Effects of palmityl coenzyme A and palmitylcarnitine on phosphorylating respiration in heart mitochondria

Glutamate-supported respiration in mitochondria is inhibited by palmityl-CoA in the presence of carnitine. Palmityl-CoA-induced lag phase and depressed state 3 rates increase with increasing ADP. Palmityl-CoA inhibition of state 3 respiration with glutamate shows an increased I₅₀ for palmityl-CoA (three to fourfold) when ADP increases and carnitine is present. ADP alone has a small effect. Glutamate-supported respiration is more profoundly inhibited by palmityl-CoA (+carnitine) than palmityl-CoA oxidation. With palmityl-CoA (+ carnitine) alone, the I₅₀ for palmityl-CoA is two-to threefold greater than when glutamate is also present. Active respiration with palmityl-CoA as substrate demonstrates a 2.5-fold greater apparent affinity for ADP than when glutamate is also present. The kinetics are competitive in both cases. Palmitylcarnitine, above 30 μm, produces inhibition of glutamate-supported respiration, concomitant with mitochondrial swelling and eventual lysis. At 15 μm palmitylcarnitine (minimal swelling), succinate (+ rotenone)-supported respiration decreases with a decrease in K_app for ADP; no effect of 15–20 μm palmitylcarnitine on glutamate-supported respiration is observed. However, palmityl-CoA (+ carnitine)-inhibited respiration with glutamate is further decreased with 15 and 20 μm palmitylcarnitine, i.e., by 13 and 29%, respectively. Inhibition is competitive with ADP. With 3 μm palmitylCoA and 20 μm palmitylcarnitine, a decrease in carnitine (1.5 to 0.25 mm) decreases the apparent K_i for palmityl-CoA from 2.6 to 1.8 μm. The results suggest that glutamate increases the palmityl-CoA available to inhibit adenine nucleotide transport. Inhibition may take place external to the inner membrane. Competition of carnitine and palmitylcarnitine for substrate sites may explain the decreased apparent K_i for palmityl-CoA as carnitine decreases.     

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 $\text{ATP}\text{ADP}$ and $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ 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.     

Palmityl-CoA and palmitylcarnitine interactions in mitochondrial fatty acid elongation

We have investigated interactions of palmityl-CoA and l-palmitylcarnitine as substrates for mitochondrial fatty acid elongation. l-Palmitylcarnitine is a more effective substrate primer for fatty acid elongation by intact mitochondria than is palmityl-CoA. Exogenous l-carnitine inhibited l-palmitylcarnitine-supported mitochondrial fatty acid elongation by both sonically disrupted and intact heart mitochondria, probably by shifting the equilibrium between palmitylcarnitine and palmityl-CoA toward palmitylcarnitine, thus removing palmityl-CoA from the reaction. d-Carnitine was without effect. d-Palmitylcarnitine inhibition of palmitylcarnitine transferase activity decreased palmitylcarnitine-stimulated mitochondrial fatty acid elongation but increased palmityl-CoA supported fatty acid elongation, presumably by increasing the effective concentration of palmityl-CoA in the assay medium. The data indicate that, although l-palmitylcarnitine is an effective substrate primer for mitochondrial fatty acid elongation, palmityl-CoA rather than palmitylcarnitine is the immediate precursor for fatty acid chain elongation.     

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.     

Suppression of the mitochondrial oxidation of (-)-palmitylcarnitine by the malate-aspartate and alpha-glycerophosphate shuttles.

