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)

The myenteric plexus of the domestic fowl (Gallus domesticus) small intestine was studied by means of silver staining, glyoxylic acid-induced fluorescence, the modified Koelle-Friedenwald method for the detection of acetylcholinesterase, NADH-diaphorase techniques and the unlabelled antibody method involving the use of an antiserum raised against GABA conjugated by glutaraldehyde to bovine serum albumin. The majority of the perikarya were in the ganglia, with an average density of 3370 +/- 942 nerve cells/cm2. Cholinesterase-positive and a few GABA-immunoreactive nerve cell bodies were seen in the myenteric ganglia, while fluorescent ganglion cells were not observed. In addition to AChE and GABA-positive nerve fibres, a rich fluorescent network of varicose and nonvaricose nerve fibres was detected, pointing to the presence of an extrinsic aminergic system in the domestic fowl myenteric plexus. Electron microscopic observations on nerve cells, axon profiles and varicosites with various vesicle populations were in good agreement with the histochemical findings.
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PMID:Histochemical characterization of myenteric plexus in domestic fowl small intestine. 207 64

A fluorometric flow-injection method for determining carnitine with use of immobilized enzymes carnitine dehydrogenase (EC 1.1.1.108) and diaphorase (EC 1.8.1.4) was developed and applied to the assay of carnitine in serum of patients treated with valproic acid. After fractionation and hydrolysis of carnitines in serum samples by perchloric acid and potassium hydroxide, liberated carnitine was converted to resorufin by immobilized carnitine dehydrogenase and diaphorase in the presence of beta-NAD+ (1.0 mmol/L), resazurin (12.5 mumol/L), and Tris acetate (0.6 mol/L, pH 9.0) at 37 degrees C. The fluorescence intensity of resorufin was monitored at lambda Ex 560 nm and lambda Em 580 nm. The calibration curve was linear for carnitine amounts from 0.1 to 1.0 nmol. Quantitative analytical recovery and satisfactory within- and between-run imprecision of carnitine in each carnitine fraction were obtained. Interference by bilirubin, serum albumin, and hemoglobin was negligible. Carnitine deficiencies were detected in about 20% of the valproic acid-treated patients (n = 198). The present method should be useful for monitoring carnitine deficiencies in clinical laboratories.
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PMID:Fluorometric determination of carnitine in serum with immobilized carnitine dehydrogenase and diaphorase. 225 48

Mitochondria were isolated from whole homogenates of normal liver and Novikoff hepatomas using reorienting rate zonal centrifugation on sucrose gradients. The activities of several mitochondrial-specific enzymes and ultrastructure were compared in the two tissues. Our results indicate that cytochrome oxidase, lipoamide dehydrogenase, malate dehydrogenase, and succinate dehydrogenase activities are all higher in liver homogenates than in Novikoff hepatoma homogenates. Mitochondrial hexokinase, however, is much greater in the hepatoma than in liver. The activity of these enzymes in isolated mitochondria displayed a much different pattern. Both cytochrome oxidase and succinate dehydrogenase activities were higher in hepatoma mitochondria than in liver mitochondria. Lipoamide dehydrogenase and malate dehydrogenase, conversely, were higher in liver mitochondria. Hexokinase was found to be virtually absent in liver mitochondria but plentiful in hepatoma mitochondria. Ultrastructural studies have shown that the hepatoma mitochondria are much smaller in size, which results in a decreased rate of migration into the gradient. These studies have also shown that normal liver consists of predominantly "condensed" forms of mitochondria, whereas hepatoma contained a majority of "twisted" species. Experiments using 1% bovine serum albumin in the homogenization procedures and in the gradient have confirmed earlier observations that bovine serum albumin is essential for optimal isolation of neoplastic mitochondria.
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PMID:Characteristics of mitochondria isolated by rate zonal centrifugation from normal liver and Novikoff hepatomas. 624 94

Copper Fenton systems (Cu(II)/H2O2 and Cu(II)/Asc) inactivated the lipoamide reductase and enhanced the diaphorase activity of pig-heart lipoamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H2O2 treatment, the number of titrated thiols in LADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD+ or ATP) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H2O2. Dihydrolipoamide, dihydrolipoic acid, Captopril, acetylcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanothione prevented LADH inactivation. 100 microM GSH, DL-dithiothreitol, N-(2-mercaptopropionylglicine) and penicillamine protected LADH against Cu(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also protected LADH against Cu(II)/H2O2. Allopurinol provided partial protection against Cu(II)/H2O2. Ethanol, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H2O2 or Cu(II). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H2O2. It is concluded that site-specifically generated HO, radicals were responsible for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injury.
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PMID:Inactivation of heart dihydrolipoamide dehydrogenase by copper Fenton systems. Effect of thiol compounds and metal chelators. 775

