Intramitochondrial NAD Research Articles

Sensitivity of mitochondrial peptidyl-prolyl cis-trans isomerase, pyridine nucleotide hydrolysis and Ca 2+ release to cyclosporine a and related compounds

Prooxidants activate a specific Ca 2+ release pathway from mitochondria. Here we investigate the inhibitory potency of cyclosporine A and six related compounds with respect to peptidyl-prolyl cis-trans isomerase (PPIase), pyridine nucleotide hydrolysis and Ca 2+ release. Whereas the absolute inhibitory potency of the compounds varies by about three orders of magnitude, a given compound is always most effective on PPIase, followed by pyridine nucleotide hydrolysis, and least effective in Ca 2+ release inhibition. The data show that pyridine nucleotide hydrolysis is a prerequisite but not a consequence of Ca 2+ release. They also strongly suggest that PPIase participates in the Ca 2+ release mechanism from intact mitochondria by regulating the intramitochondrial NAD + glycohydrolase, and thereby ascribe a physiological function to the protein. Furthermore, a complete lack of correlation between the inhibitory potencies described here and the reported immunosuppressive activities of the drugs is evident.

Deviant energetic metabolism of glycolytic cancer cells

The central glycolytic and oxidative pathways and the ATP-producing mechanisms differ in sane and malignant cells by their regulation and dynamics. Fast-growing, poorly-differentiated cancer cells characteristically show high aerobic glycolysis. In the same way, cholesterol biosynthesis, which occurs by normal pathways in tumors, is deficient in feed-back regulation and in sterol-transport mechanism. Other metabolic ways are deficient, as for example, intramitochondrial aldehyde catabolism, at the origin of a possible acetaldehyde toxicity, which can be circumvented by the synthesis of an unusual and neutral product for mammalian cells acetoin, through tumoral pyruvate dehydrogenase. If most of the glycolytic pyruvate is deviated to lactate production, little of the remaining carbons enter a truncated Krebs cycle where citrate is preferentially extruded to the cytosol where it feeds sterol synthesis. Glutamine is the major oxidizable substrate by tumor cells. Inside the mitochondrion, it is deaminated to glutamate through a phosphate-dependent glutaminase. Glutamate is then preferentially transminated to α-ketoglutarate that enters Krebs cycle. Glutamine may be completely oxidized through the abnormal Krebs cycle only if a way of forming acetyl CoA is present: cytosolic malate entering mitochondria is preferentially oxidized to pyruvate + CO 2 through an intramitochondrial NAD(P) + -malic enzyme, whereas intramitochondrial malate is preferentially oxidized to oxaloacetate through malate dehydrogenase, thus providing a high level of intramitochondrial substrate compartmentation. These and other regulatory aberrations in tumor cells appear to be reflections of a complex set of non-random phenotypic changes, initiated by expression of oncogenes.

Uncoupling action of antibiotic sporaviridins with rat-liver mitochondria.

The effects of the glycoside antibiotic sporaviridins (SVDs) on oxidative phosphorylation of rat-liver mitochondria were examined. SVDs released state 4 respiration, dissipated transmembrane electrical potential, and accelerated ATPase activity. These facts demonstrated that SVDs are potent uncouplers of oxidative phosphorylation. During the uncoupling caused by SVDs, large amplitude swelling and oxidation of intramitochondrial NAD(P)H occurred, suggesting that SVDs greatly enhanced nonspecific permeability of the inner mitochondrial membrane. It is suggested that the uncoupling action of SVDs might be caused by dissipation of proton electrochemical potential due to an increase in the permeability of inner mitochondrial membrane.

Quinone toxicity in hepatocytes: Studies on mitochondrial Ca 2+ release induced by benzoquinone derivatives

