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At least 379 records · Page 21Linked to original sources
PubMed1993

Multiple acyl-coenzyme A dehydrogenation disorder responsive to riboflavin: substrate oxidation, flavin metabolism, and flavoenzyme activities in fibroblasts.

Multiple acyl-CoA dehydrogenation disorders result from generalized defects in intramitochondrial acyl-CoA dehydrogenation. Fibroblasts from a riboflavin-responsive multiple acyl-CoA dehydrogenation disorder patient catabolized 14C-butyrate, -octanoate, and -leucine normally after culture in riboflavin-supplemented medium (2 mg/L). After culture in riboflavin-depleted medium (< or = 1.4 micrograms/L), his cells oxidized the same substrates poorly at 20 to 33% of control (p < 0.05). Patient cells incubated in a wide range of D-[2-14C]riboflavin concentrations (3, 31.4, and 100 micrograms/L) synthesized 14C-flavin mononucleotide and 14C-flavin adenine dinucleotide (FAD) normally and had normal cytosolic 14C-flavin mononucleotide and 14C-FAD contents, which argues against defects in cellular riboflavin uptake and conversion to flavin mononucleotide and FAD. After culture in 31.4 micrograms 14C-riboflavin/L for 2 wk, 14C-FAD specific radioactivities plateaued and were similar in patient and control cells. However, culturing these uniformly labeled cells in riboflavin-depleted medium for 2 wk lowered the patient's cellular 14C-FAD content to only 23% of control levels. Similarly, after incubation in low 14C-riboflavin concentrations (4.4 micrograms/L), the patient's mitochondrial 14C-FAD content was only 51% of control after 1 h and 29% of control at 4 h. After a 4-h incubation in a high physiologic concentration of 14C-riboflavin (31.4 micrograms/L), which raised the patient's cellular 14C-FAD levels 3- to 4-fold, his mitochondrial 14C-FAD content rose to normal; control values did not change. We also investigated possible defective FAD binding to flavoenzymes essential for acyl-CoA dehydrogenation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1999

Glutathione reductase activity and flavin concentration in guinea-pig tissues.

Glutathione reductase (GR) activity and flavin concentration were studied in systemic tissues (brain, heart, lung, liver, spleen, stomach, pancreas, muscle, kidney, testis) and blood components (erythrocytes and plasma) from male guinea-pigs. GR activity and the flavin concentration were high in kidney and liver, and low in muscle. GR activity in erythrocytes was found in a range of tissues, but flavin concentration in erythrocytes was lower than in any tissues. GR was saturated with flavin adenine dinucleotide (FAD) in almost all tissues, but not in muscle or erythrocytes.

Animals↗
PubMed2001

Mechanism of NAD(P)H:quinone reductase: Ab initio studies of reduced flavin.

NAD(P)H:quinone oxidoreductase type 1 (QR1, NQO1, formerly DT-diaphorase; EC 1.6.99.2) is an FAD-containing enzyme that catalyzes the nicotinamide nucleotide-dependent reduction of quinones, quinoneimines, azo dyes, and nitro groups. Animal cells are protected by QR1 from the toxic and neoplastic effects of quinones and other electrophiles. Alternatively, in tumor cells QR can activate a number of cancer chemotherapeutic agents such as mitomycins and aziridylbenzoquinones. Thus, the same enzyme that protects the organism from the deleterious effects of quinones can activate cytotoxic chemotherapeutic prodrugs and cause cancer cell death. The catalytic mechanism of QR includes an important initial step in which FAD is reduced by NAD(P)H. The unfavorable charge separation that results must be stabilized by the protein. The details of this charge stabilization step are inaccessible to easy experimental verification but can be studied by quantum chemistry methods. Here we report ab initio quantum mechanical calculations in and around the active site of the enzyme that provide information about the fine details of the contribution of the protein to the stabilization of the reduced flavin. The results show that (1) protein interactions provide approximately 2 kcal/mol to stabilize the planar conformation of the reduced flavin isoalloxazine ring observed in the X-ray structure; (2) the charge separation present in the reduced planar form of the flavin is stabilized by interactions with groups of the protein; (3) even after stabilization, the reduction potential of the cofactor remains more negative than that of the free flavin, making it a better reductant for a larger variety of quinones; and (4) the more negative reduction potential may also result in faster kinetics for the quinone reduction step.

