Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

Arrhenius plots of the respiration rates of mitochondria isolated from chilling sensitive plant tissues (tomato and cucumber fruit, and sweet potato roots) showed a linear decrease from 25 C to about 9 to 12 C (with Q(10) values of 1.3 to 1.6), at which point there was a marked deviation with an increased slope as temperatures were reduced to 1.5 C (Q(10) of 2.2 to 6.3). The log of the respiration rate of mitochondria from chilling resistant tissues (cauliflower buds, potato tubers, and beet roots) showed a linear decrease over the entire temperature range from 25 to 1.5 C with Q(10) values of 1.7 to 1.8. Phosphorylative efficiency of mitochondria from all the tissues, as measured by ADP:O and respiratory control ratios, was not influenced by temperatures from 25 to 1.5 C. These results indicate that an immediate response of sensitive plant tissues to temperatures in the chilling range (0 to 10 C) is to depress mitochondrial respiration to an extent greater than that predicted from Q(10) values measured above 10 C. The results are also consistent with the hypothesis that a phase change occurs in the mitochondrial membrane as the result of a physical effect of temperature on some membrane component such as membrane lipids.     

Effects of Purified Helminthosporium maydis Race T Toxin on the Structure and Function of Corn Mitochondria and Protoplasts

A toxin preparation from Helminthosporium maydis Race T containing several closely related molecules with apparently identical biological activities was highly active against mitochondria and protoplasts from Texas male-sterile (T) cytoplasm corn (T mitochondria and T protoplasts, respectively) but had no effect on their male-fertile (N) cytoplasm counterparts. The toxin preparation caused multiple changes in isolated T mitochondria, including uncoupling of oxidative phosphorylation, stimulation of succinate and NADH respiration, inhibition of malate respiration, increased swelling, loss of matrix density, and unfolding of the inner membrane. Only 6 to 7 nanograms toxin per milligram mitochondrial protein (1.8 nanogram per milliliter) were required to fully uncouple oxidative phosphorylation and to completely inhibit malate respiration in isolated T mitochondria. Similar low concentrations of toxin caused collapse of T protoplasts after several days of culture. Severe ultrastructural damage to mitochondria in T protoplasts was observed within 20 minutes; no changes in other cellular components were observed at this time. These observations on the cytoplasmic specificity, multiple effects, and high activity of the toxin at the mitochondrial and cellular levels highlight its biological significance and potential usefulness in determining the molecular basis of southern corn leaf blight disease.     

Oxidative activity in mitochondria isolated from rat liver at different stages of development

The purpose of this study was to evaluate the oxidative capacities in hepatic mitochondria isolated from prepubertal, young adult and adult rats (40, 90 and 180 days of age, respectively). In these rats, mitochondrial respiratory rates using FAD- and NAD-linked substrates as well as mitochondrial protein mass were measured. The results show that only the oxidative capacity of FAD-linked pathways significantly declined in mitochondria from 180-day-old rats compared with those from younger animals. When we consider FAD-linked respiration expressed per g liver, no significant difference was found among rats of different ages because of an increased mitochondrial protein mass found in 180-day-old rats. However, when FAD-linked and lipid-dependent respiratory rates were expressed per 100 g body weight, significant decreases occurred in 180-day-old rats. Therefore, the decrease in liver weight expressed per 100 g body weight rather than an impaired hepatic cellular activity may be the cause of body energy deficit in 180-day-old rats. Copyright © 1998 John Wiley & Sons, Ltd.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

Heterogeneity of plant mitochondrial responses underpinning respiratory acclimation to the cold in Arabidopsis thaliana leaves

In this study, we investigated whether changes in mitochondrial abundance, ultrastructure and activity are involved in the respiratory cold acclimation response in leaves of the cold-hardy plant Arabidopsis thaliana. Confocal microscopy [using plants with green fluorescence protein (GFP) targeted to the mitochondria] and transmission electron microscopy (TEM) were used to visualize changes in mitochondrial morphology, abundance and ultrastructure. Measurements of respiratory flux in isolated mitochondria and intact leaf tissue were also made. Warm-grown (WG, 25/ 20 degrees C day/night), 3-week cold-treated (CT) and cold-developed (CD) leaves were sampled. Although CT leaves exhibited some evidence of acclimation (as evidenced by higher rates of respiration at moderate measurement temperatures), it was only the CD leaves that were able to re-establish respiratory flux within the cold. Associated with the recovery of respiratory flux in the CD leaves were: (1) an increase in the total volume of mitochondria per unit volume of tissue in epidermal cells; (2) an increase in the ratio of cristae to matrix within mesophyll cell mitochondria; and (3) an increase in the capacity of the energy-producing cytochrome pathway in mitochondria isolated from whole leaf homogenates. Regardless of growth temperature, we found that contrasting cell types exhibited distinct differences in mitochondrial ultrastructure, morphology and abundance. Collectively, our data demonstrated the diversity and tissue-specific nature of mitochondrial responses that underpin respiratory acclimation to the cold, and revealed the heterogeneity of mitochondrial structure and abundance that exists within leaves.     