Palmitylcarnitine oxidation by isolated liver mitochondria has been used to investigate the interaction of fatty acid oxidation with malate, glutamate, succinate, and the malate-aspartate shuttle. Mitochondria preincubated with fluorocitrate were added to a medium containing 2mM ATP and ATPase. This system, characterized by a high energy change, allowed titration of respiration to any desired rate between States 4 and 3 (Chance, B., and Williams, G. R. (1956) Adv. Enzymol. Relat. Areas Mol. Biol. 17, 65-134). When respiration (reference, with palmitylcarnitine and malate as substrates) was set at 75% of State 3, the oxidation of palmitylcarnitine was limited by acetoacetate formation. The addition of malate or glutamate approximately doubled the rate of beta oxidation. Malate circumvented this limitation by citrate formation, but the effect of glutamate apparently was due to enhancement of the capacity for ketogenesis. The rate of beta oxidation was curtailed when malate and glutamate were both present. This curtailment was more pronounced when the malate-aspartate shuttle was fully reconstituted. Among the oxidizable substrates examined, succinate was most effective in inhibiting palmitylcarnitine oxidation. Mitochondrial NADH/NAD+ ratios were correlated positively with suppression of beta oxidation. The degree of suppression of beta oxidation by the malate-aspartate shuttle (NADH oxidation) or by succinate oxidation was dependent on the respiratory state. Both substrates extensively reduced mitochondrial NAD+ and markedly suppressed beta oxidation as respiration approached State 4. Calculations of the rates of flux of hydrogen equivalents through beta oxidation show that the suppression of beta oxidation by glutamate or by the malate-aspartate shuttle is accounted for by increased flux of reducing equivalents through mitochondrial malic dehydrogenase. This increased Flux is accompanied by an increase in the steady state NADH/NAD+ ratio and a marked decrease in the synthesis of citrate. The alpha-glycerophosphate shuttle was reconstituted with mitochondria isolated from rats treated with L-thyroxine. This shuttle was about equal to the reconstructed malate-aspartate shuttle in supression of palmitylcarnitine oxidation. This interaction could not be demonstrated in euthyroid animals owing to the low activity of the mitochondrial alpha-glycerol phosphate dehydrogenase. It is concluded that beta oxidation can be regulated by the NADH/NAD+ ratio. The observed stimulation of flux through malate dehydrogenase both by glutamate and by the malate-aspartate shuttle results in an increased steady state NADH/NAD+ ratio, and is linked to a stoichiometric outward transport of aspartate. We suggest, therefore, that some of the reducing pressure exerted by the malate-aspartate shuttle and by glutamate plus malate is provided through the energy-linked, electrogenic transport of aspartate out of the mitochondria. These results are discussed with respect to the mechanism of the genesis of ethanol-induced fatty liver.     

Cellular energy metabolism during fetal development. II. Fatty acid oxidation by the developing heart

In view of the importance of fatty acids as substrates for the mature heart, fatty acid oxidation by fetal and calf heart mitochondria has been investigated. Free fatty acids of 10 carbon units or less which exhibit carnitine-independent transport into mitochondria were effective substrates for oxidative phosphorylation in both fetal and calf heart mitochondria. Efficient oxidative phosphorylation with these substrates was dependent upon the presence of bovine serum albumin in the assay medium to reverse the uncoupling effects of the fatty acids. In the presence of bovine serum albumin, ADP/0 ratios were in the range of 3 when short-chain fatty acids and carnitine esters of short- and long-chain fatty acids were substrates. Compared with calf heart mitochondria, fetal heart mitochondria showed decreased carnitine-dependent oxidation of palmityl-CoA. However, the oxidation of palmitylcarnitine was identical in both. These data suggest that the formation of palmitylcarnitine is rate limiting for palmityl-CoA oxidation by the fetal heart mitochondria and that long-chain fatty acids are not readily oxidized by the fetal heart.     

Cellular energy metabolism during fetal development. IV. Fatty acid activation, acyl transfer and fatty acid oxidation during development of the chick and rat

We have investigated developmental profiles of ATP-dependent palmityl-CoA synthetase, acetyl-CoA synthetase, palmitylcarnitine transferase, and fatty acid oxidation in heart and liver of developing chicks and rats. Palmityl-CoA synthetase activity of rat liver and heart homogenates increased 6- to 10-fold during the first postnatal week. Chick embryo heart activity peaked between 13 and 16 days of development. The activity of embryonic chick livers was bimodal with highest activity seen at 7 and 16 days of development. Posthatching values were approximately 50–75% of the peak embryonic levels. Acetyl-CoA synthetase activity of rat liver and heart homogenates was low but also showed developmental increases following birth. Acetyl-CoA synthetase activity of chick embryonic hearts was greatest at 16 days of development. Palmitylcarnitine transferase activity of rat liver and heart homogenates showed a striking increase during the first week of life. Chick heart activity was similar to that observed for palmityl-CoA synthetase with a peak between 13 and 16 days of embryonic development. Coincident with the postnatal rise in fatty acid activation and palmitylcarnitine transferase activity in developing rats, the oxidation of palmityl-CoA plus carnitine and of palmitylcarnitine increased from barely measurable levels at birth to adult levels by 30 days of age. The increases that we observe probably relate to changes in the specific activity of the enzymes as well as to an increase in the absolute number of mitochondria during development.     

