Activation of NAD-linked malic enzyme in intact plant mitochondria by exogenous coenzyme A

O2 uptake by potato and cauliflower bud mitochondria oxidizing malate was progressively inhibited as the pH of the external medium was increased, in response to accumulation of oxaloacetate. Adding 0.5 mM coenzyme A to the medium reversed this trend by stimulating intramitochondrial NAD-linked malic enzyme at alkaline pH. In intact potato mitochondria, coenzyme A stimulation of malic enzyme was not observed when the external pH was above 7.5; in cauliflower mitochondria, coenzyme A stimulated even at pH 8. This difference in the response of intact mitochondria was attributed to an inherent difference in the properties of malic enzyme from the two tissues. Malic enzyme solubilized from potato mitochondria was inactive at pH values above 7.8, while that from cauliflower mitochondria retained its activity at pH 8 in the presence of coenzyme A. In potato mitochondria, coenzyme A stimulation of O2 uptake at alkaline pH was only observed when NAD+ was also provided exogenously. The results show that coenzyme A can be taken up by intact mitochondria and that pH, NAD+, and coenzyme A levels in the matrix act together to regulate malate oxidation.     

The action of the sesquiterpenic benzoquinone, perezone, on electron transport in biological membranes

Perezone (2-(1,5-dimethyl-4-hexenyl)-3-hydroxymethyl-p-benzoquinone) is a sesquiterpenic benzoquinone isolated from roots of plants of the genus Perezia. It exhibits oxido-reduction characteristics which suggest that the compound can be used for studies of the electron transfer chain of rat liver mitochondria. Perezone at 50 microM inhibits mitochondrial electron transport through a process which differs from that of rotenone, amytal, and Antimycin A. The inhibition is temperature dependent; at 35 degrees C it fails to inhibit valinomycin-induced mitochondrial respiration, but at 20 degrees C it inhibits respiration by 80-90%. Perezone is an electron-donor and electron-acceptor compound that behaves similarly to naphtoquinone. It mediates electron transport from a reaction center preparation isolated from Rhodopseudomonas sphaeroides and added cytochrome c. The low respiration of rat liver mitochondria depleted of coenzyme Q10 (CoQ) is increased by perezone. The electron transport activity of perezone was also demonstrated with CoQ-deficient yeast mutant E3-24.     

The Utilization of Choline and Acetyl Coenzyme A for the Synthesis of Acetylcholine

Acetylcholine synthesis in rat brain synaptosomes was investigated with regard to the intracellular sources of its two precursors, acetyl coenzyme A and choline. Investigations with α-cyano-4-hydroxycinnamate, an inhibitor of mitochondrial pyruvate transport, indicated that pyruvate must be utilized by pyruvate dehydrogenase located in the mitochondria, rather than in the cytoplasm, as recently proposed. Evidence for a small, intracellular pool of choline available for acetylcholine synthesis was obtained under three experimental conditions. (1) Bromopyruvate competitively inhibited high-affinity choline transport, perhaps because of accumulation of intracellular choline which was not acetylated when acetyl coenzyme A production was blocked. (2) Choline that was accumulated under high-affinity transport conditions while acetyl coenzyme A production was impaired was subsequently acetylated when acetyl coenzyme A production was resumed. (3) Newly synthesized acetylcholine had a lower specific activity than that of choline in the medium. These results indicate that the acetyl coenzyme A that is used for the synthesis of acetylcholine is derived from mitochondrial pyruvate dehydrogenase and that there is a small pool of choline within cholinergic nerve endings available for acetylcholine synthesis, supporting the proposal that the high-affinity transport and acetylation of choline are kinetically coupled.     

Fatty acyl coenzyme A-sensitive adenine nucleotide transport in a reconstituted liposome system

The adenine nucleotide translocase was purified from bovine heart mitochondria and incorporated into membranes of phospholipid liposomes. The rate of transport of the adenine nucleotides was competitively inhibited by oleoyl coenzyme A with an approximate Ki of 1.0 microM. Significant inhibition was limited to those fatty acyl coenzyme A esters which are carnitine dependent for their oxidation in isolated mitochondria. Octanoyl coenzyme A was almost completely inactive as was palmitic acid and palmitoyl carnitine. By comparing the inhibitory characteristics of carboxyatractylate and bongkrekic acid with those of oleoyl-CoA, it was determined that the fatty acyl-CoA esters could produce inhibition whether the carrier was inserted into the liposome in either the conventional (65%) or reverse (30%) orientation. The results demonstrate that the interaction of long chain fatty acyl-CoA esters with the ADP/ATP carrier in a purified reconstituted system mimics their effects with isolated mitochondria and inverted submitochondrial particles. In general, these findings are consistent with the role of acyl-CoA esters acting as natural ligands and biological effectors of the translocator.     

Identification of the site where the electron transfer chain of plant mitochondria is stimulated by electrostatic charge screening.

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge-screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady-state reduction level of coenzyme Q, and by a small decrease in the steady-state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc1, cytochrome c and cytochrome c oxidase). It was not a consequence of any effect on quinone reduction (by external NADH or NADPH dehydrogenase). This explains the salt-induced decrease in the steady-state reduction level of coenzyme Q. Analysis around cytochrome c revealed that stimulation by salts was due to a dual effect on the respiratory chain. The kinetic curves for the oxidation and reduction pathways of cytochrome c revealed that they were both activated by salt, the simultaneity explaining the small variation observed in the steady-state reduction level of cytochrome c. A simple kinetic core model is used to show that changes in the rate of dissociation of cytochrome c from the membrane can explain the observed kinetic changes in both cytochrome c reduction and cytochrome c oxidation. The stimulation is proposed to be the result of an increase in the rate constant of cytochrome c dissociation from the membrane induced by cation screening. We conclude that this type of modular kinetic analysis is a powerful tool to identify and quantitatively characterize multiple-site effects on the mitochondrial respiratory chain.     

Coenzyme Q analogues reconstitute electron transport and proton ejection but not the antimycin-induced "red shift" in mitochondria from coenzyme Q deficient mutants of the yeast Saccharomyces cerevisiae

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase activity but contained normal amounts of cytochromes b and c1 by spectral analysis. Addition of the exogenous coenzyme Q derivatives including Q2, Q6, and the decyl analogue (DB) restored the rate of antimycin- and myxothiazole-sensitive cytochrome c reductase with both substrates to that observed with reduced DBH2. Similarly, addition of these coenzyme Q analogues increased 2-3-fold the rate of cytochrome c reduction in mitochondria from wild-type cells, suggesting that the pool of coenzyme Q in the membrane is limiting for electron transport in the respiratory chain. Preincubation of mitochondria from the Q-deficient yeast cells with DBH2 at 25 degrees C restored electrogenic proton ejection, resulting in a H+/2e- ratio of 3.35 as compared to a ratio of 3.22 observed in mitochondria from the wild-type cell. Addition of succinate and either coenzyme Q6 or DB to mitochondria from the Q-deficient yeast cells resulted in the initial reduction of cytochrome b followed by a slow reduction of cytochrome c1 with a reoxidation of cytochrome b. The subsequent addition of antimycin resulted in the oxidant-induced extrareduction of cytochrome b and concomitant oxidation of cytochrome c1 without the "red" shift observed in the wild-type mitochondria. Similarly, addition of antimycin to dithionite-reduced mitochondria from the mutant cells did not result in a red shift in the absorption maximum of cytochrome b as was observed in the wild-type mitochondria in the presence or absence of exogenous coenzyme Q analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of cytochrome b in mitochondria from yeast lacking coenzyme Q

Mitochondria isolated from coenzyme Q deficient yeast cells had no detectable NADH:cytochrome c reductase or succinate:cytochrome c reductase but had comparable amounts of cytochromes b and c1 as wild-type mitochondria. Addition of succinate to the mutant mitochondria resulted in a slight reduction of cytochrome b; however, the subsequent addition of antimycin resulted in a biphasic reduction of cytochrome b, leading to reduction of 68% of the total dithionite-reducible cytochrome b. No "red" shift in the absorption maximum was observed, and no cytochrome c1 was reduced. The addition of either myxothiazol or alkylhydroxynaphthoquinone blocked the reduction of cytochrome b observed with succinate and antimycin, suggesting that the reduction of cytochrome b-562 in the mitochondria lacking coenzyme Q may proceed by a pathway involving cytochrome b at center o where these inhibitors block. Cyanide did not prevent the reduction of cytochrome b by succinate and antimycin the the mutant mitochondria. These results suggest that the succinate dehydrogenase complex can transfer electrons directly to cytochrome b in the absence of coenzyme Q in a reaction that is enhanced by antimycin. Reduced dichlorophenolindophenol (DCIP) acted as an effective bypass of the antimycin block in complex III, resulting in oxygen uptake with succinate in antimycin-treated mitochondria. By contrast, reduced DCIP did not restore oxygen uptake in the mutant mitochondria, suggesting that coenzyme Q is necessary for the bypass. The addition of low concentrations of DCIP to both wild-type and mutant mitochondria reduced with succinate in the presence of antimycin resulted in a rapid oxidation of cytochrome b perhaps by the pathway involving center o, which does not require coenzyme Q.     

Mechanism of Cold Injury in Sweet Potatoes

The biochemical mechanism of cold injury occurring in sweet potatoes stored at 0°C was studied. Oxygen uptake and RC ratio of mitochondria from sweet potatoes kept at 0°C for about 15 days declined when succinate or malate was used as substrate. As sweet potatoes suffered slight cold injury, a decrease in the respiratory rate of state 3 of mitochondria was observed. This decrease could be restored approximately to the level of that of healthy sweet potato mitochondria by the addition of cytochrome c when succinate was used as substrate. When sweet potatoes suffered severe damage, only partial recovery was observed with cytochrome c. While it was found that the respiratory rate in state 3 of mitochondria from chilled sweet potatoes was less inhibited by cyanide than that of healthy sweet potato mitochondria, the inhibition could be restored to that of healthy sweet potato mitochondria by the addition of cytochrome c. When malate was used as substrate, no effect of cytochrome c and NADH₂ was observed. There was no difference between chilled and healthy sweet potato mitochondria in enzyme activities of the electron transport system except for malate dehydrogenase.     

Liver mitochondrial respiratory function and coenzyme Q content in rats on a hypercholesterolemic diet treated with atorvastatin

Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are effective drugs in the treatment of hypercholesterolemia, however, their undesirable actions are not fully known. We investigated the effects of atorvastatin on the oxidative phosphorylation and membrane fluidity in liver mitochondria, and also on the coenzyme Q (CoQ) content in the mitochondria, liver tissue, and plasma of rats on a standard (C) and hypercholesterolemic (HCh) diet. Atorvastatin was administered at either low (10 mg kg(-1)) or high dose (80 mg kg(-1)) for four weeks. The high dose of the drug decreased the concentrations of total cholesterol and triacylglycerols in the plasma and liver of rats on a HCh diet. Administration of atorvastatin was associated with decreased oxygen uptake (state 3), and oxidative phosphorylation rate in the mitochondria of both C and HCh rats. Further, the drug influenced mitochondrial membrane fluidity and dose-dependently reduced concentrations of oxidized and reduced forms of CoQ in the mitochondria. Our findings point to an association between in vivo administration of atorvastatin and impaired bioenergetics in the liver mitochondria of rats, regardless of diet, in conjunction with simultaneous depletion of oxidized and reduced CoQ forms from the mitochondria. This fact may play a significant role in the development of statin-induced hepatopathy.     

Role of coenzyme Q in the mitochondrial respiratory chain. Reconstitution of activity in coenzyme Q deficient mutants of yeast.

The reduction of cytochrome c by the reduced form of the 6-decyl analogue of coenzyme Q follows first-order kinetics with respect to cytochrome c and increases in a linear manner with added mitochondrial protein. The activity is completely sensitive to antimycin A in whole cell extracts of yeast as well as in isolated mitochondria and fractionates with markers for the mitochondrial electron-transport chain. The presence of both cytochrome b and c1 in an approximately 2:1 ratio appears essential for enzymatic activity. Reduced coenzyme Q-cytochrome c reductase obeys Michaelis-Menten kinetics when assayed in mitochondria obtained from a yeast strain lacking coenzyme Q. Both reduced nitotinamide adenine dinucleotide and succinate:cytochrome c reductase activities were not detectable in six coenzyme Q deficient strains tested, but were restored after addition of the oxidized form of the coenzyme Q analogue. No marked difference in the concentration of the analogue required to restore the two activities was observed.     

Coupling of "malic" enzyme and NADPH:NAD transhydrogenase in the energetics of Hymenolepis diminuta (Cestoda)

Acetylpyridine NADP replaced NADP in promoting the Mn2+ ion-requiring mitochondrial "malic" enzyme of Hymenolepis diminuta. Disrupted mitochondria displayed low levels of an apparent oxaloacetate-forming malate dehydrogenase activity when NAD or acetylpyridine NAD served as the coenzyme. Significant malate-dependent reduction of acetylpyridine NAD by H. diminuta mitochondria required Mn2+ ion and NADP, thereby indicating the tandem operation of "malic" enzyme and NADPH:NAD transhydrogenase. Incubation of mitochondrial preparations with oxaloacetate resulted in a non-enzymatic decarboxylation reaction. Coupling of malate oxidation with electron transport via the "malic" enzyme and transhydrogenase was demonstrated by polarographic assessment of mitochondrial reduced pyridine nucleotide oxidase activity.     

Alteration of plant mitochondrial proton conductance by free fatty acids. Uncoupling protein involvement

We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.     

Calmodulin is not involved in the regulation of exogenous NADH oxidation by plant mitochondria

The oxidation of exogenous NADH by mitochondria from potato ( Solanum tuberosum L., cv. Bintje) tubers, measured with different electron acceptors, oxygen, cytochrome c , duroquinone and ubiquinone 1, was greatly enhanced under high salt conditions compared to low salt conditions, confirming the stimulatory effect of electrostatic screeening of negative membrane charges by cations. In addition to this nonspecific stimulation, the oxidation of exogenous NADH showed a specific dependence on Ca²⁺. Results presented here suggest that calmodulin was not directly involved in the regulation of exogenous NADH oxidation by potato mitochondria: (1) Calmodulin antagonists were found to inhibit electron flow at several sites in a nonspecific manner. (2) Using a phenothiazine-Affi Gel column, it was not possible to demonstrate the presence of calmodulin in Triton X-100 solubilized mitochondria. (3) Fractions eluted from a calmodulin-Sepharose column with EGTA [ethyleneglycolbis (β-aminoethylether)-N, N, N', N'-tetraacetic acid] did not display any activity related to mitochondrial electron transport, suggesting that NADH dehydrogenase had no specific affinity for calmodulin. The possible indirect involvement of calmodulin in the regulation of exogenous NADH oxidation by Ca²⁺ is discussed.     

Inhibition of mitochondrial electron transport by hydrophilic metal chelators. Determination of dehydrogenase topography

The topography of the inner mitochondrial membrane was investigated using inhibitors of electron transport on preparations of beef heart mitochondria and electron transport particles of opposite orientation. Reductions of juglone, ferricyanide, indophenol, coenzyme Q, duroquinone, and cytochrome c by NADH are inhibited to different extents on both sides of the membrane by the impermeant hydrophilic chelators bathophenanthroline sulfonate and orthophenanthroline. The extent of inhibition for each acceptor increased in the order given. At least two chelator-sensitive sites are present on each membrane face between the flavoprotein and coenzyme Q and a chelator-sensitive site is present on the matrix face between the sites of coenzyme Q and duroquinone interaction. Duroquinol oxidation in mitochondria only is stimulated by bathophenanthroline sulfonate. Juglone reduction is stimulated in electron transport particles (only) by p-hydroxymercuribenzenesulfonate, but after mercurial treatment, juglone reduction in both particles and mitochondria is more sensitive to bathophenanthroline sulfonate.Succinate dehydrogenase components are inhibited by hydrophilic orthophenanthroline or bathophenanthroline sulfonate in mitochondria only. Electron flow between the dehydrogenases of succinate and NADH occurs via a chelator-sensitive site located on the matrix face of the membrane. Inter-complex electron flow is prevented by rotenone or thenoyltrifluoroacetone. The lack of succinate-indophenol reductase inhibition by bathophenanthroline sulfonate in the presence of rotenone or thenoyltrifluoroacetone indicates that the rotenone-sensitive site may be located on the matrix face and demonstrates that electrons flow between the NADH and succinate dehydrogenases via a hydrophilic chelator and rotenone-thenoyltrifluoroacetone-sensitive site on the matrix face of the membrane. Inhibition by hydrophilic chelators only in mitochondria indicates that succinate dehydrogenase as well as NADH dehydrogenase has a transmembranous orientation.     

Comparison of structural properties of cyclosporin A and its analogue alisporivir and their effects on mitochondrial bioenergetics and membrane behavior

The paper considers the effect of the MPT pore inhibitor cyclosporin A (CsA) and its non-immunosuppressive analogue alisporivir (Ali) on the functioning of rat skeletal muscle mitochondria. We have shown that both agents at a standard in vitro concentration of 1 μM increase the calcium capacity of organelles and have no effect on the parameters of oxidative phosphorylation. However, an increase in their concentration to 5 μM leads to the suppression of oxygen consumption by mitochondria, which is more pronounced in the case of Ali. This effect is accompanied by a decrease in the membrane potential of organelles and, apparently, is based on the inhibition of electron transport along the mitochondrial respiratory chain due to limited mobility of coenzyme Q. We have noted that both agents do not affect the production of hydrogen peroxide by isolated mitochondria. NMR spectroscopy and molecular dynamics simulation did not reveal significant differences in the structure and backbone flexibility of CsA and Ali. Both agents decrease the overall fluidity of the membrane of DPPC liposomes, inducing an increase in laurdan generalized polarization parameter. A similar effect was also found in the case of mitochondrial membranes. We suggested that these effects of CsA and Ali, associated with their lipophilic nature and the ability to accumulate in the lipid phase of membranes, may cause a decrease in the efficiency of electron transport in the respiratory chain of mitochondria and suppression of the bioenergetics of these organelles.     

Mitochondrial Respiratory Electron Carriers Are Involved in Oxidative Stress during Heat Stress in Saccharomyces cerevisiae

In the present study we sought to determine the source of heat-induced oxidative stress. We investigated the involvement of mitochondrial respiratory electron transport in post-diauxic-phase cells under conditions of lethal heat shock. Petite cells were thermosensitive, had increased nuclear mutation frequencies, and experienced elevated levels of oxidation of an intracellular probe following exposure to a temperature of 50 degrees C. Cells with a deletion in COQ7 leading to a deficiency in coenzyme Q had a much more severe thermosensitivity phenotype for these oxidative endpoints following heat stress compared to that of petite cells. In contrast, deletion of the external NADH dehydrogenases NDE1 and NDE2, which feed electrons from NADH into the electron transport chain, abrogated the levels of heat-induced intracellular fluorescence and nuclear mutation frequency. Mitochondria isolated from COQ7-deficient cells secreted more than 30 times as much H(2)O(2) at 42 as at 30 degrees C, while mitochondria isolated from cells simultaneously deficient in NDE1 and NDE2 secreted no H(2)O(2). We conclude that heat stress causes nuclear mutations via oxidative stress originating from the respiratory electron transport chains of mitochondria.     