Isolation and characterization of metabolically competent mitochondria from spinach leaf protoplasts

Intact mitochondria were prepared from spinach (Spinacia oleracea L. var. Kyoho) leaf protoplasts and purified by Percoll discontinuous gradient centrifugation. Assays of several marker enzymes showed that the final mitochondrial preparations obtained are nearly free from other contaminating organelles, e.g. chloroplasts, peroxisomes, and endoplasmic reticulum. These mitochondria oxidized malate, glycine, succinate, and NADH, tightly coupled to oxidative phosphorylation with high values of ADP to O ratio as well as respiratory control ratio. The rate of NADH oxidation was 331 nmoles O₂ per milligram mitochondrial protein per minute, which is comparable to that obtained by highly purified potato or mung bean mitochondria. However, the activity of glutamine synthetase was barely detectable in the isolated mitochondrial fraction. This finding rules out a hypothetical scheme (Jackson, Dench, Morris, Lui, Hall, Moore 1971 Biochem Soc Trans 7: 1122) dealing with the role of the mitochondrial glutamine synthetase in the reassimilation of NH₃, which is released during the step of photorespiratory glycine decarboxylation in green leaf tissues, but it is consistent with the photosynthetic nitrogen cycle (Keys, Bird, Cornelius, Lea, Wallsgrove, Miflin 1978 Nature (Lond) 275: 741), in which NH₃ reassimilation occurs outside the mitochondria.     

Reversible effects of fatty acids on respiration, oxidative phosphorylation, and heat production of rat liver mitochondria.

1. Oleic acid at low concentrations (0--70 nmol/mg protein) stimulated mitochondrial state 4 respiration 4-fold, increased the apparent enthalpy change of the respiration per gram atom of oxygen consumed from -112 to -208 kJ/O and completely inhibited ATP synthesis without significant effect on the Mg-ATPase activity of mitochondria. 2. Similar effects on mitochondrial respiratory activities were observed with other fatty acids. 3. Bovine serum albumin (BSA) protected mitochondria from the effects of oleic acid irrespective of the order of addition of oleic acid and BSA to mitochondria. The capacity of BSA to bind oleic acid was calculated to be 3.6--7.1 (mean, 4.9) mol of oleic acid/mol of BSA. 4. The response time of mitochondrial respiration to added oleic acid or BSA was 20--25 s.     

Citrate and succinate uptake by potato mitochondria

The uptake of [¹⁴C]citrate and [¹⁴C]succinate was studied in potato mitochondria (Solanum tuberosum var. Russet Burbank) using cellulose pore filtration and was found to occur by the same mechanisms as described for mammalian mitochondria. Potato mitochondria, in the absence of respiration, have a very low capacity for uptake by exchange with endogenous anions, taking up only 2.4 nanomoles citrate and 2.0 nanomoles succinate per milligram protein. Maximum citrate uptake of over 17 nanomoles per milligram protein occurs in the presence of inorganic phosphate, a dicarboxylic acid, and an external energy source (NADH), conditions where net anion accumulation proceeds, mediated by the interlinking of the inorganic phosphate, dicarboxylate, and tricarboxylate carriers. Maximum succinate uptake in the absence of respiratory inhibitors requires only added inorganic phosphate.     

Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation

Discontinuous Percoll density gradients have been developed for the purification of mitochondria, permitting rapid separation under isosmotic and low viscosity conditions. Mitochondria from several etiolated tissues have been successfully separated from contaminating subcellular structures by this method. For potato tuber the ratio of washed to purified mitochondrial protein was 2.6, similar to the increase in specific activity of cytochrome c oxidase following separation. The purification of mitochondria from green leaf tissues on Percoll gradients has reduced chlorophyll contamination of spinach mitochondria from about 70 micrograms chlorophyll per milligram protein to approximately 8 micrograms chlorophyll per milligram protein.     