Hepatocyte cytotoxicity caused by substituted benzoquinones was associated with increased cytosolic Ca 2+ concentration. p-Benzoquinone-induced hepatotoxicity was enhanced when the hepatocytes were loaded with Ca 2+ by preincubation with ATP. A similar order of potency of the substituted benzoquinones in releasing Ca 2+ from isolated mitochondria and inducing hepatocyte cytotoxicity was found; in decreasing order, this was 2-Br-, unsubstituted-, 2-CH 3-, 2,6-(CH 3O) 2-, 2,6-(CH 3) 2-, 2,5-(CH 3) 2-, 2,3,5-(CH 3) 3-, and 2,3,5,6-(CH 3) 4-benzoquinones (duroquinone). The cellular products of quinone metabolism, hydroquinones and glutathione conjugates, did not cause mitochondrial Ca 2+ release. Benzoquinone-induced mitochondrial Ca 2+ release was preceded by GSH conjugate formation and NAD(P)H oxidation but followed by mitochondrial swelling. With duroquinone, a slow GSH and NADPH oxidation preceded Ca 2+ release, but GSH oxidation did not occur with Se-deficient mitochondria lacking glutathione peroxidase activity. Cyanide-insensitive respiration was also observed with duroquinone but not with benzoquinone, suggesting that duroquinone undergoes redox cycling. GSH was depleted by both arylation and oxidation with 2,6-(CH 3O) 2-, 2,6-(CH 3) 2-, 2,5(CH 3) 2-, and 2,3,5-(CH 3) 3-benzoquinones. Benzoquinone concentrations that totally depleted GSH did not cause Ca 2+ release until intramitochondrial NAD(P)H was oxidized. Ca 2+ release was also prevented when NAD(P)H generation was stimulated by the presence of isocitrate or 3-hydroxybutyrate. This suggests that mitochondrial Ca 2+ release is associated with NAD(P)H oxidation catalyzed by NADH dehydrogenase with benzoquinone or by the glutathione peroxidase-glutathione reductase system with duroquinone.

Reye's syndrome: Salicylates and mitochondrial functions

The effects of aspirin (acetylsalicylate, ASA) and related compounds in the presence of Ca 2+ on the oxidative metabolism of isolated rat liver mitochondria were studied. Intact mitochondrial preparations preincubated with ASA + Ca 2+ exhibited a transient stimulation of the state 4 respiratory rate with NAD +-linked substrates, followed by an inhibition which could not be released by the addition of ADP or uncoupler. Maximum respiratory rates were achieved by subsequent addition of NAD + or succinate. The Ca 2+-transport inhibitors ruthenium red and ethylene glycol-bis-(β-aminoethyl ether) N, N′-tetraacetic acid (EGTA) prevented these effects. Five brands of commercial aspirin were tested and were as effective as purified ASA. Tylenol (acetaminophen) could reproduce these effects only at much higher (≥ 10-fold) concentrations. Other salicyl derivatives showed results qualitatively similar to ASA, with potencies in the order: acid ⋙ASA > alcohol ≥ catechol > amide, salicylate being approximately 10-fold more potent than ASA. The magnitude of the effect seen depended on the Ca 2+ (endogenous + exogenous) and salicylate concentrations/mg mitochondrial protein, and on the length of the preincubation. Added inorganic phosphate was also required. That salicylate + Ca 2+ induces an increase in the permeability of the mitochondrial inner membrane was demonstrated by the observation that 90% of the intramitochondrial NAD(P) + was released into the surrounding medium upon preincubation of intact mitochondria with these agents. Salicylate + Ca 2+ had virtually no effect on respiration with succinate (+ rotenone) as substrate at salicylate concentrations which markedly affected NAD +-linked substrate oxidation. The presence of rotenone in the preincubation mixture prevented the damaging effects of salicylate + Ca 2+ on the mitochondrial membrane, suggesting that the redox state of intramitochondrial pyridine nucleotides can modulate these effects. The results reported here are similar to those reported previously by our laboratory for the effects of Reye's plasma and allantoin + Ca 2+, and indicate that, like these agents, salicylate and salicyl compounds can potentiate the Ca 2+-induced damage to the mitochondrial inner membrane and may be another factor responsible for Reye's syndrome.

Carrier mediated GABA translocation into rat brain mitochondria

GABA added to rat brain mitochondria causes oxidation of intramitochondrial NAD(P)H as well as inducing glutamate efflux from the mitochondrial matrix. The rate of NAD(P)H oxidation shows saturation characteristics, depends on GABA transport across the mitochondrial membrane and is inhibited by non-penetrant compounds and by the metal-complexing agent bathophenanthroline. These results show the existence of a specific GABA carrier. Inhibition studies strongly suggest the existence of two separate binding sites, namely the GABA binding site and the dicarboxylates binding site, as well as suggest the presence of a metal ion (ions) at GABA binding site. The occurrence of a GABA/GLUTAMATE antiport is proposed which allows a cyclical route to account for GABA synthesis and degradation in brain.

