Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NADH:ubiquinone reductase (complex I) of the mitochondrial inner membrane respiratory chain binds a number of mitochondrial matrix NAD-linked dehydrogenases. These include pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, mitochondrial malate dehydrogenase, and beta-hydroxyacyl-CoA dehydrogenase. No binding was detected between complex I and cytosolic malate dehydrogenase, glutamate dehydrogenase, NAD-isocitrate dehydrogenase, lipoamide dehydrogenase, citrate synthase, or fumarase. The dehydrogenases that bound to complex I did not bind to a preparation of complex II and III, nor did they bind to liposomes. The binding of pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase complex, and mitochondrial malate dehydrogenase to complex I is a saturable process. Based upon the amount of binding observed in these in vitro studies, there is enough inner membrane present in the mitochondria to bind the dehydrogenases in the matrix space. The possible metabolic significance of these interactions is discussed.
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PMID:Complex I binds several mitochondrial NAD-coupled dehydrogenases. 643 16

Branched-chain alpha-ketoacid dehydrogenase has been purified to homogeneity from bovine liver mitochondria. The isolated complex has a specific activity of 5-8 mumol of reduced nicotinamide adenine dinucleotide min-1 (mg of protein)-1 as isolated and does not require the addition of exogenous lipoamide dehydrogenase for activity. Addition of porcine heart lipoamide dehydrogenase stimulated complex activity by no more than 20%. Four subunits copurify with the complex with molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 55 000, 52 000, 46 500, and 37 500. Here we show that the 52 000-dalton subunit is the lipoyl-containing transacylase component of the complex. Data are presented to support the hypothesis that the branched-chain ketoacid dehydrogenase complex is physically and catalytically similar to, but separate from, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The transacylase of the branched-chain ketoacid dehydrogenase complex has an exposed trypsin-sensitive region. Proteolytic action of trypsin separates a lipoyl-containing component from the remainder of the protein. Data from our laboratory presented here and elsewhere define a specific function for three of the four subunits.
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PMID:Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase. 665 74

The lipoic acids of the alpha-ketoglutarate dehydrogenase multienzyme complex from Escherichia coli have been modified with two fluorescent probes, N-(1-pyrenyl)-maleimide and 5-[[[(iodoacetyl)amino]ethyl]amino]-naphthylene-1-sulfonic acid. Time-resolved fluorescence polarization of partially labeled complexes (18-77% inhibition of enzyme activity) reveals a complex depolarization process: one component of the anisotropy is characterized by a rotational correlation time much longer than the time scale of the measurements (less than or equal to 400 ns), reflecting the overall rotation of the complex, while a second component of the anisotropy decays with a rotational correlation time of 320 (+/- 50) ns. This decay is essentially independent of viscosity and is consistent with a model in which the depolarization is due to the dissociation from and rotation of lipoic acids between binding sites on the multienzyme complex. The sum of the rate constants characterizing the association and dissociation with the binding sites is approximately 3 x 10(6) s-1. In addition, approximately 5% of the anisotropy of the N-(1-pyrenyl)maleimide-labeled complex decays with a rotational correlation time of 25 ns; this can be attributed to local motion of the probe. At high extents of N-(1-pyrenyl)maleimide labeling (90-95% inhibition of enzyme activity), the anisotropy decay can be described by a constant term plus a rotational correlation time of about 1 microseconds. The increase in the correlation time probably reflects interactions between pyrene moieties. The N-(1-pyrenyl)maleimide-labeled dihydrolipoyl transsuccinylase core of the multienzyme complex has been isolated, and the anisotropy is constant over the observed time range of 300 ns. This suggests that the native structure is necessary for observation of lipoic acid movement within the complex. Fluorescent-labeled limited trypsin digestion fragments of the alpha-ketoglutarate dehydrogenase complex also have been isolated, and anisotropy measurements reveal substantial mobility of the label within the fragments. The time-resolved anisotropy of FAD in the native complex and in the isolated dihydrolipoyl dehydrogenase indicates some rapid local mobility of the FAD (rotational correlation time of 12 ns) that is viscosity independent, as well as a component of the anisotropy that is constant over the 35-ns time scale of the experiments.
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PMID:Fluorescence polarization study of the alpha-ketoglutarate dehydrogenase complex from Escherichia coli. 675 46

