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PubMedDate not supplied

Effect of atebrine on glucose blood levels and on flavinic nucleotides content in rat liver in deficiency state and in satisfied demand of riboflavine.

Changes in glucose levels in blood, excretion of riboflavine in urine, FAD and FMN flavinic nucleotides and a total riboflavine content in liver homogenates were determined in rats. Both riboflavine deficiency and atebrine do not effect the glucose, blood levels. Atebrine (ATB) significantly inhibits the urinary excretion of riboflavine both in deficiency state and in satisfied demand of this vitamin. The lack of riboflavine in food results in a decrease of total riboflavine and FA- in liver, while during the first period of deficiency the amount of FMN increases. When the riboflavine demand is satisfied, low doses of ATP do not influence the total riboflavine and flavinic nucleotides content in the liver, while at high doses, the total riboflavine increases and the amount of FMN initially significantly exceeds the amount of FAD. ATB markedly inhibits the growth of rats both in the deficiency state and in the satisfied demand of riboflavine.

Animals↗
PubMed1977

[The influence of biophysical factors on biological oxidation and redox processes. 16. The possibility of influencing flavine coenzymes in liver mitochondria by means of ultrasonic treatment (author's transl)].

The quantity of FAD and FMN in mitochondria isolated from the liver of albino rats was determined before and after application of ultrasonic energy with the intensities 0,2,0,6 and 1,0 w/cm2 in a short-term test, and the intensity 0,2 w/cm2 applied at ten successive days in a long-term test. The total amount of the two coenzymes as well as their percentages were ascertained. The following results derive from our experiments: 1. A quantitative increase of FAD in liver mitochondria. This suggests that the capacity of the shortened respiratory chain has increased, since redox systems represent a factor determining its stage. 2. The flavine-mononucleotide responds to ultrasonics as a relatively instable biological macromolecule. 3. The total amount of flavine coenzymes is influenced especially by the quantity of FAD, and, correspondingly, by changes of the latter.

Animals↗
PubMed2006

Investigation of the pathways of excess electron transfer in DNA with flavin-donor and oxetane-acceptor modified DNA hairpins.

Oxetane is a potential intermediate that is enzymatically formed during the repair of (6-4) DNA lesions by special repair enzymes (6-4 DNA photolyases). These enzymes use a reduced and deprotonated flavin to cleave the oxetane by single electron donation. Herein we report synthesis of DNA hairpin model compounds containing a flavin as the hairpin head and two different oxetanes in the stem structure of the hairpin. The data show that the electron moves through the duplex even over distances of 17 A. Attempts to trap the moving electron with N2O showed no reduction of the cleavage efficiency showing that the electron moves through the duplex and not through solution. The electron transfer is sequence dependent. The efficiency is reduced by a factor of 2 in GC rich DNA hairpins.

Circular Dichroism↗
PubMed2005

Probing the N(5)-H bond of the isoalloxazine moiety of flavin radicals by X- and W-band pulsed electron-nuclear double resonance.

An X- (9.7 GHz and W-band (94 GHz) pulsed electron-nuclear double resonance (ENDOR) study of the flavin cofactor of Escherichia coli DNA photolyase in its neutral radical form is presented. Through proton and deuteron ENDOR measurements at T = 80 K, we detect and characterize the full anisotropy of the hyperfine coupling (hfc) tensor of the proton or deuteron bound to N(5) of the isoalloxazine ring. Scaling of the anisotropic proton hfc components by multiplication with the quotient of the magnetogyric ratio of a deuteron and a proton, chiD/chiH, reveals subtle differences compared to the respective deuteron couplings obtained by 95-GHz deuterium ENDOR spectroscopy on an H-->D buffer-exchanged sample. These differences can be attributed to the different lengths of N(5)-H and N(5)-D bonds arising from the different masses of protons and deuterons. From the R(-3) dependence of the dipolar hyperfine splitting, we estimated that the N(5)-D bond is about 2.5% shorter than the respective N(5)-H bond. That such subtle bond-length differences can be resolved by pulsed ENDOR spectroscopy suggests that this method may be favorably used to probe the geometry of hydrogen bonds between the H(5) of the paramagnetic flavin and the protein backbone. Such information is only obtained with difficulty by other types of spectroscopy.

Anisotropy↗
PubMed1983

Flow-cytometric monitoring of intracellular flavins simultaneously with NAD(P)H levels.

