Calcium uptake by two preparations of mitochondria from heart

Ca²⁺ transport and respiratory characteristics of two preparations of cardiac mitochondria (Palmer, J.W., Tandler, B. and Hoppel, C.L. (1977) J. Biol. Chem. 252, 8731–8739) isolated using polytron homogenization (subsarcolemmal mitochondria) and limited Nagarse exposure (intermyofibrillar mitochondria) are described.The Nagarse procedure yields mitochondria with 50% higher rates of oxidative phosphorylation than the polytron-prepared mitochondria in both rat and dog. Rat hear intermyofibrillar mitochondria contain 50% more cytochrome aa₃ than the polytron preparation, whereas in the dog, cytochrome aa₃ content is not significantly different. Cytochrome oxidase activities and cytochrome c, c₁ and b contents were comparable in both populations of rat and dog heart mitochondria.The V of succinate-supported Ca²⁺ accumulation for Nagarse-prepared mitochondria from rat heart was 1.8-fold higher than the polytron-prepared mitochondria. In dog heart, the Nagarse preparation showed a 3.0-fold higher V for Ca²⁺ uptake compared to the polytron preparation. A lower apparent affinity for Ca²⁺ was demonstrated in the intermyofibrillar mitochondria for both species (K_m is 2–2.5-fold higher). The Hill coefficient was 1 both mitochondrial types. Subsarcolemmal mitochondria from both species were treated with Nagarse to determine the role of this treatment on the observed differences. Nagarse did not alter any kinetic parameter of Ca²⁺ uptake.The properties of these mitochondria with reference to their presumed intracellular location may pertain to the role of mitochondria as an intracellular Ca²⁺ buffering mechanism in contractile tissue.     

Calcium transport by ehrlich ascites cell mitochondria in vitro and in situ

The rate, maximum extent of accumulation, and passive release of Ca²⁺ by mitochondria within Ehrlich ascites tumor cells treated with digitonin and by isolated tumor mitochondria have been compared. The mitochondrial protein content of Ehrlich cells was determined by cytochrome and cytochrome oxidase analyses. The Ca²⁺ uptake rate in situ is approximately one-half the rate in vitro whereas maximum Ca²⁺ accumulation by mitochondria within the cell is about twice the value for isolated mitochondria. When isolated tumor mitochondria were supplemented with exogenous ATP the maximum uptake (approximately 3.0 μeq Ca²⁺/mg protein) was about the same as in situ. Adenine nucleotides retained in digitonized cells may account for the observed differences. The rate of uncoupler stimulated Ca²⁺ release from mitochondria within the cell (ca. 10 neq Ca²⁺/min · mg mitochondrial protein for Ca²⁺ loads up to 800 neq Ca²⁺/mg protein) agrees exceptionally well with previous estimates for isolated tumor mitochondria. Therefore the capacity for extensive Ca²⁺ accumulation without uncoupling and attenuation of Ca²⁺ efflux are virtually the same in the cell as in vitro.     

Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis

Addition of NAD⁺ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O₂ uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD⁺ (NAP₄-NAD⁺), an inhibitor of NAD⁺ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD⁺-stimulated malate-cytochrome c reductase activity, and reduction of added NAD⁺ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD⁺ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD⁺ by storing on ice for 72 hours, was completely dependent on added NAD⁺, and the effect of NAD⁺ on these mitochondria was prevented by incubating them with NAP₄-NAD⁺. External NAD⁺ reduction by these mitochondria was not affected by NAP₄-NAD⁺. We conclude that all effects of exogenous NAD⁺ on plant mitochondrial respiration can be attributed to net uptake of the NAD⁺ into the matrix space.     