Dependence of gluconeogenesis, urea synthesis, and energy metabolism of hepatocytes on intracellular pH.

The relationship of intracellular pH to extracellular pH has been measured in suspensions of isolated hepatocytes at 25 degrees C. The internal pH was found to be a linear function of external pH and it changed by 0.45 pH unit per 1.0 unit change in external pH. The internal [H+] was equal to the external [H+] at approximately pH 7.1. Gluconeogenesis, urea synthesis, and oxidative phosphorylation showed different dependencies on the intracellular pH. Gluconeogenesis was the most sensitive to changes in [H+] and it declined by 80% when the intracellular pH decreased from 7.1 to 6.9. Urea synthesis was less pH-dependent, decreasing by about 30% for the same change in the intracellular [H+] whereas respiratory rate showed very little dependence on pH at this temperature. Intracellular [ATP]/[ADP] decreased linearly from 8.5 to 1.5 as the intracellular pH increased from 6.8 to 7.6, while intracellular [Pi] was essentially constant at 3.2 nmol/mg of cells, wet weight. Cytochrome c became more reduced with increasing intracellular pH, from less than 10% at pH 6.8 to 35% at pH 7.7. The calculated free energy of hydrolysis of ATP was nearly independent of pH as was the free energy of electron transfer from the intramitochondrial NAD couple (calculated from the [acetoacetate]/[3-OH-butyrate] ratio) to cytochrome c.

Regulation of citrate efflux from mitochondria of oleaginous and non-oleaginous yeasts by adenine nucleotides.

The regulation of mitochondrial citrate metabolism has been investigated in oleaginous and non-oleaginous yeasts to ascertain its importance in controlling the rate of citrate efflux from mitochondria. The following observations were made: 1. Citrate efflux from mitochondria of the oleaginous yeast Candida curvata D, in the presence of L-malate and pyruvate, was stimulated by adding ATP and reduced by AMP. In the non-oleaginous yeast, Candida utilis 359, there was very little stimulation of citrate efflux by ATP but it was reduced by AMP. These effects appeared to be generalized as similar results were obtained in an examination of eight further yeasts (seven oleaginous and one non-oleaginous). 2. The effects of ATP and AMP were not observed in mitochondria whose metabolism had been inhibited by antimycin A and rotenone indicating the direct regulation of the citrate translocase was not involved. 3. In C. curvata D, ATP increased the total mitochondrial citrate content and reduced that of 2-oxoglutarate whereas AMP had the reverse effect. In C. utilis 359, AMP had a similar effect but that of ATP was much smaller. 4. To explain these observations the mitochondrial NAD+-dependent isocitrate dehydrogenase was studied in a number of yeasts. The enzyme from oleaginous yeasts had a requirement for AMP for activity and was inhibited by ATP. In non-oleaginous yeasts the enzyme was active in the absence of AMP and increased in activity as the isocitrate concentration increased. 5. The enzyme in C. curvata D was constantly more sensitive to increasing energy charge than that of the non-oleaginous yeast. These results indicate that the supply of citrate (and hence acetyl-CoA) to the cytosol is controlled by the activity of the intramitochondrial NAD+-dependent isocitrate dehydrogenase which in turn is regulated by adenine nucleotides. The sensitivity of this enzyme to the ATP/AMP ratio during lipogenesis is therefore an important control in the accumulation of lipid by yeasts.

Reconstruction of steady state in cell-free systems: Interactions between glycolysis and mitochondrial metabolism: Regulation of the redox and phosphorylation states