Trypanosoma brucei S427cl1 organisms made 6 divisions in modified minimal essential medium (BMEM) supplemented with fetal bovine serum (FBS)-low or high density lipoprotein (LDL, HDL) and fatty acid-free bovine serum albumin (FAF-BSA). Omission of lipoproteins or FAF-BSA from the medium caused the parasites to accumulate in G1 of the cell cycle and to lose the ability to replicate at 37 degrees C. Proteinase K-treated LDL or HDL, which did not have detectable apolipoprotein, supported the G1 to S cell cycle transition of T. brucei S427cl1 organisms in BMEM supplemented with FAF-BSA. Addition of C6:0, C7:0 or fatty C8:0 fatty acid (1 mol fatty acid mol-1 FAF-BSA in the incubation mixture) to serum-free medium supplemented with LDL or HDL and FAF-BSA prevented T. brucei S427cl1 organisms from progressing through G1 into S of the cell cycle. T. brucei S427cl1 organisms became stumpy-like forms during plateau phase growth under axenic conditions. Stumpy-like T. brucei S427cl1 organisms were mainly in G1 of the cell cycle, expressed raised levels of NAD diaphorase activity, were unable to replicate at 37 degrees C, but were able to differentiate to replicating procyclic organisms. Medium collected from plateau phase cultures of T. brucei S427cl1 did not support the G1 to S cell cycle transition of exponentially growing T. brucei organisms. The capacity of plateau phase medium to support G1 to S transition of T. brucei S427cl1 organisms was restored by addition of FAF-BSA and its capacity to support 4 cycles of replication of the parasites was restored by addition of FAF-BSA and LDL or HDL.
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PMID:Control of G1 to S cell cycle progression of Trypanosoma brucei S427cl1 organisms under axenic conditions. 843 15

Nitrate reductase from the yeast Candida nitratophila was found to contain one molecule of cytochrome b557 and one atom of molybdenum per subunit. FAD/haem-dependent diaphorase activity (haem domain) was associated with a 40 kDa tryptic fragment of the subunit. The 50 amino-terminal residues of this fragment were determined, and the sequence did not show significant similarity to deduced sequences of other nitrate reductases previously published. Increasing ionic strength in vitro had a stimulatory effect on enzymic activity via stimulation of the molybdenum-dependent terminal nitrate-reducing activity. Stimulation of activity by exogenous protein (bovine serum albumin or casein) also appeared to be an ionic effect. Stimulation of catalytic activity by phosphate was a separate effect.
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PMID:Further characterization of the assimilatory nitrate reductase from the yeast Candida nitratophila. 847 56

Inactivation of lipoamide dehydrogenase (LipDH) by the Cu(II)/H2O2 Fenton system (SF-Cu(II): (5.0 microM Cu(II), 3.0 mM H2O2) was enhanced by catecholamines (CAs), namely, epinephrine, levoDOPA (DOPA), DOPAMINE, 6-hydroxyDOPAMINE (OH-DOPAMINE) and related compounds (DOPAC, CATECHOL, etc.). After 5 min incubation with the Cu(II)/H2O2/CA system (0.4 mM CA), the enzyme activity decayed as indicated by the following percentage values (mean +/- S.D.; in parenthesis, number of determinations): SF-Cu(II) alone, 43 +/- 10 (18); SF-Cu(II) + epinephrine, 80 +/- 9 (5); SF-Cu(II) + DOPA, 78 +/- 2 (4); SF + Cu(II) + DOPAMINE, 88 +/- 7 (5); SF-Cu(II) + OH-DOPAMINE 87 +/- 6 (7); SF-Cu(II) +/- DOPAC, 88 +/- 3 (6); SF-Cu(II) + catechol, 85 +/- 6 (5). In all cases P < 0.05, with respect to the SF-Cu(II) control sample. CAs effect was concentration-dependent and at the 0-100 microM concentration range, it varied with the CA structure. Above the 100 microM concentration, CAs were equally effective and produced 90-100% enzyme, inactivation (Figure 2). In the absence of oxy-radical generation, the enzyme specific activity (mean +/- S.D.) was 149 +/- 10 (24) mumol NADH/min/mg protein. Assay of HO. production by the Cu(II)/H2O2/CA system in the presence of deoxyribose (TBA assay) yielded values much greater than those obtained omitting CA. Hydroxyl radical production depended on the presence of Cu(II) and H2O2 and significant H. values were obtained with OH-DOPAMINE, DOPAC, epinephrine, DOPAMINE, DOPA and catecol supplemented systems (Table 2). LipDH (1.0 microM) inhibited 50-80% deoxyribose oxidation, the inhibition depending on the CA structure (Table 2). Native catalase (20 micrograms/ml) and bovine serum albumin (40 micrograms/ml) effectively prevented LipDH inactivation by the Cu(II)/H2O2/CA system; denaturated catalase, SOD, 0.3 M mannitol, 6.0 mM ethanol and 0.2 M benzoate were less effective or did not protect LipDH (Table 3). Incubation of CAs with the Cu(II)/H2O2 system produced a time and Cu(II)-dependent destruction of CAs, the corresponding o-quinone, production as illustrated with epinephrine (figures 6 and 7), as illustrated with epinephrine and DOPAMINE (Table 4). These results support LipDH inactivation by (a) reduction of Cu(II) to Cu(I) by CAs followed by Cu-catalyzed production of HO. from H2O2; (b) CA oxidation followed by the corresponding o-quinone interaction with LipDH. CAPTOPRIL, N-acetylcysteine, mercaptopropionylglycine and penicillamine prevented to various degree LipDH inactivation by the Cu(II)/H2O2/CA systems (Table 1). The former was the most effective and 0.4 mM CAPTOPRIL prevented about 95-100% the effect of Cu(II)/H2O2/CA systems supplemented with epinephrine, DOPAMINE and OH-DOPAMINE (Figures 3 and Table 1). LipDH increased and CAPTOPRIL inhibited epinephrine oxidation by Cu(II)/H2O2 (Figures 4 and 5). Since un-physiological concentrations of CAs and Cu(II) may be released in the myocardium after ischemia-reperfusion, the summarized observations may contribute to explain myocardial damage in that condition.
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PMID:[Inactivation of the myocardial lipoamide dehydrogenase by catecholamines. Prevention by captopril and other thiol compounds]. 872 69

