Succinate Oxidation In Mitochondria Research Articles

Melatonin inhibits peroxide production in plant mitochondria

The effect of melatonin on respiration and production (release) of hydrogen peroxide during succinate oxidation in mitochondria isolated from lupine cotyledons and epicotyls of pea seedlings was studied. It has been shown for the first time that melatonin (10-7-10-3 M) had a significant inhibitory effect on the production of peroxide by plant mitochondria, which was characterized by concentration dependence and species specificity. At the same time, melatonin (at a concentration of up to 100 microns) had virtually no effect on mitochondrial respiration rate and respiratory control coefficient. The results confirm the antioxidant function of melatonin and indicate that it is involved in the regulation of ROS levels and maintenance of redox balance in plant mitochondria.

Melatonin Inhibits Peroxide Production in Plant Mitochondria.

The effect of melatonin on respiration and production (release) of hydrogen peroxide during succinate oxidation in mitochondria isolated from lupine cotyledons and epicotyls of pea seedlings was studied. It was shown for the first time that melatonin (10-7-10-3 M) had a significant inhibitory effect on the production of peroxide by plant mitochondria, which was characterized by concentration dependence and species specificity. At the same time, melatonin (at a concentration of up to 100 μM) had virtually no effect on mitochondrial respiration rate and respiratory control coefficient. The results confirm the antioxidant function of melatonin and indicate that it is involved in the regulation of ROS levels and maintenance of redox balance in plant mitochondria.

Propofol Prevents Oxidative Stress by Decreasing the Ischemic Accumulation of Succinate in Focal Cerebral Ischemia-Reperfusion Injury.

Oxidative stress caused by mitochondrial dysfunction during reperfusion is a key pathogenic mechanism in cerebral ischemia-reperfusion (IR) injury. Propofol (2,6-diisopropylphenol) has been proven to attenuate mitochondrial dysfunction and reperfusion injury. The current study reveals that propofol decreases oxidative stress injury by preventing succinate accumulation in focal cerebral IR injury. We evaluated whether propofol could attenuate ischemic accumulation of succinate in transient middle cerebral artery occlusion in vivo. By isolating mitochondria from cortical tissue, we also examined the in vitro effects of propofol on succinate dehydrogenase (SDH) activity and various mitochondrial bioenergetic parameters related to oxidative stress injury, such as the production of reactive oxidative species, membrane potential, Ca2+-induced mitochondrial swelling, and morphology via electron microscopy. Propofol significantly decreased the ischemic accumulation of succinate by inhibiting SDH activity and inhibited the oxidation of succinate in mitochondria. Propofol can decrease membrane potential in normal mitochondria but not in ischemic mitochondria. Propofol prevents Ca2+-induced mitochondrial swelling and ultrastructural changes to mitochondria. The protective effect of propofol appears to act, at least in part, by limiting oxidative stress injury by preventing the ischemic accumulation of succinate.

Metabolites-mitochondria-macrophages (MMM): new therapeutic avenues for inflammation and muscle atrophy

Mills et al. (1) have published an important article to demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages in mice change to a pro-inflammatory state, producing ATP instead by oxidative phosphorylation (OX-PHOS) to glycolysis pathway. They demonstrate that increased mitochondrial oxidation of succinate through succinate dehydrogenase (SDH) brings high mitochondrial membrane potential and increases production of reactive oxygen species (ROS). Blocking ROS production with rotenone, a complex I inhibitor and uncoupling mitochondria, results in an inhibition of this macrophagic pro-inflammatory phenotype. Also, expressing alternative oxidase, which inhibits the inflammatory phenotype, protects mice from LPS lethality.

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine todrive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing thealternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPSlethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Maintenance of homeostasis in the aging hypothalamus: the central and peripheral roles of succinate.

Aging is the phenotype resulting from accumulation of genetic, cellular, and molecular damages. Many factors have been identified as either the cause or consequence of age-related decline in functions and repair mechanisms. The hypothalamus is the source and a target of many of these factors and hormones responsible for the overall homeostasis in the body. With advanced age, the sensitivity of the hypothalamus to various feedback signals begins to decline. In recent years, several aging-related genes have been identified and their signaling pathways elucidated. These gene products include mTOR, IKK-β/NF-κB complex, and HIF-1α, an important cellular survival signal. All of these activators/modulators of the aging process have also been identified in the hypothalamus and shown to play crucial roles in nutrient sensing, metabolic regulation, energy balance, reproductive function, and stress adaptation. This illustrates the central role of the hypothalamus in aging. Inside the mitochondria, succinate is one of the most prominent intermediates of the Krebs cycle. Succinate oxidation in mitochondria provides the most powerful energy output per unit time. Extra-mitochondrial succinate triggers a host of succinate receptor (SUCN1 or GPR91)-mediated signaling pathways in many peripheral tissues including the hypothalamus. One of the actions of succinate is to stabilize the hypoxia and cellular stress conditions by inducing the transcriptional regulator HIF-1α. Through these actions, it is hypothesized that succinate has the potential to restore the gradual but significant loss in functions associated with cellular senescence and systemic aging.

