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PubMed2004

Lipoic acid-dependent oxidative catabolism of alpha-keto acids in mitochondria provides evidence for branched-chain amino acid catabolism in Arabidopsis.

Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2002

Crystallization and preliminary X-ray analysis of the full-size cubic core of pig 2-oxoglutarate dehydrogenase complex.

The full-length (untruncated) dihydrolipoamide succinyltransferase from pig heart was crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction patterns indicate that the crystal belongs to space group I432, with unit-cell parameter a = 189.9 A. The crystal structure has been preliminarily solved at 7 A resolution by the molecular-replacement method. The unit cell contains two cubic cores, in each of which 24 subunits of E2 are associated according to crystallographic 432 symmetry. At the corners of each cubic core, the catalytic domains of E2s form a trimer through tight interactions around the crystallographic threefold axes. In the electron-density maps, many small broad peaks are observed in regions expected to contain the remaining N-terminal domains (the E1/E3-binding domain and the lipoyl domain), suggesting flexibility of these domains relative to the core. The architecture of the cubic core is similar to that of the other truncated E2s. In the unit cell, however, the core-core contact occurs in a different direction from that found for the truncated proteins.

Animals↗
PubMed1984

Cytochrome b of cob revertants in yeast. Bioenergetic characterization of revertants with reduced content and shifted maximum absorption wavelength of cytochrome b.

22 revertants of Saccharomyces cerevisiae with intragenic suppressors (supa) of cob exon mutations (G. Burger, Mol. Gen. Genet., in the press) were analyzed. They display either a reduced amount of cytochrome b, or a shifted maximum absorption wavelength of total cytochrome b or a reduced growth rate on glycerol. The relationship of physico-chemical properties (content, light absorption and midpoint potential of cytochromes bK and bT) and functional properties (electron transport and energy yield) has been examined. In seven of eight revertants with a shifted maximum absorption wavelength of cytochrome b neither growth rate nor electron transfer activity was affected. In 13 of 14 revertants, reduced content of cytochrome b corresponds to a reduced electron transport rate through the cytochrome bc1 segment. A lower enzymatic activity, which is not due to a quantitative but to a qualitative alteration of cytochrome b was found in two revertants. Two revertants show electron transport rates of wild-type level concomitant with a reduced growth rate on glycerol, obviously due to a less efficient energy coupling. All revertants were shown to contain a high and a low potential cytochrome b, referred to as bK and bT. Those cob-/supa mutations which shift the maximum absorption wavelength or diminish the content of cytochrome b affect both b cytochromes in all cases. The results support that electron transport and energy conservation are catalyzed by the unity of cytochrome bK and bT and that both heme centers are bound to an identical apoenzyme. Comparing electron flow rates of succinate:cytochrome c oxidoreductase and NADH:cytochrome c oxidoreductase in cob- mutants and two revertants provides evidence that ubiquinone does not constitute a homogeneous pool, suggested by the dissimilar interaction of both dehydrogenases with the bc1 segment.

Cytochrome b Group↗
PubMed1986

On the oxidation pathways of the mitochondrial bc1 complex from beef heart. Effects of various inhibitors.

We have investigated the oxidation of the reduced ubiquinol:cytochrome c reductase (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate-reduced bc1 complex is not a first-order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron-sulphur center. Among the several inhibitors tested, only 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high-potential centers of the beef heart bc1 complex. The oxidation of cytochrome b by cytochrome c in both the succinate-reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneously with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors. The oxidation of cytochrome b by exogenous ubiquinones is stimulated by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c. Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron-sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone-10. This electron pathway is sensitive to all the inhibitors of the enzyme.

Animals↗
PubMed1995

New inhibitors of the ubiquinol oxidase of higher plant mitochondria.

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1. A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.

Electron Transport Complex III↗
PubMed1997

New insights into the genomic organization and origin of the major histocompatibility complex: role of chromosomal (genome) duplication in the emergence of the adaptive immune system.

Recently, it became clear that the human and mouse genomes contain at least three regions paralogous to the major histocompatibility complex (MHC) region. This observation led us to the proposal that the MHC region emerged as a result of chromosomal duplication that took place at an early stage of vertebrate evolution. Here I briefly review this proposal. Accumulating evidence indicates that (a) genome-wide duplication(s) took place close to the origins of vertebrates. Taking this and others into account, I suggest that the duplication(s) involving the MHC region probably took place as a part of the genome-wide duplication(s). The human T cell receptor (TCR) and immunoglobulin (Ig) genes also appear to be located on paralogous chromosomal segments. These findings raise the possibility that the genome-wide duplication provided a major impetus not only to the emergence of the full-fledged MHC system, but also to the appearance of other key molecules of the adaptive immune system such as TCR and Ig.