A cytofluorimeter is described, using the combination of Argon UV (351-363 nm) and Argon Blue (488 nm) lasers. The dual excitation makes it possible to monitor simultaneously the redox state of flavins and NAD(P)H as indicative of cell metabolic state. Light scatter, absorption, and staining with exogenous fluorescent dyes can add additional information. Thus, a five-parameter flow analysis becomes possible. The present paper describes flavin and NAD(P)H measurements on isolated rat liver cells and mouse bone marrow.

Animals↗
PubMed2005

Determination of the g-matrix orientation in flavin radicals by high-field/high-frequency electron-nuclear double resonance.

A high-microwave-frequency/high-magnetic-field pulsed electron-nuclear double resonance (ENDOR) study performed at 94 GHz on the flavin semiquinone cofactor of Xenopus laevis (6-4) photolyase in its neutral radical state is presented. Although the principal values of the flavin radical's g-matrix are not fully resolved in the 94-GHz EPR spectrum in a nonoriented sample, the orientation of the principal axes of g is obtained by exploiting the orientation selection of the proton ENDOR signals from the methyl protons at C-8alpha and the deuteron ENDOR signals from D-5 in an enzyme sample in deuterated buffer. This procedure for assigning the orientation of g relative to the molecular frame makes use of commercially available ENDOR instrumentation without the necessity to perform single-crystal studies.

Animals↗
PubMed1994

Two-dimensional NMR studies of the flavin binding site of Desulfovibrio vulgaris flavodoxin in its three redox states.

The riboflavin 5'-monophosphate (FMN) binding site of Desulfovibrio vulgaris flavodoxin in the diamagnetic oxidized and two-electron reduced form was investigated using two-dimensional proton NMR. The NMR results are compared to existing X-ray crystallographic data. In the paramagnetic one-electron reduced redox state resonances of protons which are close to the FMN ring are strongly broadened due to the paramagnetic properties of the flavin ring. From comparison of the NMR spectra of the three redox states it could be concluded that outside the FMN binding site no structural changes occur upon reduction. Strong hydrogen bonds are observed between the N(1) and C(2) carbonyl of the isoalloxazine ring and the amide protons of D95 and C102, respectively. The amide resonances of D95 and C102 are strongly downfield shifted upon two-electron reduction, caused by the negative charge in the N(1)-C(2) carbonyl region in the two-electron reduced FMN. It is suggested that the ring current of the central pyrazine ring of the FMN molecule in the two-electron reduced flavodoxin is decreased compared to the oxidized flavodoxin. The decrease in ring current is apparently caused by the loss of aromaticity of this pyrazine moiety due to protonation of N(5). Strong hydrogen bonds between the flavin phosphate group and amide and hydroxyl protons of the apoprotein are observed. Resonances of protons involved in this hydrogen bonding network are downfield shifted up to 3.5 ppm. It is suggested that the negative charges of the dianionic FMN phosphate group are stabilized by local peptide dipoles. On reduction of the protein from the oxidized to the one-electron reduced form, a conformational change occurs in the FMN binding region. No conformational change can be observed between the one-electron and the two-electron reduced state.

Amino Acid Sequence↗
PubMed1994

Flavin substrate specificity of the vitamin B2-aldehyde-forming enzyme from Schizophyllum commune.

Vitamin B2-aldehyde-forming enzyme from Schizophyllum commune catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have monitored enzyme activity by spectrophotometrically measuring the reduction of 2,6-dichlorophenol-indolphenol as electron acceptor to assess 35 riboflavin analogs as potential substrates or competitive inhibitors with the purpose of delimiting structural requirements of the substrate binding site. Analogs with side chains of two- to six-carbon length modified by deletion of secondary hydroxyls or by changes in their epimeric configuration are not oxidized. The omega-hydroxyalkyl-flavins (n = 2-6) are competitive inhibitors (Ki = 7-16 microM) of riboflavin oxidation, as are some analogs with L-secondary hydroxyls in the side chain. Analogs with bulky substituents on the isoalloxazine ring are also not substrates. The enzyme does not significantly bind flavins with an 8 alpha-N-imidazole; diethylamino, methylethylamino, dimethylamino, ethylamino, or ethoxy groups at position 8; methyl at 6; and beta-hydroxyethylamino at position 2. Also the replacement of N with CH in 1-deazariboflavin disallows substrate reaction. Analogs with fluoro, chloro, methyl, amino, or methylamino at position 8; chloro at 7; methyl or carboxylmethyl at 3; thio at 2, and C replacing N at positions 3 or 5 are substrates with relative Vmax values ranging from 27 to 110% that of riboflavin. The Km values for the analogs oxidized are all found to be in the micromolar range (22-176 microM). Overall specificity of the enzyme for riboflavin is found to be rather narrow and sterically limited, which suggests that the vitamin is the natural substrate.