Amino Acid Sequence↗
PubMed2004

Capillary liquid chromatographic determination of cellular flavins.

A capillary LC system was set up and optimized, in which a UV absorbance detector was used and a monolithic silica-ODS column as the separation column. Two on-line concentration techniques, namely, gradient elution mode and in-tube solid-phase ion-pair microextraction (SPIPME), were combined with the capillary LC system, which proved to be beneficial to enhance the concentration sensitivity by enabling the injection of large volumes of samples. The limits of detection at ppb levels for the flavins [riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)] were achieved using the two techniques. For in-tube SPIPME, a monolithic silica-ODS column was employed as the extraction column, on which FAD and FMN were retained b y interaction with an ion-pair reagent, tetrabutylammonium phosphate, resulting in greater than 110-fold enhancement in their concentration sensitivities relative to conventional injection method. The reproducibility and linearity of the two methods were investigated. The two methods were applied to analyze trace amounts of flavins in bacterial Escherichia coli cell extracts and recombinant flavoenzymes.

Chromatography, Liquid↗
PubMed1975

Flavin nucleotides, glutathione reductase and assessment of riboflavin status.

The in vitro effects of flavin nucleotides on human erythrocyte glutathione reductase were studied. Flavin mononucleotide was found to be a potent inhibitor of glutathione reductase in haemolysates, having an effect at a concentration of 1 muM. Flavin adenine dinucleotide inhibited at a concentration at least ten fold this, lower levels producing the expected stimulation of enzyme activity. Different assay conditions also altered the activity of the enzyme, although the flavin mononucleotide inhibition remained. The implications of these findings in our understanding of glutatione reductase and in using the enzyme to assess riboflavin status are discussed.

Erythrocytes↗
PubMedDate not supplied

[Effect of the combined use of flavin coenzyme preparations and pyridoxine on the body vitamin balance in animals].

The effect of combined administration of flavin coenzymes and pyridoxine on B2-avitaminosis rats' supply with these vitamins has been studied. It has been disclosed that pyridoxine promotes more effective normalization of riboflavin and pyridoxine balance in the body, this balance being measured from the excretion of riboflavin and 4-pyridoxin acid with urine as well as from the content of total flavins in blood and tissues. In vitamin B2 lack, it is recommended that pyridoxin be combined with flavin mononucleotide and in particular with flavin adenine dinucleotide.

Animals↗
PubMed1993

Further characterization and purification of the flavin-dependent S-benzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes.

Previously, we provided evidence that cysteine conjugate S-oxidase (S-oxidase) activities of rat liver and kidney microsomes may be associated with flavin-containing monooxygenases (FMOs). In this study, the biochemical properties of these activities were further investigated. When NADPH was replaced by NADH, the S-oxidase activities were reduced significantly. Removal of the flavin moiety from microsomes significantly reduced the S-oxidase activities; however, addition of exogenous FAD or FMN restored the activities of the flavin-depleted microsomes. Solubilization of hepatic or renal microsomes with Emulgen 911, Nonidet P-40, Triton X-100, or 3-[(3-cholamidopropyl)dimethyl-ammonio]-1- propane sulfate or inclusion of the sulfhydryl-reactive agents Hg2+, N-ethylmaleimide, or iodoacetamide did not affect the S-oxidase activities, whereas solubilization of either hepatic or renal microsomes by cholate or heating of renal microsomes in the absence of NADPH significantly reduced the S-oxidase activities. In addition to male rat hepatic and renal microsomes, the S-oxidase activities were detected in lung microsomes of male rats and hepatic and renal microsomes of male mice and female rats and rabbits. The male rat kidney maintained the highest S-oxidase activity of all species and tissues examined. Whereas the aforementioned results provided further evidence for the S-oxidase activities being associated with FMOs, unambiguous evidence for this hypothesis was provided by the purification of the activities from rat liver (580-fold) and kidney (700-fold) microsomes and by the use of the isolated proteins in polyacrylamide gel electrophoresis, flavin content determinations, amino-terminal amino acid sequence analysis, amino acid composition analysis, and substrate kinetic studies. The findings that the S-oxidases were immunoreactive with antibodies raised against the pig liver 1A1 isozyme but not with antibodies raised against the rabbit lung 1B1 isozyme and that the liver S-oxidase amino-terminal amino acid sequence was more comparable to the amino-terminal amino acid sequences of pig and rabbit liver 1A1 isozymes than to those of rabbit lung 1B1 and liver 1D1 isozymes provide evidence that the S-oxidases are related to the known FMO 1A1 isozymes.