Methylmalonate inhibits succinate‐supported oxygen consumption by interfering with mitochondrial succinate uptake

The effect of methylmalonate (MMA) on mitochondrial succinate oxidation has received great attention since it could present an important role in energy metabolism impairment in methylmalonic acidaemia. In the present work, we show that while millimolar concentrations of MMA inhibit succinate-supported oxygen consumption by isolated rat brain or muscle mitochondria, there is no effect when either a pool of NADH-linked substrates or N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD)/ascorbate were used as electron donors. Interestingly, the inhibitory effect of MMA, but not of malonate, on succinate-supported brain mitochondrial oxygen consumption was minimized when nonselective permeabilization of mitochondrial membranes was induced by alamethicin. In addition, only a slight inhibitory effect of MMA was observed on succinate-supported oxygen consumption by inside-out submitochondrial particles. In agreement with these observations, brain mitochondrial swelling experiments indicate that MMA is an important inhibitor of succinate transport by the dicarboxylate carrier. Under our experimental conditions, there was no evidence of malonate production in MMA-treated mitochondria. We conclude that MMA inhibits succinate-supported mitochondrial oxygen consumption by interfering with the uptake of this substrate. Although succinate generated outside the mitochondria is probably not a sig-nificant contributor to mitochondrial energy generation, the physiopathological implications of MMA-induced inhibition of substrate transport by the mitochondrial dicarboxylate carrier are discussed.

Protective role of pinacidil against adrenaline-induced myocardium injury in guinea pig liver mitochondria

AbstractWe investigated the role of the ATP-sensitive potassium channel opener pinacidil and blocker glibenclamide on guinea pig liver mitochondrial function, and a possible significance of pinacidil in the pharmacological treatment during myocardium dystrophy. First, a series of experiments was performed to determine the effect of pinacidil and glibenclamide on mitochondrial oxygen consumption. We found that pinacidil increased the rate of mitochondrial respiration for FAD-generated substrate (succinate oxidation), but was most effective for α-ketoglutarate oxidation with enhancement of respiratory control ratio. Oxidation of FAD-generated substrate inhibited efficiency of phosphorylation for α-ketoglutarate oxidation in pinacidil-treated animals. Glibenclamide decreased the rate of respiration with the lowest value of efficiency of phosphorylation, especially for α-ketoglutarate oxidation. A second series of experiments was performed to determine the effects of pinacidil and glibenclamide on oxidative phosphorylation during adrenaline-induced myocardium dystrophy. The increase in respiratory control ratio and efficiency of phosphorylation for α-ketoglutarate oxidation was greater than for succinate oxidation in mitochondria of pinacidil-pretreated animals during myocardium dystrophy. Inhibitory analysis with malonate suggested that endogenous succinate increased oxidation of NADH-generated substrates in mitochondria. Pinacidil is mainly involved in the adrenaline-induced alterations of mitochondrial function due to elevation of phosphorylation efficiency for α-ketoglutarate oxidation and a decreased level of lipid peroxidation.

A highly sensitive colorimetric microplate ferrocyanide assay applied to ascorbate-stimulated transplasma membrane ferricyanide reduction and mitochondrial succinate oxidation

Ferricyanide reduction frequently is analyzed to determine the activity of membraneous reductases. An improved, highly sensitive, and rapid method for quantitative endpoint determination of ferrocyanide is presented. Ferrocyanide is oxidized by Fe 3+ in the presence of Ferene-S under acid conditions to form a chromogenic Ferene-S/Fe 2+ complex. The latter is quantitated at 593 nm with a sensitivity of 33.2 mM −1 · cm −1. The assay is 60% more sensitive to ferrocyanide (and with a 50% lower detection limit) than the prevailing method of Avron and Shavit, which employs sulfonated bathophenanthroline as the ferrous chromogen. Both pH dependence and potential sources of interference are discussed. Using the method, a sulfhydryl-sensitive, ascorbate-stimulated transplasma membrane ferricyanide reductase was assayed in human chronic myeloid (K562) leukemia cells. Furthermore, malonate-sensitive succinate dehydrogenase activity of heart mitochondria was easily assayed with ferricyanide as terminal electron acceptor. The current method will suit routine applications demanding high throughput, robustness, and sensitivity in a 96-well plate format.

