Inhibition of 2-oxoglutarate oxidation in plant mitochondria by pyruvate

Oxidation of 2-oxoglutarate (in the presence of malonate) by mitochondria isolated from turnip, pea leaf and cauliflower tissue was dramatically inhibited by micromolar concentrations of pyruvate. Pyruvate, however, had little or no effect on 2-OG oxidation when carried out in the absence of malonate. The inhibition was reversed by alpha-cyano-4-hydroxycinnamic acid, indicating pyruvate uptake into the matrix was required for the inhibitory effect. In contrast, pyruvate had no effect on 2-oxoglutarate oxidation by mitochondria isolated from rat heart. The possible significance of the effect in terms of the control of 2-oxoglutarate dehydrogenase activity during the operation of a malate/aspartate shuttle in plant mitochondria is discussed.     

A squared michaelis-menten function of substrate concentration for plant mitochondrial respiration

Dry and Wiskich ([1987] Arch Biochem Biophys 257: 92-99) have published data showing the response of plant mitochondrial respiration to increasing additions of oxaloacetate or malate when these substrates have been depleted by inhibition of succinate dehydrogenase by malonate, and coenzyme A (CoA) has been sequestered as acetyl-CoA by pyruvate dehydrogenase. In the presence of 2-oxoglutarate, it is shown that the response is given by a Michaelis-Menten curve, but in its absence, when malate has to supply substrate for dehydrogenation as well as to liberate CoA via malate dehydrogenase and citrate synthase, the response is presumably the product of two Michaelis-Menten functions, which can be approximated by the square of a single function.     

Effect of fatty acids and ketones on the activity of pyruvate dehydrogenase in skeletal-muscle mitochondria.

The presence of palmitoyl-L-carnitine and acetoacetate (separately) decreased flux through pyruvate dehydrogenase in isolated mitochondria from rat hind-limb muscle. The effect of acetoacetate was dependent on the presence of 2-oxoglutarate and Ca2+. Palmitoylcarnitine, but not acetoacetate, also decreased the mitochondrial content of active dephospho-pyruvate dehydrogenase (PDHA). This effect was large only in the presence of EGTA. Addition of Ca2+-EGTA buffers stabilizing pCa values of 6.48 or lower gave near-maximal values of PDHA content, irrespective of the presence of fatty acids or ketones when mitochondria were incubated under the same conditions used for the flux studies, i.e. at low concentrations of pyruvate. There was, however, a minor decrement in PDHA content in response to palmitoylcarnitine oxidation when the substrate was L-glutamate plus L-malate. Measurement of NAD+, NADH, CoA and acetyl-CoA in mitochondrial extracts in general showed decreases in [NAD+]/[NADH] and [CoA]/[acetyl-CoA] ratios in response to the oxidation of palmitoylcarnitine and acetoacetate, providing a mechanism for both decreased PDHA content and feedback inhibition of the enzyme in the PDHA form. However, only changes in [CoA]/[acetyl-CoA] ratio appear to underlie the decreased PDHA content on addition of palmitoylcarnitine when mitochondria are incubated with L-glutamate plus L-malate (and no pyruvate) as substrate. The effect of palmitoylcarnitine oxidation on flux through pyruvate dehydrogenase and on PDHA content is less marked in skeletal-muscle mitochondria than in cardiac-muscle mitochondria. This may reflect the less active oxidation of palmitoylcarnitine by skeletal-muscle mitochondria, as judged by State-3 rates of O2 uptake. In addition, Ca2+ concentration is of even greater significance in pyruvate dehydrogenase interconversion in skeletal-muscle mitochondria than in cardiac-muscle mitochondria.     

Acetyl-coenzyme a can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon K_m and K_i relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

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.     

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.     

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

A method is presented for the preparation of pure phthalonic acid (PTA) in high yields. This PTA was used to determine the capacity of the malate/aspartate shuttle in pea (Pisum sativum) leaf mitochondria. The inhibition of glycine-dependent O2 uptake in the combined presence of 5 mM-aspartate and 5 mM-2-oxoglutarate (2-OG) was decreased by 55 +/- 22% (n = 13) in washed and 50 +/- 2% (n = 11) in purified mitochondria by 0.23 mM-PTA. This concentration of PTA had no effect on the oxidation of 5 mM-2-OG, suggesting that part of the observed inhibition of O2 uptake in the presence of aspartate and 2-OG was due to the production of oxaloacetate (OAA) by aspartate aminotransferase external to the mitochondrial inner membrane. Levels of external aspartate aminotransferase were estimated to be 24 +/- 1% (n = 4) and 13 +/- 1% (n = 4) of the total mitochondrial activity in washed and purified mitochondria respectively. Malate/aspartate-shuttle activity was estimated directly by measuring rates of malate efflux from isolated mitochondria and was found to match estimates of shuttle activity based on the PTA-insensitive inhibition of O2 uptake. Comparisons of malate/aspartate- and malate/OAA-shuttle activities indicated potentially similar rates of NADH export from pea leaf mitochondria under conditions in vivo. These extrapolated to whole-tissue rates of 5-11 mumol of NADH.h-1.mg of chlorophyll-1. The potential role of the malate/aspartate shuttle in the support of photorespiratory glycine oxidation in leaf tissue is discussed.     

