Concentrations Of 2-oxoglutarate Research Articles

Activation of respiration of liver mitochondria by catecholamines

Catacholamines activate in vivo and in vitro the rat liver mitochondria respiration (VO2) at high piruvate, 2-oxoglutarate and succinate concentrations. The effect was equally characteristic of free oxidation (state 4), phosphorylation (state 3), and even of VO2 uncoupled by DNP. EDTA and bovine serum albumin addition failed to eliminate the effect of the oxidoreductases activation by catecholamines.

Anti-ketogenic effect of glucose in the lactating cow deprived of food.

1. The aim of this study was to determine the effect of a constant infusion of glucose on the ketosis that is observed when dairy cows are deprived of food in early lactation. 2. Cows in early lactation were first deprived of food for 4 days (96h) to induce a 'fasting ketosis'. Glucose was then infused intravenously at a constant rate of 0.75 g/min for 48h while deprivation of food was maintained. At the end of this 48 h period, blood and liver ketone-body concentrations had decreased to values well below those found in healthy fed cows. 3. On the assumption that the anti-ketogenic effect of glucose was mainly due to suppression of hepatic ketogenesis, it was concluded that two anti-ketogenic mechanisms had been identified. These were (a) a decrease in the availability of free fatty acids for hepatic oxidation, and (b) anti-ketogenic changes within the liver itself. 4. These latter anti-ketogenic changes were twofold. The first was a major increase in the hepatic concentrations of citrate and 2-oxoglutarate. The second was an increase in the degree of oxidation of the hepatic cytosol. It was proposed that both these intrahepatic changes might indicate an augmentation of the quantity of oxaloacetate available for condensation with acetyl-CoA derived from fat oxidation. 5. Hepatic glycerol 1-phosphate concentration fell substantially after glucose infusion. 6. Glucose infusion into fed cows produced qualitatively similar effects to those observed in the unfed cows. However, blood and liver ketone-body concentrations were not decreased to the same extent in the fed cows as in the unfed cows.

Activation of aspartate transaminase by pyridoxal 5′-phosphate in the serum of normal subjects

Preincubation of 16 normal sera with pyridoxal 5′-phosphate resulted in a 56% increase of aspartate transminase activity. The magnitude of aspartate transaminase activation by PP is dependent on the concentration of L-aspartate and 2-oxoglutarate in the final reaction mixture.

Influence of Auxiliary Enzymes on the Spectrophotometric Measurement of Alanine Aminotransferase and Aspartate Aminotransferase Activities

We investigated the enzyme activity of the blank in the spectrophotometric determination of the aminotransferase activities and aspartate aminotransferase activity. 6 lactate dehydrogenase and 3 malate dehydrogenase preparations from different manufactures and from different organs showed additional and contaminating activity. The additional activity depends upon the 2-oxoglutarate concentration. The contaminating activity is caused by alanine aminotransferase and aspartate aminotransferase in the auxiliary enzymes. We propose that exact definitions must be given for the auxiliary enzymes in the recommendations of standard determinations for enzyme activities.

A Kinetic Study of the α-Keto Acid Dehydrogenase Complexes from Pig Heart Mitochondria1

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.

Activation of pyruvate dehydrogenase during metabolism of ammonium ions in hemoglobin-free perfused rat liver.