Animals↗
PubMed1994

Stereospecific acyl transfers on the erythromycin-producing polyketide synthase.

During assembly of complex polyketide antibiotics like erythromycin A, molecular recognition by the multienzyme polyketide synthase controls the stereochemical outcome as each successive methylmalonyl-coenzyme A (CoA) extender unit is added. Acylation of the purified erythromycin-producing polyketide synthase has shown that all six acyltransferase domains have identical stereospecificity for their normal substrate, (2S)-methylmalonyl-CoA. In contrast, the configuration of the methyl-branched centers in the product, that are derived from (2S)-methylmalonyl-CoA, is different. Stereoselection during the chain building process must, therefore, involve additional epimerization steps.

Acetyl Coenzyme A↗
PubMed2003

Role of defective apoptosis in type 1 diabetes and other autoimmune diseases.

Lymphocyte development, selection, and education are strictly controlled to prevent autoimmunity, with potentially autoreactive cells being removed by apoptosis. Dysregulation of apoptosis is a central defect in diverse murine autoimmune diseases. In murine models of autoimmune lupus, for example, mutations in the death receptor Fas (CD95) or in its ligand, FasL (CD95L), have been identified and shown to render lymphoid cells resistant to apoptosis. In contrast, select lymphoid subpopulations of mice with autoimmune diabetes manifest an increased susceptibility to apoptosis as a result of impaired activation of the transcription factor nuclear factor-kappa B (NF-kappaB), which normally protects cells against tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis. The genetic basis of this defect in NF-kappaB activation is a mutation in the promoter-enhancer region of a gene that encodes an essential subunit (LMP2) of the proteasome. Although no specific genetic defects have been identified in most common forms of human autoimmune disease, functional assays consistently demonstrate heightened apoptosis attributable to multiple death signaling pathways.

Animals↗
PubMed1997

Activation of mitochondrial 2-oxoacid dehydrogenases by thioredoxin.

The regulation of mitochondrial dehydrogenases of 2-oxoacids by thioredoxin is established. It is found that at low NAD+ and saturating concentrations of 2-oxoacids and CoA, inactivation of 2-oxoacid dehydrogenase complexes takes place, preventing NAD+ reduction under such conditions. However, addition of oxidized E. coli thioredoxin to the reaction medium without dithiothreitol allows effective NAD+ reduction at this substrate ratio. Product accumulation curves show that thioredoxin activates the complexes by protecting them from the inactivation observed in the conditions when the complex-bound dihydrolipoate is accumulated. Disappearance of the activatory effect of thioredoxin after its treatment with SH-specific reagents indicates the involvement of the redox-active cysteine couple of thioredoxin in its activation of 2-oxoacid dehydrogenase complexes. The redox-inactive thioredoxin not only shows no activation, but in fact exerts an inhibitory effect. The inhibition manifests the complex formation between SH-modified thioredoxin and dehydrogenase systems, involving amino acid residues of thioredoxin other than cysteine. High efficiency of thioredoxin from E. coli as compared to chloroplast thioredoxin f and glutathione disulfide is revealed. This indicates the importance of specific protein structure also for the influence of the redox-active thioredoxin upon the 2-oxoacid dehydrogenase complexes. The results obtained suggest that these key enzyme systems of mitochondrial metabolism represent previously unidentified targets for the action of mitochondrial thioredoxin, which is known to resemble the E. coli counterpart studies in this work.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1999

Impaired energy metabolism in hearts of septic baboons: diminished activities of Complex I and Complex II of the mitochondrial respiratory chain.

