Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.5.2 (NQO1)
 6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An enzymatic method for the determination of plasma formate concentration is described. Formate dehydrogenase is used to reduce NAD+ to NADH in the presence of formate. The resulting NADH then reduces the dye resazurin to resorufin, a reaction catalyzed by the endogenous diaphorase of the plasma. The generated resorufin is then measured fluorimetrically by exciting it at 565 nm and quantitating the emitted light at 590 nm. The method uses the patient's plasma as the blank and as the matrix for the construction of a patient-specific formate calibration curve. The blank contains all components of the assay system except the formate dehydrogenase. Formate concentration is determined from the calibration curve, constructed by adding known quantities of sodium formate to the plasma base, and plotting the fluorescence intensity against formate concentration. The assay which is sensitive to a formate level of 7 mg/L should find application in cases where formate is a metabolite.
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PMID:An enzymatic method for the analysis of formate in human plasma. 654 98

Two procedures have been developed for the solubilization of vitamin K epoxide reductase from rat liver microsomal membranes using the detergent Deriphat 160 at pH 10.8. The methods are applicable to both normal and Warfarin-resistant-strain rat liver microsomes and yield material suitable for further purification. The preparations retain dithiothreitol-dependent vitamin K quinone reductase activity as well as vitamin K epoxide reductase and are free of vitamin K-dependent carboxylase and epoxidase activities. Optimal epoxide reductase activity is obtained at 0.1 M KCl and pH 9 in the presence of sodium cholate. Artifactual formation of vitamin K metabolites was eliminated through the use of mercuric chloride to remove excess dithiothreitol prior to extraction and metabolite assay. Using the solubilized enzyme, valid initial velocities were measured, and reproducible kinetic data was obtained. The substrate initial velocity patterns were determined and are consistent with a ping-pong kinetic mechanism. The kinetic parameters obtained are a function of the cholate concentration, but do not vary drastically from those obtained using intact microsomal membranes. At 0.8% cholate, the enzymes solubilized from normal Warfarin-sensitive- and Warfarin-resistant-strain rat livers exhibit respective values of Vmax = 3 and 0.75 nmol/min/g liver; Km for vitamin K epoxide = 9 and 4 microM; and Km for dithiothreitol of 0.6 and 0.16 mM.
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PMID:Solubilization and characterization of vitamin K epoxide reductase from normal and warfarin-resistant rat liver microsomes. 669 43

The enzyme ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2) from whole filaments of Anabaena cylindrica can be separated into four major fractions by chromatography on phosphocellulose; chromatography using ferredoxin-Sepharose 4B proved to be less satisfactory in separating the fractions. The purified fractions, designated 1, 2, 3 and 4, all showed diaphorase and ferredoxin-dependent cytochrome c reductase activity. The major fractions present were 2 and 3 which were each obtained in an electrophoretically homogeneous state (forms 2 and 3) and represented 30-37% and 30-42%, respectively, of the total enzyme activity. Each was a monomeric species with a molecular weight of approx. 33 000 as determined by gel filtration and sodium dodecyl (SDS)-polyacrylamide gel electrophoresis. Evidence for the presence of a 70 000 molecular weight dimer was also obtained. Forms 2 and 3 had isoelectric points of 5.75 and 6.0, respectively, had similar kinetic properties and were flavoproteins. Extracts of isolated heterocysts showed no form 2 or 3 activity but contained a single form which closely resembled one of the species present in fraction 4; fraction 1 may have been a purification artifact because it was not detected in crude extracts of the cyanobacterium.
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PMID:Molecular heterogeneity of ferredoxin:NADP+ oxidoreductase from the cyanobacterium Anabaena cylindrica. 678

In this simple and direct method for determining total bile acids in serum, the serum was mixed with sodium pyruvate, a lactate dehydrogenase blocker, and bile acids were then measured spectrophotometrically after the following enzyme reaction. Bile acids are converted to 3-oxo bile acids with 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) with concomitant reduction of NAD+ to NADH. The hydrogen in the NADH generated is transferred by diaphorase (EC 1.6.4.3) to nitrotetrazolium blue to yield diformazan 540 nm). Analytical recovery of the various bile acids in serum averaged 96.2%. The CV for the day-to-day variation was 4.3%. Normal values are less than 7 mumol/L. Total serum bile acids were estimated by this method in 118 fasting patients with various liver diseases. This determination is clearly shown to be useful as a liver-function test.
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PMID:Direct spectrophotometry of total bile acids in serum. 689 53

In the mammalian retina there are two populations of nitric oxide synthase-containing amacrine cells that stain with the nicotinamide adenine dinucleotide phosphate-diaphorase reaction. To determine the response of these neurons to light, immunoreactivity to Fos proteins was used as a marker of synaptic activation. Fos immunoreactivity is absent in dark-adapted retinas, but 70% of large, Type I nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells and 5-10% of the smaller but more numerous Type II nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells contain Fos proteins after light stimulation. To localize putative cellular targets of nitric oxide in the retina, retinas were stained immunocytochemically for cyclic GMP after the local administration of the nitric oxide donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine. Both compounds induce strong cyclic GMP immunoreactivity in ON cone bipolar cells. The data suggest that the light-induced inward current in ON cone bipolar cells is enhanced by a nitric oxide-cyclic GMP pathway and that the major source of nitric oxide is the nicotinamide adenine dinucleotide phosphate-diaphorase-reactive amacrine cells in the rabbit retina.
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PMID:NADPH-diaphorase (nitric oxide synthase)-reactive amacrine cells of rabbit retina: putative target cells and stimulation by light. 750 76

