Hyperoxia-induced clonogenic killing of HeLa cells associated with respiratory failure and selective inactivation of Krebs cycle enzymes

Cellular intoxication by elevated concentrations of O2 may be considered as a model for accelerated cellular aging processes resulting from excessive free radical production by normal metabolic pathways. We describe here that exposure of HeLa cell cultures to 80% O2 for 2 days causes progressive growth inhibition and loss of reproductive capacity. This intoxication was correlated with inhibition of cellular O2 consumption and inactivation of 3 mitochondrial flavoproteins, i.e., partial inactivation of NADH and succinate dehydrogenases and total inactivation of alpha-ketoglutarate dehydrogenase. As alpha-ketoglutarate dehydrogenase controls the influx of glutamine/glutamate into the Krebs cycle, which is the major pathway for oxidative ATP generation in HeLa cells, the inactivation of alpha-ketoglutarate dehydrogenase was expectedly correlated with a net fall in glutamine/glutamate utilization. Furthermore, a simultaneous increase in glucose consumption and lactate production was observed, indicating that the cellular response to respiratory failure is to generate more ATP from glycolysis. In spite of this response, extensive depletion of ATP was observed. Thus, hyperoxia-induced growth inhibition and loss of clonogenicity seem to be due primarily to an impairment of mitochondrial energy metabolism resulting from inactivation of SH-group-containing flavoprotein enzymes localized at or near the inner mitochondrial membrane. These observations may be relevant for theories implicating loss of mitochondrial function as a prime factor in the aging process.     

Effect of Different Nutritional Conditions on the Synthesis of Tricarboxylic Acid Cycle Enzymes

The effect of various nutritional conditions on the levels of Krebs cycle enzymes in Bacillus subtilis, B. licheniformis, and Escherichia coli was determined. The addition of glutamate, alpha-ketoglutarate, or compounds capable of being catabolized to glutamate, to a minimal glucose medium resulted in complete repression of aconitase in B. subtilis and B. licheniformis. The synthesis of fumarase, succinic dehydrogenase, malic dehydrogenase, and isocitric dehydrogenase was not repressed by these compounds. It is postulated that glutamate or alpha-ketoglutarate is the true corepressor for the repression of aconitase. A rapidly catabolizable carbon source and alpha-ketoglutarate or glutamate must be simultaneously present for complete repression of the formation of aconitase. Conditions which repress the synthesis of aconitase in B. subtilis restrict the flow of carbon in the sequence of reactions leading to alpha-ketoglutarate but do not prevent glutamate oxidation in vivo. The data indicate that separate and independent mechanisms regulate the activity of the anabolic and catabolic reactions of the Krebs cycle in B. subtilis and B. licheniformis. The addition of glutamate to the minimal glucose medium results in the repression of aconitase, isocitric dehydrogenase, and fumarase, but not malic dehydrogenase in E. coli K-38.     

Effects of aluminum on activity of krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate.

Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: alpha-ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.     

Mitochondrial complex I,aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.     

Intracellular distribution of tricarboxylic acid cycle enzymes in liver of rainbow trout Salmo gairdneri

The intracellular distribution of enzymes of the TCA cycle was investigated in liver of rainbow trout. All enzymes of the cycle apart from succinyl thiokinase were detected. Citrate synthase, alpha-ketoglutarate dehydrogenase and succinate dehydrogenase were wholly mitochondrial. Fumarase, malate dehydrogenase, aconitase and NADP-isocitrate dehydrogenase were detected in both cytosol and mitochondria.     

Preconditioning prevents loss in mitochondrial function and release of cytochrome c during prolonged cardiac ischemia/reperfusion

Loss in mitochondrial function and induction of mitochondrial-mediated apoptosis occur as a result of cardiac ischemia/reperfusion. Brief and repeated cycles of ischemia/reperfusion, termed ischemic preconditioning, prevent or minimize contractile dysfunction and apoptosis associated with prolonged episodes of cardiac ischemia and reperfusion. The effects of preconditioning on various indices of ischemia/reperfusion-induced alterations in mitochondrial function and structure were therefore explored. Utilizing an in vivo rat model data is provided indicating that preconditioning completely prevents cardiac ischemia/reperfusion-induced: (1) loss in the activity of the redox sensitive Krebs cycle enzyme alpha-ketoglutarate dehydrogenase; (2) declines in NADH-linked ADP-dependent mitochondrial respiration; (3) insertion of the pro-apoptotic Bcl-2 protein Bax into the mitochondrial membrane; and (4) release of cytochrome c into the cytosol. The results of the current study indicate that preconditioning prevents specific alterations in mitochondrial structure and function that are known to impact cellular viability and provide insight into the collective benefits of preconditioning.     