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.
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PMID:Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition. 689 47

NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.
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PMID:Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification. 746 25

The mitochondrion is the only extranuclear organelle containing DNA (mtDNA). As such, genetically determined mitochondrial diseases may result from a molecular defect involving the mitochondrial or the nuclear genome. The first is characterized by maternal inheritance and the second by Mendelian inheritance. Ragged-red fibers (RRF) are commonly seen with primary lesions of mtDNA, but this association is not invariant. Conversely, RRF are seldom associated with primary lesions of nuclear DNA. Large-scale rearrangements (deletions and insertions) and point mutations of mtDNA are commonly associated with RRF and lactic acidosis, e.g. Kearns-Sayre syndrome (KSS) (major large-scale rearrangements), Pearson syndrome (large-scale rearrangements), myoclonus epilepsy with RRF (MERRF) (point mutation affecting tRNA(lys) gene), mitochondrial myopathy, lactic acidosis, and stroke-like episodes (MELAS) (two point mutations affecting tRNA(leu)(UUR) gene) and a maternally-inherited myopathy with cardiac involvement (MIMyCa) (point mutation affecting tRNA(leu)(UUR) gene). However, RRF and lactic acidosis are absent in Leber hereditary optic neuropathy (LHON) (one point mutation affecting ND4 gene, two point mutations affecting ND1 gene, and one point mutation affecting the apocytochrome b subunit of complex III), and the condition associated with maternally inherited sensory neuropathy (N), ataxia (A), retinitis pigmentosa (RP), developmental delay, dementia, seizures, and limb weakness (NARP) (point mutation affecting ATPase subunit 6 gene). The point mutations in MELAS, MIMyCa, and MERRF, and the large-scale mtDNA rearrangements in KSS and Pearson syndrome have a broader biochemical impact since these molecular defects involve the translational sequence of mitochondrial protein synthesis. The nuclear defects involving mitochondrial function generally are not associated with RRF. The biochemical classification of mitochondrial diseases principally catalogues these nuclear defects. This classification divides mitochondrial diseases into five categories. Primary and secondary deficiencies of carnitine are examples of a substrate transport defect. A lipid storage myopathy is often present. Disturbances of pyruvate or fatty acid metabolism are examples of substrate utilization defects. Only four defects of the Krebs cycle are known: fumarase deficiency, dihydrolipoyl dehydrogenase deficiency, alpha-ketoglutarate dehydrogenase deficiency, and combined defects of muscle succinate dehydrogenase and aconitase. Luft disease is the singular example of a defect in oxidation-phosphorylation coupling. Defects of respiratory chain function are manifold. Two clinical syndromes predominate, one involving limb weakness, and the other primarily affecting brain function. Leigh syndrome may result from different enzyme defects, most notably pyruvate dehydrogenase complex deficiency, cytochrome c oxidase deficiency, complex I deficiency, and complex V deficiency associated with the recently described NARP point mutation. A new group of mitochondrial diseases has emerged.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The expanding clinical spectrum of mitochondrial diseases. 833 7

To determine whether the reduction in brain alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease (AD) is associated with an abnormality in one of its three constituent enzyme subunits, we measured protein levels of alpha-ketoglutarate dehydrogenase (El), dihydrolipoamide succinyltransferase (E2), and dihydrolipoamide dehydrogenase (E3), in postmortem brain of 29 patients with AD (mean age, 73 years; age range of onset, 50-78 years) and 29 control subjects. In the AD group protein levels of all three subunits were significantly reduced by 23 to 41% in the temporal cortex, whereas in the parietal cortex (El: -28%; E3: -32%) and hippocampus (E3: -33%) significant changes were limited to El and E3. alpha-Ketoglutarate dehydrogenase complex activities were more markedly reduced (by 46-68%) and did not correlate with protein levels, suggesting that decreased enzyme activity cannot be primarily explained by loss of alpha-ketoglutarate dehydrogenase complex protein. We did not find two E2 immunoreactive forms in the brain of any patient, as has been reported in fibroblasts of patients with very-early-onset chromosome 14-linked AD. We conclude that brain protein and activity levels of alpha-ketoglutarate dehydrogenase complex are reduced in patients with AD who have onset after 50 years and suggest that these changes, which are also observed in other human brain disorders, may represent a nonspecific consequence of different neurodegenerative processes. Nevertheless, reduced levels of this rate-limiting enzyme of the Krebs cycle could contribute to the brain neurodegenerative mechanisms of AD.
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PMID:Brain protein and alpha-ketoglutarate dehydrogenase complex activity in Alzheimer's disease. 861 44