A reconstituted “open” system comprising respiring mitochondria and actively glycolyzing muscle extract was devised for studies of vectorially mediated interactions. Glycogen particles were the substrate for the glycolyzing enzymes. Purified soluble (F 1) ATPase was added in varying quantities to establish a range of energetic steady states. The data generally confirm our recent conclusions (Wu and Davis, (1981) Arch. Biochem., Biophys. 208, 85–89) on the relative efficacy of the adenine nucleotides and their ratios, and of inorganic phosphate on flux through rate-controlling steps of glycolysis. When mitochondrial ATP synthesis was blocked, glycolytic flux was relatively rapid, and the lactate/pyruvate ratio increased with time to values up to greater than 300. If functional mitochondria were present, glycolytic flux was very strongly suppressed, provided the energy state ( ATP ADP ) was high, and the phosphate concentration[ Pi] was low. Adenine nucleotide control of glycolysis was to a large extent lost when the steady-state ATP ADP was below about 10, or if [ Pi] was elevated. In the two-phase system containing respiring mitochondria and components of the malate-aspartate shuttle, the ATP ADP and both extra- and intramitochondrial NAD +/NADH ratios were maintained constant, and to various perturbable levels with varying energy load (ATPase). The gradient in reduction potentials attained values (Δ G rmredox ) of up to about 2.5 kcal. The extramitochondrial redox state was not positively correlated with the external phosphorylation potential ([ATP]/[ADP] × [ Pi]). The following conclusions are drawn on the basis of the present data, together with other reports (Davis, Bremer, and Åkerman (1980) J. Biol. Chem. 255, 2277–2283) and (Klingenberg and Rottenberg (1977) Eur. J. Biochem. 73, 125–130) : (a) the gradient in reduction potential is driven by the membrane potential (Δψ), mediated by the electrogenic glutamate-aspartate exchange, and the poise or set point of this gradient is a function of Δψ; and (b) the gradient of ATP ADP ratios across the membrane is also driven principally by Δψ, mediated by the electrogenic ATP-ADP exchange. Hence, segregation of phosphorylation and reduction potentials is linked through a mutually shared electrical driving force.

Reye's syndrome: Plasma-induced alterations in mitochondrial structure and function

Preincubation of rat liver mitochondria with plasma from Reye's syndrome (RS) patients induces a transient stimulation of the State 4 respiratory rate of the oxidation of NAD-linked substrates which is followed by inhibition. A loss of nearly 90% of the intramitochondrial NAD + and NADP + is also seen. The respiratory rate cannot be stimulated upon subsequent addition of ADP, but can be fully restored upon the addition of either NAD + or succinate (plus rotenone). The degree of effectiveness depends on the incubation time and the ratio of RS-plasma/mitochondrial protein. The RS-plasma effects can be eliminated by an inhibitor of mitochondrial Ca 2+ transport (ruthenium red) or by a Ca 2+ chelator (ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid). Control plasma at a concentration of 2 mg dry wt per milligram of mitochondrial protein, or 30 μ m Ca 2+ gives no effect, but can reproduce the RS-plasma effects completely when a minute amount of allantoin (10 −11 mol/mg mitochondrial protein) is also present. We conclude that allantoin and Ca 2+ can increase the permeability of mitochondrial membrane, and may be the key components responsible for the mitochondrial injuries produced by RS-plasma.

Mitochondrial myopathy: Biochemical studies revealing a deficiency of NADH-cytochrome b reductase activity

This paper presents biochemical data upon a young male with a mitochondrial myopathy characterised by weakness, severe exercise intolerance, muscle wasting and exercise-induced lactic acidaemia. Two similar cases have been previously documented (Morgan-Hughes et al. 1979). This report more precisely locates the mitochondrial defect. In vitro mitochondrial studies show markedly decreased respiratory rates with all NAD-linked substrates whilst that with flavin-linked succinate is normal. Oxidative phosphorylation is normally coupled. Mitochondrial cytochrome components as determined by low temperature spectroscopy are normal. NADH-ferricyanide reductase and primary dehydrogenase activities are present at levels far in excess of that required to support normal NAD-linked substrate oxidation rates. Intramitochondrial NAD levels are similar to those found in other mammalian muscle. It is proposed therefore that the mitochondrial defect is situated between NADH dehydrogenase and the CoQ-Cytochrome b complex; possibly being a derangement of a non-haem iron sulphur centre.