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx) muscles

The precise mechanisms underlying skeletal muscle damage in Duchenne muscular dystrophy (DMD) remain ill-defined. Functional ischemia during muscle activation, with subsequent reperfusion during rest, has been documented. Therefore, one possibility is the presence of increased oxidative stress. We applied a model of acute hindlimb ischemia/reperfusion (I/R) in mdx mice (genetic homolog of DMD) to evaluate dynamic in vivo responses of dystrophic muscles to this form of oxidative stress. Before the application of I/R, mdx muscles showed: 1) decreased levels of total glutathione (GSH) with an increased oxidized (GSSG)-to-reduced (GSH) glutathione ratio; 2) greater activity of the GSH-metabolizing enzymes glutathione peroxidase (GPx) and glutathione reductase; and 3) lower activity levels of NADP-linked isocitrate dehydrogenase (ICDH) and aconitase, two metabolic enzymes that are sensitive to inactivation by oxidative stress and also implicated in GSH regeneration. Interestingly, nondystrophic muscles subjected to I/R exhibited similar changes in total glutathione, GSSG/GSH, GPx, ICDH, and aconitase. In contrast, all of the above remained stable in mdx muscles subjected to I/R. Taken together, these results suggest that mdx muscles are chronically subjected to increased oxidative stress, leading to adaptive changes that attempt to protect (although only in part) the dystrophic muscles from acute I/R-induced oxidative stress. In addition, mdx muscles show significant impairment of the redox-sensitive metabolic enzymes ICDH and aconitase, which may further contribute to contractile dysfunction in dystrophic muscles.     

Endotoxin challenge reduces aconitase activity in myocardial tissue

Sepsis impairs mitochondrial respiration but the mechanisms responsible are incompletely understood. We propose that Krebs cycle enzymes are inhibited in sepsis, contributing to reduced rates of oxidative phosphorylation. Hypothesis. The activities of Krebs cycle enzymes are decreased in endotoxemia and contribute to reduced rates of oxidative phosphorylation. Methods. Adult male rats received an intraperitoneal injection of either endotoxin or saline. Cardiac mitochondria were subsequently isolated and measures of mitochondrial respiration and enzyme activities performed. Main results. By 24 h post endotoxin administration, there was a 28% reduction in mitochondrial respiration (P = 0.0005) and a 24% reduction in aconitase activity (P = 0.001). Functional activity of the electron transport chain was unaffected. Conclusion. Our data demonstrate that in the heart, the administration of endotoxin significantly and selectively decreased aconitase activity in association with reduced rates of oxidative phosphorylation. We conclude that decreased activity of aconitase contributes to the endotoxin-stimulated reduction in mitochondrial respiration.     

Global transcription analysis of Krebs tricarboxylic acid cycle mutants reveals an alternating pattern of gene expression and effects on hypoxic and oxidative genes

To understand the many roles of the Krebs tricarboxylic acid (TCA) cycle in cell function, we used DNA microarrays to examine gene expression in response to TCA cycle dysfunction. mRNA was analyzed from yeast strains harboring defects in each of 15 genes that encode subunits of the eight TCA cycle enzymes. The expression of >400 genes changed at least threefold in response to TCA cycle dysfunction. Many genes displayed a common response to TCA cycle dysfunction indicative of a shift away from oxidative metabolism. Another set of genes displayed a pairwise, alternating pattern of expression in response to contiguous TCA cycle enzyme defects: expression was elevated in aconitase and isocitrate dehydrogenase mutants, diminished in alpha-ketoglutarate dehydrogenase and succinyl-CoA ligase mutants, elevated again in succinate dehydrogenase and fumarase mutants, and diminished again in malate dehydrogenase and citrate synthase mutants. This pattern correlated with previously defined TCA cycle growth-enhancing mutations and suggested a novel metabolic signaling pathway monitoring TCA cycle function. Expression of hypoxic/anaerobic genes was elevated in alpha-ketoglutarate dehydrogenase mutants, whereas expression of oxidative genes was diminished, consistent with a heme signaling defect caused by inadequate levels of the heme precursor, succinyl-CoA. These studies have revealed extensive responses to changes in TCA cycle function and have uncovered new and unexpected metabolic networks that are wired into the TCA cycle.     

