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PubMed2003

Effects of cannabinoids in the rat model of Huntington's disease generated by an intrastriatal injection of malonate.

Cannabinoids could provide neuroprotection in neurodegenerative disorders. In this study, we examined whether a treatment with Delta9-tetrahydrocannabinol, a non-selective cannabinoid receptor agonist, or with SR141716, a selective antagonist for the cannabinoid CB(1) receptor subtype, could affect the toxicity of the complex II reversible inhibitor malonate injected into the striatum, which replicates the mitochondrial complex II deficiency seen in Huntington's disease patients. As expected, malonate injection produced a significant reduction in cytochrome oxidase activity in the striatum consistent with the expected neurodegeneration caused by this toxin. The administration of Delta9-tetrahydrocannabinol increased malonate-induced striatal lesions compared to vehicle and, surprisingly, SR141716, far from producing effects opposite to those of Delta9-tetrahydrocannabinol, also enhanced malonate effects, and to an even greater extent. In summary, our results are compatible with the idea that manipulating the endocannabinoid system can modify neurodegeneration in Huntington's disease, and suggest that highly selective CB(1) receptor agonists might be necessary to produce neuroprotective effects against indirect excitotoxicity.

Animals↗
PubMed2005

Development and characterization of human constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies.

Delta (Y), MB1 (X), and Z are the three catalytic beta-subunits located in the inner rings of the constitutive proteasome, an intracellular multicatalytic complex responsible for the generation of peptides presented by human leukocyte antigen (HLA) class I antigens to T cells. When cells are incubated with interferon-gamma, delta (Y), MB1 (X), and Z are replaced by LMP2, LMP7, and LMP10, respectively, leading to the expression of immunoproteasome which generates peptides with increased affinity for HLA class I antigens. The characterization of the expression of constitutive proteasome and immunoproteasome subunits in cells, normal tissues, and malignant lesions has been hampered by the lack or limited availability of constitutive proteasome and immunoproteasome subunit-specific monoclonal antibodies (mAbs), which are suitable for immunohistochemical staining. To overcome this limitation, we generated human delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10-specific mAb-secreting hybridomas from BALB/c mice immunized with peptides and recombinant fusion proteins. The mAbs SY-5, SJJ-3, NB-1, SY-1, HB-2, and TO-7 were shown to be specific for delta (Y), MB1 (X), Z, LMP2, LMP7, and LMP10, respectively, as they react specifically with the corresponding molecules when tested with a human B lymphoid LG2 cell lysate in Western blotting and with the peptide derived from each molecule in enzyme-linked immunosorbent assay. The reactivity of the six mAbs with the corresponding intracellular antigens resulted in intracellular staining when the mAbs were tested with microwave-treated and saponin-permeabilized cells in indirect immunofluorescence and with formalin-fixed, paraffin-embedded tissue sections in immunohistochemical reactions. These results suggest that the constitutive proteasome and immunoproteasome subunit-specific mAbs we have developed are useful probes to characterize the expression of proteasome subunits in normal tissues and in pathological lesions.

Animals↗
PubMed1982

ATP synthetase (F1F0) of Escherichia coli K-12. High-yield preparation of functional F0 by hydrophobic affinity chromatography.

1. The purified ATP synthetase complex (F1F0) from Escherichia coli was adsorbed to immobilized poly-(L-lysine)-deoxycholic acid. About 0.7 mg F1F0 were bound per ml of settled gel. The hydrophilic F1 part was dissociated from the complex by treatment with 7 M urea. F0 was eluted in high yield either with deoxycholate (6 mM) or taurodeoxycholate (10 mM). About 14% of the total protein bound to the column was eluted as F0, which corresponds to 64% of the total F0 in the F1F0 complex. 2. The purified F0 preparation obtained was composed of three different kinds of subunits with apparent molecular weights of 24000 (a), 19000 (b) and 8300 (c), respectively as determined by sodium dodecyl sulfate gel electrophoresis. 3. After incorporation into liposomes and the generation of a potassium diffusion potential by valinomycin, the F0 preparation mediated H+ translocation. This H+ uptake is inhibited by either dicyclohexylcarbodiimide or purified F1 ATPase. 4. Incubation of F0-containing liposomes with F1 led to the reconstitution of an ATP-driven quenching of acridine-dye fluorescence. The quenching was abolished by uncoupler and prevented by dicyclohexylcarbodiimide.

ATP Synthetase Complexes↗
PubMed1985

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing.

The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.

Amino Acid Sequence↗
PubMed1995

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations.

Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo'-deficient mutants of Escherichia coli K-12 with the following order of potency: Zn(II) > Cd(II) >> Co(II). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.

Binding Sites↗
PubMed1999

Oxidative phosphorylation at the fin de siècle.

Mitochondria produce most of the energy in animal cells by a process called oxidative phosphorylation. Electrons are passed along a series of respiratory enzyme complexes located in the inner mitochondrial membrane, and the energy released by this electron transfer is used to pump protons across the membrane. The resultant electrochemical gradient enables another complex, adenosine 5'-triphosphate (ATP) synthase, to synthesize the energy carrier ATP. Important new mechanistic insights into oxidative phosphorylation have emerged from recent three-dimensional structural analyses of ATP synthase and two of the respiratory enzyme complexes, cytochrome bc1 and cytochrome c oxidase. This work, and new enzymological studies of ATP synthase's unusual catalytic mechanism, are reviewed here.

Adenosine Triphosphate↗
PubMed1989

Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum.

The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine. In cell extracts four radioactive protein fractions labeled with D-[2-14C]riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity. The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band. NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase. This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations. The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity. Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.

