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Results for “Flavin-Adenine Dinucleotide”

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At least 19 recordsLinked to original sources
PubMed1976

Some properties of human platelet monoamine oxidase in iron-deficiency anaemia.

1. Monoamine oxidase activity in platelets prepared from the blood of patients with iron-deficiency anaemia was significantly lowered when compared with that in platelets from normal subjects. 2. The Km values of the platelet enzyme for the substrates dopamine, 5-hydroxytryptamine, phenylethylamine and kynuramine were similar for the platelet enzyme from iron-deficient and normal groups. 3. Heat-in-activation studies showed that the platelet monoamine oxidase from iron-deficient subjects was more labile to this treatment, when compared with the platelet enzyme from normal subjects. 4. The sensitivity of platelet monoamine oxidase to the inhibitors, clorgyline and deprenil, was increased in iron-deficiency anaemia. 5. Binding studies with the 14C-binding irreversible monoamine oxidase inhibitor, deprenil, showed that the amount of enzyme capable of binding this inhibitor was lowered by 48% in platelets from iron-deficient patients when compared with platelets from normal subjects. 6. The results show that there is a lowered amount of active enzyme in platelets from iron-deficient subjects. It is suggested that iron is necessary either for the synthesis of monoamine oxidase apoenzyme or is a cofactor for an enzyme which attaches flavin-adenine dinucleotide covalently to the monoamine oxidase apoenzyme.

Adult↗
PubMed1976

An automated flavin adenine dinucleotide-dependent glutathione reductase assay for assessing riboflavin nutriture.

An automated AutoAnalyzer method using 5:5'-dithiobis-2-nitrobenzoic acid is described for determining whole blood glutathione reductase (BGR) activity and for measuring in vitro activation of BGR with flavin adenine dinucleotide (FAD). BGR activity is expressed as mumoles glutathione regenerated from oxidized glutathione per ml of whole blood (WB) or per g of hemoglobin. The stimulatory effect of FAD on BGR activity divided by the activity without FAD determined the activity coefficient (AC). We found that NADPH and oxidized glutathione assay concentrations of 0.100 mmole/liter and 0.250 mmole/liter, respectively, in 0.1 mole/liter phosphate buffer, pH 7.4, gave consistent results when WB, before assay, was diluted 20-fold. WB samples to be stored are initially diluted 10-fold with distilled water and frozen. Prior to assay, two aliquots of the sample are diluted 2-fold, one aliquot with distilled water and another with 46 mumole/liter FAD. With sample and manifold dilutions the assay FAD concentrations is 1.0 mumole/liter: assay concentrations greater than 5.0 mumole FAD/liter were shown to be inhibitory. We examined blood samples from 617 children in the age range 6 to 60 months and determined the normal AC range to be between 1.00 and 1.35. Six weaned rats (23 days of age), maintained on a riboflavin-deficient diet, showed a mean AC of 1.23, 1.54, 2.02, and 2.41 at 23, 26, 30, and 36 days of age, respectively. Six control rats maintained an AC of 1.23 +/- 0.05 (SD) during the same period.

Animals↗
PubMed1975

Fluorescence of oxidized flavoproteins from perifused isolated pancreatic islets.

In perifused pancreatic islets, the fluorescence of oxidized flavoproteins (FAD) was recorded continuously. Elevation of glucose concentration in the medium form 0 or 5 mM to 20 mM led to decrease in FAD-fluorescence beginning 10 sec after change of medium. L-leucine (10 mM), (+/-)-B-BCH (20 mM) and alpha-ketoisocaproic acid (10 mM) caused typical kinetics of FAD-fluorescence decrease. The results are interpreted to indicate rapid changes of the functional state of B-cell mitochondria induced by the above-mentioned stimulators of insulin release.

Amino Acids↗
PubMed1976

The existence of three types of acetohydroxy acid synthetase in an isoleucine-requiring mutant of Aerobacter aerogenes.

