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PubMed2002

Ceramide-induced apoptosis of D283 medulloblastoma cells requires mitochondrial respiratory chain activity but occurs independently of caspases and is not sensitive to Bcl-xL overexpression.

Ceramides are potent lipid second messengers that are involved in apoptotic and hypoxic/ischaemic neurone death. We investigated the role of mitochondria and the mitochondrial apoptosis pathway in ceramide-induced cell death using human D283 medulloblastoma cells with a reduced mitochondrial DNA copy number (rho- cells) and a corresponding defect in mitochondrial respiration. Treatment with the complex I inhibitor rotenone, C2- or C8-ceramide induced cell death in D283 control cells, while rho- cells were significantly protected. In contrast, activation of the mitochondrial apoptosis pathway by transient overexpression of the pro-apoptotic Bax protein or exposure to the kinase inhibitor staurosporine induced apoptosis to a similar extent in control and rho- cells. Overexpression of the antiapoptotic protein Bcl-xL failed to inhibit the toxic effect of C2-ceramide in D283 control cells, and no significant increase in caspase-3-like protease activity could be detected during the death process. Despite this, C2-ceramide induced significant chromatin condensation and cell shrinkage in D283 control cells, reminiscent of apoptosis. These morphological alterations were associated with the activation of calpains. Both apoptotic morphology and calpain activation were attenuated in rho- cells. Our data indicate that the apoptosis-inducing effect of C2-ceramide may require mitochondrial respiratory chain activity and can occur independently of the mitochondrial apoptosis pathway, but involves the activation of calpains.

Apoptosis↗
PubMed1998

A simple procedure for the isolation of rat kidney lysosomes.

BACKGROUND: A procedure for the isolation of highly purified lysosomes from normal rat kidney is described. METHODS: The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 2 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation. RESULTS: The lysosomal fraction obtained by our method was enriched more than 30-fold in terms of marker enzymes with a yield of about 11%. Electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles. CONCLUSION: We believe that this procedure for isolating kidney lysosome will be useful in the study of the mechanisms of specific modification, processing and catabolism of proteins.

Albumins↗
PubMed1996

Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EPF and RAD6 are recognized by 26 S proteasome subunit 5.

Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys-->Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd < or = 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1998

The quinone-binding site in succinate-ubiquinone reductase from Escherichia coli. Quinone-binding domain and amino acid residues involved in quinone binding.

When purified ubiquinone (Q)-depleted succinate-ubiquinone reductase from Escherichia coli is photoaffinity-labeled with 3-azido-2-methyl-5-methoxy-[3H]6-geranyl-1,4-benzoquinone ([3H]azido-Q) followed by SDS-polyacrylamide gel electrophoresis, radioactivity is found in the SdhC subunit, indicating that this subunit is responsible for ubiquinone binding. An [3H]azido-Q-linked peptide, with a retention time of 61.7 min, is obtained by high performance liquid chromatography of the protease K digest of [3H]azido-Q-labeled SdhC obtained from preparative SDS-polyacrylamide gel electrophoresis on labeled reductase. The partial N-terminal amino acid sequence of this peptide is NH2-TIRFPITAIASILHRVS-, corresponding to residues 17-33. The ubiquinone-binding domain in the proposed structural model of SdhC, constructed based on the hydropathy plot of the deduced amino acid sequence of this protein, is located at the N-terminal end toward the transmembrane helix I. To identify amino acid residues responsible for ubiquinone binding, substitution mutations at the putative ubiquinone-binding region of SdhC were generated and characterized. E. coli NM256 lacking genomic succinate-Q reductase genes was constructed and used to harbor the mutated succinate-Q reductase genes in a low copy number pRKD418 plasmid. Substitution of serine 27 of SdhC with alanine, cysteine, or threonine or substitution of arginine 31 with alanine, lysine, or histidine yields cells unable to grow aerobically in minimum medium with succinate as carbon source. Furthermore, little succinate-ubiquinone reductase activity and [3H]azido-Q uptake are detected in succinate-ubiquinone reductases prepared from these mutant cells grown aerobically in LB medium. These results indicate that the hydroxyl group, the size of the amino acid side chain at position 27, and the guanidino group at position 31 of SdhC are critical for succinate-ubiquinone reductase activity, perhaps by formation of hydrogen bonds with carbonyl groups of the 1,4-benzoquinone ring of the quinone molecule. The hydroxyl group, but not the size of the amino acid side chain, at position 33 of SdhC is also important, because Ser-33 can be substituted with threonine but not with alanine.

