOG fibres, nor is it always correlated with capillary supply. The positive myotrophic effect of Durabolin represents the sum of modest changes at different levels of organisation.

Limiting factor of nitrogenase system mediating hydrogen production of Rhodobacter sphaeroides S

To explain the decrease of hydrogen production rate in the batch culture of Rhodobacter sphaeroides S, the activities of enzymes related to the TCA cycle, nitrogenase in cell-free extracts, ATP generation by chromatophores, and ferredoxin were examined at the beginning, middle and end of the hydrogen production phase of batch culture. The activities of TCA cycle enzymes, nitrogenase and ATP generation were found to remain at almost the same level throughout the culture, while ferredoxin activity decreased linearly with time. In addition, by bubbling N 2 gas into the culture broth at the end of the culture, the hydrogen production rate was restored to the initial level through the increase of the ferredoxin activity. Although the decrease of ferredoxin activity and its restoration by bubbling N 2 gas remained unexplained, ferredoxin activity was considered to be a key function in the nitrogenase system for H 2 production by this photosynthetic bacterium.

Activities of tricarboxylic acid cycle enzymes in an aflatoxigenic strain of Aspergillus parasiticus after a peptone to glucose carbon source shift

A sequential technique involving the transfer of mycelia from a peptone-based, aflatoxinnonsupporting medium to a glucose-based, aflatoxin-supporting medium was used to study possible relationships between tricarboxylic acid cycle activity and aflatoxin biosynthesis in Aspergillus parasiticus . Analysis indicated that the specific activities of various TCA cycle enzymes are influenced by duration of post-transfer incubation and an apparent carbon catabolite repression. The results support the hypothesis that aflatoxin biosynthesis occurs during a period of depressed TCA activity, which is dependent on the catabolism of a suitable carbohydrate.

Coordination of energy and carbon metabolism in lysine producing Brevibacterium strains

The present paper deals with the coordination of energy metabolism, glucose consumption rate, glycolytic and TCA cycle enzyme activities in the lysine-producing bacterium Brevibacterium flavum. It is shown, that inhibition of the elctron transport chain causes changes of the following sequence: at first, TCA cycle enzymes are activated; secondly, TCA cycle enzyme activity decreases, and glycolytic enzyme activities as well as glucose transport rate increase; there is a slight increase in Qo2 and a considerable one of O2 consumption in cyanide-resistant respiration pathway; thirdly, TCA cycle enzyme activities and glucose transport rate decrease.

Terminal oxidations in Bacillus brevis ATCC 10068. 3. Evidence of TCA cycle enzyme activity.

1. Evidence is presented that contrary to previous reports a number of enzymes implicated in the TCA cycle are functional in an antibiotic producing strain of Bacillus brevis. 2. The level of enzyme activites recorded was found to be influenced by the permeability state of the cell membrane and by the presence of an NAD+/NADH degrading enzyme. These factors are discussed in relation to the previously reported negative findings of other workers. 3. The distribution and specific activities of TCA cycle enzymes in membrane and supernatant fractions are reported. 4. The results, by comparison with other organisms known to possess a functional TCA cycle, suggest that the cycle provides a major pathway of energy metabolism in B. brevis.

Effects of Low Oxygen Gas Respiration on the TCA Cycle Enzyme Activities of Rat Tissues.

Haldane and Barcroft's postulate in 1920 (Lancet) that anoxemia not only stops the machine, but wrecks the machinery, leads to the concept that the enzyme activities of tissues should be damaged in anoxemia. There are reports that activities of enzymes which metabolize substrates located near the entrance to the TCA cycle, namely pyruvate and citrate, are depressed due to hypoxia, especially with studies on myocardial tissues (Kimura, S., from our department; Jap. Circulation J. 21 : 376, 1957). However, information on tissues of other organs is relatively lacking. Also, comparative studies concerning the influence of hypoxemia, caused by low percentage oxygen respiration under normal atmospheric pressure, on the above mentioned enzyme activities of the heart and other organs are lacking. Furthermore, there is a question unclarified, whether acute hypoxia can sometimes increase respiratory enzyme activity, and also the relation between the changes in the enzyme activity and the degree of hypoxia is left unclarified.The present study was undertaken to elucidate the above points, with experiments on tissues of the heart, brain, liver and kidney.Materials and Methods Male albino rats, weighing about 150 grams, were placed in gas mixtures containing 10, 5 or 3±0.2% O2 with adequate ventilation at normal atmospheric pressure for several minutes to 5 hours and decapitated.Estimations of dehydrogenase activities were made on tissues homogenates of myocardium, cerebral cortex, liver and kidney, by the Thunberg technique. Sodium salts of lactic, pyruvic, citric and succinic acids were used as substrates. The decolorizing time of methylene blue was used for statistical analyses, but the ratio-methylene blue reduced (γ)/decolorizing time (minutes)- was considered an indicator of methylene blue reducing, i.e. substrate oxidizing, capacity of tissue homogenates, and used for the calculation of percentage changes.The endogenous respirations of homogenates of myocardium and cerebral cortex, were determined by the Lemley and Meneely's method. Also, the Qo2 of homogenates with pyruvate, citrate or succinate as substrate were obtained.Results Obtained 1. Normal controls The oxidizing capacities of tissue homogenates when pyruvate, citrate or succinate is used as substrate are-myocardium>kidney>liver>brain-the brain being especially weak. Also, with these substrates, the Qo2 of myocardial homogenates are larger than that of the brain. The addition of citrate had little or no effect on the oxidizing capacity and Qo2 of brain homogenates. It was felt, as reported by others, that the TCA cycle activity was weak in the brain tissue.2. Effects of various degrees of hypoxia a) Relatively mild hypoxia (10 % O2) for 2 hours : The homogenates of myocardium and brain showed no significant change in their oxidizing capacity, but significant increases were observed with liver and kidney homogenates. Especially with the liver, compared to control values, the endogenous oxidizing capacity was increased by +47 % (P<0.01), and with lactate, pyruvate or citrate as substrate, respective increases of +39 % (P<0.01), +23 % (P<0.05) and +34 % (P<0.02) were noted.It was concluded that relatively mild hypoxia of this degree and duration, could cause increases in the oxidizing capacities of liver and kidney tissues, while those of the myocardium and brain were maintained.b) Moderate hypoxia (5 % O2) for 10 minutes to 5 hours : The oxidizing capacity of tissue homogenates showed no significant change after 10 minutes of 5 % O2 respiration. One hour of hypoxia caused decreases in the oxidizing capacities of myocardial homogenates, only when pyruvate (-22%, P<0.01) or citrate (-33%, P<0.01) were used as substrates.After 2 hours of hypoxia, significant decreases were found in all tissues studied. [the rest omitted]