Thyroxine-induced changes in rat liver mitochondrial ubiquinone

Ubiquinone was extracted from liver mitochondria isolated from euthyroid and hyperthyroid rats. The redox state of ubiquinone was determined during States III and IV respiration with succinate or glutamate-malate substrates. Ubiquinone was more reduced during State III or IV in the hyperthyroid mitochondria with either substrate. Furthermore, the concentration of ubiquinone increased in the hyperthyroid rats.     

Effects of short-term ozone exposure on lung mitochondrial oxidative and energy metabolism

Ozone effects on lung mitochondrial oxidative metabolism were examined after short-term exposure of rats and monkeys to O₃. Exposure of animals to 2 ppm O₃ for 8 hr or to 4 ppm O₃ for 4 hr caused a 15–27% (P < 0.05) depression of lung mitochondrial O₂ consumption, using 2-oxoglutarate, succinate, and glycerol-1-phosphate. but not ascorbate plus Wurster's blue as substrates. Under these exposure conditions (4 ppm 4 hr) the ADP:O ratios dropped 25–36% (P < 0.05) and the respiratory control indices decreased 27–33% (P < 0.02) for oxidation of all substrates examined. Lung mitochondria from control animals were relatively impermeable to added NADH, but those from O₃-exposed animals showed an increased permeability as judged from NADH oxidation at a rate 3-fold higher than the control. Likewise, added cytochrome c caused a 22% (P < 0.01) stimulation of succinate oxidation in exposed lung mitochondria as against 5% (nonsignificant) in controls. Ozone exposure also caused a 20% (P < 0.01) oxidation of thiol groups in lung mitochondria, but no lipid peroxidation products were detectable in O₃-exposed lung tissue. The depression of substrate utilization, coupled phosphorylation and respiratory control observed in lung mitochondria of O₃-exposed animals might be related to alteration of membrane permeability, and inhibition of respiratory enzymes (dehydrogenases) due to oxidation of functional thiol groups.     

Oxidations in kidney mitochondria of heat-exposed rats: Regulation by cytochromec

Exposure of rats to higher environmental temperature (36–37°C) decreased the capacity of their kidney mitochondria to oxidize succinate. The decrease was corrected on the addition of exogenous cytochromec. Kidney mitochondria of heat-exposed animals showed decreased rates of H₂O₂ generation when -glycerophosphate, but not succinate, was used as electron donor. These mitochondria also showed decreased activity of -glycerophosphate dehydrogenase but not of succinate dehydrogenase. The content of cytochromec in kidney mitochondria of heat-exposed animals was low even though the concentration of the pigment in the whole tissue did not decrease. Starvation as well as administration of an antithyroid agent like propylthiouracil simulated some of the effects of heat exposure on kidney mitochondria, but the cytochromec-dependent reversal of inhibition of oxidation was obtained only in heat exposure.     

Metabolism of calcium and effect of divalent cations on respiratory activity of yeast mitochondria

The adsorption of Ca²⁺ to the mitochondria ofSaccharomyces cerevisiae was investigated and it was found that, in contrast with animal mitochondria, Ca²⁺ is not accumulated through an energydependent process but is more probably adsorbed to mitochondrial membranes. The adsorption magnitude depends both on the amount of added calcium and on the ionic composition of the medium. It was found by study of the effect of divalent cations on the respiratory activity of yeast mitochondria that (a) Ca²⁺ and Mg²⁺ inhibit their oxidation competitively with succinate or citrate, the oxidation of NADH not being affected; (b) stimulation of oxidation of NADH and inhibition of oxidation of citrate and succinate may be observed with Ca²⁺ in the mitochondria ofTorulopsis utilis and with Co²⁺ in the mitochondria ofSaccharomyces cerevisiae; (c) Zn²⁺ inhibits the oxidation of NADH and of citrate; (d) the rate of oxidation of NADH in the presence of Cd²⁺ is several-fold greater than State 3 activity—on the other hand, oxidation of suceinate and citrate is inhibited by cadmium. In comparison with animal mitochondria, the fate of Ca²⁺ as well as the effects of other divalent cations on the respiratory activity of yeast mitochondria are different.     