Alcohol Oxidoreductases↗
PubMed1995

A site-directed mutagenesis study at Lys-113 of NAD(P)H:quinone-acceptor oxidoreductase: an involvement of Lys-113 in the binding of the flavin adenine dinucleotide prosthetic group.

NAD(P)H: quinone-acceptor oxidoreductase (EC 1.6.99.2), also referred to as DT-diaphorase, is a flavoprotein that catalyzes the two-electron reduction of quinones and quinonoid compounds to hydroquinones, using either NADH or NADPH as the electron donor. Using an Escherichia coli expression system developed previously, we prepared three mutants of the rat liver quinone reductase. These mutants are Lys-113-His (K113H), Lys-113-Asp (K113D), and Lys-113-Ala (K113A). While the mutant K113H was readily purified using the same procedure as for the purification of the wild-type quinone reductase and found to have an activity similar to that of the wild-type enzyme, K113D and K113A were purified only in very small quantities, mainly in the form of apoprotein, and had very low activities. The results suggest that a positively charged amino acid at this position is important for the binding of the flavin adenine dinucleotide (FAD) prosthetic group. Flavin spectral studies of 6-mercapto-FAD-reconstituted mutants revealed that mutation at Lys-113 affects the protein environment around position-6 of the isoalloxazine ring.

Amino Acid Sequence↗
PubMed1996

The interaction of NADPH-P450 reductase with P450: an electrochemical study of the role of the flavin mononucleotide-binding domain.

The electrochemically reduced mediator cobalt sepulchrate requires the presence of a flavoprotein for the rapid transfer of electrons to cytochrome P450. This electrochemical method has been used here to show the interaction of NADPH-P450 reductase (either the detergent-solubilized form, d-OR, or the proteolytic-cleaved truncated form, t-OR), as well as Escherichia coli flavodoxin (FLD), with P450c17 by measuring the rate of 17 alpha-hydroxylation of progesterone. When NADPH is used as electron donor with a reconstituted system composed of d-OR and P450c17, the addition of t-OR, flavodoxin, or cytochrome c inhibited the rate of formation of 17 alpha-hydroxyprogesterone. These results suggest the presence of a common protein binding site on the surface of d-OR, t-OR, and flavodoxin which plays a role in the interaction of the flavoproteins with the P450. It is speculated that a domain composed of acidic amino acids, located near the flavin mononucleotide-binding region of the flavoproteins, may serve as this site. No inhibition by t-OR, flavodoxin, or cytochrome c is observed when comparable experiments are carried out using the artificial recombinant fusion protein rF450[mBov17A/mRatOR]L1 containing the heme-domain of P450c17 linked to the flavin-domains of NADPH-P450 reductase.

Amino Acid Sequence↗
PubMed1999

Methionine S-oxidation in human and rabbit liver microsomes: evidence for a high-affinity methionine S-oxidase activity that is distinct from flavin-containing monooxygenase 3.

Methionine has previously been shown to be S-oxidized by flavin-containing monooxygenase (FMO) forms 1, 2, and 3. The most efficient catalyst was FMO3, which has a Km value for methionine S-oxidation of approximately 4 mM, and exhibits high selectivity for formation of the d-diastereoisomer of methionine sulfoxide. The current studies provide evidence for an additional methionine S-oxidase activity in liver microsomes. Human and rabbit liver microsomes exhibited a biphasic response to methionine at concentrations ranging from 0.05 to 10 mM, as indicated by both Eadie-Hofstee plots and nonlinear regression. The low-affinity component of the biphasic response had Km values of approximately 3 and 5 mM for humans and rabbits, respectively, as well as high diastereoselectivity for methionine sulfoxide formation. The low-affinity activity in rabbit liver microsomes was inhibited by methimazole, S-allyl-l-cysteine, and by mild heat treatment, suggesting the activity is FMO3. The high-affinity component of the biphasic response had Km values of approximately 0.07 and 0.04 mM for humans and rabbits, respectively, as well as lower diastereoselectivity for methionine sulfoxide formation. Further characterization of the high-affinity activity in rabbit liver microsomes indicated lack of involvement of cytochrome P450s or reactive oxygen species. The high-affinity activity was inhibited 25% by potassium cyanide and greater than 50% by methimazole and S-allyl-l-cysteine. Mild heat treatment produced 85% inhibition of the low-affinity activity, but only 30% inhibition of the high-affinity activity. Both high- and low-affinity activities were decreased by 85% in flavin-depleted microsomes. Because these results suggested the additional S-oxidase activity has characteristics of an FMO, recombinant human FMO4 was evaluated as a potential catalyst of this activity. Recombinant FMO4 catalyzed S-oxidation of both methionine and S-allyl-l-cysteine, with similar diastereoselectivity to the high-affinity microsomal S-oxidase; however, the Km values for both reactions appeared to be greater than 10 mM. In summary, these studies provide evidence for two microsomal methionine S-oxidase activities. FMO3 is the predominant catalyst at millimolar concentrations of methionine. However, at micromolar methionine concentrations, there is an additional S-oxidase activity that is distinct from FMO3.

