Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT02288 (NADH)
 21,914 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Glutamate dehydrogenase from pig kidney has been purified to homogeneity by means of affinity chromatography on matrix bound Cibacron Blue F3G-A and gel chromatography on Sepharose 6B. The enzyme exhibits allosteric properties with the substrates alpha-ketoglutarate, ammonium, and NADH, respectively. GTP is a strong inhibitor which strengthened the cooperative interactions between the ammonium binding sites. ADP as an activator relieves the inhibition by GTP. Like glutamate dehydrogenase from bovine liver, glutamate dehydrogenase from pig kidney shows the ability of self-association, too. The sedimentation coefficient increases from 13.5 S at 0.07 mg protein/ml to 19.4 S at 1.32 mg protein/ml. In the sodium dodecylsulphate gel electrophoresis the enzyme migrates as a single band with a molecular-weight at 51000.
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PMID:Purification and properties of pig kidney glutamate dehydrogenase. 20 75

Chromatography on columns of immobilized Cibacron Blue (Blue Dextran--agarose) can be used as a major step in the purification of quinonoid dihydropterin reductase. The reductase has been isolated from fractions of beef kidney by selective binding to the immobilized Cibacron in the presence of tetrahydropterin. The binding of the reductase to Blue Dextran and its specific elution from columns of Blue Dextran--agarose indicate that the reductase possesses the dinucleotide (NAD+) binding domain. The results of kinetic experiments give validity to both our affinity chromatography of the reductase and to an ordered mechanism for the formation of tetrahydropterin. Chromatography on Blue Dextran--agarose has been used to show that folate or amethopterin can compete with Cibacron Blue for the dinucleotide domain of the reductase. The p-aminobenzoyl-glutamate moiety of the folates competes with Cibacron Blue for the NADH site of the reductase. A stable binary complex of dihydropterin reductase with NADH has been detected by gel electrophoresis.
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PMID:Purification of dihydropterin reductase using immobilized Cibacron Blue. 45 16

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)] mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5'-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5'- and 3'-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promotor. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10(-5)M. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.
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PMID:Expression of mammalian DT-diaphorase in Escherichia coli: purification and characterization of the expressed protein. 170 98

Difference spectroscopy and enzyme kinetics were employed to study the interaction of lactate dehydrogenase (LDH) from rabbit muscle with the azo-dye Procion Red HE-3B and two of its structural variants in order to follow the significance of the sulphonated terminal rings for the strength and specificity of binding. Procion Red HE-3B possesses a significantly higher affinity to LDH compared to the dye Cibacron Blue F3G-A, a well characterized pseudo-biospecific ligand of dehydrogenases. Moreover, Procion Red HE-3B showed competition towards the cofactor NAD+/NADH. The enzyme-dye complex is mainly stabilized by hydrophobic interactions, but other binding forces cannot be excluded. LDH possesses one dye-binding site per subunit. As a binding region the active center of LDH, preferentially the hydrophobic nicotinamide pocket is involved. Removal of the negatively charged sulphonic acid group from the terminal rings of Procion Red HE-3B decreases the affinity to LDH significantly but does not change the type of binding. Addition of an anilino group to the terminal rings of Procion Red HE-3B does not affect the affinity to the active site significantly but enables the binding on other sites with lower affinity in dependence on the dye concentration.
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PMID:Difference spectroscopic and kinetic studies on the interaction of lactate dehydrogenase with structurally related triazine dyes. 182 35

The dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled-pore glass (CPG) beads (200 nm pore diameter) and aminohexyl-Cibacron Blue, was 44 microM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 microM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH.NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 microM) and that determined for the soluble complex (KD = 2.4 microM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self-association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 microM vs 22 microM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 microM and 43 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography. 209 89

