Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.
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PMID:Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases. 2 27

Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH in the presence of molecular oxygen, an 02-stable semiquinone containing one flavin free radical per two flavins was formed, in agreement with previous work on purified trypsin-solubilized reductase. The reduction of oxidized enzyme by NADPH, and autoxidation of NADPH-reduced enzyme by air, proceeded by both one-electron equivalent and two-electron equivalent mechanisms. The reductase reduced cytochrome P-450 (from phenobarbital-treated rabbits) and cytochrome P-448 (from 3-methylcholanthrene-treated rabbits). The rate of reduction of cytochrome P-450 increased in the presence of a substrate, benzphetamine, but that of cytochrome P-448 did not.
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PMID:Studies on the microsomal mixed function oxidase system: redox properties of detergent-solubilized NADPH-cytochrome P-450 reductase. 2 10

Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.
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PMID:Characterization of human platelet glutathione reductase. 3 11

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.
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PMID:Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin. 3 75

Washed microsomes from rabbit liver reduced 1-nitrosoadamantane to N-hydroxy-1-aminoadamantane in the presence of a cofactor solution under aerobic conditions; no further reduction of the hydroxylamino metabolite to 1-aminoadamantane (amantadine) occurred. Reduced pyridine nucleotide cofactors are needed for the metabolic reduction. The rate of formation of N-hydroxy-1-aminoadamantane depended upon the microsomal protein content, the time of incubation and the concentration of 1-nitrosoadamantane incubated. The metabolic reduction occurred in air as well as under nitrogen or carbon monoxide. Cupric chloride, mercuric chloride, cysteamine, FAD, and FMN decreased significantly the C-nitroso reductase. The properties of the C-nitroso reductase differed from those of other microsomal reductive pathways.
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PMID:Metabolic reduction of 1-nitrosoadamantane by rabbit liver microsomes. Properties of a C-nitroso reductase system. 3 89

A strain of the obligate anaerobe, Eubacterium lentum, isolated from human feces, catalyzes the 21-dehydroxylation of 11-deoxycorticosterone to progesterone. A quantitative radiochromatographic assay was developed to measure 21-dehydroxylase activity in cell extracts. Maximum enzyme activity in cell extracts required both a reduced pyridine nucleotide and an oxidized flavin coenzyme. However, photochemically reduced flavin (FMNH2) could replace the requirement for NAD(P)H plus oxidized flavin. NAD(P)H : flavin (either FMN or FAD) oxidoreductase activity was detected spectrophotometrically in cell extracts assayed under anaerobic conditions. 21-Dehydroxylase was active from pH 5.4 to 8.5 with an apparent optimum between 6.4 and 6.8 using mixtures of NADH plus FMN as coenzymes. The substrate concentration at half-maximal reaction velocity was 8.0 microM and a specific acitivity of 5.8 nmol [3H]progesterone formed . h-1 . mg-1 protein was determined using [3th]deoxycorticosterone as substrate. Atabrine, rotenone, acriflavin, and 2,4-dinitrophenol (all at 1 mM) inhibited 21-dehydroxylase activity in cell extracts by 25, 24, 35 and 84%, respectively. These results suggest that 21-dehydrogenase may be coupled to a NAD(P)H : flavin oxidoreductase system in E. lentum.
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PMID:Characterization of a C21 neutral steroid hormone transforming enzyme, 21-dehydroxylase, in crude cell extracts of Eubacterium lentum. 3 50

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.
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PMID:8-Mercaptoflavins as active site probes of flavoenzymes. 3 28

The uptake hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) from the bacteroids of soybean root nodules infected with Rhizobium japonicum 110 has been purified and characterized. Bacteroids were prepared, then broken by sonication. The particulate enzyme was solubilized by treatment with Triton X-100 and further purified by polyethylene glycol fractionation, DEAE-cellulose and Sephadex G-100 chromatography. The specific activity has been increased 196-fold to 19.6 units/mg protein. The molecular weight is 63 300 as determined by gel filtration and 65 300 as determined by SDS-polyacrylamide gel electrophoresis, indicating that the enzyme is a monomer. The enzyme is O2 sensitive, with a half-life of 70 min when exposed to air. The pH optimum of the solubilized enzyme is near 5.5; the Km for H2 is 1.4 microM. Suitable electron acceptors are methylene blue, ferricyanide, 2,6-dichlorophenolindophenol, and cytochrome c. Benzyl viologen is reduced slowly; methyl viologen, NAD(P)+, FAD, FMN, and O2 are not reduced. The optimum temperature for activity is 65-70 degrees C with an activation energy of 9.2 kcal. H2 evolution by the enzyme has been demonstrated. The hydrogenase is well-suited to function in an environment where all the available H2 is generated in situ.
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PMID:Purification and properties of the particulate hydrogenase from the bacteroids of soybean root nodules. 4 Jun 1

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C1--C10 tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD+, NADP+, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.
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PMID:Microbial oxidation of methane and methanol: purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora. 4 58

NAD(P)H: FMN oxidoreductase (flavin reductase) couples in vitro to bacterial luciferase. This reductase, which is also postulated to supply reduced flavin mononucleotide in vivo as a substrate for the bioluminescent reaction, has been partially purified and characterized from two species of luminous bacterial. From Photobacterium fischeri the enzyme has a M. W. determined by Sephadex gel filtration, of 43,000 and may have a subunit structure. The turnover number at 20 degrees C, based on a purity estimate of 20 percent, is 1.7 times 10-4 moles of NADH oxidized per min per mole of reductase. The reductase isolated from Beneckea harveyi has an apparent molecular weight of 23,000; its purity was too low to permit estimation of specific activity. Using a spectrophotometric assay at 340 nm with the P. fischeri reductase, both NADH (Km, 8 times 10-5 M) and NADPH (Km, 4 times 10-4 M) were enzymatically oxidized, the Vmax with NADH being approximately twice that of NADPH. Of the flavins tested in this assay, only FMN (Km, 7.3 times 10-5 M) and FAD (Km, 1.4 times 10-4 M) were effective, FMN having a Vmax three times that of FAD. In the coupled assay, i.e., measuring the bioluminescence intensity of the reaction with added luciferase, the optimum FMN concentration was nearly 100 times less than in the spectrophotometric assay. The studies reported suggest the existence of a functional reductase-luciferase complex.
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PMID:Flavin mononucleotide reductase of luminous bacteria. 4 4


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