Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 A, compared to a value of 81 A for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1-2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a Mr 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of Mr 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.
Arch Biochem Biophys 1984 Sep
PMID:Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase. 654 59

The effect of NAD+ on lipoamide dehydrogenase from pig heart was investigated physicochemically. The observed and theoretical oxidation-reduction mid-point potentials for the oxidized lipoamide dehydrogenase (E)/two-electron-reduced lipoamide dehydrogenase (EH2) couple in the presence on NAD+ were -218 mV and -251 mV, respectively, at pH 6.0. Therefore, unexpectedly the mid-point potential of the enzyme became more positive on NAD+ binding. Decreases in the fluorescence lifetime and intensity and increase in the degree of polarization of enzyme-bound FAD were observed in the presence of NAD+. Fluorescence quenching of bound FAD by NAD+ was released by phenobarbital. The results suggest that NAD+ strengthens the intramolecular dynamic interaction between the isoalloxazine moiety and adenine moiety of bound FAD, and so alters the mid-point potential of the enzyme. These findings indicate that NAD+ acts not only as an acceptor of electrons from EH2, but also as an effector in the flavin-disulfide interaction of EH2.
J Biochem 1984 Sep
PMID:Effect of nicotinamide adenine dinucleotide on the oxidation-reduction potentials of lipoamide dehydrogenase from pig heart. 654 41

Many preparations of flavin nucleotides contain nucleotide isomers of the natural compounds which are difficult to remove or separate. The method of dynamic complex-exchange (or paired-ion) chromatography has been used with high-performance liquid chromatography to achieve resolution and purification of isomers. A solution of nucleotide in water was chromatographed isocratically on a C18-substituted silica column with a mobile phase of methanol, water, and tetrabutylammonium phosphate at neutral pH. Commercial preparations of FMN and FAD contained multiple components. The purified isomers were subjected to ion-exchange chromatography directly on a quaternary nitrogen-substituted silica column to remove methanol and tetrabutylammonium cation, and thus obtain pure nucleotide in aqueous buffer suitable for use with proteins. With analytical equipment, a milligram of pure FMN or FAD was produced in 1 day. The same procedure was useful for the rapid identification and quantitation of flavin nucleotides in proteins. After exposure of a protein solution to heat treatment, the supernatant was subjected to dynamic complex-exchange chromatography, as described above.
Anal Biochem 1983 Sep
PMID:The purification and identification of flavin nucleotides by high-performance liquid chromatography. 663 2

Respiratory chain-linked L-glycerol 3-phosphate (G3P) dehydrogenase [EC 1.1.99.5] of marine bacterium, Vibrio alginolyticus, was extracted from the membrane fraction by treatment with Tween 20, and fractionation on DEAE-Sephacel and QAE-Sephadex in the presence of 0.05% Liponox DCH(alkyl polyoxyethylene ether) yielded a preparation having a specific activity of 22.1 units/mg protein when assayed by phenazine methosulfate (PMS)-coupled reduction of thiazolyl blue tetrazolium (MTT). The purified enzyme had an apparent molecular weight of 300,000 as determined by chromatography on Sepharcyl S-300 in 0.05% Liponox DCH, and had noncovalently bound FAD as its coenzyme. The enzyme had a pH optimum of 8.5-9.0, and required 200 mM NaCl or KCl and an appropriate detergent (such as Tween, Brij or Liponox DCH) for maximum activation. The activating effect of NaCl was due to a decrease in Km for G3P and that of Tween 20 was due to both a decrease in Km and an increase in Vm. Triton X-100 could not activate the enzyme and was inhibitory in the presence of phospholipids. The reaction followed a ping-pong mechanism. IN addition to PMS, 2,6-dichlorophenol indophenol and duroquinone, the enzyme could reduce ubiquinone-5 (Q-5) in the presence of Liponox DCH at a rate of 46% of the PMS reductase activity. The enzyme was strongly inhibited by heavy metal ions and by p-chloromercuribenzoate. The activity for Q-5, but not for PMS, was inhibited by o-phenanthroline and bathophenanthroline, suggesting the participation of nonheme iron protein in the Q-5 reduction.
J Biochem 1981 Sep
PMID:Partial purification and properties of respiratory chain-linked l-glycerol 3-phosphate dehydrogenase from a marine bacterium, Vibrio alginolyticus. 679 67

Isolated intestinal mucosa cells of the guinea pig were employed to study the intestinal transport of riboflavin. This method provided a direct approach to characterize the mechanism of riboflavin absorption. Riboflavin was taken up by both jejunal and ileal cells in a temperature-dependent process. Uptake was energy-dependent and exhibited saturation kinetics with an apparent Km ranging between 70 and 87 nM and Vmax of 1.6 and 1.82 pmol/(mg cell protein x minute) for jejunal and ileal cells, respectively. Absence of Na+ or presence of ouabain in the incubation medium strongly decreased the rate and extent of transport. Riboflavin uptake was inhibited by the flavins FAD and FMN, but not by ribose. These results indicate that riboflavin is absorbed in all parts of the small intestine in an active, carrier-mediated process.
J Nutr 1983 Sep
PMID:Riboflavin uptake by isolated enterocytes of guinea pigs. 688 19