Subcellular fractionation of bovine thyroid tissue by differential pelleting and isopycnic gradient centrifugation in a zonal rotor indicated that NAD(+) glycohydrolase is predominantly located and rather uniformly distributed in the plasma membrane. Comparison of NAD(+) glycohydrolase activities of intact thyroid tissue slices, functional rat thyroid cells in culture (FRT(l)) and their respective homogenates indicated that most if not all of the enzyme (catalytic site) is accessible to extracellular NAD(+). The reaction product nicotinamide was predominantly recovered from the extracellular medium. The diazonium salt of sulphanilic acid, not penetrating into intact cells, was able to decrease the activity of intact thyroid tissue slices to the same extent as in the homogenate. Under the same conditions this reagent almost completely abolished NAD(+) glycohydrolase activity associated with intact thyroid cells in culture. The triazine dye Cibacron Blue F3GA and its high-M(r) derivative Blue Dextran respectively completely eliminated or caused a severe depression in the NAD(+) glycohydrolase activity of FRT(l) cells. The enzyme could be readily solubilized from bovine thyroid membranes by detergent extraction, and was further purified by gel filtration and affinity chromatography on Blue Sepharose CL-6B. The overall procedure resulted in a 1940-fold purification (specific activity 77.6mumol of nicotinamide released/h per mg). The purified enzyme displays a K(m) of 0.40mm for beta-NAD(+), a broad pH optimum around pH7.2 (0.1 m-potassium phosphate buffer) and an apparent M(r) of 120000. Nicotinamide is an inhibitor (K(i) 1.9mm) of the non-competitive type. The second reaction product ADP-ribose acts as a competitive inhibitor (K(i) 2.7mm). The purified enzyme splits beta-NAD(+), beta-NADP(+), beta-NADH and alpha-NAD(+) at rates in the relative proportions 1:0.75:<0.02:<0.02 and exhibits transglycosidase (pyridine-base exchange) activity. Anionic phospholipids such as phosphatidylinositol and phosphatidylserine inhibit the partially purified enzyme. A stimulating effect was observed upon the addition of histones.
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PMID:Topography, purification and characterization of thyroidal NAD+ glycohydrolase. 298 95

Cytosolic NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed, FAD-containing enzyme that catalyzes the obligatory two-electron reduction of quinones. Cibacron Blue is an inhibitor of this enzyme comparable in potency to dicoumarol. Pure quinone reductase was obtained from the livers of Sudan II (1-[2,4-dimethylphenylazo]-2-naphthol)-treated rats in a single step by Cibacron Blue-agarose chromatography. Cibacron Blue is a competitive inhibitor with respect to NADH (Ki = 170 nM) and is a noncompetitive inhibitor with respect to menadione (Ki = 540 nM). Addition of Cibacron Blue to quinone reductase resulted in a decrease and red shift of the enzyme-bound FAD peak at 450 nm. The titration of the absorbance changes for both FAD and Cibacron Blue could be fitted to curves describing an equilibrium binding equation with a KD of 300 nM and one binding site per enzyme subunit. Furthermore, the Cibacron Blue difference spectrum that resulted from binding to quinone reductase was abolished by dicoumarol. Significant amino acid homology between quinone reductase and the nucleotide binding regions of enzymes that bind to Cibacron Blue was found. These data indicate that Cibacron Blue is a useful ligand for the purification of quinone reductase and a new probe for its NAD(P)H binding site. Conditions for crystallizing rat liver quinone reductase are also described.
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PMID:Purification and crystallization of rat liver NAD(P)H:(quinone-acceptor) oxidoreductase by cibacron blue affinity chromatography: identification of a new and potent inhibitor. 321 67