Affinity Labels↗
PubMed2001

The Quinone-binding sites of the Saccharomyces cerevisiae succinate-ubiquinone oxidoreductase.

The Saccharomyces cerevisiae succinate dehydrogenase (SDH) of the mitochondrial electron transport chain oxidizes succinate and reduces ubiquinone. Using a random mutagenesis approach, we identified functionally important amino acid residues in one of the anchor subunits, Sdh4p. We analyzed three point mutations (F69V, S71A, and H99L) and one nonsense mutation (Y89OCH) that truncates the Sdh4p subunit at the third predicted transmembrane segment. The F69V and the S71A mutations result in greatly impaired respiratory growth in vivo and quinone reductase activities in vitro, with negligible effects on enzyme stability. In contrast, the Y89OCH and the H99L mutations elicit large structural perturbations that impair assembly as evidenced by reduced covalent FAD levels, membrane-associated succinate-phenazine methosulfate reductase activities, and thermal stability. We propose that the Phe-69 and the Ser-71 residues are involved in the formation of a quinone-binding site, whereas the His-99 residue is at the interface of the peripheral and the membrane domains. In addition, the properties of the Y89OCH mutation are consistent with the interpretation that the third transmembrane segment is not involved in catalysis but rather plays an important structural role. The mutant enzymes are differentially sensitive to a quinone analog inhibitor, providing further evidence for a two-quinone binding model in the yeast SDH.

2,4-Dinitrophenol↗
PubMed2001

Effect of hypoenergetic feeding and refeeding on muscle and mononuclear cell activities of mitochondrial complexes I--IV in enterally fed rats.

BACKGROUND: Previous studies suggested that cell energetics are altered by malnutrition. OBJECTIVE: We hypothesized that nutritional manipulations influence mitochondrial enzyme activities of the electron transport chain in both skeletal muscle and blood mononuclear cells. DESIGN: After a gastrostomy tube was inserted, 44 rats were randomly assigned to 1 of 4 experimental groups: control fed (CF; 364 kJ/d for 7 d), hypoenergetic fed (HF; 92 kJ/d for 7 d), hypoenergetic protein refed (HPR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d), and hypoenergetic glucose refed (HGR; 92 kJ/d for 7 d and then 129 kJ/d for 1 d). The protein and glucose contents of the liquid formulas were different for the HPR and HGR groups. After mitochondria were isolated from the soleus muscle, the activities of complexes I--IV were measured spectrophotometrically. Because of the lack of available tissue, only the activity of complex I was measured in the mononuclear cell extract. RESULTS: The recovery of complex activities in the CF and HF groups was not significantly different in the mitochondrial fraction of the soleus muscle. Compared with that in the CF group, the activities of complexes I--III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly lower in the HF group. The activities of complexes I--III in the mitochondrial fraction of the soleus muscle and the activity of complex I in mononuclear cells were significantly higher in the HPR than in the HF group. The activity of complex IV was generally not affected by nutritional manipulations. CONCLUSION: Malnutrition decreases activities of mitochondrial complexes, which are restored by protein but not glucose refeeding.

Adenosine Triphosphate↗
PubMed1985

Acetate catabolism by Methanosarcina barkeri: evidence for involvement of carbon monoxide dehydrogenase, methyl coenzyme M, and methylreductase.