低压缺氧对大鼠脑线粒体腺苷酸转运体特性的影响

本文探讨低压缺氧对大鼠脑线粒体内膜腺苷酸转运体（adenine nucleotide translocator,ANT）转运特性的影响。实验将雄性Wistar大鼠随机分为常氧对照组和缺氧组,后者分别连续暴露于模拟5000m高原1、5、15、30d（23h／d）。分别于平原和模拟4000m高原断头处死动物,分离脑线粒体,用抑制剂终止法测定线粒体对。H-ADP的转运效率,抑制剂滴定法测定ANT密度,HPLC测定线粒体内腺苷酸含量。结果显示：缺氧后ANT转运活性均明显低于常氧组,缺氧不同天数线粒体内膜ANT分布密度无显著改变,线粒体内（ATP＋ADP）含量下降与转运活性变化一致。以上观察结果表明,低压缺氧暴露可显著抑制ANT转运活性,降低能量产生和利用的周转率,但不改变ANT密度,提示ANT活性改变是低压缺氧时细胞能量代谢障碍的重要机制。     

Preservation of energy-linked functions of insect mitochondria by simple freezing methods.

Attempts are made to preserve some energy-linked functions of mitochondria isolated from the fat body of the cockroach Nauphoeta cinerea. The ability of mitochondria to sustain appreciable levels of oxidation, phosphorylation, respiratory control and 2, 4-DNP-stimulation of respiration which are normally lost during incubation at 30°C for 2 hrs or during aging at 0–2°C for 48 hrs can be preserved effectively by storing mitochondrial suspensions in sucrose - EDTA medium for 1 week at - 20°C and at least 3 weeks at -196°C (liquid nitrogen temperature). DMSO (dimethyl sulfoxide; 10% v/v) was found to be ineffective as an aid to preservation at 0–2°C, a significant aid at -20°C and unnecessary at -196°C. With slight variations (for flight muscle mitochondria which requires DMSO at -196°C), this procedure is also effective for mitochondria isolated from other tissues of various insect species. EDTA was found to be an essential ingredient of the isolation medium and therefore for all storage procedures. Both lyophilization and the preincubation of mitochondria with nupercaine (400 μM) have deleterious effects.     

Metabolic control analysis of mitochondrial aconitase: influence over respiration and mitochondrial superoxide and hydrogen peroxide production

Abstract

The Fe-S cluster of mitochondrial aconitase is rapidly and selectively inactivated by oxidants, yielding an inactive enzyme that can be reactivated by reductants and iron in vivo. In order to elucidate the metabolic impact of oxidant-dependent aconitase inhibition over the citric acid cycle, the respiratory chain reactions, and reactive species formation, we performed a metabolic analysis using isolated mitochondria from different rat tissues. Titrations with fluorocitrate showed IC₅₀ for aconitase inhibition ranging from 7 to 24 μM. The aconitase inhibition threshold in mitochondrial oxygen consumption was determined to range from 63 to 98%. Of the tissues examined, brain and heart exhibited the highest values in the flux control coefficient (> 0.95). Aconitase-specific activity varied widely among tissues examined from ?60 mU/mg in liver to 321 mU/mg in kidney at 21% O₂. In brain and heart, aconitase-specific activity increased by 42 and 12%, respectively, at 2% O₂ reflecting aconitase inactivation by oxygen-derived oxidants at 21% O₂. Both mitochondrial membrane potential and hydrogen peroxide production significantly decreased upon aconitase inhibition in heart and brain mitochondria. These results indicate that aconitase can exert control over respiration (with tissue specificity) and support the hypothesis that inactivation of aconitase may provide a control mechanism to prevent O₂^●? and H₂O₂ formation by the respiratory chain.     

Effects of the Proline Analog l-Thiazolidine-4-carboxylic Acid on Proline Metabolism

The effect of various proline analogs on proline oxidation in mitochondria isolated from etiolated barley (Hordeum vulgare) shoots was investigated. Of the analogs tested, only l-thiazolidine-4-carboxylic acid (T4C) was an effective inhibitor. T4C (1 millimolar) inhibited proline (10 millimolar) -dependent 0₂ uptake an average of 67%. T4C was also oxidized to some degree (12.9 nanoatoms oxygen per minute per milligram protein for 10 millimolar). The effect of T4C on the oxidation of other mitochondrial substrates was also tested. T4C inhibited ¹-pyrrolidine-5-carboxylic acid-dependent oxygen uptake slightly (13%), the oxidation of malate plus pyruvate even less (6%), and stimulated the oxidation of succinate (+11%), exogenous NADH (+19%), and citrate (+20%). Thus, inhibition by T4C in mitochondria is relatively specific to proline oxidation. T4C was found to inhibit proline dehydrogenase and not the transport of proline into the matrix.     

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv `Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O₂ uptake rates were determined. O₂ uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O₂ uptake in tissues more than 9 days old. Mitochondria in which O₂ uptake was coupled to ATP synthesis were isolated from all tissues. O₂ uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.     