Valinomycin induced energy-dependent mitochondrial swelling,cytochrome <Emphasis Type="Italic">c</Emphasis> release,cytosolic NADH/cytochrome <Emphasis Type="Italic">c</Emphasis> oxidation and apoptosis

In valinomycin induced stimulation of mitochondrial energy dependent reversible swelling, supported by succinate oxidation, cytochrome c (cyto-c) and sulfite oxidase (Sox) [both present in the mitochondrial intermembrane space (MIS)] are released outside. This effect can be observed at a valinomycin concentration as low as 1 nM. The rate of cytosolic NADH/cyto-c electron transport pathway is also greatly stimulated. The test on the permeability of mitochondrial outer membrane to exogenous cyto-c rules out the possibility that the increased rate of exogenous NADH oxidation could be ascribed either to extensively damaged or broken mitochondria. Accumulation of potassium inside the mitochondria, mediated by the highly specific ionophore valinomycin, promotes an increase in the volume of matrix (evidenced by swelling) and the interaction points between the two mitochondrial membranes are expected to increase. The data reported and those previously published are consistent with the view that “respiratory contact sites” are involved in the transfer of reducing equivalents from cytosol to inside the mitochondria both in the absence and the presence of valinomycin. Magnesium ions prevent at least in part the valinomycin effects. Rather than to the dissipation of membrane potential, the pro-apoptotic property of valinomycin can be ascribed to both the release of cyto-c from mitochondria to cytosol and the increased rate of cytosolic NADH coupled with an increased availability of energy in the form of glycolytic ATP, useful for the correct execution of apoptotic program.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

Bioenergetics of mitochondria from flight muscles of aging blowflies: partial reactions of oxidation and phosphorylation.

Mitochondria from flight muscles of senescent blowflies, Phormia regina, which exhibit decreased rates of coupled, or ADP-stimulated, oxidation of α-glycerolphosphate or pyruvate show similar decreased rates of uncoupled, or carbonylcyanide-p-trifluoromethoxyphenylhydrazone-stimulated oxidation. Thus, uncoupled α-glycerolphosphate oxidation is decreased by 20% and uncoupled pyruvate oxidation by 39% in mitochondria isolated from 31- to 33-day-old blowflies as compared with mitochondria from 7- to 9-day-old flies. The finding of nearly equal decreases with age in coupled and uncoupled respiration suggests that the age-dependent defect lies within the oxidative or electron transport pathway, and not associated with phosphorylation. However, no such change with age is observed in any of the partial reactions of electron transport that were examined. These include the following partial reactions: (a) pyruvic dehydrogenase; (b) pyruvate-ferricyanide reductase: (c) α-glycerolphosphate dehydrogenase; (d) α-glycerolphosphate-ferricyanide reductase; and (e) cytochrome oxidase. In addition, no age-associated decline is observed in the content of cytochromes b, c + c₁, a, or a₃ or in the specific activity of the fully activated mitochondrial ATPase (assayed in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone). However, the specific activity of the masked ATPase (assayed in the absence of uncoupler) is increased 38% in mitochondria from senescent blowflies.     

Effect of Aging on the Metabolism of Pyruvate and 3-Hydroxybutyrate in Nonsynaptic and Synaptic Mitochondria from Rat Brain

Abstract: Age-dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24-month-old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12-and 24-month-old rats compared with 3-month-old animals. A similar age-dependent reduction in the oxidation of [1-¹¹C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24-month-old rats. When DL-3-hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24-month-old rats compared with 3- month-old animals. Similarly, an age-dependent reduction in the oxidation of 3-hydroxy[3-¹¹C]butyrate was also observed only in synaptic mitochondria from 12-and 24-month-old rats. However, a significant reduction in the activities of ketone body-metabolizing enzymes, namely, 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA transferase, and acetoacetyl-CoA thiolase was observed in both mitochondrlal populations from 12- and 24-month-old rats compared with 3 month-old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.     