The synthesis of the three types of acetolactate synthase (EC 4.1.3.18) which are responsible for the biosynthesis os isoleucine and valine, was observed in Aerobacter aerogenes I-12, an isoleucine-requiring mutant, when grown on the four kinds of media. When the cells were grown on isoleucine-rich medium, acetolactate synthase sensitive to feedback inhibition and having an optimum pH at 8.0 was formed. By increasing the amount of potassium phosphate in the medium, the catabolite repression of the enzyme having an optimum pH at 6.0 and which is insensitive to feedback inhibition, was released. In contrast, acetolactate synthase having an optimum pH at 8.0 and insensitive to feedback inhibition was formd when isoleucine was limited, irrespective of phosphate concentrations. Two insensitive enzymes were not regulated by isoleucine, leucine and valine, although sensitive pH 8.0 enzyme was repressed by them. Thus, it may be assumed that the synthesis of insensitive pH 8.0 enzyme were repressed by limiting the amount of isoleucine is still open.

Acetolactate Synthase↗
PubMed1975

Some characteristics of two azoreductase systems in rat liver. Relevance to the activity of 2-[4'-di(2"-bromopropyl)-aminophenylazo]benzoic acid (CB10-252), a compound possessing latent cytotoxic activity.

The system involved in the reduction of 2-[4'-di(2''-bromopropyl) aminophenylazolbenzoic acid (CB10-252), an agent designed for treating primary liver cell cancer, has been demonstrated to be localised mainly in the 108 000 X g supernatant fraction of rat liver homogenate. It is also present in other organs particularly in the spleen. DAB-azoreductase as shown previously is present almost entirely in the microsomal fraction and is found in high concentration only in liver. The pH maximum for CB10-252-azoreductase implying the importance of the 2'-carboxyl group in determining substrate specificity. The use of enzyme inhibitors and other additives showed that CB10-252 WAS NOT AXANTHINE OXIDASE OR DIHYDROFOLATE REDUCTASE. Its activity was not affected by carbon monoxide, phenobarbitone (PB), or 3-methylcholanthrene (MC) pretreatment. Enhancement of the activity by ferrous ions and FAD indicated that at least part of the reduction system could involve a flavoprotein with FAD as the prosthetic group. The activity of CB10-252-azoreductase and methylred-azoreductase was reduced by menadione (vitamin K3), cyanide and propylgallate. A diaphorase preparation from pig heart reduced both CB10-252 and methylred with both NADPH- and NADH-generating systems.

Animals↗
PubMed1976

Preparation, characterization, and chemical properties of the flavin coenzyme analogues 5-deazariboflavin, 5-deazariboflavin 5'-phosphate, and 5-deazariboflavin 5'-diphosphate, 5'leads to5'-adenosine ester.

In order to facilitate interpretation of the deazaisoalloxazine system as a valid mechanistic probe of flavoenzyme catalysis, we have examined some of the fundamental chemical properties of this system. The enzymatic synthesis, on a micromole scale, of the flavin coenzyme analogues 5-deazariboflavin 5'-phosphate (deazaFMN) and 5-deazariboflavin 5'-diphosphate, 5' leads to 5'adenosine ester (deazaFAD) has been achieved. This latter synthesis is accomplished with a partially purified FAD synthetase complex (from Brevibacterium ammoniagenes), containing both phosphorylating and adenylylating activities, allowing direct conversion of the riboflavin analogue to the flavin adenine dinucleotide level. The structure of the reduced deazaflavin resulting from enzymatic and chemical reduction is established as the 1,5-dihydrodeazaflavin by proton magnetic resonance. Similarly, the C-5 position of the deazaflavins is demonstrated to be the locus for hydrogen transfer in deazaflavin redox reactions. Preparation of 1,5-dihydrodeazaflavins by sodium borohydride reduction stabilized them to autoxidation (t 1/2 approximately 40 h, 22 degrees C) although dihydrodeazaflavins are rapidly oxidized by other electron acceptors, including riboflavin, phenazine methosulfate, methylene blue, and dichlorophenolindophenol. Mixtures of oxidized and reduced deazaflavins undergo a rapid two-electron disproportionation (k = 22 M-1 S-1 0 degrees C), and oxidized deazaflavins form transient covalent adducts with nitroalkane anions at pH less than 5. Generalized methods for the synthesis of isotopically labeled flavin and deazaflavin coenzymes and their purification by adsorptive chromatography are given.

Brevibacterium↗
PubMed1976

Isolation and some properties of NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii.

NAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder0 452, 411, and 385 nm (the 411 nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119,000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen...

2,6-Dichloroindophenol↗
PubMed1976

Bacterial metabolism of resorcinylic compounds: purification and properties of orcinol hydroxylase and resorcinol hydroxylase from Pseudomonas putida ORC.