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

Mitochondria from skeletal muscle, heart and liver of strain 129/ReJ-dy dystrophic mice and their littermate controls were characterized with respect to their respiratory and phosphorylating activities. Skeletal muscle mitochondria from dystrophic mice showed significantly lower state 3 respiratory rates than controls with both pyruvate + malate and succinate as substrates (P < 0.01). ADP/O and Ca2+/O ratios were found to be normal. A decreased rate of NADH oxidation (0.01 <P < 0.05) by sonicated mitochondrial suspensions from dystrophic mice was also seen. High respiratory rates with ascorbate + phenazine methosulfate as substrates indicated that cytochrome oxidase was not rate limiting in the oxidation of either pyruvate + malate or succinate. Skeletal muscle mitochondria from dystrophic mice showed no deficiency in any of the cytochromes or coenzyme Q. Mg2+-stimulated ATPase activity was higher in dystrophic muscle mitochondria than in controls, but basal and oligomycin-insensitive activities were virtually identical to those of controls. A significant reduction in the intramitochondrial NAD+ content (0.01 <P < 0.02) was seen in dystrophic skeletal muscle as compared to controls. Heart mitochondria from dystrophic mice showed similar, though less extensive abnormalities while liver mitochondria were essentially normal. We concluded from these results that skeletal muscle mitochondria from strain 129 dystrophic mice possess impairments in substrate utilization which may result from (1) an abnormality in the transfer of electrons on the substrate side of coenzyme Q in the case of succinate oxidation; (2) a defect on the path of electron flow from NADH to cytochrome c, and (3) a deficiency of NAD+ in the case of NAD+-linked substrates.

Energy Metabolism of Spermatozoa. VII. Interactions Between Lactate, Pyruvate and Malate as Oxidative Substrates for Rabbit Sperm Mitochondria

Rabbit spermatozoa, like other mammalian spermatozoa, have part of their lactate dehydrogenase in the mitochondrial matrix. This enables lactate to be utilized directly as a NAD-linked oxidizable substrate by the mitochondria, but it also enables pyruvate to act as an oxidant of intramitochondrial NADH which should inhibit 02 uptake. These reactions have been investigated in hypotonically treated rabbit epididymal sperm (HTRES) by means of 0, uptake rate determinations and fluorimetric measurements of the reduction level of intramitochondrial NAD. Pyruvate is a potent inhibitor of lactate oxidation; the inhibition is overcome by malate. Pyruvate alone is oxidized slowly but is oxidized rapidly when malate is present. Lactate alone is oxidized rapidly but malate is required for maximal 02 uptake. Fluorimetric measurements show a high reduction level for intramitochondrial NAD with lactate as substrate; this level is increased by malate. Pyruvate reoxidizes intramitochondrial NAD reduced by lactate and inhibits 0, uptake; reversal of this inhibition by malate partially restores the NAD reduction level. Malate plus pyruvate maintain a NAD reduction level sufficient for rapid 02 uptake. We conclude that malate is essential to pyruvate and lactate oxidation by rabbit sperm mitochondria and functions by 2 mechanisms: a) The NADH generated from malate dehydrogenase does not have access to LDH and is made directly available to the respiratory chain. b) The oxaloacetate from malate oxidation stimulates pyruvate dehydrogenase by reaction with the product acetyl C0A. These mechanisms appear to act in concert to prevent inhibition of respiration by pyruvate oxidation of intramitochondrial NADH catalyzed by LDH.

The regulation of pyruvate dehydrogenase by fatty acids in isolated rabbit heart mitochondria