Recent findings support the view that the bioenergetic part of septic organ failure is not caused by insufficient supply of oxygen but by disturbances of the mitochondrial function. Therefore, the aim of the present study was to investigate key enzymes of energy metabolism in septic hearts to answer the question whether or not impairment of mitochondrial or glycolytic enzymes occur under these conditions. For this purpose the well established model of septic baboons was used. Baboons under general anesthesia were made septic by infusion of Escherichia coli. Single challenge with infusion of high amounts of bacteria was compared with a multiple challenge protocol (less bacteria infused). Some animals obtained no E. coli (sham). The hearts of the baboons were removed after 72 h (survival: yes) or after death (survival: no) of the animals, frozen in liquid nitrogen, and stored at -80 degrees C until spectrophotometrical measurement of nine mitochondrial and glycolytic enzymes. A reduction of the activity of NADH:cytochrome-c-reductase (Complex I + III) to 67% and succinate:cytochrome-c-reductase (Complex II + III) to 45% was found in the hearts of surviving animals after infusion of high amounts of bacteria. After multiple challenge with lesser amounts of bacteria, no significant changes in enzyme activity were detectable. After lethal septic shock, activities of Complex I + III (12%) and Complex II + III (13%) as well as of phosphofructokinase (16%) were found to be strongly diminished. Decylubiquinol:cytochrome-c-reductase (Complex III, 59%), cytochrome-c-oxidase (51%), succinate dehydrogenase (60%), glucosephosphate isomerase (61%), lactate dehydrogenase (61%), and citrate synthase (120%) were less or unaffected. Similar but less pronounced effects were found after infusion of lesser amounts of bacteria. By means of inhibitor titrations of succinate: cytochrome-c-reductase, it was shown that the loss of activity is not caused by Complex III but by disturbances in Complex II. It is concluded that E. coli-induced sepsis causes decreased activities of Complex I and Complex II in baboon heart mitochondria in a dose-dependent manner.

Animals↗
PubMed2002

[Complex II].

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Diagnosis, Differential↗
PubMedDate not supplied

[Supermolecular organization of aminotransferases and dehydrogenases of alpha-ketoacid from rat brain mitochondria].

A fraction of coarse mitochondria from the rat brain was deposited after short-term effect of supersound to obtain "metabolones". Activity of dehydrogenases of alpha-ketoacids, succinate dehydrogenase, aspartate-, alanine- and GABA-alpha-ketoglutarate amino-transferases has been determined in the supernatant liquid and in "metabolones". It is shown that dehydrogenase activity is mainly (93-100%) localized in "metabolones", while the level of aminotransferase activity in the latter is lower (72-94%). Nonproportionally high activity of aminotransferases in the supernatant liquid is found to considerably suprpass a decrease in activity of these enzymes in "metabolones" against a background of extremely scanty losses of protein (within 5%) induced by the supersound effect. A hypothetic model of a "metabolone" containing the enzymes of the Krebbs cycle and GABA-shunt is suggested.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1981

Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain.

Almost complete phospholipid depletion has been achieved for Complex I and III of the mitochondrial respiratory chain using a technique that involves elution on Sephadex LH-20 in the presence of Triton X-100. Enzymic activity may be regenerated by replenishment with phospholipid. However, restoration of enzymic activity in phospholipid-depleted Complex I and III has been shown to require the presence of cardiolipin. These results are, therefore, similar to findings on the absolute catalytic requirement of cardiolipin for cytochrome oxidase activity (Fry, M., and Green, D. E. (1980) Biochem. Biophys. Res. Commun. 93, 1238-1246). At least two roles for phospholipid involvement in electron transfer processes are proposed, a catalytic role provided specifically by cardiolipin and a dispersive role that may be provided by various phospholipids or detergents. The absolute requirement of enzymic activity for cardiolipin suggests that this phospholipid plays a crucial role in the coupled electron transfer process.

Animals↗
PubMed1982

The effect of cyclophosphamide on the myocardial oxidase system of mitochondria.

The authors studied the effect of immunosuppressive doses of cyclophosphamide (5 mg/kg b. w.) on the activity of myocardial NADH, succinate and cytochrome oxidases in the mitochondria of rabbit myocardium on two experimental models: a) after a single dose of cyclophosphamide (acute model); b) after cyclophosphamide given daily for a period of 10 weeks (chronic model). A single dose of cyclophosphamide did not affect the myocardial oxidase systems of mitochondria, whereas its chronic administration significantly increased the succinate oxidase activity and decreased cytochrome oxidase activity. The drug did not interfere with NADH- oxidase activity. It is concluded that long-term administration of cytostatic and immunosuppressive doses of cyclophosphamide may result in severe myocardial lesions on subcellular level. This should be taken into account in clinical practice.

Animals↗
PubMed1984

Characterization of purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26.