Adenine Nucleotides↗
PubMedDate not supplied

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

Aliivibrio fischeri↗
PubMed2005

Flavins are source of visible-light-induced free radical formation in cells.

BACKGROUND AND OBJECTIVES: It has been hypothesized that reactive oxygen species (ROS) are created by an endogenous photosensitizer during low energy visible light illumination of tissue, and these ROS are responsible for reported biostimulative effects. This study aims to identify the endogenous photosensitizer responsible for ROS production by visible light. STUDY DESIGN/MATERIALS AND METHODS: Electron paramagnetic resonance (EPR) spectroscopy was used to detect oxyradicals produced in cardiac and sperm cells during illumination by a halogen lamp. Oxyradical production was determined as a function of illumination wavelength, cell fraction, and molecular weight. RESULTS: Oxyradicals were created solely by the 400-500 nm range of visible light. The endogenous photosensitizer is found predominantly in the cytosol and is smaller than 12 kD. Flavin mononucleotide produces the same signal at concentrations consistent with reported intracellular free flavin concentrations. CONCLUSIONS: A small, water soluble photosensitizer active only at wavelengths shorter than 500 nm is consistent with a flavin, which reproduces cellular signals at physiological concentrations. Thus, flavins are responsible for the photosensitization of the observed oxyradicals in cells.

Animals↗
PubMed1997

Evidence for two conformational states of thioredoxin reductase from Escherichia coli: use of intrinsic and extrinsic quenchers of flavin fluorescence as probes to observe domain rotation.

Thioredoxin reductase (TrxR) from Escherichia coli consists of two globular domains connected by a two-stranded beta sheet: an FAD domain and a pyridine nucleotide binding domain. The latter domain contains the redox-active disulfide composed of Cys 135 and Cys 138. TrxR is proposed to undergo a conformational change whereby the two domains rotate 66 degrees relative to each other (Waksman G, Krishna TSR, Williams CH Jr, Kuriyan J, 1994, J Mol Biol 236:800-816), placing either redox active disulfide (FO conformation) or the NADPH binding site (FR conformation) adjacent to the flavin. This domain rotation model was investigated by using a Cys 138 Ser active-site mutant. The flavin fluorescence of this mutant is only 7% that of wild-type TrxR, presumably due to the proximity of Ser 138 to the flavin in the FO conformation. Reaction of the remaining active-site thiol, Cys 135, with phenylmercuric acetate (PMA) causes a 9.5-fold increase in fluorescence. Titration of the PMA-treated mutant with the nonreducing NADP(H) analogue, 3-aminopyridine adenine dinucleotide phosphate (AADP+), results in significant quenching of the flavin fluorescence, which demonstrates binding adjacent to the FAD, as predicted for the FR conformation. Wild-type TrxR, with or without PMA treatment, shows similar quenching by AADP+, indicating that it exists mostly in the FR conformer. These findings, along with increased EndoGluC protease susceptibility of PMA-treated enzymes, agree with the model that the FO and FR conformations are in equilibrium. PMA treatment, because of steric limitations of the phenylmercuric adduct in the FO form, forces the equilibrium to the FR conformer, where AADP+ binding can cause fluorescence quenching and conformational restriction favors proteolytic susceptibility.

