Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To provide for bioluminescence measurements of the enzymatic activities of dehydrogenases, disturbing contaminants were removed from a bacterial luciferase extract by chromatography, using Blue Sepharose CL-6B, a cross-linked agarose to which Cibacrone Blue F3G-A is covalently attached. This compound has a strong affinity to the dinucleotide fold, which is a region in enzymes binding NAD(H) or NADP(H). In contrast to the absorbed dehydrogenases, both luciferase and oxidoreductase were easily eluted and appeared close to the main bulk of UV-absorbing but analytically less important material. A rapid recording of the elution of luciferase was accomplished with a new electrochemical bioluminescence assay. Due to this and the early elution of the desired material, it could be chromatographed, recognized and collected in less than two hours. Thereby the light-yielding capacity of the sensitive material was well preserved. For bioluminescence assay solutions composed of pooled oxidoreductase-luciferase fractions, FMN and a long chain aldehyde were prepared and supplemented with NAD+ and either lactate, malate or 3-hydroxybutyrate. The analyses were carried out in a single step performance by adding the enzyme sample to the luciferase solution. Minute amounts of lactate dehydrogenase, malate dehydrogenase and 3-hydroxybutyrate dehydrogenase yielded a linear light response permitting assay in the lower part of the femtomole region. In case a dehydrogenase does not occur as a contaminant of a commercial luciferase preparation, purification with Cibacrone Blue can be omitted as demonstrated for glucose-6-phosphate dehydrogenase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Single-step bioluminescence analyses of enzymes, using Cibacrone Blue chromatography for removal of interfering dehydrogenases. 663 14

Reduced flavin mononucleotide dependent luciferase (EC 1.14.14.3) from Photobacterium fischeri has been used to measure NAD(P) dependent enzymes in submicrogram quantities of tissue homogenates and isolated structures of rabbit and human kidney. The procedure for measuring NAD(P)H was optimized, with internal standardization, to give a linear constant signal between 1 and 100 pmol. This method was applied to the measurement of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) along the various structures of the rabbit nephron, microdissected from fresh tissue slices. Blank and recovery measurements were performed on each structure, and enzyme catalytic activities were calculated on the basis of tubular length and protein. Glucose-6-phosphate dehydrogenase was found to be present in all nephron structures with highest catalytic activities in glomeruli, thin limbs of Henle's loop and medullary collecting tubules. Lowest catalytic activities were detected in the pars recta of proximal tubules, the distal convoluted and the connecting tubule. The mitochondrial 3-hydroxybutyrate dehydrogenase exhibited a different distribution pattern: Highest catalytic activities were found in the cortical ascending limbs of Henle's loop, the proximal and distal convoluted tubule. An unusual internephron heterogeneity for this enzyme was found in the distal convoluted tubule. Catalytic activities in thin limbs of Henle's loop, the medullary ascending limb, and the cortical and medullary collecting tubule were not significantly different from blank activities. The results obtained in isolated nephron segments agreed with those calculated from cortex and medullary homogenates. In a preliminary experiment on human kidney it could be demonstrated that the procedures can be applied to fresh human biopsy samples. The bioluminescence method offers several advantages (simplicity, rapidity) in comparison with the classical techniques used for ultramicro analysis of tissue enzymes (enzymatic cycling, radiochemical tests). The results confirm the biochemical heterogeneity of nephron structures and allow conclusions about species dependent functional differences of the pentose-phosphate cycle and renal ketone body metabolism along the nephron.
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PMID:Bioluminescence procedures for the measurement of NAD(P) dependent enzyme catalytic activities in submicrogram quantities of rabbit and human nephron structures. 671 53

Vibrio harveyi aldehyde dehydrogenase, which catalyzes the oxidation of long chain aliphatic aldehydes to acids, has been discovered to have both acyl-CoA reductase and thioesterase activities. Tetradecanoyl-CoA was reduced to tetradecanal in the presence of NAD(P)H, as monitored by the stimulation of luciferase activity by the aldehyde product (acyl-CoA reductase). In the absence of NADPH, [3H]tetradecanoyl-CoA was hydrolyzed to the hexane-soluble fatty acid (thioesterase). Inhibition data with N-ethylmaleimide suggest that a single active site on aldehyde dehydrogenase is responsible for all three enzymatic activities. The acyl-CoA reductase activity was maximal at low NADPH concentration (about 1 microM), whereas much higher concentrations of NADH (greater than 100-fold) were required for optimal activity. Further increases in NADPH or NADH concentrations inhibited both the acyl-CoA reductase and thioesterase reactions. On the basis of the specificity of aldehyde dehydrogenase for NADP(H), an improved purification procedure employing affinity chromatography on 2', 5'-ADP-Sepharose is described. Although fatty acid reductase activity could not be reconstituted, aldehyde dehydrogenase specifically stimulated the rate of acylation of the acyl protein synthetase component from the Photobacterium phosphoreum fatty acid reductase system. This observation, combined with the partial reversal of aldehyde oxidation described above, suggests a possible role for aldehyde dehydrogenase in aldehyde biosynthesis for the luminescent reaction in V. harveyi.
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PMID:Vibrio harveyi aldehyde dehydrogenase. Partial reversal of aldehyde oxidation and its possible role in the reduction of fatty acids for the bioluminescence reaction. 672 83