The pathway of acetate catabolism in Methanosarcina barkeri strain MS was studied by using a recently developed assay for methanogenesis from acetate by soluble enzymes in cell extracts. Extracts incubated with [2-14C]acetate, hydrogen, and ATP formed 14CH4 and [14C]methyl coenzyme M as products. The apparent Km for acetate conversion to methane was 5 mM. In the presence of excess acetate, both the rate and duration of methane production was dependent on ATP. Acetyl phosphate replaced the cell extract methanogenic requirement for both acetate and ATP (the Km for ATP was 2 mM). Low concentrations of bromoethanesulfonic acid and cyanide, inhibitors of methylreductase and carbon monoxide dehydrogenase, respectively, greatly reduced the rate of methanogenesis. Precipitation of CO dehydrogenase in cell extracts by antibodies raised to 95% purified enzyme inhibited both CO dehydrogenase and acetate-to-methane conversion activity. The data are consistent with a model of acetate catabolism in which methylreductase, methyl coenzyme M, CO dehydrogenase, and acetate-activating enzymes are components. These results are discussed in relation to acetate uptake and rate-limiting transformation mechanisms in methane formation.

Acetates↗
PubMed2003

Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature.

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.

Animals↗
PubMed2005

Sepsis induces diaphragm electron transport chain dysfunction and protein depletion.

RATIONALE: Sepsis significantly alters skeletal muscle mitochondrial function, but the mechanisms responsible for this abnormality are unknown. OBJECTIVES: We postulated that endotoxin elicits specific changes in electron transport chain proteins that produce derangements in mitochondrial function. To examine this issue, we compared the effects of endotoxin-induced sepsis on mitochondrial ATP (adenosine triphosphate) formation and electron transport chain protein composition. METHODS AND MEASUREMENTS: Diaphragm mitochondrial oxygen consumption and mitochondrial nicotinamide adenine dinucleotide, reduced form, oxidase assays were measured in control rats (n=13) and rats given endotoxin (8 mg/kg/d) for 12 (n=14), 24 (n=14), 36 (n=14), and 48 h (n=13). Electron transport chain subunits from Complexes I, III, IV, and V were isolated using Blue Native polyacrylamide gel electrophoresis techniques. MAIN RESULTS: Endotoxin administration: 1) elicited large reductions in mitochondrial oxygen consumption (e.g., 201+/-3.9 SE natoms O/min/mg for controls and 101+/-4.5 SE natoms O/minutes/mg after 48 h endotoxin, p<0.001), in nicotinamide adenine dinucleotide, reduced form, oxidase activity (p<0.002), and in uncoupled respiration (p<0.001) and 2) induced selective reductions in two subunits of Complex I, three subunits of Complex III, one subunit of Complex IV, and one subunit of Complex V. The time course of depletion of protein subunits mirrored alterations in oxygen consumption. CONCLUSIONS: Our data indicate that endotoxin selectively depletes critical components of the electron transport chain that diminishes electron flow, reduces proton pumping and decreases ATP formation.

Adenosine Triphosphate↗
PubMedDate not supplied

Chronic stavudine exposure induces hepatic mitochondrial toxicity in adult Erythrocebus patas monkeys.

OBJECTIVES: To understand the mitochondrial mechanisms underlying the lactic acidosis and hepatic steatosis seen in some HIV-1-infected individuals after long-term stavudine (d4T) exposure, we have explored mitochondrial integrity in adult monkeys (Erythrocebus patas) given a daily human equivalent dose of d4T for 78 days. STUDY DESIGN/METHODS: Three Erythrocebus patas (patas) monkeys were given 3 mg d4T orally twice daily (total 6 mg d4T), or approximately 1.2 mg d4T/kg body weight per day, for 78 days and compared with 3 unexposed animals. Blood taken from controls and from treated monkeys before and after drug exposure was subjected to a complete clinical chemistry profile. Liver and skeletal muscles were examined for oxidative phosphorylation enzyme specific activities, mitochondrial deoxyribonucleic acid (mtDNA) quantity by slot blot, and mtDNA integrity by Southern blot. RESULTS: Clinical chemistry assays demonstrated few significant differences; however, one d4T-exposed monkey had a serum lactate of 8.1 mmol/L after 78 days of oral d4T ingestion. Specific activities of oxidative phosphorylation Complexes I, II, and IV were significantly altered in both livers and skeletal muscles from the d4T-exposed animals, compared with the controls (p < or = 0.05). Significant depletion of mitochondrial DNA was observed in livers of drug-exposed monkeys, but not in skeletal muscle (p < or = 0.05). Further examination of liver DNA by Southern blot confirmed hepatic mtDNA depletion in drug exposed animals. CONCLUSIONS: The data suggest that direct examination of the liver may be required to elucidate clinical d4T-induced hepatotoxicity related to mitochondrial damage.