Temporary Sequestration of Potassium by Mitochondria in Astrocytes

Increases in extracellular potassium concentration ([K⁺]_o), which can occur during neuronal activity and under pathological conditions such as ischemia, lead to a variety of potentially detrimental effects on neuronal function. Although astrocytes are known to contribute to the clearance of excess K⁺_o, the mechanisms are not fully understood. We examined the potential role of mitochondria in sequestering K⁺ in astrocytes. Astrocytes were loaded with the fluorescent K⁺ indicator PBFI and release of K⁺ from mitochondria into the cytoplasm was examined after uncoupling the mitochondrial membrane potential with carbonyl cyanide m-chlorophenylhydrazone (CCCP). Under the experimental conditions employed, transient applications of elevated [K⁺]_o led to increases in K⁺ within mitochondria, as assessed by increases in the magnitudes of cytoplasmic [K⁺] ([K⁺]_i) transients evoked by brief exposures to CCCP. When mitochondrial K⁺ sequestration was impaired by prolonged application of CCCP, there was a robust increase in [K⁺]_i upon exposure to elevated [K⁺]_o. Blockade of plasmalemmal K⁺ uptake routes by ouabain, Ba²⁺, or a mixture of voltage-activated K⁺ channel inhibitors reduced K⁺ uptake into mitochondria. Also, reductions in mitochondrial K⁺ uptake occurred in the presence of mito-K_ATP channel inhibitors. Rises in [K⁺]_i evoked by brief applications of CCCP following exposure to high [K⁺]_o were also reduced by gap junction blockers and in astrocytes isolated from connexin43-null mice, suggesting that connexins also play a role in K⁺ uptake into astrocyte mitochondria. We conclude that mitochondria play a key role in K⁺_o handling by astrocytes.     

Effects of doxorubicin and its aglycone metabolite on calcium sequestration by rabbit heart, liver, and kidney mitochondria

Previous investigators have shown that following doxorubicin treatment heart mitochondria appear swollen and contain intramitochondrial dense inclusion bodies identified as calcium phosphate. In vitro studies have shown that similar morphological changes occur in mitochondria previously loaded with excess calcium. The present studies were performed to determine the effects of doxorubicin and its aglycone metabolite on ⁴⁵Ca²⁺ uptake by mitochondria isolated from the heart, liver, and kidney of the rabbit. Doxorubicin (100 μM) significantly inhibited the initial rate of ⁴⁵Ca²⁺ accumulated by mitochondria isolated from the three tissues. In contrast, the aglycone metabolite (100 μM) induced the reverse effect. In preloaded mitochondria the aglycone stimulated the release of calcium while doxorubicin was without effect. Mitochondria from the heart were significantly more sensitive to the effects of these anthracyclines than were mitochondria from the other two tissues. If these in vitro effects also occur in vitro, then the aglycone metabolite would be a more likely candidate in explaining the morphological changes in heart mitochondria previously described.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Mechanisms for Intracellular Calcium Regulation in Heart : I. Stopped-Flow Measurements of Ca++ Uptake by Cardiac Mitochondria

Initial velocities of energy-dependent Ca⁺⁺ uptake were measured by stopped-flow and dual-wavelength techniques in mitochondria isolated from hearts of rats, guinea pigs, squirrels, pigeons, and frogs. The rate of Ca⁺⁺ uptake by rat heart mitochondria was 0.05 nmol/mg/s at 5 µM Ca⁺⁺ and increased sigmoidally to 8 nmol/mg/s at 200 µM Ca⁺⁺. A Hill plot of the data yields a straight line with slope n of 2, indicating a cooperativity for Ca⁺⁺ transport in cardiac mitochondria. Comparable rates of Ca⁺⁺ uptake and sigmoidal plots were obtained with mitochondria from other mammalian hearts. On the other hand, the rates of Ca⁺⁺ uptake by frog heart mitochondria were higher at any Ca⁺⁺ concentrations. The half-maximal rate of Ca⁺⁺ transport was observed at 30, 60, 72, 87, 92 µM Ca⁺⁺ for cardiac mitochondria from frog, squirrel, pigeon, guinea pig, and rat, respectively. The sigmoidicity and the high apparent K_m render mitochondrial Ca⁺⁺ uptake slow below 10 µM. At these concentrations the rate of Ca⁺⁺ uptake by cardiac mitochondria in vitro and the amount of mitochondria present in the heart are not consistent with the amount of Ca⁺⁺ to be sequestered in vivo during heart relaxation. Therefore, it appears that, at least in mammalian hearts, the energy-linked transport of Ca⁺⁺ by mitochondria is inadequate for regulating the beat-to-beat Ca⁺⁺ cycle. The results obtained and the proposed cooperativity for mitochondrial Ca⁺⁺ uptake are discussed in terms of physiological regulation of intracellular Ca⁺⁺ homeostasis in cardiac cells.     

