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)

This study was undertaken to search for the endogenous dithiol cofactor of the reductases of the vitamin K cycle. As a starting point, the redox-active lipophilic endogenous compounds lipoic acid and lipoamide were looked at. The study shows that microsomes contain NADH-dependent lipoamide reductase activity. Reduced lipoamide stimulates microsomal vitamin K epoxide reduction with kinetics comparable with those for the synthetic dithiol dithiothreitol (DTT). Reduced lipoic acid shows higher (4-fold) Km values. No reductase activity with lipoic acid was found to be present in microsomes or cytosol. The reduced-lipoamide-stimulated vitamin K epoxide reductase is as sensitive to warfarin and salicylate inhibition as is the DTT-stimulated one. Both vitamin K epoxide reductase and lipoamide reductase activity are recovered in the rough microsomes. NADH/lipoamide-stimulated vitamin K epoxide reduction is uncoupled by traces of Triton X-100, suggesting that microsomal lipoamide reductase and vitamin K epoxide reductase are associated. The results suggest that the vitamin K cycle obtains reducing equivalents from NADH through microsomal lipoamide reductase.
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PMID:Microsomal lipoamide reductase provides vitamin K epoxide reductase with reducing equivalents. 829 31

Mammalian pyruvate dehydrogenase complex (PDC) contains a subunit, protein X, which mediates high-affinity binding of dihydrolipoamide dehydrogenase (E3)to the dihydrolipoamide acetyltransferase (E2) core. Precise stoichiometric determinations on bovine heart PDC, by means of two approaches, indicate the presence of 12 mol protein X/mol PDC and 60 mol E2/mol PDC. Studies of the organisation of collagenase-modified PDC by means of covalent cross-linking of N,N'-1,2-phenylenedimaleimide to lipoamide thiols on protein X, reveal that the main cross-linked products have Mr values corresponding to homodimers of protein X. However, significant formation of higher-Mr aggregates indicates that lipoyl domains of protein X can form an interacting network independent of E2 lipoyl domains. These data suggest that either 12 interacting X monomers or 6 interacting X dimers are involved in the binding of six E3 homodimers to the E2/X core. The presence of 60 E2 subunits/complex also supports proposals for a non-integrated external position of protein X. Collagenase-treated PDC possesses residual activity (15 %), indicating that protein-X-linked lipoamide groups can substitute for the lipoyl domains of E2 in overall complex catalysis. Protein-X-mediated diacetylation of dihydrolipoamide moieties is also performed by the modified complex which raises the possibility of a unique catalytic function for protein X.
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PMID:Stoichiometry, organisation and catalytic function of protein X of the pyruvate dehydrogenase complex from bovine heart. 861 88

Catecholamines (CAs: epinephrine, norepinephrine, dopamine, L-DOPA, 6-hydroxydopamine) and o-diphenols (DOPAC and catechol) enhanced dihydrolipoamide dehydrogenase (LADH) inactivation by Cu(II)/H2O2 (Cu-Fenton system). The inhibition of LADH activity correlated with Cu(II), H2O2 and CA concentrations. Similar inhibitions were obtained with the assayed CAs and o-diphenols. CAs enhanced HO. radical production by Cu(II)/H2O2, as demonstrated by benzoate hydroxylation and deoxyribose oxidation; LADH counteracted the pro-oxidant effect of CAs by scavenging hydroxyl radicals. Captopril, dihydrolipoamide, dihydrolipoic acid, DL-dithiothreitol, GSSG, trypanothione and histidine effectively preserved LADH from oxidative damage, whereas N-acetylcysteine, N-(2-mercaptopropionylglycine) and lipoamide were less effective protectors. Catalase (though neither bovine serum albumin nor superoxide dismutase) protected LADH against the Cu(II)/H2O2/CAs systems. Denatured catalase protected less than the native enzyme, its action possibly depending on Cu-binding. LADH increased and Captopril inhibited epinephrine oxidation by Cu(II)/H2O2 and Cu(II). The summarized evidence supports the following steps for LADH inactivation: (1) reduction of LADH linked-Cu(II) to Cu(I) by CAs; (2) production of HO. from H2O2 by LADH-linked Cu(I) (Haber-Weiss reaction) and (3) oxidation of aminoacid residues at the enzyme active site by site-specifically generated HO. radicals. Hydrogen peroxide formation from CAs autoxidation may contribute to LADH inactivation.
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PMID:Catecholamines enhance dihydrolipoamide dehydrogenase inactivation by the copper Fenton system. Enzyme protection by copper chelators. 873 Oct 15