Adenine Nucleotides↗
PubMed2001

Versatility and specificity in flavoenzymes: control mechanisms of flavin reactivity.

Flavoenzymes are characterized by their remarkable versatility and strict specificity. The former can be grasped when flavoenzymes are treated as a whole, while the latter refers to each flavoenzyme in which the broad versatility of flavin is specifically controlled. The versatility stems from the variety of the redox, ionic, and electronic states that the flavin ring system can adopt. Versatility of flavoenzymes is reflected in their classification, which has generally been based on substrates and reactions catalyzed. A different classification is presented according to the number of electrons transferred in the reductive and oxidative half reactions. Specificity of each flavoenzyme is understood in terms of the regulatory mechanism of the broad reactive potentiality of flavin. The elements of this regulatory mechanism include hydrogen-bonding network, electrostatic effect, charge-transfer interaction, positioning between a substrate/ligand and flavin, and modulation of resonance hybridization, each of which is explained with relevant examples provided mainly by studies from the author's group.

Enzymes↗
PubMed1980

Modulation of the flavin redox potential as mode of regulation of succinate dehydrogenase activity.

The redox properties of flavin in active and non-active (oxaloacetate reacted) soluble succinate dehydrogenase were studied. Quantitative analysis of reductive activation titrations of redox titrations of active and non-active enzyme reveal that the redox potential of the histidyl-flavin in the active enzyme (-3 +/- 15 mV) is high enough to allow reduction by succinate, whereas in the non active enzyme it is -196 +/- 19 mV, far to low to be reduced by substrate. The flavin radical in the active enzyme attains 60% of total flavin at a poised redox potential of about +60 mV, upon addition of oxaloacetate the magnitude of the signal is diminished and the potential where it reaches maximal concentration is shifted by about -200 mV. A mechanism is proposed which ascribes the fundamental difference between active and non-active enzyme to the inability of the latter to be reduced by substrate.

Animals↗
PubMed1983

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase.

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.

Animals↗
PubMed1988

Inhibition of flavin metabolism by adriamycin in skeletal muscle.

Adriamycin (ADR), a potent antineoplastic agent, has been shown to interact with flavin derivatives and to compete with flavin coenzymes for their respective binding sites on flavin-containing enzymes. The present investigation determined that ADR administration inhibited flavin adenine dinucleotide (FAD) biosynthesis from riboflavin in rat skeletal muscle in a dose-related manner compared to results in pair-fed controls. Five groups of adult Holtzman rats of both sexes were given twice daily intraperitoneal injections of ADR for 3 days, representing cumulative doses of 6, 12, 18, 24, and 30 mg/kg body weight. At the cumulative dose of 6 mg/kg, there was no significant effect, but at 12, 18, 24, and 30 mg/kg levels significant increases in [14C]FAD formation from [14C]riboflavin occurred. ADR-induced myopathy may be due, at least in part, to inhibition of FAD formation, ultimately leading to changes in energy metabolism and oxidative capacity.

Animals↗
PubMed1994

NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein.

The NAD(P)H-flavin oxidoreductase gene from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, was expressed in Escherichia coli, and the enzyme purified using Cibacron Blue 3G-A affinity column chromatography from crude extracts in a single step. The purified enzyme had a typical flavoprotein absorption spectrum and flavin mononucleotide (FMN) was identified as a prosthetic group, non-covalently bound in a molar ratio of 1:1. The enzyme catalyzed the electron transfer from NADH via FMNH2 to various other electron acceptors. Reduced flavin produced by flavin reductase participated non-enzymatically in the following reactions: H2O2-forming NADH oxidase-like, oxygen-insensitive nitroreductase-like, diaphorase (quinone reductase)-like and bacterial luciferase reactions.

Amino Acid Sequence↗
PubMed1989

Reduction of aryl-nitroso compounds by pyridine and flavin coenzymes.