Changes in mitochondrial electron transport chain activity during insect metamorphosis

The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.

The Saccharomyces cerevisiae COQ10 Gene Encodes a START Domain Protein Required for Function of Coenzyme Q in Respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q2. Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q6, the coq10 mutant has near normal amounts of Q6 in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the "aromatic-rich protein family" Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q6/mol of protein. We propose that Coq10p is a Q6-binding protein and that in the coq10 mutant Q6 it is not able to act as an electron carrier, possibly because of improper localization.

Effects of KCN, SHAM and Oxygen Concentrations on Respiratory Properties of Purified Mitochondria Isolated from Ananas comosus (Pineapple) and Kalanchoë daigremontiana

Effects of potassium cyanide (KCN), salicylhydroxamic acid (SHAM), and oxygen concentrations on mitochondrial respiration were investigated in purified mitochondria of a typical phosphoenolpyruvate carboxykinase (PCK) crassulacean acid metabolism (CAM) plant Ananas comosus (pineapple) and a typical malic enzyme (ME)-CAM plant Kalanchoë daigremontiana. Mitochondria of A. comosus oxidized succinate and NADH faster than that of K. daigremontiana. Succinate and NADH oxidations in mitochondria of both species were partially inhibited by KCN and SHAM, indicating that these oxidations were connected to cytochrome and alternative pathways in their electron transport chain (ETC). NADH oxidation was more sensitive to KCN than succinate oxidation, suggesting that the ETC from NADH oxidation was less connected to the alternative pathway than that from succinate in mitochondria of both species. Concurrent oxidation of succinate and NADH resulted in much higher rates of cytochrome and alternative respirations than each individual oxidation alone in both species. NADH oxidation in A. comosus mitochondria was more connected to the cytochrome pathway, so A. comosus could produce much more ATP than K. daigremontiana. This capacity might be one of the fitting mechanisms of A. comosus to produce a sufficient amount of ATP for cytosolic PCK in the daytime. In addition, the reduction of oxygen concentrations decreased not only the cytochrome respiration, but also the alternative respiration on succinate oxidation in mitochondria of both species, and the decrease was greater in K. daigremontiana than in A. comosus.

Changes in midgut active ion transport and metabolism during the fifth instar of the tobacco hornworm (Manduca sexta)

During the last larval instar of Manduca sexta (tobacco hornworm), the insect enters the wandering phase, in which the animal ceases feeding and the structural reorganization of the midgut begins. Midgut metabolism and ion transport in midguts of feeding and wandering (day 5) fifth-instar tobacco hornworms were compared by measuring midgut short-circuit current (Isc), enzyme activities, tissue respiration, and mitochondrial oxidation of substrates. The midgut Isc of wandering larvae was 80% lower than the Isc of midguts from younger larvae. The activities of midgut citrate synthase and 3-hydroxyacyl CoA dehydrogenase declined significantly at wandering, indicating that aerobic capacity and fatty oxidation are diminished at this phase of development. This view was supported by the low rate of fatty acyl carnitine oxidation by mitochondria isolated from the midguts of wandering larvae. Although the activity of midgut pyruvate kinase, an index of glycolysis, did not change between feeding and wandering, lactate dehydrogenase activity was highest in the midguts of wandering larvae. This finding indicates that anaerobic glycolysis may play a role in midgut metabolism during wandering. One day prior to wandering there is a significant decline in tissue respiration and mitochondrial succinate oxidation. These metabolic changes may be early indicators of the more dramatic changes in midgut function that are to occur upon reaching the wandering phase. Because changes in midgut function occur abruptly over a 24-hr period prior to wandering, it is argued that midgut function is modulated by the commitment peak of ecdysteroids, which induces the external and behavioral characteristics associated with the wandering phase. J. Exp. Zool. 280:135–141, 1998. © 1998 Wiley-Liss, Inc.

Is complex II involved in the inhibition of mitochondrial respiration by N-methyl-4-phenylpyridinium cation (MMP+) and N-methyl-beta-carbolines?