Reduction of the antioxidant lipoic acid has been proposed to be catalyzed in vivo by lipoamide dehydrogenase (LipDH) or glutathione reductase (GR). We have found that thioredoxin reductase (TR) from calf thymus, calf liver, human placenta, and rat liver efficiently reduced both lipoic acid and lipoamide with Michaelis-Menten type kinetics in NADPH-dependent reactions. In contrast to LipDH, lipoic acid was reduced almost as efficiently as lipoamide. Under equivalent conditions at 20 degrees C, pH 8.0, mammalian TR reduced lipoic acid by NADPH 15 times more efficiently than the corresponding NADH dependent reduction catalyzed by LipDH (297 min-1 for TR vs. 20.3 min-1 for LipDH). Moreover, TR was 2.5 times faster in reducing lipoic acid with NADPH than in catalyzing the reverse reaction (oxidation of dihydrolipoic acid with NADP+). In contrast, LipDH was only 0.048 times as efficient in the forward reaction as compared to the reverse reaction (using NADH and NAD+). We conclude that all or part of the previously described NADPH-dependent lipoamide dehydrogenase (diaphorase) activities in mammalian systems should be attributed to TR. Our results suggest that in mammalian cells a significant part of the therapeutically important reduction of lipoic acid is catalyzed by thioredoxin reductase.
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PMID:Efficient reduction of lipoamide and lipoic acid by mammalian thioredoxin reductase. 876 29

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

The occurrence of NADH --> NAD transhydrogenation and lipoamide dehydrogenase activities was demonstrated for cysticercoids of the intestinal cestode, Hymenolepis diminuta. In addition, both activities were catalyzed by the mitochondria of 6-, 10-, and 14-day H. diminuta and by the mitochondria from immature, mature, and pregravid/gravid regions of the adult cestode. A developmentally related increase in NADH --> NAD activity was suggested and the levels of both activities in the immature region of the helminth were consistent with it being a region of high metabolic activity. Adult H. diminuta mitochondrial lipoamide dehydrogenase was purified to homogeneity. The native enzyme was a homodimer with a monomeric and dimeric molecular mass of 47 and 93 kDa, respectively. Spectral analyses revealed that the enzyme contained flavin. More importantly, the purified enzyme catalyzed appreciable NADH --> NAD transhydrogenation activity, a premier finding for the phylum Platyhelminthes. The ratio of NADH --> NAD transhydrogenation to lipoamide reduction was 1:5. Both activities were inhibited by Cu2+ and Cd2+ with the NADH --> NAD activity being more resistant to inhibition. Interestingly, aside from NADH diaphorase activity, the cestode enzyme displayed NADH-ferricyanide reductase and, to a lesser degree, NADPH --> NAD transhydrogenation activities. The partial amino acid sequence of H. diminuta lipoamide dehydrogenase indicated that this enzyme was most similar to the corresponding enzymes of other parasitic helminths. Moreover, the phenylalanine for leucine substitution found in the redox-active disulfide site of the lipoamide dehydrogenases of some anaerobic systems was noted for the H. diminuta enzyme.
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PMID:Hymenolepis diminuta: mitochondrial NADH --> NAD transhydrogenation and the lipoamide dehydrogenase system. 903 Jun 66

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full-length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50,066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively. An N-terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N-terminus of the mature protein. This system allowed the synthesis of 1000 U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4-precipitation and affinity chromatography on 5' AMP-Sepharose. The K(m) values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite. LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redoxcycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure-based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.
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PMID:Cloning, sequencing and functional expression of dihydrolipoamide dehydrogenase from the human pathogen Trypanosoma cruzi. 905 40

In spite of well-known ability of lipoamide/dihydrolipoamide (LipS2NH2/Lip(SH)2NH2) and oxidized/reduced glutathione (GSSG/GSH) couples to scavenge singlet oxygen (1O2), the possible protective effects of these compounds against photodynamical damage by Alphtalocyanine tetrasulfonate (Al-PcS4) were examined. Using erythrocyte glutathione reductase, pig heart lipoamide dehydrogenase and hamster kidney fibroblast culture as model systems, we have found that protective effects of Lip(SH)2NH2 and LipS2NH2 were close to that of azide, far exceeding the effects of GSH and GSSG, and paralleling the rates of Al-PcS4-sensitized photooxidation of these compounds. We have failed to observe a previously described (Devasagayam, T.P.A., et al. (1991) Biochim Biophys. Acta 1088, 409-412) enhancement of damaging action of 1O2 by GSH. These findings point out to the possibility of LipS2NH2/Lip(SH)2NH2 to neutralize the side-effects of photodynamic therapy, and to a minor but definitely protective role of GSH.
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PMID:The protective effects of dihydrolipoamide and glutathione against photodynamic damage by Al-phtalocyanine tetrasulfonate. 911 32