1. A systematic kinetic investigation of the reduction of aryl-nitroso compounds by pyridine and flavin coenzymes and their analogs, in enzymatic and nonenzymatic systems, has been reported. 2. Two main groups of nitroso compounds have been investigated, representatives nitroso-benzene and 1-nitroso-2-naphthol; in all enzymatic and nonenzymatic systems, the former was always reduced to phenyl-hydroxyl-amine and the latter to 1-amino-2-naphthol. 3. Pyridine compounds included NADH, APAD-4H2 and DBNA-4H2 in nonenzymatic systems, and liver alcohol dehydrogenase. Flavin compounds included 1,5-dihydrolumiflavin and various forms of reduced 5-ethyl-lumiflavin, in nonenzymatic systems, and the flavoenzymes glucose-oxidase and NADPH-cytochrome P450 reductase. 5. Pyridine coenzymes and their analogs reduced nitroso compounds by a direct hydride transfer, with a primary kinetic isotope of 9.5 +/- 2.2. 6. All flavin compounds (glucose-oxidase and its nonenzymatic analog 1,5-dihydrolumiflavin and NADPH-cytochrome P450 reductase and its analog 5-ethyl-1,5-dihydrolumiflavin) reduced aryl-nitroso compounds with high efficiency (k2 greater than 10(5)M(-1) min(-1)). 7. The flavin compounds have been shown to be much more efficient reductans of nitroso compounds, compared to pyridine coenzymes, both in enzymatic and nonenzymatic systems; the only exception to this rule presented the extremely efficient reduction of p-substituted aryl-nitroso compounds by liver alcohol dehydrogenase.

Anaerobiosis↗
PubMed1998

Flavin fluorescence dynamics and photoinduced electron transfer in Escherichia coli glutathione reductase.

Time-resolved polarized flavin fluorescence was used to study the active site dynamics of Escherichia coli glutathione reductase (GR). Special consideration was given to the role of Tyr177, which blocks the access to the NADPH binding-site in the crystal structure of the enzyme. By comparing wild-type GR with the mutant enzymes Y177F and Y177G, a fluorescence lifetime of 7 ps that accounts for approximately 90% of the fluorescence decay could be attributed to quenching by Y177. Based on the temperature invariance for this lifetime, and the very high quenching rate, electron transfer from Y177 to the light-excited isoalloxazine part of flavin adenine dinucleotide (FAD) is proposed as the mechanism of flavin fluorescence quenching. Contrary to the mutant enzymes, wild-type GR shows a rapid fluorescence depolarization. This depolarization process is likely to originate from a transient charge transfer interaction between Y177 and the light-excited FAD, and not from internal mobility of the flavin, as has previously been proposed. Based on the fluorescence lifetime distributions, the mutants Y177F and Y177G have a more flexible protein structure than wild-type GR: in the range of 223 K to 277 K in 80% glycerol, both tyrosine mutants mimic the closely related enzyme dihydrolipoyl dehydrogenase. The fluorescence intensity decays of the GR enzymes can only be explained by the existence of multiple quenching sites in the protein. Although structural fluctuations are likely to contribute to the nonexponential decay and the probability of quenching by a specific site, the concept of conformational substates need not be invoked to explain the heterogeneous fluorescence dynamics.

Binding Sites↗
PubMed2002

An N1-hydrogen bonding model for flavin coenzyme.

A model flavin possessing a specific hydrogen bond to the N1-position has been synthesized. The redox potential has been measured in aqueous buffer and found to be shifted +21 mV as compared to a similar flavin lacking this hydrogen bond. The reaction of the N1-hydrogen-bonding model with sulfite and 1-benzyl-dihydronicotinamide were examined and compared with the non-hydrogen-bonded flavin. The N1-hydrogen bond did not accelerate the rate of sulfite ion or hydride addition to N5, however the N5-sulfite complex was stabilized by nearly 4-fold over a non-hydrogen-bonding model. The model flavins were also studied computationally.

Coenzymes↗