The hydroxylase activities observed in extracts of Pseudomonas putida ORC after growth on orcinol and resorcinol as sole source of carbon have been purified to homogeneity. Both enzymes were shown to be flavoproteins and to contain approximately 1 mol of FAD for each polypeptide chain, S20,W values for each enzyme are 4.1 +/- 0.1 and are independent of the presence of their aromatic substrates. Molecular weight determinations under native (approximately 68000) and denaturing (approximately 70000) conditions indicated that they are monomeric. The visible absorption spectra identical but the circular dichroic spectra of the two proteins can be distinguished. Although each protein catalyzes the NAD(P)H and O2-dependent hydroxylation of both orcinol and resorcinol, the efficiency of the transformations of the substrates by the two enzymes is radically different; furthermore resorcinol hydroxylase is much more versatile in the aromatic compounds it can utilize as substrates and effectors. Other properties of the enzymes which clearly establish their own identity include their serological characteristics and amino acid composition; the latter property is particularly evident when the quantities of valine and alanine residues are compared. The synthesis of each enzyme is also under different regulatory constraints, being controlled by the substrate used for growth.

Amino Acids↗
PubMed1976

Purification and properties of cyclopentanone oxygenase of Pseudomonas NCIB 9872.

1. Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2. The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three of four subunits of molecular weight 54000-58000. 3. The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein.

Amino Acids↗
PubMed1976

Identification and properties of 8-hydroxyflavin--adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii.

1. A new flavin prosthetic group has been isolated in pure form from the electron-transferring flavoprotein of Peptostreptococcus elsdenni. Its structure has been established as the FAD derivative of 7-methyl-8-hydroxyisoalloxazine: (see article). Proof of this structure has been obtained by chemical syntehsis of 7-methyl-8-hydroxyisoalloxazine models, and by stepwise degradation of the native compound to 7-methy-8-hydroxyalloxazine. The orange chromophore is characterized by a strong absorption band with a maximum at 472 nm (xi = 41 000 M-1 CM-1) and a pK at 4.8 due to the ionisation of the C(8)-OH group. 2. The properties of a series of functionally substituted derivatives of 8-hydroxy flavins and lumichromes have been investigated to provide a basis for interpreting the effects of pH on the spectroscopic properties of the 8-hydroxy derivatives of FAD and FMN. 3. The 8-hydroxy derivative of FAD is bound by apo-D-amino acid oxidase; the complex shows no catalytic activity. The 8-hydroxy derivative of FMN is bound by apoflavodoxin to give a complex which has catalytic activity similar to that of native flavodoxin. The complex is reversibly reduced by dithionite, first to a relatively stable semiquinone and further to the dihydroflavin form.

Binding Sites↗
PubMed1976

[Nitrates and nitrites in plants].

The formation of aminoacids and proteins from the nitrogen which enters the roots as nitra t involves a complex reaction requiring energy. The first step requires a metalloflavoprotein, the nitrate reductase and the successive intervention of NADPH, FAD and reduced molybdenum which transfers electrons to nitrate and reduces it to nitrite. The following steps involve NADPH, FAD, Copper, Iron and Manganese, the last steps of the successive reductions being ammonia, needed for the aminoacids synthesis. The activity of the different enzymes are under the dependence of the genetic equipment of the plant, of the nitrogen and oligo-element nutrition and of the different factors acting on the photosynthesis.

Amino Acids↗
PubMed1975

Affinity labeling of D-amino acid oxidase with an acetylenic substrate.

The acetylenic substrate, D-2-amino-4-pentynoic acid (D-propargylglycine), was oxidatively deaminated by hog kidney D-amino acid oxidase[EC 1.4.3.3], with accompanying inactivation of the enzyme. The flavin which was extracted by hot methanol from the inactivated enzyme was identical with authentic FAD by thin-layer chromatography and circular dichroism. The excitation spectrum of emission at 520 nm of the released flavin was very similar to the absorption spectrum of oxidized FAD. The released flavin was reduced by potassium borohydride. The apoenzyme prepared after propargylglycine treatment did not show restored D-amino acid oxidase activity on adding exogenous FAD. The absorption spectrum of this inactivated apoenzyme showed absorption peaks at 279 and 317 nm, and a shoulder at about 290 nm. These results strongly indicate that the inactivation reaction is a dynamic affinity labeling with D-propargylglycine which produces irreversible inactivation of the enzyme by a covalent modification of an amino acid residue at the active site.

Affinity Labels↗