The two catalytic components of the 2-oxoglutarate dehydrogenase complex in rat cerebral synaptic and nonsynaptic mitochondria: Comparison of the response to in vitro treatment with ammonia,hyperammonemia, and hepatic encephalopathy

The effects of in vitro treatment with ammonium chloride, hepatic encephalopathy (HE) due to thioacetamide (TAA) induced liver failure and chronic hyperammonemia produced by i.p. administration of ammonium acetate on the two components of the multienzyme 2-oxoglutarate dehydrogenase complex (OGDH): 2-oxoglutarate decarboxylase (E1) and lipoamide dehydrogenase (E3), were examined in synaptic and nonsynaptic mitochondria from rat brain. With regard to E1 the response to ammonium ions in vitro (3 mM NH₄Cl) was observed in nonsynaptic mitochondria only and was manifested by a 21% decrease of Vmax and a 35% decrease of Km for 2-oxoglutarate (2-OG). By contrast, both in vivo conditions primarily affected the synaptic mitochondrial E1: TAA-induced HE produced an 84% increase of Vmax and a 38% increase of Km for 2-OG. Hyperammonemia elevated Vmax of E1 by 110% and Km for 2-OG by 30%. HE produced no effect at all in nonsynaptic mitochondria while hyperammonemia produced a 35% increase of Vmax and a 30% increase of Km for 2-OG of E1. Both in vivo conditions produced a 20% increase of E3 activity in synaptic mitochondria, but no effect at all in nonsynaptic mitochondria. The preferential sensitivity of E1 to ammonium chloride in vitro in nonsynaptic mitochondria and hyperammonemic conditions in vivo in synaptic mitochondria may play a crucial role in the compartmentation of OGDH responses under analogous conditions. These results confirm the intrinsic differences between the OGDH properties in the synaptic and nonsynaptic brain compartments.     

A method of determining 2-oxoglutarate dehydrogenase activity in intact mitochondria

A rather simple method is suggested for measuring the activity of 2-oxoglutarate dehydrogenase of intact mitochondria. The method is based on the determination of the rate of exogenic 2-oxoglutarate decrease in the mitochondrial suspension. Experiments with sodium arsenite and comparison of kinetic parameters of the 2-oxoglutarate, dehydrogenase reaction and transport of 2-oxoglutarate to mitochondria have shown that the measurable exogenic 2-oxoglutarate oxidation rate corresponds to the 2-oxoglutarate dehydrogenase activity in intact mitochondria. The method made it possible to establish the stimulating effect of ADP on the 2-oxoglutarate dehydrogenase activity of intact mitochondria and the absence of such an effect in destructed mitochondria.     

Brain cytochrome oxidase activity of synaptic and nonsynaptic mitochondria during aging

The activity of cytochrome c oxidase was studied in aging brain on non-synaptic and intra-synaptic mitochondria from frontal cerebral cortex, hippocampus and striatum of 4, 8, 12, 16, 20 and 24 month-old Sprague-Dawley rats. Specific activities of cytochrome oxidase were significantly higher in light synaptic mitochondria than in non-synaptic or heavy ones at all the ages examined. However, enzyme activity in light mitochondria from cerebral cortex remains unchanged during aging, being increased in hippocampus and striatum. These results indicate that aging affected not only the various cerebral area (macroheterogeneity), but also the different mitochondrial populations (subcellular heterogeneity).     

Inactivation of 2-oxoglutarate dehydrogenase in rat liver mitochondria by its substrate and t-butyl hydroperoxide

Ketoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.     

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.     

Calcium sensitive isocitrate and 2-oxoglutarate dehydrogenase activities in rat liver and AS-30D hepatoma mitochondria

NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase in extracts of mitochondria from the highly malignant AS-30D rat hepatoma cell line demonstrate Ca2+ sensitivities and affinities for substrates similar to those of normal liver mitochondria. However, the maximal activities of NAD+- and NADP+-dependent isocitrate dehydrogenase were found to be 8 and 3.5 fold higher in hepatoma mitochondrial extracts than those of liver mitochondria, whereas maximal activities of succinate and 2-oxoglutarate dehydrogenases were similar in the two tissues. At pyridine nucleotide concentrations giving the lowest physiological NADH/NAD+ ratio, NAD+-isocitrate dehydrogenase activity in hepatoma mitochondrial extracts was completely inhibited at subsaturating concentrations of Ca2+, substrate, and NAD+, in contrast to rat liver mitochondrial extracts which retained significant activity.     