Modified oscillation behavior and decreased D-3-hydroxybutyrate dehydrogenase activity in diabetic rat liver mitochondria

One month after induction of diabetes in adult white rats with streptozotocin or 4–10 months after its induction by pancreatectomy (in every case glycemia was over 3 g/liter), the following alterations were observed in liver mitochondria: (a) a decrease of amplitude and an increase of the damping factor of volume oscillations induced by potassium ions and valinomycin; (b) a 50% decrease of d-3-hydroxybutyrate dehydrogenase (HBD) activity in mitochondria disrupted by repeated freeze-thawing; (c) a similar decrease in the rate of d-3-hydroxybutyrate oxidation by intact mitochondria; (d) a significant increase of cytochrome oxidase activity and cytochrome aa₃ content. Measurement of succinate dehydrogenase and NADH dehydrogenase activity, the cytochrome b, c₁, and c content, and the P:O ratio for mitochondria oxidizing d-3-hydroxybutyrate did not reveal significant differences between control and diabetic rat mitochondria. In the streptozotocin-injected rats, the variation of HBD activity and the modification of the mitochondrial oscillation pattern were time-dependent phenomena, both effects reaching their maximal expression about 1 month after the onset of diabetes. The variation of HBD activity followed a biphasic course, since it rose to above the control level during the first 2 weeks of diabetes, then fell progressively to about half the control value after the third week. Treatment of diabetic rats with NPH insulin (5 IU twice daily, for 3 days, reinforced by the same dose 45 min before sacrifice) restored the mitochondrial oscillation pattern, HBD activity, and rate of d-3-hydroxybutyrate oxidation by intact mitochondria to their normal values.     

Augmentation of mitochondrial oxidative metabolism in lung tissue during recovery of animals from acute ozone exposure

After O₃-mediated lung injury in rats (3 ppm O₃ exposure for 4 hr) recovery was studied in terms of alteration in lung mitochondrial oxidative metabolism. As judged from O₂ consumption, succinate oxidation in lung homogenate exhibited a 20% (P < 0.05) decrease at 0 hr but attained the control rate (0.6 μmole O₂/min/lung) within 12 hr and the peak rate (55% over control, P < 0.001) within 48 hr of recovery. Thereafter, the rate plateaued and at about the fifth day began to decline, exhibiting only a 15% (P < 0.05) increase over control after 21 days. The half-life for duration of this augmentation appeared to be 10 days. During recovery, the yield of isolated mitochondria was increasingly greater for exposed lungs relative to control (viz., 25–30% increase after 96 hr) as viewed from mitochondrial packed volume and protein content. Mitochondria from exposed lungs exhibited a 17–24% (P < 0.05) increase in activity (per mg of protein) for oxidation of 2-oxoglutarate, succinate, glycerol-1-phosphate, and ascorbate-Wurster's blue. The over-all augmentation of O₂ consumption observed in exposed rat lungs, therefore, would be attributable primarily to increase in population of mitochondria. Enhanced mitochondrial metabolism might serve as an index for assessing the repair process of injured lung.     

A durohydroquinone oxidation site in the mitochondrial transport chain

Although duroquinone had little effect upon NADH oxidation in neutral lipid depleted mitochondria, durohydroquinone was oxidized by ETP at a rate sensitive to antimycin A. Fractionation of mitochondria into purified enzyme systems showed durohydroquinone: cytochromec reductase to be concentrated in NADH: cytochromec reductase, absent in succinate:cytochromec reductase, and decreased in reduced coenzyme Q:cytochromec reductase. Durohydroquinone oxidation could be restored by recombining reduced coenzyme Q:cytochromec reductase with NADH:coenzyme Q reductase. Pentane extraction had no effect upon either durohydroquinone or reduced coenzyme Q₁₀ oxidation, indicating lack of a quinone requirement between cytochromesb andc. Both chloroquine diphosphate and acetone (96%) treatment irreversibly inhibited NADH but not succinate oxidation. Neither reagents had any effect upon durohydroquinone oxidation but both inhibited reduced coenzyme Q₁₀ oxidation 50%, indicating a site of action between Q₁₀ and duroquinone sites. Loss of chloroquine sensitive reduced coenzyme Q₁₀ oxidation after acetone extraction suggests two sites for Q₁₀ before cytochromeb.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and -ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1–50 μM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers

We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III₂-IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria.