Fuscin,an inhibitor of respiration and oxidative phosphorylation in ox-neck muscle mitochondria

1. The effect of fuscin on the mitochondrial oxidation of pyruvate plus malate, of succinate and of ascorbate plus tetramethyl-p-phenylenediamine (TMPD) and on the redox changes of succinate-reducible cytochromes b and c was investigated using tightly-coupled ox-neck muscle mitochondria.     

Effects of kaempferol on the oxidative properties of intact plant mitochondria

The effects of kaempferol on the oxidative and phosphorylative properties of plant mitochondria from potato tubers and etiolated mung bean (Phaseolus aureus Roxb.) hypocotyls were investigated. Kaempferol inhibited the state 3 oxidation rate of malate, NADH, and succinate, but was without effect on the ascorbate-tetramethyl p-phenylenediamine oxidation rate. The inhibition was almost the same whether the mitochondria were in state 3 or in an uncoupled state 3. When 180 micromolar kaempferol was added during state 4, the tight coupling of succinate or NADH oxidation was not released. The results obtained indicate that kaempferol inhibits the mitochondrial electron flow at, or just after, the flavoprotein site.     

The phosphorylation potentials generated by respiring Ehrlich ascites tumor mitochondria

The extra- and intramitochondrial phosphorylation potentials (ΔG_p(out) and ΔG_p(in), respectively) generated by respiring Ehrlich ascites tumor mitochondria were determined, using succinate, pyruvate + malate, ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, and ascorbate + ferrocyanide as substrate systems. Values of ΔG_p(out) exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) in post-ADP state 4 respiration were found with succinate as substrate, in agreement with data on normal rat liver mitochondria. ΔG_p(out) values exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) were also observed with ascorbate + TMPD or ascorbate + ferrocyanide as substrates. Slightly lower values of ΔG_p(out) were found with the NAD-linked substrates pyruvate + malate. The intramitochondrial ΔG_p(in) developed by respiring Ehrlich ascites tumor mitochondria respiring on succinate approached 12 kcal mol^?1 (50.2 kJ mol^?1), in agreement with reported values on rat liver mitochondria. The prior accumulation of Ca²⁺ and phosphate by the Ehrlich cell mitochondria did not lower the extramitochondrial ΔG_p(out) developed after a subsequent addition of ADP. Although the rate of oxidative phosphorylation of Ehrlich ascites tumor cells is reduced by intramitochondrial Ca²⁺ and phosphate (Villalobo and Lehninger (1980) J. Biol. Chem., 255, 2457–2464) they are still capable of generating ATP in the suspending medium against a high thermodynamic gradient, as expressed by the [ATP]/[ADP][P_i]mass action ratio.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Inhibition of cyanide-resistant respiration in pea cotyledon mitochondria by chloroquine

The action on mitochondrial respiration of a ubiquinone analog, chloroquine, has been studied using purified mitochondria from the cotyledons of germinating peas (Pisum sativum L. var. Homesteader). Chloroquine at 3 millimolar did not inhibit malate or succinate oxidation at pH 7.2, but it did inhibit malate (but not succinate) oxidation at pH 8.2. Cyanide-resistant respiration was also inhibited.     

Action of the antitumor and antispermatogenic agent lonidamine on electron transport in ehrlich ascites tumor mitochondria

The effect of lonidamine, an antispermatogenic and antitumor drug, on the oxygen consumption, ATPase activity, and redox state of the electron carriers of Ehrlich ascites tumor mitochondria has been studied. Lonidamine inhibits ADP- and uncoupler-stimulated respiration on various NAD- and FAD-linked substrates, but does not affect state 4 respiration. Experiments to determine its site of action showed that lonidamine does not significantly inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c₁ complex, also was unaffected by lonidamine, which failed to inhibit the oxidation of duroquinol. Moreover, inhibition of electron flow through site 2 was also excluded because of the inability of the N,N,N′,N′-tetramethyl-p-phenylenediamine bypass to relieve the lonidamine inhibition of the oxidation of pyruvate + malate. The F₀F₁ATPase activity and vectorial H⁺ ejection are also unaffected by lonidamine. The inhibition of succinate oxidation by lonidamine was found to take place at a point between succinate and iron-sulfur center S3. Spectroscopic experiments demonstrated that lonidamine inhibits the reduction of mitochondrial NAD⁺ by pyruvate + malate and other NAD-linked substrates in the transition from state 1 to state 4. However, lonidamine does not inhibit reduction of added NAD⁺ by submitochondrial vesicles or by soluble purified NAD-linked dehydrogenases. These observations, together with other evidence, suggest that electron transport in tumor mitochondria is inhibited by lonidamine at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state. The action of lonidamine in several respects resembles the selective inhibition of electron transport in tumor cells produced by cytotoxic macrophages.     

Effect of butanedioic Acid mono (2,2-dimethylhydrazide) on the activity of membrane-bound succinate dehydrogenase

Mitochondria isolated from hypocotyls of five-day-old bean (Phaseolus vulgaris L. `Black Valentine') seedlings rapidly oxidized succinate, malate, and NADH. Oxidation rates, respiratory control, and ADP:O ratios obtained with saturating concentrations of all three substrates indicated that the mitochondria were tightly coupled. The mitochondrial preparation was then employed to investigate the respiration-inhibiting effects of butanedioic acid mono (2,2-dimethyl-hydrazide) (daminozide) a plant growth retardant having structural similarity to an endogenous respiratory substrate (succinate). Daminozide markedly inhibited the activity of membrane-bound succinate dehydrogenase. Inhibition was of the competitive type (apparent K_i, 20.2 millimolar) with respect to succinate. Although not excluding other hypotheses, the results support an active role for daminozide in the suppression of respiration as an important metabolic site of its action as a plant growth regulator.     

The determination of the proton-motive force during cyanide-insensitive respiration in plant mitochondria

The magnitude of the components of the proton-motive force (Δp) generated in the presence of antimycin A has been determined for potato, mung bean, skunk cabbage, and Arum spadix mitochondria, Δp was calculated from the distribution of rubidium, methylamine, and 5,5′-dimethyl-2,4-oxazolodine-dione. In the presence of antimycin A, the oxidation of succinate generates a Δp of 40–50 mV, and this value is independent of the degree of antimycin A insensitivity of the various mitochondria. Under such conditions, the addition of ADP failed to either stimulate the respiratory rate or reduce Δp. Although oxygen consumption via the alternative pathway was sensitive to hydroxamic acids, no change in the components of the proton motive force was detected. The addition of an uncoupler in the presence of antimycin A and succinate reduced Δp to zero while respiration remained unaltered. The oxidation of malate in the presence of antimycin A generates a Δp of 150 mV, which was reduced to 144 mV under State 3 conditions. The addition of salicylhydroxamic acid inhibited oxygen uptake and reduced Δp to 40 mV. It is concluded that the oxidation of succinate by the alternative respiratory pathway does not generate a proton-motive force and is not coupled to ATP synthesis. The oxidation of malate by the alternative pathway, however, can conserve energy as ATP presumably via coupling Site I of the main respiratory chain.     

Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity

Poliovirus infection of COS-1 and T47D cells caused a rapid decrease in total cell respiration, and this was attributed to an inhibition of mitochondrial respiration. The stimulation of mitochondrial respiration by pyruvate plus malate or succinate was impaired in saponin-permeabilised cells. However, this inhibition could be overcome by the addition of N,N,N',N'-tetramethyl-1, 4-phenylenediamine and ascorbate. The activity of succinate dehydrogenase was impaired in parallel with the inhibition of mitochondrial respiration during poliovirus infection. This shows that mitochondrial function is profoundly altered during poliovirus infection and that this occurs primarily through inhibition of electron flow at complex II of the mitochondrial respiratory chain.     

Some effects of decenylsuccinic Acid on isolated corn mitochondria

The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 × M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6-dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N′, N′-tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.     

O2-Polarographic Studies on Soluble and Mitochondrial Enzymes of Crithidia fasciculata; Glycerophosphate Enzymes*

SYNOPSIS. Mitochondrial and supernatant fractions were isolated from Crithidia fasciculata by grinding with neutral alumina and differential centrifugation. Supernatant fractions contained at least 2 NAD-linked enzymes: an α-glycerophosphate dehydrogenase and a malate dehydrogenase. The properties of these enzymes were investigated polarographically with phenazine ethosulfate acting as electron acceptor. Agaricic acid, cinnamic acid and p-NO₂-cinnamic acid were specific inhibitors of the α-glycerophosphate dehydrogenase. Succinate, malate, DL-α-glycerophosphate and NADH stimulated respiration of mitochondrial preparations; O₂ uptake was greatest with succinate. KCN and antimycin A inhibited succinate respiration more than α-glycerophosphate respiration. Amytal did not affect succinate, α-glycerophosphate or NADH oxidation. The trypanocide suramin inhibited mitochondrial respiration at least 77% with each substrate. The relevance of these results to other members of the Trypanosomatidae is discussed.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

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.     

Oxidations of various substrates and effects of the inhibitors on purified mitochondria isolated from <Emphasis Type="Italic">Kalanchoë pinnata</Emphasis>

Kalanchoë pinnata mitochondria readily oxidized succinate, malate, NADH, and NADPH at high rates and coupling. The highest respiration rates usually were observed in the presence of succinate. The high rate of malate oxidation was observed at pH 6.8 with thiamine pyrophosphate where both malic enzyme (ME) and pyruvate dehydrogenase were activated. In CAM phase III of K. pinnata mitochondria, both ME and malate dehydrogenase (MDH) simultaneously contributed to metabolism of malate. However, ME played a main function: malate was oxidized via ME to produce pyruvate and CO₂ rather than via MDH to produce oxalacetate (OAA). Cooperative oxidation of two or three substrates was accompanied with the dramatic increase in the total respiration rates. Our results showed that the alternative (Alt) pathway was more active in malate oxidation at pH 6.8 with CoA and NAD⁺ where ME operated and was stimulated, indicating that both ME and Alt pathway were related to malate decarboxylation during the light. In K. pinnata mitochondria, NADH and NADPH oxidations were more sensitive with KCN than that with succinate and malate oxidations, suggesting that these oxidations were engaged to cytochrome pathway rather than to Alt pathway and these capacities would be desirable to supply enough energy for cytosol pyruvate orthophosphate dikinase activity.     

Properties of Wheat Mitochondria. Study of Substrates, Cofactors and Inhibitors

Isolated mitochondria of wheat shoots oxidize α- ketoglutarate, DL-malate succinate and NADH with good relative respiration control and ADP: O ratio. They have high affinity for α-ketoglutarate and NADH as substrates and utilize malate and succinate with a respiration ratio of about one-half of α-ketoglutarate. The average ADP : O ratios approach the expected theoretical values, i.e., 3.6 ± 0.2 for α-ketoglutarate, 1.8 ± 0.2 for succinate, and 2.8 ± 0.2 for malate. The ADP: O ratio with NADH is 1.8 ± 0.2. The maximum coupling of oxidation and phosphorylation is obtained at concentrations of 10 mM, 2 mM, 10 mM and 8 mM for α-ketoglutarate, NADH, malate and succinate, respectively. — Wheat mitochondria have little or no dependence on added cofactors. Mitochondria prepared by our procedure apparently retain sufficient amounts of endogenous cofactors required for NAD-linked systems. FAD⁺ is found to improve succinate oxidation. Cytochrome c does not have any significant effect on respiratory parameters of wheat mitochondria. — Wheat mitochondria are some -what resistant to DNP at 1.7 × 10^-5M. Malonate seems to improve coupling of α-ketoglutarate oxidation. Other Krebs cycle intermediates have been tested on three major substrates of TCA cycle, i.e., α-ketoglutarate, malate and succinate.     

Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola

Beffa, T., Pezet, R. and Turian, G. 1987. Multiple-site inhibition by colloidal elemental sulfur (S°) of respiration by mitochondria from young dormant α spores of Phomopsis viticola. Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra-methyl-p-phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α-ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple-site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°-inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg-acetate or p - chloromercuribenzoate. In contrast to S° these SH-group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH-groups and to self-reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux.     

Fermentation and anaerobic respiration by Rhodospirillum rubrum and Rhodopseudomonas capsulata

Rhodospirillum rubrum and Rhodopseudomonas capsulata were able to grow anaerobically in the dark either by a strict mixed-acid fermentation of sugars or, in the presence of an appropriate electron acceptor, by an energy-linked anaerobic respiration. Both species fermented fructose without the addition of accessory oxidants, but required the initial presence of bicarbonate before fermentative growth could begin. Major products of R. rubrum fermentation were succinate, acetate, propionate, formate, hydrogen, and carbon dioxide; R. capsulata produced major amounts of lactate, acetate, succinate, hydrogen, and carbon dioxide. R. rubrum and R. capsulata were also capable of growing strictly through anaerobic, respiratory mechanisms. Nonfermentable substrates, such as succinate, malate, or acetate, supported growth only in the presence of an electron acceptor such as dimethyl sulfoxide or trimethylamine oxide. Carbon dioxide and dimethyl sulfide were produced during growth of R. rubrum and R. capsulata on succinate plus dimethyl sulfoxide. Molar growth yields from cultures grown anaerobically in the dark on fructose plus dimethyl sulfoxide were 3.8 to 4.6 times higher than values obtained from growth on fructose alone and were 56 to 60% of the values obtained from aerobic, respiratory growth with fructose. Likewise, molar growth yields from anaerobic, respiratory growth conditions with succinate plus dimethyl sulfoxide were 51 to 54% of the values obtained from aerobic, respiratory growth with succinate. The data indicate that dimethyl sulfoxide or trimethylamine oxide as a terminal oxidant is approximately 33 to 41% as efficient as O₂ in conserving energy through electron transport-linked respiration.     

Effect of phosphate and uncouplers on substrate transport and oxidation by isolated corn mitochondria

A study was made to determine conditions under which malate oxidation rates in corn (Zea mays L.) mitochondria are limited by transport processes. In the absence of added ADP, inorganic phosphate increased malate oxidation rates by processes inhibited by mersalyl and oligomycin, but phosphate did not stimulate uncoupled respiration. However, the uncoupled oxidation rates were inhibited by butylmalonate and mersalyl. When uncoupler was added prior to substrate, subsequent O₂ uptake rates were reduced when malate and succinate, but not exogenous NADH, were used. Uncoupler and butylmalonate also inhibited swelling in malate solutions and malate accumulation by these mitochondria, which were found to have a high endogenous phosphate content. Addition of uncoupler after malate or succinate produced an initial rapid oxidation which declined as the mitochondria lost solute and contracted. This decline was not affected by addition of ADP or AMP, and was not observed when exogenous NADH was substrate. Increasing K⁺ permeability with valinomycin increased the P-trifluoromethoxy (carboxylcyanide)phenyl hydrazone inhibition. Kinetic studies showed the slow rate of malate oxidation in the presence of uncoupler to be characterized by a high Km and a low V_max, probably reflecting a diffusion-limited process.     

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl₄ administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V₃) and 4 (V₄) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V₃ and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl₄ resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).     

Cyanide-Resistant Respiration in Suspension Cultured Cells of Nicotiana glutinosa L

The respiration of dark-grown Nicotiana glutinosa L. cells in liquid suspension culture was found to be highly cyanide resistant and salicylhydroxamic acid (SHAM) sensitive, indicative of an active alternative respiratory pathway. This was especially true during the lag and logarithmic phases of the 14-day growth cycle. Mitochondria isolated from logarithmically growing cells exhibited active oxidation of malate, succinate, and exogenous NADH. Oxidation of all three substrates had an optimum pH of 6.5 and all were highly resistant to inhibited by cyanide and sensitive to SHAM. Respiratory control was exhibited by all three substrates but only if SHAM was present to block the alternative pathway and divert electrons to the phosphorylating cytochrome pathway. The cyanide-resistant oxidation of exogenous NADH has previously only been associated with Arum spadix mitochondria. Coemergence during evolution of the alternative respiratory pathway and the exogenous NADH dehydrogenase in plant mitochondria as a possible mechanism for removal of cytoplasmic NADH is proposed. Evidence is presented which suggests that mitochondrial assays should be performed at pH 6.5.