1 Ammonium chloride addition to perfused rat liver results in a shift of the steady state of the pyruvate dehydrogenase system towards the active (dephospho) form. This is evidenced by direct measurement of enzyme activity in freeze-stopped liver tissue, by net uptake of pyruvate from the perfusate, and also by an increased release of labeled CO2 from [1-14C]pyruvate. Half-maximal concentration of NH4Cl for extra pyruvate uptake is 0.5–0.7 mM. The effect is readily reversible. 2 Extra urea formation upon ammonia addition is matched by an extra O2 uptake as that required theoretically for the two ATP-consuming steps of urea synthesis. At 1.5 mM NH4Cl, there is no significant difference of ATP levels compared to the controls. 3 When extra urea formation and the concomitant O2 uptake are suppressed due to limitation at carbamoylphosphate synthesis in carbon-dioxide-free media, the activation of pyruvate dehydrogenase by NH4Cl is still observed. 4 When ammonia is generated intracellularly from glutamine, an extra uptake of pyruvate is not observed. 5 The NH4Cl-induced activation of pyruvate dehydrogenase is increased in presence of acetoacetate, and decreased in presence of 3-hydroxybutyrate. 6 Possible mechanisms are discussed. While some of the observations could also be explained by a direct interaction of ammonium ions with the pyruvate dehydrogenase interconversion system, the effect of ammonium ions on pyruvate dehydrogenase is interpreted to be mediated in an indirect manner by changes of the mitochondrial concentration of 2-oxoglutarate. As 2-oxoglutarate decreases during reductive amination to glutamate, a pool of high-energy compounds (eg. GTP or related substances) dependent upon 2-oxoglutarate oxidation is depleted, resulting in a de-inhibition of pyruvate dehydrogenase by the interconversion system. The detailed mechanism of the linkage between 2-oxoglutarate oxidation and pyruvate oxidation remains to be elucidated.

Purification and studies on some properties of the 4-aminobutyrate : 2-oxoglutarate transaminase from rat brain.

Using various chromatographic procedures, 4-aminobutyrate : 2-oxoglutarate transaminase from rat brain has been purified 2400 times with respect to the initial brain homogenate. The purified protein, which has a specific activity of 10 mumol times min -1, x mg-1 gave a single band by acrylamide gel electrophoresis and isoelectric focusing. It has a molecular weight of 105000 +/- 5000 and an isoelectric point of 6.8. In the presence of 0.1% sodium dodecylsulphate, a single protein band is seen on polyacrylamide gel, corresponding to a molecular weight of 57000 +/- 5000. N-terminal analysis reveals two chains with the same N-terminal amino acid, thus the enzyme may be considered as a dimer consisting of two identical subunits. The pH optimum for enzyme activity is 8.5. Studies of the enzymic reaction show that the general mechanism is of the ping-pong bi-bi model. The Km for 2-oxoglutarate at saturating 4-aminobutyrate extrapolated to saturating 2-oxoglutarate concentration is 4 mM. 2-Oxoglutarate competitively inhibits the enzyme with respect to 4-aminobutyrate, with a Ki of 1.8 times 10(-4) M. The same phenomenon is seen for the reverse reaction where the Ki is 6.6 times 10(-4) M for succinic semi-aldehyde.

Inducible accumulation of alpha-ketoglutaric acid in cultures of Streptomyces hygroscopicus JA 6599 producing a macrolide antibiotic

The excessive production of pyruvic and 2-oxoglutaric acid by S. hygroscopicus JA 6599 grown on a medium rich in complex carbon and nitrogen sources was studied. Towards the end of the first day of batch cultivation a maximum level of both keto acids in the medium was observed. By diluting the complete culture with water at 22nd hour, however, a further increase in 2-oxoglutarate concentration was induced and the antibiotic production was slightly stimulated. In diluted cultures the oxygen saturation was found to be distinctly higher than in non-diluted ones and, on the other hand, the mycelial activities of both pyruvate and 2-oxoglutarate decarboxylases were decreased. Since the 2-oxoglutarate level was strongly influenced by inhibitors of glycolysis and of citric acid cycle, it is suggested that the metabolite accumulation in diluted cultures is mainly caused by modifications of the metabolic control of carbohydrate catabolism due to an improved aeration. Furthermore, the macrolide antibiotic A 6599 produced by S. hygroscopicus JA 6599 itself was shown to interfere with the accumulation of 2-oxoglutaric acid.

Glucose tolerance and metabolic changes in human viral hepatitis.