Aconitase and ATP synthase are targets of malondialdehyde modification and undergo an age-related decrease in activity in mouse heart mitochondria

The main purpose of this study was to identify mitochondrial proteins that exhibit post-translational oxidative modifications during the aging process and to determine the resulting functional alterations. Proteins forming adducts with malondialdehyde (MDA), a product of lipid peroxidation, were identified by immunodetection in mitochondria isolated from heart and hind leg skeletal muscle of 6-, 16-, and 24-month-old mice. Aconitase, very long chain acyl coenzyme A dehydrogenase, ATP synthase, and alpha-ketoglutarate dehydrogenase were detected as putative targets of oxidative modification by MDA. Aconitase and ATP synthase from heart exhibited significant decreases in activity with age. Very long chain acyl coenzyme A dehydrogenase and alpha-ketoglutarate dehydrogenase activities were unaffected during aging in both heart and skeletal muscle. This suggests that the presence of a post-translational oxidative modification in a protein does not a priori reflect an alteration in activity. The biological consequences of an age-related decrease in aconitase and ATP synthase activities may contribute to the decline in mitochondrial bioenergetics evident during aging.     

The tricarboxylic acid cycle of Helicobacter pylori.

The composition and properties of the tricarboxylic acid cycle of the microaerophilic human pathogen Helicobacter pylori were investigated in situ and in cell extracts using [1H]- and [13C]-NMR spectroscopy and spectrophotometry. NMR spectroscopy assays enabled highly specific measurements of some enzyme activities, previously not possible using spectrophotometry, in in situ studies with H. pylori, thus providing the first accurate picture of the complete tricarboxylic acid cycle of the bacterium. The presence, cellular location and kinetic parameters of citrate synthase, aconitase, isocitrate dehydrogenase, alpha-ketoglutarate oxidase, fumarate reductase, fumarase, malate dehydrogenase, and malate synthase activities in H. pylori are described. The absence of other enzyme activities of the cycle, including alpha-ketoglutarate dehydrogenase, succinyl-CoA synthetase, and succinate dehydrogenase also are shown. The H. pylori tricarboxylic acid cycle appears to be a noncyclic, branched pathway, characteristic of anaerobic metabolism, directed towards the production of succinate in the reductive dicarboxylic acid branch and alpha-ketoglutarate in the oxidative tricarboxylic acid branch. Both branches were metabolically linked by the presence of alpha-ketoglutarate oxidase activity. Under the growth conditions employed, H. pylori did not possess an operational glyoxylate bypass, owing to the absence of isocitrate lyase activity; nor a gamma-aminobutyrate shunt, owing to the absence of both gamma-aminobutyrate transaminase and succinic semialdehyde dehydrogenase activities. The catalytic and regulatory properties of the H. pylori tricarboxylic acid cycle enzymes are discussed by comparing their amino acid sequences with those of other, more extensively studied enzymes.     

Endogenous mitochondrial oxidative stress: neurodegeneration, proteomic analysis, specific respiratory chain defects, and efficacious antioxidant therapy in superoxide dismutase 2 null mice

Oxidative stress and mitochondrial dysfunction have been linked to neurodegenerative disorders such as Parkinson's and Alzheimer's disease. However, it is not yet understood how endogenous mitochondrial oxidative stress may result in mitochondrial dysfunction. Most prior studies have tested oxidative stress paradigms in mitochondria through either chemical inhibition of specific components of the respiratory chain, or adding an exogenous insult such as hydrogen peroxide or paraquat to directly damage mitochondria. In contrast, mice that lack mitochondrial superoxide dismutase (SOD2 null mice) represent a model of endogenous oxidative stress. SOD2 null mice develop a severe neurological phenotype that includes behavioral defects, a severe spongiform encephalopathy, and a decrease in mitochondrial aconitase activity. We tested the hypothesis that specific components of the respiratory chain in the brain were differentially sensitive to mitochondrial oxidative stress, and whether such sensitivity would lead to neuronal cell death. We carried out proteomic differential display and examined the activities of respiratory chain complexes I, II, III, IV, V, and the tricarboxylic acid cycle enzymes alpha-ketoglutarate dehydrogenase and citrate synthase in SOD2 null mice in conjunction with efficacious antioxidant treatment and observed differential sensitivities of mitochondrial proteins to oxidative stress. In addition, we observed a striking pattern of neuronal cell death as a result of mitochondrial oxidative stress, and were able to significantly reduce the loss of neurons via antioxidant treatment.     