Adult↗
PubMed2001

Thioester enolate stabilization in the acyl-CoA dehydrogenases: the effect of 5-deaza-flavin substitution.

The redox-inactive thioester analog 3-thia-octanoyl-CoA blocks transfer of a hydride equivalent to the flavin prosthetic group of the medium-chain acyl-CoA dehydrogenase with the accumulation of a stable enolate intermediate not encountered with normal substrates. Substitution of the normal flavin with 5-deaza-FAD would thus be expected to lead to enolate formation with both normal and 3-thia-substrate analogs, because reduction of the 5-deaza-enzyme is thermodynamically highly unfavorable. However, spectrophotometric titrations show that neither ligand forms significant enolate species with the 5-deaza-FAD enzyme. Similarly, the substituted dehydrogenase catalyzes undetectable alpha-proton exchange with octanoyl-CoA and ca. 1% of the corresponding rate with 3-thia-octanoyl-CoA when compared to the native enzyme. This inability to stabilize enolate species is not simply due to impaired binding of CoA-thioester analogs, because binding of a range of ligands is weakened by only 2- to 10-fold with the 5-deaza-enzyme. 4-Thia-trans-2-enoyl-CoA product is polarized normally on binding to the substituted protein, showing that this critical aspect of catalysis is apparently normal. These data, together with studies with CoA-persulfide and acetoacetyl- and p-nitrophenylacetyl-CoA, suggest that 5-deaza-FAD substitution exerts subtle, unanticipated, effects on the reductive half-reaction of the medium-chain acyl-CoA dehydrogenase. The involvement of charge-transfer interactions in the acidification of weakly acidic acyl-CoA thioesters is discussed.

Acyl-CoA Dehydrogenase↗
PubMed1997

Perils of partitioning: A case study of flavins and flavokinase.

Partitioning is a common procedure for the separation of two solutes from a solution based on their differential solubility in an immiscible solvent. This has been widely used to quantitate riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide in aqueous samples by extraction with water-saturated benzyl alcohol. Here we report that the partitioning of riboflavin and FMN is affected by the presence of each other in a concentration-dependent manner, thus rendering this procedure unsuitable for quantitation. Direct quantitation of FMN formed in assays for flavokinase from Vigna radiata shows that kinetic analyses using a partition-based assay lead to erroneous conclusions.

Adenosine Triphosphate↗
PubMed2000

NF- kappa B independent suppression of endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expression by inhibition of flavin binding proteins and superoxide production.

Oxidation-reduction (redox) coupled mechanisms play an important role in the regulation of cell surface adhesion molecule expression. In endothelial cells membrane-bound NADH/NADPH oxidase is a significant source of intracellular superoxide (O(2)(-)) production. We explored the role of flavin containing proteins such as NADH/NADPH oxidase in the induction of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in human aortic endothelial cells (HAECs) and human dermal microvascular endothelial cells (HMECs). Treatment of HAECs by tumor necrosis factor- alpha (TNF- alpha, 100 U/ml) for 1 h induced a 31% increase in O(2)(-)production within 5 min as determined by lucigenin chemiluminescence analysis of whole cells (n=4, P<0.05). Pretreatment with the NADH/NADPH oxidase inhibitor diphenylene iodonium (DPI, 40 microm) for 1 h inhibited O(2)(-)production. DPI also inhibited TNF and LPS-induced VCAM-1 and ICAM-1 cell surface expression and TNF- alpha, LPS, or IL-1 beta induced VCAM-1 and ICAM-1 mRNA accumulation. However, DPI did not inhibit TNF- alpha -induced activation of nuclear NF- kappa B-like binding activity in HAECs and HMECs. Furthermore, DPI did not inhibit TNF- alpha induced transactivation of NF- kappa B-driven VCAM-1 and HIV-LTR promoter gene constructs in transiently transfected HMECs. These data suggest that flavin binding proteins such as NADH/NADPH oxidase can regulate VCAM-1 gene expression independent of NF- kappa B. Furthermore, intracellular O(2)(-)generation is not necessary for NF- kappa B activation or for transactivation of NF- kappa B driven promoters.