Enzyme activities of a alpha-ketoglutarate dehydrogenase complex (alpha KGDHC) and one of its constituent subunits, dihydrolipoamide dehydrogenase (E3), are reported to be reduced in non-CNS tissues of some patients with Friedreich's ataxia (FA); however, the results are highly conflicting. To determine whether an enzyme abnormality occurs in brain, we measured immunoreactive levels of the three alpha KGDHC subunits, namely, alpha-ketoglutarate dehydrogenase (E1), dihydrolipoamide succinyltransferase (E2) and E3 in postmortem frontal, occipital and cerebellar cortices of 18 control subjects, 9 patients with FA and, for comparison, 12 patients with spinocerebellar ataxia type 1 (SCA1). Decreased (-20 to -31%) levels of E3 were observed in all three examined areas of the patients with FA with the changes statistically significant in cerebellar and frontal cortices. The E3 reduction could be explained by a loss of alpha KGDHC or other dehydrogenase complexes (e.g. pyruvate dehydrogenase complex) which utilize this subunit. In SCA1, enzyme changes were limited to E2 in cerebellar (-26%) and frontal (-19%) cortices. Although the E3 and E2 reductions are only slight, and may represent secondary events, the changes in this key Krebs cycle enzyme could exacerbate degenerative processes in both of the spinocerebellar ataxia disorders.
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PMID:Immunoreactive levels of alpha-ketoglutarate dehydrogenase subunits in Friedreich's ataxia and spinocerebellar ataxia type 1. 873 79

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid dependent or independent manner. The alpha-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of alpha-lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, alpha-lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an alpha-lipoic acid, coenzyme A, and pyruvate or alpha-ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an alpha-lipoic acid (K0.5 = 1.4 +/- 0.8 mM) and lipoamide (K0.5 = 0.9 +/- 0.3 mM) dependent manner.
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PMID:alpha-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria. 878 42

Dihydrolipoamide dehydrogenase is a common component of mammalian multienzyme complexes that decarboxylate alpha-ketoacids and catabolize glycine. The common function is to reoxidize a reduced lipoate component of each complex, thereby preparing that lipoate for another round of catalysis. Regions within dihydrolipoamide dehydrogenase involved in association with other proteins of the complexes are poorly defined, and despite high amino acid sequence conservation through evolution, it is unknown if dihydrolipoamide dehydrogenases are functionally equivalent across species. To address this issue, we asked whether the human enzyme could restore function to the alpha-ketoacid dehydrogenase complexes in a yeast strain deficient in endogenous dihydrolipoamide dehydrogenase. This dihydrolipoamide dehydrogenase null mutant will not grow on non-fermentable carbon sources. The human enzyme expressed from a CEN plasmid complemented the growth phenotype and restored full activity to the pyruvate and alpha-ketoglutarate dehydrogenase complexes. Human dihydrolipoamide dehydrogenases with selected amino acid substitutions were then tested in the null strain for their ability to restore function. Substitutions tested represented naturally occurring candidate mutations identified in an individual with inactive dihydrolipoamide dehydrogenase. A K37E change had full function while a P453L change resulted in reduced dihydrolipoamide dehydrogenase abundance in the mitochondria and no detectable catalytic activity.
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PMID:Functional analysis in Saccharomyces cerevisiae of naturally occurring amino acid substitutions in human dihydrolipoamide dehydrogenase. 889 1


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