Modulation of mitochondrial function by hydrogen peroxide

During normal cellular metabolism, mitochondrial electron transport results in the formation of superoxide anion (O(2)) and subsequently hydrogen peroxide (H(2)O(2)). Because H(2)O(2) increases in concentration under certain physiologic and pathophysiologic conditions and can oxidatively modify cellular components, it is critical to understand the response of mitochondria to H(2)O(2). In the present study, treatment of isolated rat heart mitochondria with H(2)O(2) resulted in a decline and subsequent recovery of state 3 NADH-linked respiration. Alterations in NADH levels induced by H(2)O(2) closely paralleled changes in the rate of state 3 respiration. Assessment of electron transport chain complexes and Krebs cycle enzymes revealed that alpha-ketoglutarate dehydrogenase (KGDH), succinate dehydrogenase (SDH), and aconitase were susceptible to H(2)O(2) inactivation. Of particular importance, KGDH and SDH activity returned to control levels, concurrent with the recovery of state 3 respiration. Inactivation is not because of direct interaction of H(2)O(2) with KGDH and SDH. In addition, removal of H(2)O(2) alone is not sufficient for reactivation. Enzyme activity does not recover unless mitochondria remain intact. The sensitivity of KGDH and SDH to H(2)O(2)-mediated inactivation and the reversible nature of inactivation suggest a potential role for H(2)O(2) in the regulation of KGDH and SDH.     

Respiratory failure and stimulation of glycolysis in Chinese hamster ovary cells exposed to normobaric hyperoxia

Continuous exposure of Chinese hamster ovary (CHO) cells to an atmosphere of 98% O2, 2% CO2 (normobaric hyperoxia) leads within a period of several days to cytostasis and clonogenic cell death. Here we report respiratory failure as an important early symptom of oxygen intoxication in CHO cells, resulting in a more than 80% inhibition of oxygen consumption within 3 days of hyperoxic exposure. This inhibition appeared to be correlated with selective inactivation of three mitochondrial key enzymes, NADH dehydrogenase, succinate dehydrogenase, and alpha-ketoglutarate dehydrogenase. The latter enzyme controls the influx of glutamate into the Krebs cycle and is particularly critical for oxidative ATP generation in most cultured cells, which depends on exogenous glutamine rather than glucose as a carbon source. As expected, the inactivation of alpha-ketoglutarate dehydrogenase was correlated with a fall in cellular glutamine utilization, which became apparent from the first day of hyperoxic exposure. Thereafter, glucose utilization and lactate excretion started to increase, up to 3-fold, indicating a cellular response to respiratory failure aimed at increased ATP generation from glycolysis. However, in spite of this response, the cellular ATP level progressively decreased, up to 2.5-fold. Thus, killing of CHO cells by normobaric hyperoxia seems to be due to a severe disturbance of mitochondrial metabolism eventually leading to a depletion of cellular ATP pools.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Iron-sulfur enzyme mediated mitochondrial superoxide toxicity in experimental Parkinson's disease

Mitochondrial oxidative stress is thought to be an important pathological mediator of neuronal death in Parkinson's disease. However, the precise mechanism by which mitochondrial oxidative stress mediates the death of dopaminergic neurons of the substantia nigra remains unclear. We tested the idea that neuronal damage in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) model of Parkinson's disease results, in part, from superoxide radical toxicity via inactivation of an iron-sulfur (Fe-S) protein, mitochondrial aconitase. Administration of MPTP in mice resulted in inactivation of mitochondrial aconitase, but not fumarase in the substantia nigra. MPTP treatment mobilized an early mitochondrial pool of iron detectable by bleomycin chelation that coincided with mitochondrial aconitase inactivation. MPTP-induced mitochondrial aconitase inactivation, iron accumulation and dopamine depletion were significantly attenuated in transgenic mice overexpressing mitochondrial Sod2 and exacerbated in partial deficient Sod2 mice. These results suggest that mitochondrial aconitase may be an important early source of mitochondrial iron accumulation in experimental Parkinson's disease, and that superoxide radical toxicity manifested by oxidative inactivation of mitochondrial aconitase may play a pathogenic role in Parkinson's disease.     