A highly purified cytochrome b-c1 complex from Rhodopseudomonas sphaeroides R-26 was isolated by a procedure involving Triton X-100 solubilization, calcium phosphate column chromatography, and ammonium sulfate fractionation. The purified enzyme complex contains, in nanomoles/mg of protein, cytochrome b, 8.3; cytochrome c1, 8.3; iron-sulfur protein, 15; phospholipids, 182; and ubiquinone, 5. Four major polypeptides with apparent molecular weights of 48,000, 30,000, 24,000, and 12,000 were detected in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Mr = 48,000 and 30,000 proteins are cytochromes b and c1, respectively. The enzyme complex catalyzes electron transfer from ubiquinol to cytochrome c with a specific activity of 12.6 mumol of cytochrome c reduced per min/mg of protein at 23 degrees C. This is lower than that of the mitochondrial enzyme, although both systems have similar essential redox components and a similar Km for ubiquinol. The activity is fully sensitive to antimycin A and 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole. The enzyme complex is stable at neutral pH and at lower temperatures, but became less stable when the incubation temperature was raised. At 37 degrees C, the half-life is 15 min. The enzymatic activity was insensitive to treatment with N',N'-dicyclohexylcarbodiimide. No p-chloromercuriphenylsulfonate-alkylable sulfhydryl groups were detected. The major phospholipids associated with the purified enzyme complex are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol with molar per cent distributions of 25, 21, and 35, respectively. About 60% of the enzymatic activity was abolished upon treatment with phospholipase A2. The phospholipase A2-inactivated activity can be partially restored by the addition of EDTA followed with phospholipids prepared from either the cytochrome b-c1 complex of the same source or a mixture of phosphatidylglycerol and asolectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron Transport↗
PubMed1995

Isolation of Xenopus LMP-7 homologues. Striking allelic diversity and linkage to MHC.

The mammalian low molecular mass protein-7 (LMP-7) gene resides in the class II region of the MHC, and its product is most probably involved, as a component of a proteasome, in the processing of Ags to be presented by the MHC class I molecules. To elucidate the evolution of the LMP-7 gene at both the primary structure and genetic levels, we isolated LMP-7 cDNA clones from amphibian Xenopus laevis, which last shared a common ancestor with mammals 350 x 10(6) years ago. Two distinctive clones, showing an 85% predicted amino acid sequence identity with each other and 69 to 72% identity with human and mouse LMP-7, were identified from a liver cDNA library of outbred frogs and named XeLMP-7A and XeLMP-7B. XeLMP-7A- and XeLMP-7B-specific probes were used to detect the corresponding genes by using partially inbred frogs with known MHC haplotypes. DNA of the g and j haplotypes hybridized with the XeLMP-7A probe, whereas the f and r haplotype DNA hybridized with the XeLMP-7B probe. These hybridization patterns cosegregated with the MHC haplotypes among offspring of an f/f x f/g cross, and one recombinant revealed that the LMP-7 gene is linked more closely to class II than to class I or class III genes. Taken together, the data indicate that XeLMP-7A and XeLMP-7B are highly diverse alleles at a single locus in the frog MHC. The great allelic diversity can be explained either by coselection with particular class I alleles or by differential silencing of MHC genes in the polyploid X. laevis.

Alleles↗
PubMed1993

MHC-linked low-molecular mass polypeptide subunits define distinct subsets of proteasomes. Implications for divergent function among distinct proteasome subsets.

Proteasomes are 650-kDa, multisubunit endopeptidases that might be involved in the MHC class I Ag processing pathway. We demonstrate the existence of multiple structurally distinct subsets of proteasomes. Distinct forms of proteasomes share a hypothetical core to which unique subunits are added. One of these subsets, LMP2+ proteasome, contains the product of the MHC-linked Lmp-2 gene, and can be distinguished serologically and structurally from other proteasome subsets. The expression of LMP2+ and LMP2- proteasomes is variable among cell lines of different tissue types, and their relative abundance and subunit composition are regulated by IFN-gamma. LMP2+ proteasomes comprise 0 to 74% of total cellular proteasomes. Both LMP2+ and LMP2- proteasomes are proteolytically active. We suggest proteasome function might be regulated by subunit composition, and some, or all proteasome subsets, might participate in the production or delivery of peptides to MHC class I molecules. Both LMP2+ and LMP2- subsets can be further subdivided on the basis of the presence or absence of other unique subunits. Implications of the existence of structurally distinct forms of proteasomes in different tissue types is discussed.

Amino Acid Sequence↗