Animals↗
PubMed2003

Calpain is a major cell death effector in selective striatal degeneration induced in vivo by 3-nitropropionate: implications for Huntington's disease.

Striatal cell death in Huntington's Disease (HD) may involve mitochondrial defects, NMDA-mediated excitotoxicity, and activation of death effector proteases such as caspases and calpain. However, the precise contribution of mitochondrial defects in the activation of these proteases in HD is unknown. Here, we addressed this question by studying the mechanism of striatal cell death in rat models of HD using the mitochondrial complex II inhibitor 3-nitropropionic acid (3-NP). The neurotoxin was either given by intraperitoneal injections (acute model) or over 5 d by constant systemic infusion using osmotic pumps (chronic model) to produce either transient or sustained mitochondrial deficits. Caspase-9 activation preceded neurodegeneration in both cases. However, caspase-8 and caspase-3 were activated in the acute model, but not in the chronic model, showing that 3-NP does not require activation of these caspases to produce striatal degeneration. In contrast, activation of calpain was specifically detected in the striatum in both models and this was associated with a calpain-dependent cleavage of huntingtin. Finally, in the chronic model, which mimics a steady blockade of complex II activity reminiscent of HD, selective calpain inhibition prevented the abnormal calpain-dependent processing of huntingtin, reduced the size of the striatal lesions, and almost completely abolished the 3-NP-induced DNA fragmentation in striatal cells. The present results demonstrate that calpain is a predominant effector of striatal cell death associated with mitochondrial defects in vivo. This suggests that calpain may play an important role in HD pathogenesis and could be a potential therapeutic target to slow disease progression.

Acute Disease↗
PubMed1992

Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation.

Structural mitochondrial damage accompanies the cytotoxic effects of several drugs including tumor necrosis factor (TNF). Using various inhibitors of mitochondrial electron transport we have investigated the mechanism of TNF-mediated cytotoxicity in L929 and WEHI 164 clone 13 mouse fibrosarcoma cells. Inhibitors with different sites of action modulated TNF cytotoxicity, however, with contrasting effects on final cell viability. Inhibition of mitochondrial electron transport at complex III (cytochrome c reductase) by antimycin A resulted in a marked potentiation of TNF-mediated injury. In contrast, when the electron flow to ubiquinone was blocked, either at complex I (NADH-ubiquinone oxidoreductase) with amytal or at complex II (succinate-ubiquinone reductase) with thenoyltrifluoroacetone, cells were markedly protected against TNF cytotoxicity. Neither uncouplers nor inhibitors of oxidative phosphorylation nor complex IV (cytochrome c oxidase) inhibitors significantly interfered with TNF-mediated effects, ruling out the involvement of energy-coupled phenomena. In addition, the toxic effects of TNF were counteracted by the addition of antioxidants and iron chelators. Furthermore, we analyzed the direct effect of TNF on mitochondrial morphology and functions. Treatment of L929 cells with TNF led to an early degeneration of the mitochondrial ultrastructure without any pronounced damage of other cellular organelles. Analysis of the mitochondrial electron flow revealed that TNF treatment led to a rapid inhibition of the mitochondria to oxidize succinate and NADH-linked substrates. The inhibition of electron transport was dose-dependent and became readily detectable 60 min after the start of TNF treatment, thus preceding the onset of cell death by at least 3-6 h. In contrast, only minor effects were observed on complex IV activity. The different effects observed with the mitochondrial respiratory chain inhibitors provide suggestive evidence that mitochondrial production of oxygen radicals mainly generated at the ubisemiquinone site is a causal mechanism of TNF cytotoxicity. This conclusion is further supported by the protective effect of antioxidants as well as the selective pattern of damage of mitochondrial chain components and characteristic alterations of the mitochondrial ultrastructure.