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

Oxygen radical generating systems, namely, Cu(II)/ H2O2, Cu(II)/ascorbate, Cu(II)/NAD(P)H, Cu(II)/ H2O2/catecholamine and Cu(II)/H2O2/SH-compounds irreversibly inhibited yeast glutathione reductase (GR) but Cu(II)/H2O2 enhanced the enzyme diaphorase activity. The time course of GR inactivation by Cu(II)/H2O2 dependent on Cu(II) and H2O2 concentrations and was relatively slow, as compared with the effect of Cu(II)/ascorbate. The fluorescence of the enzyme Tyr and Trp residues was modified as a result of oxidative damage. Copper chelators, catalase, bovine serum albumin and HO. scavengers prevented GR inactivation by Cu(II)/H2O2 and related systems. Cysteine, N-acetylcysteine, N-(2-dimercaptopropionylglycine and penicillamine enhanced the effect of Cu(II)/H2O2 in a concentration- and time-dependent manner. GSH, Captopril, dihydrolipoic acid and dithiotreitol also enhanced the Cu(II)/H2O2 effect, their actions involving the simultaneous operation of pro-oxidant and antioxidant reactions. GSSG and trypanothione disulfide effectively protected GR against Cu(II)/H2O2 inactivation. Thiol compounds prevented GR inactivation by the radical cation ABTS.+. GR inactivation by the systems assayed correlated with their capability for HO. radical generation. The role of amino acid residues at GR active site as targets for oxygen radicals is discussed.
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PMID:Inactivation of yeast glutathione reductase by Fenton systems: effect of metal chelators, catecholamines and thiol compounds. 945 90

Dihydrolipoamide dehydrogenase (E3) from Escherichia coli, an FAD-linked homodimer, can be fully reconstituted in vitro following denaturation in 6 m guanidinium chloride. Complete restoration of activity occurs within 1-2 h in the presence of FAD, dithiothreitol, and bovine serum albumin. In the absence of FAD, the dihydrolipoamide dehydrogenase monomer forms a stable folding intermediate, which is incapable of dimerization. This intermediate displays a similar tryptic resistance to the native enzyme but is less heat-stable, because its ability to form native E3 is lost after incubation at 65 degrees C for 15 min. The presence of FAD promotes slow, additional conformational rearrangements of the E3 subunit as observed by cofactor-dependent decreases in intrinsic tryptophan fluorescence. However, after 2 h, the tryptophan fluorescence spectrum and far UV CD spectrum of E3, refolded in the absence of FAD, are similar to that of the native enzyme, and full activity can still be recovered on addition of FAD. Cross-linking studies show that FAD insertion is necessary for the monomeric folding intermediate to attain an assembly competent state leading to dimerization. Thus cofactor insertion represents a key step in the assembly of this enzyme, although its initial presence appears not to be required to promote the correct folding pathway.
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PMID:FAD insertion is essential for attaining the assembly competence of the dihydrolipoamide dehydrogenase (E3) monomer from Escherichia coli. 1097 Aug 89


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