Control of glutamate oxidation in brain and liver mitochondrial systems

1. Glutamate oxidation in brain and liver mitochondrial systems proceeds mainly through transamination with oxaloacetate followed by oxidation of the α-oxoglutarate formed. Both in the presence and absence of dinitrophenol in liver mitochondria this pathway accounted for almost 80% of the uptake of glutamate. In brain preparations the transamination pathway accounted for about 90% of the glutamate uptake. 2. The oxidation of [1-¹⁴C]- and [5-¹⁴C]-glutamate in brain preparations is compatible with utilization through the tricarboxylic acid cycle, either after the formation of α-oxoglutarate or after decarboxylation to form γ-aminobutyrate. There is no indication of γ-decarboxylation of glutamate. 3. The high respiratory control ratio obtained with glutamate as substrate in brain mitochondrial preparations is due to the low respiration rate in the absence of ADP: this results from the low rate of formation of oxaloacetate under these conditions. When oxaloacetate is made available by the addition of malate or of NAD⁺, the respiration rate is increased to the level obtained with other substrates. 4. When the transamination pathway of glutamate oxidation was blocked with malonate, the uptake of glutamate was inhibited in the presence of ADP or ADP plus dinitrophenol by about 70 and 80% respectively in brain mitochondrial systems, whereas the inhibition was only about 50% in dinitrophenol-stimulated liver preparations. In unstimulated liver mitochondria in the presence of malonate there was a sixfold increase in the oxidation of glutamate by the glutamate-dehydrogenase pathway. Thus the operating activity of glutamate dehydrogenase is much less than the `free' (non-latent) activity. 5. The following explanation is put forward for the control of glutamate metabolism in liver and brain mitochondrial preparations. The oxidation of glutamate by either pathway yields α-oxoglutarate, which is further metabolized. Since aspartate aminotransferase is present in great excess compared with the respiration rate, the oxaloacetate formed is continuously removed by the transamination reaction. Thus α-oxoglutarate is formed independently of glutamate dehydrogenation, and the question is how the dehydrogenation of glutamate is influenced by the continuous formation of α-oxoglutarate. The results indicate that a competition takes place between the α-oxoglutarate-dehydrogenase complex and glutamate dehydrogenase, probably for NAD⁺, resulting in preferential oxidation of α-oxoglutarate.     

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebro-cortical mitochondria

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxyburyrate on the flux of PDHC (as determined by [1-¹⁴C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-¹⁴C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-¹⁴C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861–1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.     

The reaction of pyruvate with saccharopine dehydrogenase.

The preceding paper in this journal has reported that pyruvate could be substituted for 2-oxo-glutarate as a substrate of saccharopine dehydrogenase [epsilon-N-(L-glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine-forming) in the direction of reductive condensation. In the present communication, the kinetic mechanism of saccharopine dehydrogenase reaction with NADH, L-lysine and pyruvate as reactants is reported. The results of initial velocity study, inhibition studies with lysine analogs and a reaction product, NAD+, are consistent with an ordered mechanism with the coenzyme binding first and pyruvate last. The reaction mechanism is at variance with that of the normal reaction in which 2-oxoglutarate is the substrate, in that the order of addition of the amino and oxo acid substrates is reversed. This fact suggests that there exists a small degree of randomness in the binding of amino and oxo acid substrates. From a product inhibition study, NAD+ was shown to be the last reactant released. Saccharopine [epsilon-N-(L-glutaryl-2)-L-lysine] was found to act as a potent dead-end inhibitor of the condensation reactions (of lysine and 2-oxoglutarate, and of lysine and pyruvate) by forming an abortive E. NADH. saccharopine complex.     

Inhibition of pyruvate carboxylase by sequestration of coenzyme A with sodium benzoate

Pyruvate-dependent CO2 fixation by isolated mitochondria was strongly inhibited by sodium benzoate. Pyruvate carboxylase was identified as a site of inhibition by limiting flux measurements to assays of pyruvate carboxylase coupled with malate dehydrogenase. Benzoate reduced pyruvate-dependent incorporation of [14C]KHCO3 into malate and pyruvate-dependent malate accumulation by 74 and 72%, respectively. Aspartate-dependent malate accumulation was insensitive to benzoate, ruling out malate dehydrogenase as a site of action. Inhibition by benzoate was antagonized by glycine, which sharply accelerated conversion of benzoate to hippurate. Assays of coenzyme A and its acyl derivatives revealed inhibition to correlate with depletion of acetyl CoA and accumulation of benzoyl CoA. Depletion of acetyl CoA was sufficient to account for greater than 50% reduction in pyruvate carboxylase activity. Competition between acetyl CoA and benzoyl CoA for the activator site on pyruvate carboxylase was insignificant. Results support the interpretation that the observed inhibition of pyruvate carboxylase occurred primarily by depletion of the activator, acetyl CoA, through sequestration of coenzyme A during benzoate metabolism.     