1. Glucose tolerance and metabolic changes after intravenous glucose were studied in fifteen patients with acute viral hepatitis and in eight patients after recovery. In the acute stage glucose tolerance was significantly impaired compared with control subjects or with patients after recovery from hepatitis. 2. Serum insulin concentrations were significantly higher in fasting acute hepatitis patients than in control subjects and the time-integrated serum insulin concentration in the first 5 min after glucose was inappropriately high for the observed glucose disappearance rate, indicating insulin resistance. Serum growth hormone concentrations in male acute hepatitis patients were significantly higher than controls and suppressed poorly. 3. Fasting blood lactate concentrations were significantly elevated and after intravenous glucose there was a variable response. 2-Oxoglutarate, glutamate and glutamine concentrations from blood of fasting subjects, were significantly higher in acute hepatitis patients than controls. 4. There were no significant differences in blood ketones, glycerol and plasma free fatty acid concentrations from fasting subjects, but plasma triglycerides were significantly increased in the acute hepatitis patients. 5. The results indicate that viral hepatitis is associated with glucose intolerance and insulin resistance. After recovery the lactate response after glucose is diminished compared with controls.

Inhibition of gluconeogenesis from lactate by phenylethylbiguanide in the perfused guinea pig liver.

1. Two hours after the intraperitoneal injection of 12 mg/kg 1-β-phenylethylbiguanide (DBI) to guinea pigs fasted 48 h, the 3-hydroxybutyrate/acetoacetate ratio and the concentration of both lactate and pyruvate were increased in the blood of the vena cava. The glucose level and the lactate/pyruvate ratio were not altered. In perfused livers taken from these animals immediately after the blood samples were withdrawn, the rate of glucose formation from lactate was not inhibited. However, the addition of 2×10−5 M DBI to the perfusate caused a 60% suppression of gluconeogenesis under these conditions. — 2. The accumulation of hepatic acetyl-S-Co A with a concomitant decrease of the citrate-and of the 2-oxoglutarate concentration in the presence of DBI indicated an impaired synthesis of citrate. A slight reduction of hepatic oxygen consumption was also consistent with an inhibition of the citric acid cycle. It is concluded that biguanides primarily suppressed hepatic cell respiration, which led to an accumulation of reducing equivalents inside the mitochondria. This may have caused a lack of oxalacetate for mitochondrial citrate synthesis. — 3. The pattern of hepatic metabolite concentrations did not clearly show the rate limiting step of glucose formation from lactate in the presence of biguanides, however, some evidence was found that the conversion of pyruvate to phosphoenolpyruvate was affected. — 4. No exact correlation was found between the effect of DBI on gluconeogenesis and on the hepatic ATP/ADP ratio.

Biochemical adaptation of rat liver in response to marginal oxygen toxicity.

1. Hepatic glucose 6-phosphate dehydrogenase activity was increased in rats exposed to 5lb/in(2) (equivalent to 27000ft), 100% O(2) when compared with control animals in a 14.7lb/in(2) (sea level), air environment. Glyceraldehyde 3-phosphate dehydrogenase, isocitrate dehydrogenase, and succinate dehydrogenase were not affected by the 5lb/in(2), 100% O(2) environment. 2. Animals exposed to the hyperoxic environment consumed food, expired CO(2) and gained weight at the same rate as normoxic control animals. Additionally, blood glucose and liver glycogen concentrations were unchanged in the hyperoxic animals. The only readily apparent physiological difference in the hyperoxic animals was a decreased haematocrit. 3. The increase in glucose 6-phosphate dehydrogenase was eliminated by the injection of actinomycin D or cycloheximide. 4. Expiration of (14)CO(2) from [1-(14)C]glucose was approximately the same in hyperoxic and normoxic rats. However, (14)CO(2) expiration from [6-(14)C]glucose was markedly decreased in the animals exposed to the hyperoxic environment. 5. Calculations of the relative importance of the pentose phosphate pathway versus the tricarboxylic acid cycle plus glycolysis indicated that the livers from animals in the 5lb/in(2), 100% O(2) environment metabolized twice as much carbohydrate by way of the pentose phosphate pathway as did those from the sea-level air control animals. 6. In livers of rats exposed to 5lb/in(2), 100% O(2) the concentrations of pyruvate, citrate and 2-oxoglutarate were increased, that of isocitrate was slightly elevated, whereas the concentrations of succinate, fumarate and malate were decreased. 7. An inactivation of both tricarboxylic acid cycle lipoate-containing dehydrogenases, pyruvate and 2-oxoglutarate, under hyperoxic conditions is proposed. 8. The adaptive significance of the induction of glucose 6-phosphate dehydrogenase and the resultant production of NADPH under hyperoxic conditions is discussed.