It has been reported that N-methyl-beta-carbolinium analogues of the neurotoxic N-methyl-4-phenylpyridinium cation (MPP+) inhibit NADH-linked mitochondrial oxidations, as well as mitochondrial respiration on succinate nearly to the same extent [Fields, Albores, Neafsey and Collins (1992) Arch. Biochem. Biophys. 294, 539-544]. Those authors further claimed that MPP+ itself also blocks respiration through succinate dehydrogenase, in addition to its well-known effect on NADH dehydrogenase (Complex I), and concluded that both effects may contribute to the development of Parkinsonian symptoms. Since N-methyl-beta-carboliniums are thought to be endogenous metabolites, these findings, if verified, would have important implications on the etiology of idiopathic Parkinsonism. We have re-examined these observations, using mitochondria after full activation of succinate dehydrogenase, as well as submitochondrial particles, in which complexities due to membrane transport are not present. We report the following observations. (1) N-Methyl-beta-carboliniums inhibit mitochondrial respiration on NAD(+)-linked substrates in a time-dependent manner, and the inhibition is potentiated by the presence of tetraphenylboron anion (TPB-), as expected for positively charged compounds. (2) Unlike MPP+ itself, however, these compounds are uncouplers at higher concentrations, so that the effects seen in State 3 cannot be assigned exclusively to inhibition of NADH oxidation. (3) The effects on succinate oxidation in mitochondria, in which the full activity of the enzyme is expressed, are 1-1.5 orders of magnitude lower than on respiration via Complex I and are thus unlikely to contribute significantly to the neurotoxicity. (4) The effect of MPP+ on mitochondrial respiration via succinate dehydrogenase is trivial, in accord with previous reports from several laboratories, but contradicting the findings of Fields et al. (cited above). (5) In submitochondrial particles the inhibition of NADH oxidation (via the complete respiratory chain) has been confirmed, but it differs markedly from the action of MPP+ in two respects. First, the enhancement by TPB- is very small; secondly, the inhibition of NADH oxidation measured using ubiquinone (Q) analogues is far lower, suggesting that Complex I is not the only target. (6) In submitochondrial particles the inhibition of succinate oxidation by either O2 or Q analogues is incomplete, trivial or absent. (7) We thus conclude that we find no basis for assigning any potential biological effect of N-methyl-beta-carboliniums to the blockade of succinate oxidation.

Effects of various inhibitors including carboxin on Botrytis cinerea mitochondria isolated from mycelium

Summary — Due to the development of resistant srains, the practical control of Botrytis cinerea remains a problem. Little is known about its respiratory chain and this paper describes the preparation and the properties of B cinerea mitochondria with the aim of testing new antifungal compounds. NADH, succinate and α-ketoglutarate as substrates provided good oxygen consumption. Respiratory control ratios were high and showed that electron transport was well coupled to oxidative phosphorylation. The phosphorylation efficiencies (ADP/O) obtained with all 3 substrates were compatible with theoretical values. The addition of cytochrome c did not greatly enhance the oxygen consumption due to ascorbate: the quality of the outer mitochondrial membrane was good enough to prevent externally added cytochrome c from interacting easily with the respiratory chain. These observations indicate that the mitochondria isolated sustain a high level of structural and functional integrity. This study showed also that they were devoid of the alternative pathway resistant to antimycin A and cyanide, but that they exhibited a rotenone-insensitive respiratory route. The action and the effective doses of oligomycin, carbonylcyanide m-chlorophenylhydrazone (CCCP) and carboxyatractyloside on B cinerea mitochondria were similar to those observed with plant mitochondria. At 10 μM, carboxin completely inhibited succinate oxidation in mitochondria from B cinerea but failed to have any effect on NADH and α

Inhibition of mitochondrial succinate oxidation—Similarities and differences between N-methylated β-carbolines and MPP +

N-Methylated β-carbolinium compounds ( N-Me-BCs), including 2- N-methyl and 2,9- N,N-dimethyl analogs, structural analogs of 1-methyl-4-phenylpyridinium (MPP +), may be endogenously bioactivated, MPP +-like toxins, capable of inducing parkinsonism. Both MPP + and selected N-Me-BCs inhibit NADH-linked mitochondrial respiration (Complex I). We now show that both also inhibit succinate-supported (Complex II) respiration, the greatest inhibition (80%) being seen for 2,9-dimethylharmanium. Complex I inhibition occurs at MPP + concentrations (IC 50 = 0.17 m m) about one order of magnitude lower than Complex II inhibition (> 1.2 m m). In contrast, Complex I and Complex II inhibition by the N-Me-BCs tested occurred at similar concentrations (I, 0.1 m m; II, 0.25 m m) and concentrations similar to Complex I inhibition by MPP +. 2,9- N,N-Dimethyl-BCs, which are the permanently charged BC analogs of MPP +, show inhibitory characteristics similar to MPP +: slow onset of inhibition, potentiation by TPB, and reversal by DNP. The fact that succinate oxidation cannot bypass the Complex II inhibition by N-Me-BCs could enhance any chronic neurotoxicity of N-Me-BCs.