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.     

In vitro 14C-leucine incorporation into nonsynaptic and synaptic rat brain mitochondria isolated by Ficoll gradients

The in vitro incorporation of 14C-leucine by nonsynaptic and synaptic rat brain mitochondria purified by means of discontinuous Ficoll gradients has been characterised. The incorporation was linear for the first 45 min for both populations. Synaptic mitochondria showed a higher rate of incorporation than the nonsynaptic mitochondria at high concentrations of leucine. The incorporation was more effective in the presence of Mg2+ and inhibited by dinitrophenol. The incorporation was sensitive to chloramphenicol and insensitive to cycloheximide. Bacterial contamination was in any case lower than 1,000 colonies per ml after the incubation period. The incorporation was carried out in the presence of either an external ATP-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase or with mitochondria respiring with oxidisable substrates plus ADP (state III). The rates obtained for incorporation in this state III were higher for all the substrates assayed (succinate, pyruvate and glutamate) than in the presence of exogenous ATP. The highest rate obtained was found when glutamate was the respiratory substrate. No significant metabolic oxidation of leucine occurs in either synaptic or nonsynaptic mitochondria in the presence of exogenous ATP. Glutamate did not increase leucine uptake in any mitochondrial populations.     

Calcium transport by rat brain mitochondria and oxidation of 2-oxoglutarate

Contrary to previous reports brain mitochondria have a substantial capacity for net Ca²⁺ uptake (approx. 1.2 μeq. Ca²⁺ per mg protein) providing succinate is the oxidizable substrate. ATP stimulates calcium uptake (to 1.8 μeq. per mg protein), but is not required. The accumulation of Ca²⁺ with NAD-linked substrates is, however, significantly less. With 2-oxoglutarate, very limited Ca²⁺ uptake occurs before respiration is inhibited. At low concentrations (10 μM), Ca²⁺ stimulates the 2-oxoglutarate dehydrogenase activity of detergent solubilized mitochondria. Millimolar [Ca²⁺] is required for inhibition. Therefore, Ca²⁺ inhibition of 2-oxoglutarate oxidation can explain the low maximum uptake with this substrate, but probably not by directly effecting the dehydrogenase. Hence, the oxidation of 2-oxoglutarate can be either enhanced or suppressed depending upon the net Ca²⁺ accumulated by brain mitochondria.     

Cyclic succinic dehydrogenase activity in rat liver mitochondria

Succinic dehydrogenase activity was assayed in isolated rat liver mitochondria. Rats had been exposed for at least two weeks to a 24 hour feeding-fasting schedule, 5 hours feeding, 19 hours fasting. Enzyme activity was determined at nine specific hours over a 24 hour period. Lowest enzyme activity occured six hours after the feeding cages had been closed. The highest activity, which was 158 percent greater than the lowest activity, occurred during the next feeding period. It is concluded that time of sacrifice is an important consideration when determining succinic dehydrogenase activity.     

Synaptic and nonsynaptic mitochondria demonstrate a different degree of calcium-induced mitochondrial dysfunction

AimsSince variety in response to Ca²⁺-induced mitochondrial dysfunction in different neuronal mitochondrial populations is associated with the pathogenesis of several neurological diseases, we investigated the effects of Ca²⁺ overload on synaptic (SM) and nonsynaptic mitochondrial (NM) dysfunction and probed the effects of cyclosporin A (CsA), 4′-chlorodiazepam (CDP) and Ru360 on relieving mitochondrial damage.Main methodsSM and NM mitochondria were isolated from rats' brains (n = 5/group) and treated with various concentrations (5, 10, 100, and 200 μM) of Ca²⁺, with and without CsA (mPTP blocker), CDP (PBR/TSPO blocker) and Ru360 (MCU blocker) pretreatments. Mitochondrial function was determined by mitochondrial swelling, ROS production and mitochondrial membrane potential changes (ΔΨm).Key findingsAt 200-μM Ca²⁺, SM presented mitochondrial swelling to a greater extent than NM. At 100 and 200-μM Ca²⁺, the ROS production of SM was higher than that of NM and ΔΨm dissipation of SM was also larger. CsA, CDP and Ru360 could reduce ROS production of SM and NM with exposure to 200-μM Ca²⁺. However, only Ru360 could completely inhibit ROS generation in both SM and NM, whereas CsA and CDP could only partially reduce the ROS level in SM. Moreover, CsA and CDP pretreatments were not able to restore ΔΨm. However, Ru360 pretreatment could protect ΔΨm dissipation in both SM and NM, with complete protection observed only in NM.SignificanceOur findings suggested that mitochondrial calcium uniporter is a possible major pathway for calcium uptake in both mitochondrial populations. However, SM might have additional pathways involved in the calcium uptake.     