大鼠脑线粒体体外蛋白翻译体系的建立及蛋白产物的鉴定

目的 :建立大鼠脑组织线粒体的体外蛋白合成体系并对其合成产物进行电泳分离和分子量鉴定。方法 :分离大鼠脑组织线粒体 ,用3 H 亮氨酸掺入法探索线粒体体外翻译的最佳条件 ,3 5S 蛋氨酸掺入并对翻译后产物经SDS 聚丙烯酰胺凝胶电泳和放射自显影进行分子量鉴定。结果 :分离的线粒体氧化磷酸化偶联程度高 ,呼吸控制率(RCR)在 3.5～ 5 .5之间 ;体外3 H 亮氨酸的掺入活性在 6 0min内近似线性增长 ,而后维持在一相对稳定水平 ;3 H 亮氨酸的掺入活性随线粒体蛋白浓度而增加 ,而单位线粒体蛋白的掺入活性在 1mg/ml时最高 ;3 5S 蛋氨酸掺入SDS 聚丙烯酰胺凝胶电泳后可观察到清晰的 8条自显影带 ,分子量分别为 (单位Kda) 86、6 6、5 6、43、33、2 9、2 5、18。结论 :用此方法建立的脑线粒体离体翻译反应体系具有高活性和翻译忠实性等特点 ,是研究脑mtDNA在翻译水平的表达及调控的有效方法     

Effect of salicylic acid on the metabolic activity of plant mitochondria

The effects of salicylic acid (SA) on mitochondrial respiration and generation of membrane potential across the inner membrane of mitochondria isolated from stored taproots of sugar beet (Beta vulgaris L.) and etiolated seedling cotyledons of yellow lupine (Lupinus luteus L.) were studied. When malate was oxidized in the presence of glutamate, low SA concentrations (lower than 1.0 mM) exerted predominantly uncoupling action on the respiration of taproot mitochondria: they activated the rate of oxygen uptake in State 4 (in the absence of ADP) and did not affect oxidation in State 3 (in the presence of ADP). In contrast, in lupine cotyledon mitochondria these SA concentrations inhibited oxygen uptake in the presence of ADP and much weaker activated substrate oxidation in State 4. Thus, SA (0.5 mM) reduced the respiratory control ratio according to Chance (RCR) by 25% in the taproots and 35% in cotyledons. When the concentration of phytohormone was increased (above 1.0 mM), malate oxidation in State 3 was inhibited and in State 4 — activated independently of the plant material used. In this case, the values of RCR and ADP/O were reduced by 50–60%. The effect of high SA concentrations (2 mM and higher) on malate oxidation depended on the duration of incubation and had a biphasic pattern: the initial activation of oxygen uptake was later replaced by its inhibition. The parallel studying the SA effect on the generation of membrane potential (ΔΨ) at malate oxidation in the mitochondria of beet taproots and lupine cotyledons showed that ΔΨ dissipation was observed because of SA uncoupling and inhibiting action on respiration. The degree of ΔΨ dissipation depended on the phytohormone concentration and duration on mitochondria treatment, especially at its high concentrations. In general, a correlation was found between the effects of SA on mitochondrial respiration and ΔΨ values in the coupling membranes. Furthermore, these results show that the responses of mitochondria to SA were determined not only by its concentration but also by treatment duration and evidently by the sensitivity to the phytohormone of mitochondria isolated from different plant tissues.     

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P_i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.     

Modulation of sulfide oxidation and toxicity in rat mitochondria by dehydroascorbic acid

Hydrogen sulfide is enzymatically produced in mammalian tissues and functions as a gaseous transmitter. However, H(2)S is also highly toxic as it inhibits mitochondrial respiration at the level of cytochrome c oxidase, which additionally is involved in sulfide oxidation. The accumulation of toxic sulfide levels contributes to the pathology of some diseases. This paper demonstrates that sulfide toxicity can be modified, and dehydroascorbic acid functions as an effector in this process. It significantly reduces the inhibitory effect of sulfide on cytochrome c oxidase, resulting in higher rates of respiration and sulfide oxidation in rat mitochondria. After the addition of dehydroascorbic acid mitochondria maintained more than 50% of the oxygen consumption and ATP production rates with different substrates in the presence of high concentrations of sulfide that would normally lead to complete inhibition. Dehydroascorbic acid significantly increased the sulfide concentration necessary to cause half maximal inhibition of mitochondrial respiration and thus completely prevented inhibition at low, physiological sulfide concentrations. In addition, sulfide oxidation was stimulated and led to ATP production even at high concentrations. The decrease in sulfide toxicity was more pronounced when analyzing supermolecular functional units of the respiratory chain than in isolated cytochrome c oxidase activity. Furthermore, the protective effect of dehydroascorbic acid at high sulfide concentrations was completely abolished by quantitative solubilization of mitochondrial membrane proteins with dodeclymaltoside. These results suggest that binding of cytochrome c oxidase to other proteins probably within respiratory chain supercomplexes is involved in the modulation of sulfide oxidation and toxicity by dehydroascorbic acid.