The rate of pyruvate oxidation by isolated rabbit heart mitochondria was inhibited by fatty acylcarnitine derivatives. The extent of inhibition by pyruvate oxidation in State 3 was greatest with palmitylcarnitine and only a minimal inhibition was observed with acetylcarnitine, while octanoylcarnitine or octanoate caused an intermediate extent of inhibition. Analyses of the intramitochondrial ATP ADP and NADH NAD + ratios under the different conditions of incubation indicated that it is unlikely that changes in either or both of these parameters were the primary negative effectors of the rate of pyruvate oxidation. A positive correlation between the decrease in the rate of pyruvate oxidation and the decrease in the level of free CoASH in the mitochondria was observed. Extraction and assay of the pyruvate dehydrogenase from rabbit heart mitochondria during the time course of the fatty acid-mediated inhibition of pyruvate oxidation indicated that pyruvate dehydrogenase was strongly inactivated when palmitylcarnitine was the fatty acid, while incubation with octanoate and acetylcarnitine resulted in less extensive inactivation of pyruvate dehydrogenase. Measurement of the effects of NADH, NAD +, acetyl-CoA, and CoASH on the inactivation of pyruvate dehydrogenase extracted from rabbit heart mitochondria indicated that NADH and acetyl-CoA activated the pyruvate dehydrogenasee kinase while CoASH strongly inhibited the kinase and NAD + was without effect. In addition, palmityl-CoA and octanoyl-CoA had little, if any, effect on the pyruvate dehydrogenase kinase activity. It was observed that palmityl-CoA but not octanoyl-CoA strongly inhibited the activity of the extracted pyruvate dehydrogenase. Hence, it is concluded that (a) decreased mitochondrial CoASH levels, which essentially remove a potent inhibitor of the pyruvate dehydrogenase kinase, (b) possibly a diminished free CoASH supply, which may be utilized as a substrate for the active complex, and (c) direct inhibitory effects of palmityl-CoA on the active form of the pyruvate dehydrogenase complex combine to make palmitylcarnitine a much more potent inhibitor of mitochondrial pyruvate oxidation than shorter chain length acylcarnitine derivatives.

Homeostatic regulation of cellular energy metabolism: experimental characterization in vivo and fit to a model

Measurements in isolated liver cells, cultured kidney cells, protozoa (Tetrahymena pyriformis), and yeast (Candida utilis) indicate that homeostatic regulation of cellular energy metabolism is of common origin. In every case near equilibrium is maintained between the transfer of reducing equivalents from the intramitochondrial NAD couple to cytochrome c and the phosphorylation of cytosolic ADP to ATP. Under conditions of constant energy demand, changes in the intracellular phosphate concentration cause an adjustment in the [ATP]/[ADP] to maintain a constant [ATP]/[ADP][Pi] and constant respiratory rate. The regulation of mitochondrial respiration occurs as part of the reactions by which reduced cytochrome c is oxidized by molecular oxygen. At similar values for the [ATP]/[ADP][Pi] the respiratory rate increases with increasing reduction of cytochrome c. A model for mitochondrial respiratory control is found to give a good fit to the data in all of the different types of cells tested.

Kinetic study of glutamate transport in rat brain mitochondria.

Abstract— The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non‐synaptosomal mitochondria, either on a glutamate‐hydroxyl antiporter or on a glutamate‐aspartate antiporter.An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N‐ethylmaleimide. Swelling of the mitochondria in iso‐osmotic NH4+‐l‐glutamate was inhibited in the presence of avenaciolide and N‐ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino‐oxyacetic acid had no effect on the glutamate‐hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double‐beam spectrophotometry, and this reduction was inhibited on the one hand by N‐ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of l‐[14C]glutamate into brain mitochondria was performed by using the centrifugation‐stop procedure. This penetration followed saturation kinetics, with a mean apparent Km of 1.56 MM at pH 7.4 and at 20°C, the value of Knax was 4.34 nmol per min per mg protein in the same conditions. IV‐Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non‐competitive with a Ki of 0.7 Mm ‐at pH 7.4 and at 20°C. The entry of glutamate was pH‐dependent and it increased 2‐fold in the pH range of 7.4 to 6.4. A temperature‐dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated EA of 12.8 kCal per mol of glutamate and a Q10 of 2.16. The activity of γ‐glutamyl transpeptidase was practically absent in these rat brain mitochondria.Oxidation of extramitochondrial NADH by the‘malate‐aspartate shuttle’reconstituted in vitro was followed in rat brain non‐synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the ‘shuttle’ did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or γ‐aminobutyrate did not replace glutamate efficiently. A high inhibition of the‘malate‐aspartate shuttle’occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of n‐ethylmaleimide, glisoxepide or n‐butylmalonate.Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.