Pyruvate transport by thermogenic-tissue mitochondria.

1. Mitochondria isolated from the thermogenic spadices of Arum maculatum and Sauromatum guttatum plants oxidized external NADH, succinate, citrate, malate, 2-oxoglutarate and pyruvate without the need to add exogenous cofactors. 2. Oxidation of substrates was virtually all via the alternative oxidase, the cytochrome pathway constituting only 10-20% of the total activity, depending on the stage of spadix development. 3. During later stages of spadix development, pyruvate oxidation was enhanced by the addition of aspartate. This was caused by acetyl-CoA condensing with oxaloacetate, produced from pyruvate/aspartate transamination, and so decreasing feedback inhibition of pyruvate dehydrogenase. 4. Pyruvate oxidation was inhibited by the long-chain acid maleimides AM5-11, but not by those with shorter polymethylene side groups, AM1-4. 5. The alpha-cyanocinnamate derivatives UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and CHCA [alpha-cyano-4-hydroxycinnamate] inhibited pyruvate-dependent O2 consumption and the carrier-mediated uptake of pyruvate across the mitochondrial inner membrane. Characteristics of non-competitive inhibition were observed for CHCA, whereas for UK5099 the results were more complex, suggesting a very low rate of dissociation of the inhibitor-carrier complex. 6. A comparison of the values of Vmax. and Km for oxidation and transport suggested that it was the latter which controls the overall rate of pyruvate oxidation by mitochondria from both tissues.     

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

(1) d(-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d- and l-lactate, n-butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD⁺ or NADH. With d-3-hydroxybutyrate as the variable reactant (K_{m app} = 0.26 mM) the inhibition constant of methylmalonate (K_is) was 0.09 mm. (3) The rate of utilization of d-3-hydroxybutyrate (78 μm) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μm methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n-octanoate in the absence of added ADP, methylmalonate (1–3 mm) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 mm) although the inhibition of total ketone formation from octanoate was more severe.     

The activities of 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase in hearts and mammary glands from ruminants and non-ruminants.

1. The activities of 2-oxoglutarate dehydrogenase (EC 1.2.4.2) were measured in hearts and mammary glands of rats, mice, rabbits, guinea pigs, cows, sheep, goats and in the flight muscles of several Hymenoptera. 2. The activity of 2-oxoglutarate dehydrogenase was similar to the maximum flux through the tricarboxylic acid cycle in vivo. Therefore measuring the activity of this enzyme may provide a simple method for estimating the maximum flux through the cycle for comparative investigations. 3. The activities of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian hearts were similar to those of 2-oxoglutarate dehydrogenase, suggesting that in these tissues the tricarboxylic acid cycle can be supplied (under some conditions) by acetyl-CoA derived from pyruvate alone. 4. In the lactating mammary glands of the rat and mouse, the activities of pyruvate dehydrogenase exceeded those of 2-oxoglutarate dehydrogenase, reflecting a flux of pyruvate to acetyl-CoA for fatty acid synthesis in addition to that of oxidation via the tricarboxylic acid cycle. In ruminant mammary glands the activities of pyruvate dehydrogenase were similar to those of 2-oxoglutarate dehydrogenase, reflecting the absence of a significant flux of pyruvate to fatty acids in these tissues.     

Specific inhibition of mitochondrial fatty acid oxidation by 2-bromopalmitate and its co-enzyme A and carnitine esters