The effects of caffeine on the ultrastructure and mitochondrial function of the embryonic chick heart.

Results from this study indicate that caffeine (at an embryotoxic dose equal to the LD40 administered to 3-day chick embryos produced both ultrastructural and functional abnormalities in embryonic cardiac mitochondria. The principal effects of caffeine on the ultrastructure of embryonic myocardial cells were clearly suggestive of cellular injury and included: (1) a marked disruption of mitochondrial cristae with formation of intramitochondrial myelin-like figures and (2) intracellular edema. A biochemical analysis of mitochondrial function revealed that caffeine inhibited the capacity of mitochondria to oxidize succinate. However, when pyruvate and malate were employed as substrates for isolated mitochondria, caffeine did not significantly alter mitochondrial function. Interference with embryonic cardiac mitochondrial succinate oxidation and/or fragmentation of mitochondrial membranes are suggested as possible events in the pathogenesis of caffeine-induced cardiac cell injury which, in turn, may lead to the embryonic death of the chick.

Effects of Temperature, pH, and CO₂ Tension on the Metabolism of Isolated Hepatic Mitochondria of the Desert Iguana, Dipsosaurus dorsalis

Mitochondria were isolated from the liver of the desert iguana, Dipsosaurus dorsalis. The kinetics (Km, Vmax) of succinate oxidation by these mitochondria were determined at 25 C and 40 C over a range of pH values (6.5-7.5) and CO₂ tensions (0.2-41 torr). Mitochondrial succinate oxidation was very closely described by Michaelis-Menten kinetics. Vmax varied directly with temperature but was not affected by variation of either pH or PCO₂. Apparent Km for succinate was affected by pH and PCO₂ as well as temperature, varying directly with pH and PCO₂ but inversely with temperature. The effects of temperature on Km for succinate were minimized when mitochondria were incubated in media whose pH and PCO₂ approximated in vivo intracellular values. The Q10 values were calculated for three different acid-base regulatory patterns over a wide range of substrate concentrations. The data support the hypothesis that the desert iguana's normal acid-base regulatory pattern serves to minimize the effect of temperature on mitochondrial metabolism. The results, however, cannot be explained solely in terms of temperature/acid-base effects on the ionization state of proteinbound dissociable groups.

Freezing injury in the yeast respiratory system

The effect of freeze-thawing on the yeast respiratory system was studied at rapid rates of cooling. Freezing of whole cells with liquid nitrogen induced decrease of respiratory activity to under 20% of that of original cells. Mitochondria harvested from freeze-thawed cells have markedly decreased succinate oxidizing activity. Activity of succinate cytochrome c reductase was reduced significantly after freeze-thawing of whole cells while activities of succinate dehydrogenase and cytochrome c oxidase were reduced slightly. By spectrophotometric analysis it was found that about one-half the amount of cytochrome c + c 1 was eluted from mitochondria to cytosol after freeze-thawing of cells. The activities of succinate oxidation in mitochondria from freeze-thawed cells were restored to normal levels by the addition of cytochrome c. Freeze-thawing of isolated mitochondria did not induce deactivation of succinate oxidizing activities and succinate cytochrome c reductase, and no elution of cytochrome c was observed. It was concluded that the decreased respiratory activities of yeast cells by freezing of cells with liquid nitrogen can be attributed primarily to the elution of cytochrome c from mitochondria.

Acute myocardial effects of chloramphenicol in newborn pigs: a possible insight into the gray baby syndrome.

Mechanical function and mitochondrial respiration were observed in newborn pig hearts in the presence of chloramphenicol. Isolated perfused hearts exposed to chloramphenicol (25, 50, or 100 micrograms/ml) demonstrated acute reductions in pressure development and cardiac output accompanied by elevated left atrial filling pressure. The effects of chloramphenicol and chloramphenicol succinate (25, 50, 100, or 200 micrograms/ml) on oxidative activity of isolated mitochondria were also investigated. With unesterified chloramphenicol, inhibition of state 3 respiration was most apparent when glutamate and palmitylcarnitine were supplied as substrates. Inhibition of mitochondrial oxidation of succinate or glutamate was only detectable at 200 micrograms/ml. Inhibition of alpha-ketoglutarate oxidation was not seen at any concentration of antibiotic studied. Chloramphenicol succinate most strongly inhibited state 3 oxidation of succinate and alpha-ketoglutarate and had relatively mild effects on oxidation of glutamate and palmitylcarnitine. Succinate oxidation by submitochondrial particles was unaffected by chloramphenicol succinate, a result suggesting interference with succinate transport.