Regulation of cellular metabolism by intracellular phosphate

1. Incubation of liver cell suspensions with 10 mM glycerol or 10 mM fructose caused a decrease in intracellular [P i] and a fall in the [ ATP] [ ADP] while the respiratory rate, the redox state of the intramitochondrial NAD couple, and the [ ATP] [ ADP] · [ P i ] remained essentially unaltered. 2. The concentration of intracellular phosphate in yeast Candida utilis was found to be dependent on phosphate concentration in the growth medium: it was 25–30 μmol/g wet weight in cells grown in the presence of 50 mM P i, 10–12 μmol/g wet weight in cells grown in 5 mM P i, and 3–4 μmol/g wet weight in cells grown with 1 mM P i. 3. The [ ATP] [ ADP] ratios were found to be highest in yeast cells grown in the presence of 50 mM P i (⩾ 30) and lowest in cells grown with 1 mM P i (8–9). The increase in [ ATP] [ ADP] was due to an increase in [ATP]. 4. The endogenous levels of glucose 6-phosphate in washed and aerated cells were lowest in cells grown in the presence of 50 mM P i and highest in those grown in the presence of 1 mM P i. A suggestion is made that the high intracellular [P i] stimulates phosphorolytic breakdown of glycogen and depletes the cellular stores of this substrate. 5. It is postulated that the cellular respiratory rate and the NADH generation rate are regulated by [ ATP] [ ADP] · [ P i ] and not by the “energy charge” as defined by Atkinson (Biochemistry 7, 4030–4034, 1968). 6. The concentration of intracellular [P i] is implicated as an important contributor to the regulatory mechanisms of cellular metabolism.

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 m m) stimulated the conversion of [1- 14C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial NADH NAD + ratio is elevated, dichloroacetate inhibited the conversion of [1- 14C]lactate to glucose. Dichloroacetate stimulated 14CO 2 production from [1- 14C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial NADH NAD + ratio, changed the metabolism of [1- 14C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 n m) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 m m [1- 14C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA 70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 m m pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.

Studies on the control of 4-aminobutyrate metabolism in ‘synaptosomal’ and free rat brain mitochondria

1. The specific activities of 4-aminobutyrate aminotransferase (EC 2.6.1.19) and succinate semialdehyde dehydrogenase (EC 1.2.1.16) were significantly higher in brain mitochondria of non-synaptic origin (fraction M) than those derived from the lysis of synaptosomes (fraction SM2). 2. The metabolisms of 4-aminobutyrate in both 'free' (non-synaptic, fraction M) and 'synaptic' (fraction SM2) rat brain mitochondria was studied under various conditions. 3. It is proposed that 4-aminobutyrate enters both types of brain mitochondria by a non-carrier-mediated process. 4. The rate of 4-aminobutyrate metabolism was in all cases higher in the 'free' (fraction M) brain mitochondria than in the synaptic (fraction SM2) mitochondria, paralleling the differences in the specific activities of the 4-aminobutyrate-shunt enzymes. 5. The intramitochondrial concentration of 2-oxoglutarate appears to be an important controlling parameter in the rate of 4-aminobutyrate metabolism, since, although 2-oxoglutarate is required, high concentrations (2.5 mM) of extramitochondrial 2-oxoglutarate inhibit the formation of aspartate via the glutamate-oxaloacetate transaminase. 6. The redox state of the intramitochondrial NAD pool is also important in the control of 4-aminobutyrate metabolism; NADH exhibits competitive inhibition of 4-aminobutyrate metabolism by both mitochondrial populations with an apparent Ki of 102 muM. 7. Increased potassium concentrations stimulate 4-aminobutyrate metabolsim in the synaptic mitochondria but not in 'free' brain mitochondria. This is discussed with respect to the putative transmitter role of 4-aminobutyrate.

Phthalonic acid, an inhibitor of α-oxoglutarate transport in mitochondria

Phthalonic acid is a powerful inhibitor of α-oxoglutarate transport in mitochondria. This conclusion is based on the following observations: 1. 1. Phthalonic acid inhibits the oxidation of α-oxoglutarate but has no effect on the oxidation of glutamate or cis-aconitate. 2. 2. With arsenite present, phthalonic acid inhibits the oxidation of glutamate plus malate and of cis-aconitate plus malate. Under these conditions α-oxoglutarate accumulates inside the mitochondria. With glutamate plus malate as substrates the inhibition is competitive with malate with a K i value of 20 μM. 3. 3. Phthalonic acid inhibits the oxidation of intramitochondrial NAD(P)H by α-oxoglutarate plus ammonia. The inhibition is competitive with respect to α-oxoglutarate with a K i of 30 μM. 4. 4. Phthalonic acid inhibits the exchange between extramitochondrial α-oxoglutarate and intramitochondrial malate.