Double staining for Fos and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-D) was used to study the distribution of activated neurons that synthesize nitric oxide in the paraventricular (PVN) and supraoptic nuclei (SON) following hypotensive stimulation in conscious rats. Fos was detected in many magno- and parvocellular NADPH-D positive neurons in response to haemorrhage or drug-evoked hypotension using i.v. infusions of sodium nitroprusside. However, quantitative analysis did not reveal any differences in the number of Fos positive PVN neurons following either mode of stimulation. These results suggest that a subpopulation of hypothalamic NADPH-D positive neurons is activated following hypotensive challenge. This activation of NADPH-D neurons may occur indirectly through other CNS structures that influence the excitability of hypothalamic SON and PVN. Furthermore, the lack of a difference in activated neurons within the PVN following either haemorrhage or nitroprusside infusion suggests that while a drop in blood pressure causes activation of neurons that produce nitric oxide, a decrease in blood volume, which accompanies haemorrhage, does not.
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PMID:Hypotension induces Fos immunoreactivity in NADPH-diaphorase positive neurons in the paraventricular and supraoptic hypothalamic nuclei of the rat. 756 85

NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
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PMID:Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. 769 24

The respiratory chain of marine and moderately halophilic bacteria requires Na+ for maximum activity, and the site of Na(+)-dependent activation is located in the NADH-quinone reductase segment. The Na(+)-dependent NADH-quinone reductase purified from marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta, and gamma, with apparent M(r) of 52, 46, and 32 kDa, respectively. The FAD-containing beta-subunit reacts with NADH and reduces ubiquinone-1 (Q-1) by a one-electron transfer pathway to produce ubisemiquinones. In the presence of the FMN-containing alpha-subunit and the gamma-subunit, Q-1 is converted to ubiquinol-1 without the accumulation of free radicals. The reaction catalyzed by the alpha-subunit is strictly dependent on Na+ and is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), which is tightly coupled to the electrogenic extrusion of Na+. A similar type of Na(+)-translocating NADH-quinone reductase is widely distributed among marine and moderately halophilic bacteria. The respiratory chain of V. alginolyticus contains another NADH-quinone reductase which is Na+ independent and has no energy-transducing capacity. These two types of NADH-quinone reductase are quite different with respect to their mode of quinone reduction and their sensitivity toward NADH preincubation.
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PMID:Na(+)-translocating NADH-quinone reductase of marine and halophilic bacteria. 822 20

Mitomycin C and porfiromycin were found to inactivate rat hepatic DT-diaphorase. Inactivation was pH dependent; little inactivation was detected at pH 5.8, but inactivation increased as the pH was raised to 7.8. Inactivation was concentration and time dependent and displayed pseudo-first-order kinetics. Inactivation was NADH dependent, indicating that reductive metabolism was necessary for inhibition. [3H]Mitomycin C was covalently bound to DT-diaphorase during inhibition, and the stoichiometry for inactivation of DT-diaphorase by mitomycin C was approximately 0.8 nmol of mitomycin C bound/nmol of enzyme. A higher molecular mass product (60 kDa) was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of DT-diaphorase preincubated with NADH and mitomycin C at pH 7.8, suggesting that mitomycin C is capable of cross-linking DT-diaphorase. The kinetics of inhibition, requirement for NADH for inhibition, covalent binding of [3H] mitomycin C to DT-diaphorase, and approximate 1:1 stoichiometry suggest that this inactivation process may be mechanism based. Inhibition of DT-diaphorase by mitomycin C and porfiromycin is not limited to a cell-free system and could also be observed in HT-29 cells in culture at pH 7.2. Bioactivation of mitomycin C or porfiromycin by DT-diaphorase is favored at lower pH, whereas at higher pH values enzyme alkylation and inactivation of DT-diaphorase occur. These data suggest that the success of attempts to exploit the elevated DT-diaphorase content of certain human tumors for improved chemotherapeutic response using mitomycin C or porfiromycin will depend on intracellular pH.
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PMID:pH-dependent inactivation of DT-diaphorase by mitomycin C and porfiromycin. 826 49

We have previously demonstrated that ibotenate (IBO) injected into the pedunculopontine tegmental nucleus (PPTg) damages all neurones there while quinolinate (QUIN) makes relatively selective lesions of cholinergic neurones. We now compare the effects of two anaesthetics, sodium pentobarbitone and Avertin (tribromoethanol/tert-amylalcohol dissolved in ethanol, saline and phosphate buffer) on three doses of IBO and QUIN in the PPTg. Diaphorase-positive cell loss after QUIN was attenuated under barbiturate, the relative selectivity of QUIN for diaphorase-positive neurones was lost and lesion volumes were uniformly small compared with lesions made under Avertin anaesthesia. IBO toxicity was unaffected by anaesthesia. These data are discussed with reference to the actions of excitotoxins at glutamate receptor subtypes and interactions of barbiturates with the GABAA receptor.
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PMID:Barbiturate anaesthesia reduces the neurotoxic effects of quinolinate but not ibotenate in the rat pedunculopontine tegmental nucleus. 841 94


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