Raised L-glutamate and 2-oxoglutarate concentrations associated with chronic hypercapnia and hypoxaemia.

Raised L-glutamate and 2-oxoglutarate concentrations associated with chronic hypercapnia and hypoxaemia.

Effect of 2-oxoglutarate on the catalytic activity and stability of glutamate dehydrogenase

1. 1. ATP-induced inhibition of the NADH oxidation reaction catalyzed by glutamate dehydrogenase ( l-glutamate:NAD(P) oxidoreducatase (deaminating), EC 1.4.1.3) is counteracted by high concentrations of 2-oxoglutarate; this holds also for the ATP activation of the NADPH oxidation reaction. 2. 2. High concentrations of both NADH and NADPH are inhibitory in the reductive amination reaction. 2-oxoglutarate and glutamate affect the catalytic activity in a different manner depending upon whether the di- or the triphosphopyridine nucleotides are coenzymes of the reaction. NADH inhibition is enhanced at high 2-oxoglutarate concentrations, while NADPH inhibition is diminished. Contrarily to the activating effect of high NAD + concentrations, high concentrations of NADP +-inhibit the oxidative deamination reaction. 3. 3. When added to the glutamate deamination assay mixture at 50 mM glutalate, low concentrations of 2-oxoglutarate exhibit competitive inhibition towards glutamate with NAD + as coenzyme, and a cooperative type of inhibition with NADP + as coenzyme of the reaction. 4. 4. It is postulated that the substrates of glutamate dehydrogenase, and especially 2-oxoglutarate, play a regulatory role in the activity of this enzyme. 5. 5. The enzyme is stabilized against inactivation in 10 mM Tris-EDTA (pH 8.0) at 25° by sub-stoichiometric concentrations of ADP and by low concentrations of 2-oxoglutarate, but not by similar or higher concentrations of glutamate. It is concluded that protection must occur through binding of the ligands, and cannot be attributed to ionic effects.

A comparison of the effects of hexobendine with those of anoxia on the concentration of myocardial metabolites in vivo

The action of hexobendine (N, N'-Dimethyl-N, N'-bis-[3(3',4',5'-trimethoxybenzoxy)-propyl]-ethyl-enediamine) was compared with the effects produced by anoxia on the concentration of various myocardial substrates in rats which had been starved for 24 hours and anaesthetized with urethane-chloralose. The following results were obtained: 1. 1) The procedure elaborated in pilot experiments to ensure minimal tissue anoxia during removal of the heart consisted of anaesthesia, artificial respiration during opening of the thorax and rapid freezing of the beating heart employing metal clamps cooled in liquid nitrogen. The myocardial concentrations of fructose-1, 6-diphosphate, glycerol-3-phosphate and lactate were taken as criteria of the virtual absence of anoxia and their concentrations in control experiments did not exceed 3.0, 1.0 and 50 μmoled/100 g wet, respectively. 2. 2) Hexobendine (1.0 mg/kg i.v.) caused a significant, but transient, increase in the myocardial concentrations of ATP, glycerol-3-phosphate, malate, citrate and free fatty acids between the first and third minute following its injection. The concentrations of glucose- and fructose-6-phosphate, glucose, glycogen, lactate, pyruvate, 2-oxoglutarate and esterified fatty acids were not significantly altered within the 10-minute period immediately following the injection of hexobendine. 3. 3) Respiratory arrest of 1 minute's duration following decapitation of the animals resulted in a significant decrease in the glycogen, ATP, citrate and 2-oxoglutarate concentrations of the heart, whereas the fructose-1, 6-diphosphate, glycerol-3-phosphate, lactate and glucose concentrations rose significantly. There was no significant alteration in the myocardial concentrations of malate, hexose phosphates, pyruvate, free fatty acids and esterified fatty acids. 4. 4) The differences between the alteration in cardiac metabolism caused by anoxia on the one hand, and the action of hexobendine on the other hand are discussed and an evaluation made of the importance of these changes in connection with the coronary dilatation resulting from anoxia and pharmacodynamic influences, respectively.