OSMOTICALLY LYSED RAT LIVER MITOCONDRIA : Biochemical and Ultrastructural Properties in Relation to Massive Ion Accumulation

Osmotically lysed rat liver mitochondria have been utilized for a study of the biochemical and ultrastructural properties in relation to divalent ion accumulation. Osmotic lysis of mitochondria by suspension and washing in cold, distilled water results in the extraction of about 50% of the mitochondrial protein, the loss of the outer mitochondrial membrane, an increase in respiration, and a marked decrease in the ability to catalyze oxidative phosphorylation. Nevertheless, except for a decrease in the ability to accumulate Sr²⁺ by an ATP-supported process, these lysed mitochondria retain full capacity to accumulate massive amounts of divalent cations by respiration-dependent and ATP-supported mechanisms. The decreased ability of osmotically lysed mitochondria to accumulate Sr²⁺ by an ATP-energized process does not appear to be due to a loss or inactivation of a specific Sr²⁺-activated ATPase. The energy-dependent accumulation processes in lysed mitochondria show an increased sensitivity to inhibition by monovalent cations. Extraction of cytochrome c from osmotically lysed mitochondria results in a complete loss of phosphorylation and the respiration-dependent accumulation of Ca²⁺; a lesser, but significant, decrease in the ATP-supported accumulation of Ca²⁺ also was observed. The addition of cytochrome c fully restores the respiration-dependent accumulation of Ca²⁺ to the level present in unextracted, osmotically lysed mitochondria. The ATP-supported process is not affected by the addition of cytochrome c to extracted mitochondria, indicating that cytochrome c is not involved in ion transport energized by ATP. The osmotically lysed mitochondria are devoid of outer membranes and contain relatively little matrix substance. The accumulation of Ca²⁺ and P_i by lysed mitochondria under massive loading conditions is accompanied by the formation of electron-opaque deposits within the lysed mitochondria associated with the inner membranes. This finding suggests that the inner membrane plays a role in the deposition of divalent ions within intact rat liver mitochondria. The relevance of these observations to those of other investigators is discussed.     

A general characteristic of tumor mitochondria: Leakage of endogenous Mg2+ on incubation with uncoupler and resultant reduction of uncoupler-stimulated ATPase activity

Previous studies showed that stimulation of mouse mitochondrial ATPase activity of tumor cells, fetal liver, and adult brain by the uncoupler 2,4-dinitrophenol was markedly suppressed during incubation of the mitochondria with the uncoupler (J.-I. Hayashi et al., 1980, Biochem. Biophys. Res. Commun.92, 261–267). The present work showed the reason for this suppression. More than half the endogenous Mg²⁺ leaked from mitochondria of all tumor cells tested, and of fetal liver and adult brain during incubation with the uncoupler, while only about 30% of the endogenous Mg²⁺ leaked from mitochondria of other normal tissues. The effect of the uncoupler on Mg²⁺ leakage from liver mitochondria changed from the fetal to the adult type within about 30 min after birth. In hypotonic medium, normal liver mitochondria also lost more than half their total Mg²⁺ and concomitantly stimulation of their ATPase activity by uncoupler was considerably reduced. Exogenously added Mg²⁺ could reverse this reduced effect of the uncoupler on ATPase activity of mitochondria from normal tissues and tumor cells. These results show that the endogenous Mg²⁺ content of mitochondria directly affects the stimulation by uncoupler of ATPase activity of mitochondria from both normal tissues and tumor cells. Thus, mitochondria of all tumor cells tested, and of fetal liver and adult brain are leaky to Mg²⁺ during incubation with uncoupler and as a result of the leakage, the stimulatory effect of the uncoupler on their ATPase activity is greatly reduced.     