Aorta↗
PubMed1994

The mammalian flavin-containing monooxygenases: molecular characterization and regulation of expression.

The flavin-containing monooxygenase (FMO) has been characterized in several mammalian species, including human. The FMO forms a stable NADP(H)- and oxygen-dependent 4 alpha-hydroperoxy flavin enzyme intermediate in the absence of an oxygenatable substrate. As such, substrate specificity appears to be controlled by access to this stabilized intermediate, resulting in this enzyme's ability to metabolize a wide variety of xenobiotics. These include tertiary and secondary alkyl- and arylamines, many hydrazines, thiocarbamides, thioamides, sulfides, disulfides, thiols, and other soft nucleophiles. Although some of these compounds are oxidized to less active derivatives, several examples of metabolic activation to potentially toxic intermediates also exist. Mercapto-pyrimidines and thiocarbamides, for example, appear to be activated predominantly by FMO. Thus, this enzyme system may play an important role in the early steps of chemical toxicity. Often, the contribution of FMO to the metabolism of a given compound can be assessed by its unique stereoselectivity relative to other oxygenases. For example, the cytochromes P450 oxidize (S)-nicotine to a mixture of cis- and trans-N-1'-oxides. In contrast, (S)-nicotine is oxidized by human FMO3 exclusively to the trans-N-1'-oxide. With the purification and cloning of FMO from multiple tissues and species it became apparent that more than one FMO exists. Further, there are considerable tissue- and species-specific differences in FMO expression that likely contribute to observed differences in detoxication competency and toxicant susceptibility.

Animals↗
PubMed1987

An EPR study of the interactions between heme and flavin in yeast flavocytochrome b2.

E.P.R. experiments and spin-lattice relaxation time measurements have been performed on Flavocytochrome b2 in the range 10 K to 100 K, to obtain information on the distance between the two prosthetic groups of the protein, flavin and heme. We have used the stabilization effect of pyruvate on the semiquinone form of the flavin, to compare the E.P.R. spectral shape and the relaxation properties of the radical when the heme is either in the ferrous form or in the ferric form. When the heme is ferric, no significant increase of the line broadening or enhancement of the relaxation rate of the radical can be detected in the range 10 K to 100 K. From these results, a minimum intercentre distance of 18 to 20 A can be estimated.

Electron Spin Resonance Spectroscopy↗
PubMed1984

Bluelight-induced, flavin-mediated transport of redox equivalents across artificial bilayer membranes.

This paper continues our studies of physico-chemical properties of vesicle-bound flavins. Based on previous results, an advanced model system was designed in order to study the mechanisms underlying bluelight-induced redox transport across artificial membranes. The lumen of single-shelled vesicles was charged with cytochrome c, and amphiphilic flavin (AF1 3, AF1 10) was bound to the membrane. Upon bluelight irradiation redox equivalents are translocated from exogeneous 1e-(EDTA)-and 2e-(BH3CN-) donors across the membrane finally reducing the trapped cytochrome c both under aerobic and anaerobic conditions. The mechanisms involved are explored and evidence for the involvement of various redox states of oxygen, dihydroflavin and flavosemiquinone is presented.

Biological Transport↗
PubMed1987

Isolation and characterization of a flavin-containing placental protein (PP3).

Placental protein 3 (PP3) has been isolated to purity from saline extracts of human term placentas. PP3 turned out to be a flavin-containing protein and thus appears to be an enzyme. The prosthetic group was identified as flavin-adenine dinucleotide (FAD) which was found to be noncovalently bound to the protein. The physical characterization of PP3 showed that the molecules are composed of two identical subunits having molecular weights of 55000 daltons which are noncovalently linked. Each subunit appears to contain one FAD group. In addition PP3 was found to have an electrophoretic mobility in between the alpha 1 and alpha 2 globulins, an isoelectric point in the range of 5.3-5.7, a sedimentation coefficient of 6.3 S and an extinction coefficient of 15.7 (E1%(1cm) 280 nm). Immunochemical methods were used to detect and quantitate PP3 in extracts of placentas and other human tissues. From one human term placenta an average of around 4 mg PP3 could be extracted; PP3 was also found to occur in extracts of adult human stomach. In concentrated extracts of other human tissues and in human body fluids this protein could not be detected, at least not in concentrations higher than 2 mg/dl.

Adult↗