Sex-related differences in the activity of hepatic FAD-containing monooxygenase (FAD-M) were found in C3H/St mice. Adult female mice had enzyme activities nearly two-fold greater than male mice and these differences, which were absent in sexually immature mice, became apparent at the onset of puberty. The sex differences in hepatic FAD-M appeared to be mediated through the suppressive effect of testosterone; castration of male mice enhanced enzyme activity, while androgenic replacement returned activities to control levels. Testosterone's suppressive effect was found to be relatively specific for hepatic FAD-M. Treatment of castrated male mice with both the anti-androgen flutamide and testosterone returned enzyme activity to control levels, suggesting that testosterone's regulation of hepatic microsomal FAD-M is receptor-mediated. Female gonadectomy had no effect on this enzyme's activity.
Life Sci 1982 Sep 06
PMID:Androgenic suppression of mouse hepatic FAD-containing monooxygenase activity. 713 54

1. Ferredoxin-NADP+ reductase resolved into apoprotein and flavin by incubation with 2.5 M CaCl2 at pH 7.5 and 2 degrees C. Essential factors to recover a reconstitutable apoprotein are dithiothreitol, glycerol and guanidine/HCl. The apoprotein is stable for at least a week at -20 degrees C. 2. The release of the prosthetic group from the protein by the Ca2+ ions is a multi-step process. Three different effects of these ions are identifiable: (a) a rapid 20-25% inhibition of catalytic activity, probably caused by an increase in the ionic strength of the medium; other cations can produce it as well; (b) a slower induction of a conformational change in the protein which causes complete loss of activity and exposure to solvent of the flavin moiety; the FAD is finally released from the protein; (c) complete conversion of FAD to FMN, which blocks reconstitution to holoenzyme, caused by the well-known hydrolytic action of Ca2+ ions on the pyrophosphate bridge of FAD. 3. Binding of FAD by the apoferredoxin-NADP+ reductase is very rapid and it is complete in a few minutes even at 0 degrees C. A Kd of 3.4 X 10(-9) M has been determined by fluorescence titration. The reconstituted holoenzyme has catalytic activity, spectral and fluorescence properties nearly identical to the native enzyme. The gel electrophoresis and isoelectrofocusing patterns of the two enzymes are very similar. Removal of factors from the apoprotein solution such as dithiothreitol and glycerol promotes the appearance of protein aggregates.
Eur J Biochem 1982 Sep 01
PMID:Preparation of apoprotein from spinach ferredoxin-NADP+ reductase. Studies on the resolution process and characterization of the FAD reconstituted holoenzyme. 714 Jul 36

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.
Clin Chem 1981 Sep
PMID:Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format. 726 29

The resonance coherent anti-Stokes Raman technique was used to obtain vibrational spectra of flavin in flavodoxins from Desulfovibrio gigas and Desulfovibrio vulgaris and of the simpler 6,7-dimethyl-8-ribityllumazine chromophore in the blue fluorescence lumazine protein from the bioluminescent bacterium Photobacterium phosphoreum. In the region examined, 1100-1700 cm-1, the Raman spectrum of the lumazine is less crowded than that of the flavin and this facilitates assignment of observed frequencies to particular vibrational modes. Certain modes are not affected by chromophore binding to the protein, but others are changed in frequency or intensity in a way that can be rationalized by expected interactions of the chromophore with the amino acid residues of the binding site. For example, a tentative assignment of change in hydrogen bonding at N(5) is suggested as the cause of the frequency shift for the chromophore in both flavodoxins (free-bound, 1582-1572 cm-1) and for C(4)=O in glucose oxidase (1359-1364 cm-1) and lumazine protein (1359-1362 cm-1). Shifts of the C(2)-N(3) mode in D2O may arise from hydrogen-bonding changes at C(2)=O in lumazine protein (1284-1291 cm-1), flavodoxin (1300-1280 cm-1), and glucose oxidase (1297-1287 cm-1). Bonding at N(3)-H may be the origin of changes in the frequency or intensity of the amide III mode in riboflavin binding protein and glucose oxidase. A stacking interaction is suggested for the change in a pyrimidine ring mode in FAD (1508 cm-1) since this mode is found at 1504 cm-1 in 30% Me2SO/H2O, where the adenine and pyrimidine are unstacked. The results clearly indicate different interactions in the binding sites of those proteins studied to date.
Biochemistry 1980 Sep 30
PMID:Protein-ligand interactions in lumazine protein and in Desulfovibrio flavodoxins from resonance coherent anti-Stokes Raman spectra. 742 21

The behaviour of cytochrome b5 reductase holoenzyme and apoenzyme toward blue-dextran--Sepharose has been studied. Holoenzyme was adsorbed at low ionic strength and could be eluted with 100 microM NADH or NAD+. Flavin-free enzyme was even more strongly bound and could be eluted with 1 M NaCl, or 100 microM NADH + 10 microM FAD. Separately the cofactors were without effect. FMN was less effective than FAD. ADP and AMP eluted nothing. Cibacron blue F3GA was found to exert a mixed inhibition on NADH oxidation. Dye binding to holoenzyme elicited a characteristic red shift in its spectrum. Comparison of the difference spectrum amplitude at 680 and 585 nm showed the presence of a second binding mode at higher dye concentrations. These results point to the existence for cytochrome b5 reductase of two binding sites with high affinity for blue-dextran--Sepharose: the NADH binding site and flavin binding site. For the latter it is clear that isoalloxazine pocket must play a role in dye binding. Cytochrome b5 reductase is the second flavoenzyme which has been shown to have affinity for immobilized dye at the flavin site, the first one being flavocytochrome b2, and FMN-dependent enzyme [D. Pompon and F. Lederer (1978) Eur. J. Biochem. 90, 563--569].
Eur J Biochem 1980 Sep
PMID:Binding of Cibacron blue F3GA to the flavin and NADH sites in cytochrome b5 reductase. 743 74


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>