1. The CoA and carnitine esters of 2-bromopalmitate are extremely powerful and specific inhibitors of mitochondrial fatty acid oxidation. 2. 2-Bromopalmitoyl-CoA, added as such or formed from 2-bromopalmitate, inhibits the carnitine-dependent oxidation of palmitate or palmitoyl-CoA, but not the oxidation of palmitoylcarnitine, by intact liver mitochondria. 3. 2-Bromopalmitoylcarnitine inhibits the oxidation of palmitoylcarnitine as well as that of palmitate or palmitoyl-CoA. It has no effect on succinate oxidation, but inhibits that of pyruvate, 2-oxoglutarate or hexanoate; however, the oxidation of these substrates (but not of palmitate, palmitoyl-CoA or palmitoyl-carnitine) is restored by carnitine. 4. In damaged mitochondria, added 2-bromopalmitoyl-CoA does inhibit palmitoylcarnitine oxidation; pyruvate oxidation is unaffected by the inhibitor alone, but is impaired if palmitoylcarnitine is subsequently added. 5. The findings have been interpreted as follows. 2-Bromopalmitoyl-CoA inactivates (in a carnitine-dependent manner) a pool of carnitine palmitoyltransferase which is accessible to external acyl-CoA. This results in inhibition of palmitate or palmitoyl-CoA oxidation. A second pool of carnitine palmitoyltransferase, inaccessible to added acyl-CoA in intact mitochondria, can generate bromopalmitoyl-CoA within the matrix from external 2-bromopalmitoylcarnitine; this reaction is reversible. Such internal 2-bromopalmitoyl-CoA inactivates long-chain beta-oxidation (as does added 2-bromopalmitoyl-CoA if the mitochondria are damaged) and its formation also sequesters intramitochondrial CoA. Since this CoA is shared by pyruvate and 2-oxoglutarate dehydrogenases, the oxidation of their substrates is depressed by 2-bromopalmitoylcarnitine, unless free carnitine is available to act as a ;sink' for long-chain acyl groups. 6. These effects are compared with those reported for other inhibitors of fatty acid oxidation.     

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.     

The effect of disulphides on mitochondrial oxidations

1. Nicotinamide nucleotide-linked mitochondrial oxidations were inhibited by the disulphides NNN′N′-tetraethylcystamine, cystamine and cystine diethyl ester, whereas l-homocystine, oxidized mercaptoethanol, oxidized glutathione, NN′-diacetylcystamine and tetrathionate were only slightly inhibitory. Mitochondrial oxidations were not blocked by the thiol cysteamine. 2. NAD-independent oxidations were not inhibited by cystamine. The oxidation of choline was initially stimulated. 3. The inactivation of isocitrate, malate and β-hydroxybutyrate oxidation of intact mitochondria could be partially reversed by external NAD. For the reactivation of α-oxoglutarate oxidation a thiol was also required. 4. A leakage of nicotinamide nucleotides from the mitochondria is suggested as the main cause of the inhibition. In addition, a strong inhibition of α-oxoglutarate dehydrogenase by cystamine was observed. A mixed disulphide formation with CoA and possibly also lipoic acid and lipoyl dehydrogenase is suggested to explain this inhibition.     

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.     

Persistence of the effect of insulin on pyruvate dehydrogenase activity in rat white and brown adipose tissue during the preparation and subsequent incubation of mitochondria.

Increases in the amount of the active non-phosphorylated form of pyruvate dehydrogenase in rat epididymal adipose tissue, as a result of incubation with insulin, persist not only during the preparation of mitochondria but also during subsequent incubation of coupled mitochondria in the presence of respiratory substrates. No effect on insulin was found if the hormone was added directly to mitochondria in the presence or absence of added plasma membranes. Concentrations of several possible regulators of pyruvate dehydrogenase kinase (ATP, ADP, NADH, NAD+, acetyl-CoA, CoA and potassium) were measured in rat epididymal-adipose-tissue mitochondria incubated under conditions where differences in pyruvate dehydrogenase activity persist as a result of insulin action. No alterations were found, and it is suggested that inhibition of the kinase is not the principal means by which insulin activates pyruvate dehydrogenase. The intramitochondrial concentration of magnesium was also unaffected. Differences in pyruvate dehydrogenase activity in interscapular brown adipose tissue associated with manipulation of plasma insulin concentrations of cold-adapted rats were also shown to persist during the preparation and subsequent incubation of mitochondria in the presence or absence of GDP. It is pointed out that the persistence of the effect of insulin on pyruvate dehydrogenase in incubated mitochondria will facilitate the recognition of the mechanism of this action of the hormone. Evidence that the short-term action of insulin involves an increase in pyruvate dehydrogenase phosphate phosphatase activity rather than inhibition of that of pyruvate dehydrogenase kinase is discussed.     

Tetraphenylphosphonium inhibits oxidation of physiological substrates in heart mitochondria

We show that tetraphenylphosphonium inhibits oxidation of palmitoylcarnitine, pyruvate, malate, 2-oxoglutarate and glutamate in heart mitochondria in the range of concentration (1–5 µM) commonly used for the determination of mitochondrial membrane potential. The inhibition of 2-oxoglutarate (but not other substrate) oxidation by tetraphenylphosphonium is dependent on the concentration of 2-oxoglutarate and on extramitochondrial free calcium, and the kinetic plots are consistent with a mixed type of inhibition. Our results indicate that tetraphenylphosphonium interacts with enzymes, specifically involved in the oxidation of 2-oxoglutarate, most possibly, 2-oxoglutarate dehydrogenase.