Trypsin-induced ATPase Activity in Potato Mitochondria

Potato mitochondria (Solanum tuberosum var. Russet Burbank), which readily phosphorylate ADP in oxidative phosphorylation, show low levels of ATPase activity which is stimulated neither by Mg²⁺, 2,4-dinitrophenol, incubation with respiratory substrates, nor disruption by sonication or treatment with Triton X-100, individually or in concert. Treatment of disrupted potato mitochondria with trypsin stimulates Mg²⁺-dependent, oligomycin-sensitive ATPase activity 10- to 15-fold, suggesting the presence of an ATPase inhibitor protein. Trypsin-induced ATPase activity was unaffected by uncoupler. Oligomycin-sensitive ATPase activity decreases as exposure to trypsin is increased. Incubation at alkaline pH or heating at 60 C for 2 minutes also activates ATPase of sonicated potato mitochondria. Disruption of cauliflower (Brassica oleracea), red sweet potato (Ipomoea batatas), and carrot (Daucus carota) mitochondria increases ATPase activity, which is further enhanced by treatment with trypsin. The significance of the tight association of the inhibitor protein and ATPase in potato mitochondria is not clear.     

The interaction of Ga2+ with mitochondria from human myometrium.

Mitochondria prepared from human myometrium contain large amounts of endogenous Ca²⁺ (up to 200 nmol/mg of protein) even if isolated in media containing ethylene glycol-bis(β-aminoethylether)-N,N′-tetraacetic acid. The endogenous Ca⁺², however, is not irreversibly sequestered, since it can be rapidly and quantitatively discharged by uncouplers. Human myometrial mitochondria are capable of efficient energy-linked Ca²⁺ transport. In the absence of phosphate, the amount of Ca²⁺ accumulated is reduced to insignificant levels. Mg²⁺ has a strong inhibitory effect, which has been exploited to develop an inhibitor-stop method which has permitted the determination of the affinity of myometrial mitochondria for Ca²⁺ (K_m, ~5 μM) and of the maximal velocity of uptake (0.55 nmol/mg of protein/s). The respiration of human myometrial mitochondria is stimulated by Ca²⁺, with respiratory control indexes of the order of 4–5. In contrast, ADP induces an insignificant stimulation, or no stimulation at all. The response of respiration to ADP is somewhat improved if mitochondria are preincubated under conditions which decrease their endogenous Ca²⁺ content. The adenine nucleotide exchange in human myometrial mitochondria is deficient with respect to liver mitochondria.     

Impaired substrate utilization in mitochondria from strain 129 dystrophic mice

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

Duchenne muscular dystrophy is associated with the inhibition of calcium uniport in mitochondria and an increased sensitivity of the organelles to the calcium-induced permeability transition