Aldehyde dehydrogenase activity in rat cerebral structures

By means of the quantitative histochemical method aldehyde dehydrogenase (AldDG; acidic phosphatase 1.2.1.3.) activity has been studied in neuronal structures of all parts of the rat CNS. The greatest activity has been revealed in cytoplasm of receptor (nucleus of the mesencephalic tract trigeminal nerve-1,100 stipulated units) and effector (all motor nuclei of the trunk and spinal cord: 500-800 stipulated units) cerebral neurons. In perikaryons and axons of most of the intercalated neurons AldDG activity is not great (200-300 stipulated units). A positive correlation is found between distribution of AldDG activity among the forebrain structures, on the one hand, and density of dopaminergic terminals, dopamine content and MAO activity of these structures--on the other. In the metencephalon similar correlation is found between AldDG activity and noradrenaline content and density of serotoninergic terminals. A direct dependence is stated of AldDG activity on phylogenic age of the cerebral structures. The data presented demonstrate that AldDG activity is connected with those cerebral structures that are supposed to possess, in the process of common and mediatory metabolism, a high level of natural synthesis of aldehydes.     

On the role of the mitochondrial 2-oxoglutarate dehydrogenase complex in amino acid metabolism

Mitochondria are tightly linked to cellular nutrient sensing, and provide not only energy, but also intermediates for the de novo synthesis of cellular compounds including amino acids. Mitochondrial metabolic enzymes as generators and/or targets of signals are therefore important players in the distribution of intermediates between catabolic and anabolic pathways. The highly regulated 2-oxoglutarate dehydrogenase complex (OGDHC) participates in glucose oxidation via the tricarboxylic acid cycle. It occupies an amphibolic branch point in the cycle, where the energy-producing reaction of the 2-oxoglutarate degradation competes with glutamate (Glu) synthesis via nitrogen incorporation into 2-oxoglutarate. To characterize the specific impact of the OGDHC inhibition on amino acid metabolism in both plant and animal mitochondria, a synthetic analog of 2-oxoglutarate, namely succinyl phosphonate (SP), was applied to living systems from different kingdoms, both in situ and in vivo. Using a high-throughput mass spectrometry-based approach, we showed that organisms possessing OGDHC respond to SP by significantly changing their amino acid pools. By contrast, cyanobacteria which lack OGDHC do not show perturbations in amino acids following SP treatment. Increases in Glu, 4-aminobutyrate and alanine represent the most universal change accompanying the 2-oxoglutarate accumulation upon OGDHC inhibition. Other amino acids were affected in a species-specific manner, suggesting specific metabolic rearrangements and substrate availability mediating secondary changes. Strong perturbation in the relative abundance of amino acids due to the OGDHC inhibition was accompanied by decreased protein content. Our results provide specific evidence of a considerable role of OGDHC in amino acid metabolism.     

A critique on the preparation and enzymatic characterization of synaptic and nonsynaptic mitochondria from hippocampus

1. In literature two interesting methods are described to obtain from whole pooled brains or areas three types of mitochondria, namely, those of perikaryal origin and those contained in synaptosomes. 2. However, for many types of studies, such "preparative" preparations are not useful; for example, in pharmacological studies only data from a single n number of animals may be of statistical usefulness and may be correctly analyzed by statistical tests. 3. Thus a method is described by which it was possible to characterize by enzyme activities three populations from single rat brain hippocampus. 4. During preparative "analytical" procedure, it was noted that the 10% Ficoll gradients previously used in the literature were unable to separate purified mitochondria-free mitochondria. This gradient should be 12% Ficoll for single areas. 5. In addition, when results are compared using the more appropriate omega 2t for calculations of gravity forces to be applied instead of the maximum or average g for different rotors, enzymatic characterization differed considerably among the various mitochondrial populations. 6. The above considerations are also true when different pestle clearances and/or pestle rotations speeds are used during omogenizations; also lysis conditions are essential. 7. Results showed that selected experimental conditions are to be used when subcellular fractions are to be analyzed biochemically.