The occurrence of soluble reduced nicotinamide nucleotide:acceptor oxidoreductases has been reported in a number of strains of the oxygen-tolerant anaerobe Trichomonas vaginalis and other trichomonad species. The quantitatively more important enzyme in most strains of T. vaginalis is an NADH oxidase which produces water from the reduction of oxygen. This enzyme has been purified by a combination of gel filtration, chromatofocusing, Cibacron Blue chromatography and high pressure gel permeation chromatography. It is a monomeric protein with an estimated molecular mass from sodium dodecyl sulphate gel electrophoresis of 98 kDa; an isoelectric point of approximately pH 5.5 and a Km for NADH of 5.4 microM. The purified NADH oxidase is significantly inactivated during turnover under air (t1/2 3.65 min) and rapidly inactivated by microM levels of hydrogen peroxide. The NADPH-dependent minor activity requires a flavin. It has been partially purified by gel filtration and chromatofocusing. The apparent molecular mass of this enzyme is 36 kDa by gel filtration; it has an isoelectric point of approximately pH 5.2 and Km values for NADPH and FMN of 16.6 microM and 6.1 microM respectively. The product of oxygen reduction by this enzyme, using FMN as acceptor is hydrogen peroxide. The possible role of these two enzymes in the cell and their affinity with related enzymes from other organisms is discussed.
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PMID:The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis. 325 11

Salmonella typhimurium histidinol dehydrogenase produces histidine from the amino alcohol histidinol by two sequential NAD-linked oxidations which form and oxidize a stable enzyme-bound histidinaldehyde intermediate. The enzyme was found to catalyze the exchange of 3H between histidinol and [4(R)-3H]NADH and between NAD and [4(S)-3H]NADH. The latter reaction proceeded at rates greater than kcat for the net reaction and was about 3-fold faster than the former. Histidine did not support an NAD/NADH exchange, demonstrating kinetic irreversibility in the second half-reaction. Specific activity measurements on [3H]histidinol produced during the histidinol/NADH exchange reaction showed that only a single hydrogen was exchanged between the two reactants, demonstrating that under the conditions employed this exchange reaction arises only from the reversal of the alcohol dehydrogenase step and not the aldehyde dehydrogenase reaction. The kinetics of the NAD/NADH exchange reaction demonstrated a hyperbolic dependence on the concentration of NAD and NADH when the two were present in a 1:2 molar ratio. The histidinol/NADH exchange showed severe inhibition by high NAD and NADH under the same conditions, indicating that histidinol cannot dissociate directly from the ternary enzyme-NAD-histidinol complex; in other words, the binding of substrate is ordered with histidinol leading. Binding studies indicated that [3H]histidinol bound to 1.7 sites on the dimeric enzyme (0.85 site/monomer) with a KD of 10 microM. No binding of [3H]NAD or [3H]NADH was detected. The nucleotides could, however, displace histidinol dehydrogenase from Cibacron Blue-agarose.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Kinetic mechanism of histidinol dehydrogenase: histidinol binding and exchange reactions. 330 6

The interaction of alkaline phosphatase (EC 3.1.3.1) from calf intestine with different dyes, especially with Procion Red HE-3B was studied by several methods. From the kinetic analysis a nonlinear noncompetitive type of inhibition with an inhibition constant Ki = 0.03 mM for Procion Red HE-3B and Cibacron Blue F3G-A was estimated. The extent of inhibition of the two dyes at constant substrate and inhibitor concentration is 10 to 20 times higher than that of natural inhibitors like L-phenylalanine and NADH. Difference spectroscopic measurements with Procion Red HE-3B showed that the enzyme dimer possesses two binding sites for the dye. The dissociation constant of the dye-enzyme complex was estimated to be Kd = 0.01 mM. The binding of Procion Red HE-3B to the enzyme is mainly stabilized by electrostatic interactions. Large aromatic parts of a dye molecule like a combination of two naphthol ring systems or an anthraquinone ring flanked by spatially arranged charged substituents are important for the extent of specificity. The elution of the enzyme from the immobilized dye and the quenching of the dye-protein difference spectral signal by the competitive inhibitor phosphate and by substrates suggest the involvement of the active center of the enzyme in the dye binding region.
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PMID:Interaction of procion red HE-3B and other reactive dyes with alkaline phosphatase: a study by means of kinetic, difference spectroscopic and chromatographic methods. 344 94


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