Duchenne muscular dystrophy (DMD) is characterized by a pronounced and progressive degradation of the structure of skeletal muscles, which decreases their strength and lowers endurance of the organism. At muscular dystrophy, mitochondria are known to undergo significant functional changes, which is manifested in a decreased efficiency of oxidative phosphorylation and impaired energy metabolism of the cell. It is believed that the DMD-induced functional changes of mitochondria are mainly associated with the dysregulation of Ca²⁺ homeostasis. This work examines the kinetic parameters of Ca²⁺ transport and the opening of the Ca²⁺-dependent MPT pore in the skeletal-muscle mitochondria of the dystrophin-deficient C57BL/10ScSn-mdx mice. As compared to the organelles of wild-type animals, skeletal-muscle mitochondria of mdx mice have been found to be much less efficient in respect to Ca²⁺ uniport, with the kinetics of Na⁺-dependent Ca²⁺ efflux not changing. The data obtained indicate that the decreased rate of Ca²⁺ uniport in the mitochondria of mdx mice may be associated with the increased level of the dominant negative subunit of Ca²⁺ uniporter (MCUb). The experiments have also shown that in mdx mice, skeletal-muscle mitochondria have low resistance to the induction of MPT, which may be related to a significantly increased expression of adenylate translocator (ANT2), a possible structural element of the MPT pore. The paper discusses how changes in the expression of calcium uniporter and putative components of the MPT pore caused by the development of DMD can affect Ca²⁺ homeostasis of skeletal-muscle mitochondria.     

Lack of the DNA glycosylases MYH and OGG1 in the cancer prone double mutant mouse does not increase mitochondrial DNA mutagenesis

Reactive oxygen species (ROS) are formed as natural byproducts during aerobic metabolism and readily induce premutagenic base lesions in the DNA. The 8-oxoguanine DNA glycosylase (OGG1) and MutY homolog 1 (MYH) synergistically prevent mutagenesis and cancer formation in mice. Their localization in the mitochondria as well as in the nucleus suggests that mutations in mitochondrial DNA (mtDNA) contribute to the carcinogenesis in the myh^?/?/ogg1^?/? double knockout mouse.In order to test this hypothesis, we analyzed mtDNA mutagenesis and mitochondrial function in young (1 month) and adult (6 months) wt and myh^?/?/ogg1^?/? mice. To our surprise, the absence of OGG1 and MYH had no impact on mtDNA mutation rates in these mice, even at the onset of cancer. This indicates that mtDNA mutagenesis is not responsible for the carcinogenesis of myh^?/?/ogg1^?/? mice. In line with these results, mitochondrial function was unaffected in the cancerous tissues liver and lung, whereas a significant reduction in respiration capacity was observed in brain mitochondria from the adult myh^?/?/ogg1^?/? mouse. The reduced respiration capacity correlated with a specific reduction (?25%) in complex I biochemical activity in brain mitochondria.Our results demonstrate that mtDNA mutations are not associated with cancer development in myh^?/?/ogg1^?/? mice, and that impairment of mitochondrial function in brain could be linked to nuclear DNA mutations in this strain. OGG1 and MYH appear to be dispensable for antimutator function in mitochondria.     

Biochemical characterization of mitochondria from adult worms and plerocercoid larvae of Spirometra mansoni shows mixed populations of anaerobic and aerobic mitochondria

The mitochondria of adult and plerocercoid Spirometra mansoni were characterized in isolated mitochondria and in situ by electron microscopic histochemistry with special attention to the respiratory chain. Although the specific activities of the constituent enzyme complexes of succinate oxidase are fairly similar in adult and plerocercoid mitochondria, those of succinate oxidase and NADH-FRD are approximately 4- and 25-fold higher in adult mitochondria than in plerocercoid mitochondria, respectively. Quinone analysis by high performance liquid chromatography and mass spectrometry showed that adult and plerocercoid mitochondria contained both rhodoquinone-10 and ubiquinone-10 at concentrations of 4.98 and 0.106 nmol mg⁻¹ for adult, and 0.677 and 0.137 nmol mg⁻¹ for plerocercoid, respectively. Inhibition studies on the succinate-oxidase system of adult mitochondria showed that they possessed both cyanide-sensitive and -insensitive succinate oxidases, the latter of which produces hydrogen peroxide. Adult mitochondria, when NADH was used as a substrate, were shown to produce hydrogen peroxide, and the production of hydrogen peroxide decreased to undetectable levels in the presence of fumarate. The specific activities of NADH-fumarate reductase and cytochrome c oxidase were significantly higher in mature proglottids than in immature and gravid proglottids. Isopycnic density-gradient centrifugation analyses and in situ electron microscopic histochemistry revealed that both adult and plerocercoid mitochondria were heterogeneous in terms of respiratory function and physicochemical properties. The physiological significance of adult and plerocercoid mitochondria is discussed in relation to the oxygen tension of their parasitic habitats.     

