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)

We have shown that the isolated sarcoplasmic reticulum from rabbit slow muscle contains cytochrome b5 which can be reduced via a flavoprotein, with FAD as the prosthetic group. In the presence of NADH and oxygen, these sarcoplasmic reticulum membranes can convert stearyl-CoA to oleyl-CoA, similarly to liver endoplasmic reticulum membranes. However, the stearyl-CoA desaturase system is virtually lacking in fast muscle sarcoplasmic reticulum. The data suggest that these differences between fast and slow twitch muscle may be related to the characteristic fatty acid composition of phospholipids and the function of the sarcoplasmic reticulum.
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PMID:Evidence for the presence of the stearyl-CoA desaturase system in the sarcoplasmic reticulum of rabbit slow muscle. 3 16

Glutathione reductase from the liver of DBA/2J mice was purified to homogeneity by means of ammonium sulfate fractionation and two subsequent affinity chromatography steps using 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose and N6-(6-aminohexyl)-adenosine 2',5'-biphosphate-Sephadex columns. A facile procedure for the synthesis of 8-(6-aminohexyl)-amino-2'-phospho-adenosine diphosphoribose is also presented. The purified enzyme exhibits a specific activity of 158 U/mg and an A280/A460 of 6.8. It was shown to be a dimer of Mr 105000 with a Stokes radius of 4.18 nm and an isoelectric point of 6.46. Amino acid composition revealed some similarity between the mouse and the human enzyme. Antibodies against mouse glutathione reductase were raised in rabbits and exhibited high specificity. The catalytic properties of mouse liver glutathione reductase have been studied under a variety of experimental conditions. As with the same enzyme from other sources, the kinetic data are consistent with a 'branched' mechanism. The enzyme was stabilized against thermal inactivation at 80 degrees C by GSSG and less markedly by NADP+ and GSH, but not by NADPH or FAD. Incubation of mouse glutathione reductase in the presence of NADPH or NADH, but not NADP+ or NAD+, produced an almost complete inactivation. The inactivation by NADPH was time, pH and concentration dependent. Oxidized glutathione protected the enzyme against inactivation, which could also be reversed by GSSG or other electron acceptors. The enzyme remained in the inactive state even after eliminating the excess NADPH. The inactive enzyme showed the same molecular weight as the active glutathione reductase. The spectral properties of the inactive enzyme have also been studied. It is proposed that auto-inactivation of glutathione reductase by NADPH and the protection as well as reactivation by GSSG play in vivo an important regulatory role.
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PMID:Mouse-liver glutathione reductase. Purification, kinetics, and regulation. 3 57

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

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.
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PMID:Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes. 12 Oct 57

The properties of electron transport systems present in soluble and particulate fractions of spores of Bacillus megaterium KM?HAVE BEEN COMPARED WIth those of similar fractions prepared from exponential-phase vegetative cells of this organism. The timing and localization of modifications of the electron transport system occurring during sporulation have been investigated by using a system for separating forespores from mother cells at all stages during development [8]. Spore membranes contained cytochromes a + a3, and o at lower concentrations than in vegetative membranes, and in addition cytochrome c, which was not found in exponential-phase vegetative membranes. An NADH oxidase activity of similar specific activity was found in both spore and vegetative membranes but DL-glycerol 3-phosphate and L-malate oxidase activities were found only in vegetative membranes. A soluble NADH oxidase of low specific activity was found in spores and vegetative cells which probably involves a flavoprotein reaction with oxygen because the activity was stimulated by FAD or FMN and difference spectra of concentrated soluble fractions showed spectra typical of a flavoprotein. Particulate NADH oxidase was sensitive to all classical inhibitors of electron transport tested whereas soluble NADH oxidase was insensitive to many of these inhibitors. Cytochrome c was formed between stage I and II of sporulation and this coincided with a five-fold increase in NADH-cytochrome c reductase activity. Forespore membranes had lower contents of cytochromes than sporangial cell membranes but similar levels of NADH and L-malate oxidases; DL-glycerol 3-phosphate oxidase activity could not be detected in either membranes by stage III of sporulation. This characterization of spore electron transport systems provides a basis for suggestions concerning initial metabolic events during spore germination and the effect of a number of germination inhibitors.
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PMID:Morphogenesis of the membrane-bound electron-transport system in sporulating Bacillus megaterium KM. 12 54

Neocarzinostatin, a protein antibiotic with anti-tumor activity was found to place single-strand scissions in DNA in an in vitro reaction. The drug's cutting activity was strongly dependent on the presence of 2-mercaptoethanol or dithiothreitol but some cutting did take place in the absence of reducing agent at very high drug levels and prolonged incubation. The requirement for reducing agents could not be replaced with NAD+, FAD, NADH or H2O2 and the strand-scission reaction was not affected by Mg2+, EDTA or intercalating agents. Similar profiles of heat-inactivation of neocarzinostatin were found whether activity was measured by the scission of DNA strand either in vitro or in HeLa cells treated with the drug. Furthermore, both of these parameters corresponded closely with the ability of the modified drug to inhibit DNA synthesis and growth of HeLa cells. By column isoelectric focusing it was shown that all four activities are associated with the same protein band (pH 3.28). From these data we conclude that the cytotoxic activity of neocarzinostatin and the nicking of DNA strands in vitro appear to reside in the same protein.
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PMID:Single-strand nicking of DNA in vitro by neocarzinostatin and its possible relationship to the mechanism of drug action. 13 67