Mitochondrial dysfunction in acute hyperammonemia

Acute hyperammonemia resulting from congenital urea cycle disorders, Reye syndrome or acute liver failure results in severe neuronal dysfunction, seizures and death. Increasing evidence suggests that acute hyperammonemia results in alterations of mitochondrial and cellular energy function resulting from ammonia-induced inhibition of the tricarboxylic acid cycle enzyme alpha-ketoglutarate dehydrogenase and by activation of the NMDA receptor. Antagonists of this receptor and NOS inhibitors prevent acute ammonia-induced seizures and mortality and prevent acute ammonia-induced changes in mitochondrial calcium homeostasis and cellular energy metabolism. Acute hyperammonemia also results in decreased activities of free radical scavenging enzymes and again, free radical formation due to ammonia exposure is prevented by either NMDA receptor antagonists or NOS inhibitors. Acute hyperammonemia also results in activation of "peripheral-type" benzodiazepine receptors and monoamine oxidase-B, enzymes which are localized on the mitochondrial membranes of astrocytes in the CNS. Activation of these receptors results in mitochondrial swelling and in increased degradation of monoamines, respectively. Alterations of mitochondrial function could contribute to the neuronal dysfunction characteristic of acute hyperammonemic syndromes.     

Enzymatic and metabolic evidence for a region specific mitochondrial dysfunction in brains of murine succinic semialdehyde dehydrogenase deficiency (Aldh5a1-/- mice)

Succinic semialdehyde dehydrogenase deficiency, a rare inherited defect of gamma-aminobutyrate (GABA) catabolism, presents with characteristic biochemical abnormalities in the central nervous system (CNS). These include elevated concentrations of GABA, gamma-hydroxybutyrate (GHB), succinic semialdehyde (SSA), 4,5-dihydroxyhexanoic acid (DHHA) and alanine as well as decreased concentrations of glutamine. GABA degradation is coupled to Krebs cycle function in mammalian CNS ("GABA shunt") through succinate and alpha-ketoglutarate. Accordingly, we hypothesized that disruption of Krebs cycle and respiratory chain function in the CNS is involved in the neuropathogenesis of this disease. For this purpose, we investigated cerebral activities of Krebs cycle and respiratory chain enzymes as well as the glutathione content in Aldh5a1(-/-) mice, a recently generated mouse model for this disease. In CNS tissue of Aldh5a1(-/-) mice, we found a significantly decreased glutathione content (hippocampus, cortex) and decreased activities of complexes I-IV (hippocampus) suggesting increased oxidative stress and mitochondrial dysfunction. However, specific activities of Krebs cycle and respiratory chain were not affected by GABA, GHB, SSA, or DHHA (up to 1 mmol/L). Although our results suggest hippocampal and cortical dysfunction in Aldh5a1(-/-) brain, we found no evidence that accumulating key metabolites of SSADH deficiency directly induce impairment of energy metabolism.     

Mitochondrial dysfunction and oxidative stress: cause and consequence of epileptic seizures

Mitochondrial dysfunction has been implicated as a contributing factor in diverse acute and chronic neurological disorders. However, its role in the epilepsies has only recently emerged. Animal studies show that epileptic seizures result in free radical production and oxidative damage to cellular proteins, lipids, and DNA. Mitochondria contribute to the majority of seizure-induced free radical production. Seizure-induced mitochondrial superoxide production, consequent inactivation of susceptible iron–sulfur enzymes, e.g., aconitase, and resultant iron-mediated toxicity may mediate seizure-induced neuronal death. Epileptic seizures are a common feature of mitochondrial dysfunction associated with mitochondrial encephalopathies. Recent work suggests that chronic mitochondrial oxidative stress and resultant dysfunction can render the brain more susceptible to epileptic seizures. This review focuses on the emerging role of oxidative stress and mitochondrial dysfunction both as a consequence and as a cause of epileptic seizures.     

Association between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase

The kinetic parameters of the individual reaction of pig heart alpha-ketoglutarate dehydrogenase complex, succinate thiokinase and the alpha-ketoglutarate dehydrogenase complex-succinate thiokinase coupled system were studied. The KCoAm of alpha-ketoglutarate dehydrogenase complex and the K-succinyl CoAm of succinate thiokinase decreased in the coupled system when compared to those of the individual enzyme reactions. This phenomenon can be explained by the interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. By means of poly(ethylene glycol) precipitation, ultracentrifugation and gel chromatography we were able to detect a physical interaction between the alpha-ketoglutarate dehydrogenase complex and succinate thiokinase. Of the seven investigated proteins only succinate thiokinase showed association with alpha-ketoglutarate dehydrogenase complex. On the other hand, succinate thiokinase did not associate with other high molecular weight mitochondrial enzymes such as pyruvate dehydrogenase complex and glutamate dehydrogenase. On this basis, the interaction between succinate thiokinase and alpha-ketoglutarate dehydrogenase complex was assumed to be specific. These in vitro data raise the possibility that a portion of the citric acid cycle enzymes exists as a large multienzyme complex in the mitochondrial matrix.