Energy transduction by the reconstituted b-c1 complex from yeast mitochondria. Inhibitory effects of dicyclohexylcarbodiimide

A purified cytochrome b-c1 complex isolated from yeast mitochondria has been reconstituted into proteoliposomes. The reconstituted comp]lex catalyzed antimycin A-sensitive electron transfer from different analogues of coenzyme Q to cytochrome c. The reconstituted complex was also capable of energy conservation as indicated by uncoupler-stimulated rates of electron transfer, electrogenic proton ejection, and reversed electron flow from cytochrome b to coenzyme Q2 in the presence of antimycin A driven by a valinomycin-induced K+-diffusion potential (negative inside). Close to four protons were ejected per two electrons transported through the reconstituted b-c1 complex with ferricyanide as an artificial and impermeable electron acceptor.l The H+/2e- ratio decreased to two in the presence of the proton-conducting agent, carbonyl cyanide m-chlorophenylhydrazone. The same processes were studied in parallel in energy-conserving site 2 of rat liver mitochondria with similar results. In the reconstituted b-c1 complex, dicyclohexylcarbodiimide (DCCD) blocked the function of the electrogenic proton translocating device in the forward direction of proton ejection as well as in the backwards direction, measured as reversed electron flow from cytochrome b to coenzyme Q2 driven by a K+-diffusion potential. The primary effect of DCCD is localized on the proton ejection process, as the low proton conductance of the proteoliposome membrane was totally preserved after DCCD treatment.     

Induction mechanism of 3-hydroxy-3-methylglutaryl-CoA reductase in potato tuber and sweet potato root tissues

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC1.1.1.34), the key enzyme in isoprenoid biosynthesis, was purified from microsomes of potato tuber tissue, and a polyclonal antibody and two monoclonal antibodies against the purified enzyme were prepared. HMGR protein content was measured by immunotitration and radioimmunoassay using these antibodies. HMGR activity was very low in the fresh tissues of both potato tuber and sweet potato root. The activity in potato tuber was increased by cutting and further by additional fungal infection of the cut tissues. In sweet potato root tissue, the activity was scarcely increased after cutting alone, but was markedly increased by additional fungal infection or chemical treatment. The HMGR protein contents in both fresh potato tuber and sweet potato root tissues were also very low, and increased markedly in response to cutting and fungal infection. From these results, we proposed a hypothesis on the induction mechanism of HMGR after cutting and fungal infection in potato tuber and sweet potato root tissues.     

The free NADH concentration is kept constant in plant mitochondria under different metabolic conditions

The reduced coenzyme NADH plays a central role in mitochondrial respiratory metabolism. However, reports on the amount of free NADH in mitochondria are sparse and contradictory. We first determined the emission spectrum of NADH bound to proteins using isothermal titration calorimetry combined with fluorescence spectroscopy. The NADH content of actively respiring mitochondria (from potato tubers [Solanum tuberosum cv Bintje]) in different metabolic states was then measured by spectral decomposition analysis of fluorescence emission spectra. Most of the mitochondrial NADH is bound to proteins, and the amount is low in state 3 (substrate + ADP present) and high in state 2 (only substrate present) and state 4 (substrate + ATP). By contrast, the amount of free NADH is low but relatively constant, even increasing a little in state 3. Using modeling, we show that these results can be explained by a 2.5- to 3-fold weaker average binding of NADH to mitochondrial protein in state 3 compared with state 4. This indicates that there is a specific mechanism for free NADH homeostasis and that the concentration of free NADH in the mitochondrial matrix per se does not play a regulatory role in mitochondrial metabolism. These findings have far-reaching consequences for the interpretation of cellular metabolism.