NADH:nitrate reductase (EC 1.6.6.1) from Chlorella vulgaris has been purified 640-fold with an over-all yield of 26% by a combination of protamine sulfate fractionation, ammonium sulfate fractionation, gel chromatography, density gradient centrifugation, and DEAE-chromatography. The purified enzyme is stable for more than 2 months when stored at minus 20 degrees in phosphate buffer (pH 6.9) containing 40% (v/v) glycerol. After the initial steps of the purification, a constant ratio of NADH:nitrate reductase activity to NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities was observed. One band of protein was detected after polyacrylamide gel electrophoresis of the purified enzyme. This band also gave a positive stain for heme, NADH dehydrogenase, and reduced methyl viologen:nitrate reductase. One band, corresponding to a molecular weight of 100, 000, was detected after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme contains FAD, heme, and molybdenum in a 1:1:0.8 ratio. One "cyanide binding site" per molybdenum was found. No non-heme-iron or labile sulfide was detected. From a dry weight determination of the purified enzyme, a minimal molecular weight of 152, 000 per molecule of heme or FAD was calculated. An s20, w of 9.7 S for nitrate reductase was found by the use of sucrose density gradient centrifugation and a Stokes radius of 89 A was estimated by gel filtration techniques. From these values, and the assumption that the partial specific volume is 0.725 cc/g, a molecular weight of 356, 000 was estimated for the native enzyme. These data suggest that the native enzyme contains a minimum of 2 molecules each of FAD, heme, and molybdenum and is composed of at least three subunits.
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PMID:Reduced nicotinamide adenine dinucleotide-nitrate reductase of Chlorella vulgaris. Purification, prosthetic groups, and molecular properties. 16 92

Labelling studies with N-ETHYLMALEIMIDE SHOW THAT EITHER IN THE PRESENCE OF Mg2+, thiamine pyrophosphate (TPP) and pyruvate or in the presence of NADH the overall activity of the pyruvate dehydrogenase complex from Azotobacter vinelandii is inhibited without much inhibition of the partial reactions. The complex undergoes a conformational change upon incubation with NADH. The inhibition by bromopyruvate is less specific. Specific incorporation of a fluorescent maleimide derivative was observed on the two transacetylase isoenzymes. Binding studies with a similar spin label analogue show that 3 molecules/FAD are incorporated by incubation of pyruvate, Mg2+ and TPP, whereas 2 molecules/FAD are incorporated via incubation with NADH. The spin label spectra support the idea that in the complex the active centres of the component enzymes are connected by rapid rotation of the lipoyl moiety. Three acetyl groups are incorporated in the complex by incubation with [2-14C]pyruvate. Time-dependent incorporation supports the view that the two transacetylase isoenzymes react in non-identical ways with the pyruvate dehydrogenase components of the complex. The results show that the complex contains 2 low-molecular-weight transacetylase molecules and 4 molecules of the high-molecular-weight isoenzyme. Mn2+-binding studies show that the complex binds 10 ions, with different affinities. 2 Mn2+ ions are bound with a 20-fold higher affinity than the remaining 8 Mn2+ ions. The latter 8 ions bind with equal affinities and are thought to reflect binding to the pyruvate dehydrogenase components of the complex. It is concluded that the complex contains 8 pyruvate dehydrogenase molecules, 4 high-molecular-weight transacetylase molecules, 2 low-molecular-weight transacetylase molecules and 1 dimeric (2-FAD-containing) symmetric molecule of lipoamide dehydrogenase. Evidence comes from pyruvate-dependent inactivation and labelling studies that the pyruvate dehydrogenase components contain either an - SH group or an S-S bridge which participates in the hydroxyethyl transfer to the transacetylase components.
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PMID:The pyruvate-dehydrogenase complex from Azotobacter vinelandii. 3. Stoichiometry and function of the individual components. 17 36

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.
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PMID:Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes. 17 49

Biosynthesis of nervonic acid by enzymatic elongation of erucyl-CoA has been studied in mouse brain microsomes. The substrate and cofactor requirements have been measured. Malonyl-CoA and reduced nicotine-adenine-dinucleotide phosphate are required, but not FMN, FAD or NADH. The effect of protein concentration, incubation time, ATP and CoA has been determined; the reaction products were checked by gas-liquid chromatography with automatic counting of the eluate. Very little activity was found in hydroxylated fatty acids. In the presence of phosphotransacetylase (which impedes the de novo microsomal system), the main reaction product was nervonic acid. It is concluded that nervonic acid is biosynthesised by elongation using a two-carbon unit from malonyl-CoA. The same enzyme biosynthesises saturated and mono-unsaturated very long chain fatty acids. The elongation capacity of "quaking" microsomes is reduced to 30% of the normal value with both erucyl-CoA and behenyl-CoA. Elongation of trans isomer (brassidyl-CoA) and poly-unsaturated homologue (clupanodonyl-CoA) was compared to elongation of erucyl-CoA in both normal and mutant mice. Both unsaturated acyl-CoAs are elongated under the same conditions as erucyl-CoA in brain: the poly-unsaturated acyl-CoA is elongated more actively than the mono-unsaturated acyl-CoA in the mutant.
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PMID:Nervonic acid biosynthesis by erucyl-CoA elongation in normal and quaking mouse brain microsomes. Elongation of other unsaturated fatty acyl-CoAs (mono and poly-unsaturated). 17 48


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