Amino Acid Oxidoreductases↗
PubMed1994

Acetoin catabolic system of Klebsiella pneumoniae CG43: sequence, expression, and organization of the aco operon.

A cosmid clone which was capable of depleting acetoin in vivo was isolated from a library of Klebsiella pneumoniae CG43 cosmids. The smallest functional subclone contained a 3.9-kb DNA fragment of the cosmid clone. Sequencing of the DNA fragment revealed three open reading frames (ORFs A, B, and C) encoding polypeptides of 34, 36, and 52 kDa, respectively. The presence of these proteins was demonstrated by expression of the recombinant DNA clone in Escherichia coli. Considerable similarities between the deduced amino acid sequences of the ORFs and those of the following enzymes were found: acetoin dissimilation enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, and branched-chain 2-oxo acid dehydrogenase complex of various origins. Activities of these enzymes, including acetoin-dependent dichlorophenolin-dohenol oxidoreductase and dihydrolipoamide acetyltransferase, were detected in the extracts of E. coli harboring the genes encoding products of the three ORFs. Although not required for acetoin depletion in vivo, a possible fourth ORF (ORF D), located 39 nucleotides downstream of ORF C, was also identified. The deduced N-terminal sequence of the ORF D product was highly homologous to the dihydrolipoamide dehydrogenases of several organisms. Primer extension analysis identified the transcriptional start of the operon as an A residue 72 nucleotides upstream of ORF A.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Consequences of aging on mitochondrial respiratory chain enzymes in cultured human fibroblasts treated with ascorbate.

The activities of mitochondrial respiratory chain enzymes with and without ascorbate pretreatment were assayed in 10- to 20-week-old cultures of human fibroblasts. Aging was associated with a significant loss of respiratory chain enzyme activities. The presence of ascorbate in the medium reduced the rate of loss of these enzymes. Free radical-mediated injuries may also contribute to aging since the changes seen in respiratory chain enzyme activities are similar to those seen in oxidatively stressed cells. This study demonstrates an age-related decline in mitochondrial respiratory chain activity as well as a protective role for ascorbate in aging.

Ascorbic Acid↗
PubMed2005

Direct transfer of NADH from malate dehydrogenase to complex I in Escherichia coli.

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH:ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate + NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate + NAD was estimated to be 1 microM, while the rate of proton translocation due to Complex I was consistent with a somewhat higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.

Binding Sites↗
PubMed1993

Hypertrophic cardiomyopathy with mitochondrial myopathy. A new phenotype of complex II defect.

Two brothers, 25 and 19 years old, were affected by asymmetrical hypertrophic cardiomyopathy. The older brother had waddling gait and weakness of the proximal girdle muscles, while the younger had a broad-based gait and weakness of selected limb girdle muscles. EMG exam was myopathic. Serum enzyme, CPK and aldolase were elevated. Histochemical reactions in muscle revealed "core-like" areas, subsarcolemmal rims of mitochondria and lipid accumulation. Succinate-dehydrogenase stain showed a lack of activity in both biopsies, with the exception of intrafusal fibers. Microphotometric quantitative measurements confirmed the defect in both biopsies. Biochemical measurements of several mitochondrial enzymes in muscle showed a reduced activity of succinate-dehydrogenase (33%) and succinate-cytochrome C reductase (36-47%) which are both components of complex II. On myocardial biopsy lipid and mitochondrial abnormalities were found. This mitochondriopathy represents a new phenotype of partial complex II defect.

Adult↗
PubMed1992

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.

Animals↗
PubMed1985

Mitochondrial cytochemistry in experimental myopathies.

In this paper, we have described mitochondrial cytochemistry (NADH oxidase, cytochrome c oxidase), in the light and electron microscopic studies of the experimental mitochondrial myopathies. DNP, oleate and crotoxin were employed to produce mitochondrial changes in the rat skeletal muscle. The DNP and oleic acid lesions showed strong NADH oxidase and cytochrome c oxidase activities in mitochondria--both at the light microscopic as well as at ultrastructural level. However, the crotoxin lesions showed marked reduction of both the enzyme activities in histochemistry and electron microscopy. Muscle necrosis was seen only in the crotoxin lesion. On the basis of these data, we propose that DNP and oleic acid treatment produced mitochondrial myopathies in which the mitochondrial structure was altered but they were enzymatically active, while the crotoxin treatment produced the structural and enzymatic alterations of the mitochondria.

2,4-Dinitrophenol↗
PubMed1988

[Malate oxidation by mitochondrial succinate:ubiquinone-reductase].

Succinate:ubiquinone reductase was shown to catalyze the oxidation of L- and D-stereoisomers of malate by artificial electron acceptors and ubiquinone. The rate of malate oxidation by succinate:ubiquinone reductase is by two orders of magnitude lower than that for the natural substrate--succinate. The values of kinetic constants for the oxidation of D- and L-stereoisomers of malate are equal to: V infinity = 0.1 mumol/min/mg protein, Km = 2 mM and V infinity = 0.05 mumol/min/mg protein, Km = 2 mM, respectively. The malate dehydrogenase activity is fully inhibited by the inhibitors of the dicarboxylate-binding site of the enzyme, i.e., N-ethylmaleimide and malonate and is practically insensitive to carboxin, a specific inhibitor of the ubiquinone-binding center. The enol form of oxaloacetate was shown to be the product of malate oxidation by succinate:ubiquinone reductase. The kinetics of inhibition of the enzyme activity by the ketone and enol forms of oxaloacetate was studied. Both forms of oxaloacetate effectively inhibit the succinate:ubiquinone reductase reaction.

Animals↗