Defects in Mitochondrial ATP Synthesis in Dystrophin-Deficient Mdx Skeletal Muscles May Be Caused by Complex I Insufficiency

Duchenne Muscular Dystrophy is a chronic, progressive and ultimately fatal skeletal muscle wasting disease characterised by sarcolemmal fragility and intracellular Ca²⁺ dysregulation secondary to the absence of dystrophin. Mounting literature also suggests that the dysfunction of key energy systems within the muscle may contribute to pathological muscle wasting by reducing ATP availability to Ca²⁺ regulation and fibre regeneration. No study to date has biochemically quantified and contrasted mitochondrial ATP production capacity by dystrophic mitochondria isolated from their pathophysiological environment such to determine whether mitochondria are indeed capable of meeting this heightened cellular ATP demand, or examined the effects of an increasing extramitochondrial Ca²⁺ environment. Using isolated mitochondria from the diaphragm and tibialis anterior of 12 week-old dystrophin-deficient mdx and healthy control mice (C57BL10/ScSn) we have demonstrated severely depressed Complex I-mediated mitochondrial ATP production rate in mdx mitochondria that occurs irrespective of the macronutrient-derivative substrate combination fed into the Kreb’s cycle, and, which is partially, but significantly, ameliorated by inhibition of Complex I with rotenone and stimulation of Complex II-mediated ATP-production with succinate. There was no difference in the MAPR response of mdx mitochondria to increasing extramitochondrial Ca²⁺ load in comparison to controls, and 400 nM extramitochondrial Ca²⁺ was generally shown to be inhibitory to MAPR in both groups. Our data suggests that DMD pathology is exacerbated by a Complex I deficiency, which may contribute in part to the severe reductions in ATP production previously observed in dystrophic skeletal muscle.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

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.

     

Dietary avocado oil supplementation attenuates the alterations induced by type I diabetes and oxidative stress in electron transfer at the complex II-complex III segment of the electron transport chain in rat kidney mitochondria

Impaired complex III activity and reactive oxygen species (ROS) generation in mitochondria have been identified as key events leading to renal damage during diabetes. Due to its high content of oleic acid and antioxidants, we aimed to test whether avocado oil may attenuate the alterations in electron transfer at complex III induced by diabetes by a mechanism related with increased resistance to lipid peroxidation. 90 days of avocado oil administration prevented the impairment in succinate-cytochrome c oxidoreductase activity caused by streptozotocin-induced diabetes in kidney mitochondria. This was associated with a protection against decreased electron transfer through high potential chain in complex III related to cytochromes c?+?c ₁ loss. During Fe²⁺-induced oxidative stress, avocado oil improved the activities of complexes II and III and enhanced the protection conferred by a lipophilic antioxidant against damage by Fe²⁺. Avocado oil also decreased ROS generation in Fe²⁺-damaged mitochondria. Alterations in the ratio of C20:4/C18:2 fatty acids were observed in mitochondria from diabetic animals that not were corrected by avocado oil treatment, which yielded lower peroxidizability indexes only in diabetic mitochondria although avocado oil caused an augment in the total content of monounsaturated fatty acids. Moreover, a protective effect of avocado oil against lipid peroxidation was observed consistently only in control mitochondria. Since the beneficial effects of avocado oil in diabetic mitochondria were not related to increased resistance to lipid peroxidation, these effects were discussed in terms of the antioxidant activity of both C18:1 and the carotenoids reported to be contained in avocado oil.