Amobarbital↗
PubMed1992

Properties of respiratory chain-linked Na(+)-independent NADH-quinone reductase in a marine Vibrio alginolyticus.

The respiratory chain of a marine Vibrio alginolyticus contains two types of NADH-quinone reductase (NQR): one is an Na(+)-dependent NQR functioning as an Na+ pump (NQR-1) and the other is an Na(+)-independent NQR (NQR-2). NQR-2 was purified about 55-fold from the membrane of mutant Nap-1 which is devoid of NQR-1, and its properties were compared with those of NQR-1. In contrast to NQR-1, the purified NQR-2 does not require any salts for activity and is not inhibited by up to 0.4 M salts. The optimum pH of NQR-2 is between 6.8 and 7.8, which is about 0.7 ph units lower than that of NQR-1. NQR-2 is insensitive to strong inhibitors of NQR-1 such as p-chloromercuribenzoate, Ag+ and 2-heptyl-4-hydroxyquinoline N-oxide. Using inverted membrane vesicles, it was confirmed that NQR-2 has no capacity to generate a membrane potential. NQR-2 reduces menadione and ubiquinone-1 by a two-electron reduction pathway. Since the NADH-reacting FAD-containing beta-subunit of NQR-1 reduces quinones by a one-electron reduction pathway, the mode of quinone reduction is closely related to energy coupling; the formation of semiquinone radicals as an intermediate is likely to be essential to functioning as an ion pump.

Electron Transport Complex II↗
PubMed1989

One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556.

Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms. The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme. The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification. In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity. This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon. The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule. The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate.

Cell Membrane↗
PubMed1987

Partial deficiency of subunits in complex I or IV of patients with mitochondrial myopathies.

Subunits of Complexes I, III, IV, and V from skeletal muscle mitochondria isolated from four patients with mitochondrial myopathies were analyzed by immunoblotting. A deficiency was observed in Complex I or IV, or in both. The patterns of deficiency of subunits of each complex were similar both in patients with deficiency of a single complex and in patients with combined deficiency of the two complexes. The deficiency of any of these subunits was only partial, and no abnormalities in the electrophoretic mobilities of the subunits were observed. These results suggest that the deficiency of Complex I or IV in these patients are based on similar molecular defects which lead to decreased levels of these subunits.

Adolescent↗
PubMed1984

Dicyclohexylcarbodiimide binds to cytochrome b and subunit VIII in soluble complex III from beef heart mitochondria.

Incubation of soluble complex III isolated from either yeast or beef heart mitochondria with 25-100 nmol of [14C]dicyclohexylcarbodiimide (DCCD)/nmol of cytochrome b followed by centrifugation through 10% sucrose or precipitation with trichloroacetic acid did not result in any changes in the appearance of the subunits of either complex. The [14C]DCCD was bound to cytochrome b and phospholipids in the yeast complex and with similar kinetics to both cytochrome b and subunit VIII (Mr = 4000-8000) plus phospholipids of the beef complex. Subunit VIII of the beef complex was partially extracted with chloroform:methanol; however, no subunit of this mobility was present in the yeast complex. Incubation of the beef complex in phosphate buffer for short times resulted in a doubling of the [14C]DCCD bound to cytochrome b relative to that to subunit VIII. Preincubation of both complexes with venturicidin prior to treatment with DCCD resulted in a 50% decrease in the binding of [14C]DCCD to cytochrome b. Reisolation of the beef complex III by precipitation with (NH4)2SO4 after incubation with [14C]DCCD resulted in the formation of a new band with an apparent molecular weight of 39,000 even in the zero time control. The [14C]DCCD was bound to subunit VIII and the core proteins but not to cytochrome b at all times, suggesting that precipitation with (NH)2SO4 in the presence of DCCD causes cross-linking of the subunits of complex III.

Ammonium Sulfate↗