Lipoamide dehydrogenase from Mycobacterium smegmatis was purified to homogeneity over 60-fold. Of 20 amino acid residues identified at the amino terminus of the enzyme, 18 and 17 were identical to the sequences of Mycobacterium leprae and Pseudomonas fluorescens lipoamide dehydrogenases, respectively. The visible spectrum of the isolated enzyme was characteristic of a flavin in apolar environment. Reduction of the enzyme with dithionite results in the appearance of an absorbance shoulder at 530-550 nm, suggesting that reducing equivalents of the two-electron reduced enzyme reside predominantly on the redox-active disulfidedithiol. The kinetic mechanism of the forward (NAD+ reducing) and reverse (NADH oxidizing) reactions proved difficult to study due to severe substrate inhibition by NAD+ and NADH. The rate of lipoamide reduction was found to depend upon the NAD+/NADH ratio, with the reaction being activated at low ratios and inhibited at high ratios. The use of 3-acetylpyridine adenine dinucleotide allowed initial velocity kinetics to be performed and revealed that the kinetic mechanism is ping pong. In addition to catalyzing the reversible oxidation of dihydrolipoamide, the enzyme displayed high oxidase activity (30% of the lipoamide reduction rate), hydrogen and t-butyl peroxide reductase activity (10% of the lipoamide reduction rate), and both naphthoquinone and benzoquinone reduction (approximately 200% of the lipoamide reduction rate). The enzyme failed to catalyze the redox cycling of nitrocompounds, but could anaerobically reduce nitrofurazone. The lipoamide-reducing reaction was reversibly inactivated by sodium arsenite, but no decrease in diaphorase activity was observed under these conditions.
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PMID:Catalytic properties of lipoamide dehydrogenase from Mycobacterium smegmatis. 914 18

Recently a newly discovered pyridine nucleotide-disulfide oxidoreductase was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bont, J. A. M., Westphal, A. & De Kok, A. (1996) J. Bacteriol. 178, 6644-6646]. The disulfide oxidoreductase has now been purified from propene-grown Xanthobacter Py2. This enzyme (component II) is a NADPH-dependent FAD-containing homodimeric protein. The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing order: 1,3-propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5,5'-dithiobis(2-nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5,5'-dithiobis(2-nitrobenzoate) and a low diaphorase and oxidase activity. Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol show that the enzyme operates by a ternary complex mechanism in the direction of NADP+ reduction. Anaerobic incubation of the enzyme with 1,3-propanedithiol resulted in slow reduction of the enzyme to yield the thiolate-FAD charge-transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP+ with the enzyme-propanedithiol mixture resulted in the formation of a complex between the reduced enzyme and NADP+ within the dead time of the instrument (5.6 ms). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)-thiol adduct, as judged from the spectral changes. This suggests that the rate-limiting step is the transfer of a hydride ion from the half-reduced enzyme to NADP+. Stopped-flow experiments involving reduction by NADPH show a biphasic behavior. The rapid formation (k(obs) = 40 s(-1)) of a transient intermediate with little absorption decrease at 460 nm and long wavelength absorption was followed by the slow formation (k(obs) = 4 s(-1)) of a species characterized as the thiolate-FAD charge-transfer complex with bound NADP+. Some formation of the FAD C(4a)-thiol adduct was also observed. Photoreduction in the presence of deazaflavin results in rapid bleaching at 450 nm, followed by the slow formation of a stable semiquinone. Full reduction could not be achieved, either by photoreduction or with NADPH, and was incomplete even with dithionite or NADPH in the presence of arsenite. The results indicate a low redox potential of the FAD and a slow rate of electron transfer from the pyridine nucleotide to the redox active disulfide and vice versa. From a sequence alignment with other disulfide reductases, it appears that the active site His-Glu diad is absent in this enzyme. The kinetic and spectral features described above will be discussed in this context.
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PMID:Purification and characterization of a flavoprotein involved in the degradation of epoxyalkanes by Xanthobacter Py2. 979 15


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