A new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.
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PMID:Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus. 673 52

Three different NAD(P)H-FMN reductases were extracted from Beneckea harveyi MB-20 cells and separated by DEAE-Sephadex A50 column chromatography. Further purification was achieved by affinity chromatography. In determinations of Km values for NADH, NADPH, and FMN, these three reductases exhibited different specificities and kinetic parameters. One reductase utilizes NADH, whereas a second one utilizes NADPH as the preferred substrate. The third, a newly described reductase species, exhibits about the same reaction rates with NADH and NADPH. The reaction mechanisms of the three enzyme forms have been deduced by steady state kinetic analysis. The highly pure (based on gel electrophoresis) NADPH-FMN reductase still exhibited a low (approximately 2%) activity for NADH, which activity was increased upon storage at 4 degrees but suppressed completely by the replacement of the phosphate buffer with sodium citrate buffer. This high specificity of NADPH-FMN reductase for NADPH under these conditions is useful for the assay of NADPH, notably in systems coupled to bacterial luciferase.
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PMID:Specificities and properties of three reduced pyridine nucleotide-flavin mononucleotide reductases coupling to bacterial luciferase. 698 Oct 58

It was shown that the luminescence of extracts prepared from luminous bacteria is stimulated by NADPH and ATP without FMN or long-chain aliphatic aldehydes, which are routinely used for producing luminescence of extracts from luminous bacteria in vitro. In these extracts an aldehyde factor, a natural analog of aliphatic aldehydes, is synthesized. The enzymatic system involved in maintaining the luminescence of NADPH and ATP is probably not coupled with the functioning of NAD(P)H: FMN oxidoreductase which has been supposed to participate in luminescence processes in vivo. It is assumed that both aldehyde factor synthesis and reduction of endogenous analog of FMN, natural substrates of bacterial luciferase, are due to the functioning of the same metabolic pathway.
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PMID:[NADPH- and ATP-dependent luminescence of extracts from luminous bacteria]. 715 22

Mineralocorticoid receptors (MRs) are nonselective in vitro, binding corticosterone, cortisol, and aldosterone with similar affinity. In the distal nephron in vivo, MRs are selectively activated by aldosterone despite much higher glucocorticoid levels. This has been suggested to reflect the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which catalyzes rapid inactivation of corticosterone to 11-dehydrocorticosterone (cortisol to cortisone). However, cellular models of this effect have not been reported, and a recent study suggested that properties intrinsic to MR contribute to aldosterone selectivity. We have screened clonal mammalian cell lines for 11 beta-HSD activity. Pig kidney epithelial LLC-PK1 cells expressed by far the greatest 11 beta-HSD activity. In cell homogenates, this was NAD-dependent, with Km for corticosterone of 34.4 nM and cortisol of 89.7 nM. Intact LLC-PK1 cells showed similar apparent Km for corticosterone (13.9 nM) and cortisol (79.4 nM); only 11 beta-dehydrogenation was detected. These biochemical data indicate the expression of the type 2 isoform, 11 beta-HSD2. Using primers to conserved regions of 11 beta-HSD2, a reverse transcriptase-polymerase chain reaction product was obtained from LLC-PK1 cell RNA. Sequence analysis revealed close homology to previously cloned 11 beta-HSD2 cDNAs from several species. LLC-PK1 cell 11 beta-HSD activity was inhibited by carbenoxolone (IC50 approximately 10(-8) M) and high concentrations of estradiol or progesterone (10(-7) and 10(-6) M), but was induced at lower estradiol concentrations (10(-8) and 10(-9) M). To examine whether the 11 beta-HSD2 activity in LLC-PK1 cells regulates corticosterone access to MR, cells were transfected with the corticosteroid-inducible mouse mammary tumor virus long terminal repeat-luciferase reporter construct. Cell transfection by a lipofection method did not alter 11 beta-HSD activity in LLC-PK1 cells. LLC-PK1 cells expressed low levels of MR (13.9 fmol/mg protein, dissociation constant (Kd) 0.3 x 10(-9) M for aldosterone) and glucocorticoid receptors (GR; 18.5 fmol/mg protein, Kd 0.3 x 10(-9) M for dexamethasone). Transfection with mouse mammary tumor virus long terminal repeat-luciferase reporter construct alone suggested that the endogenous levels of MR and GR were insufficient to affect transcription. However, cotransfection of LLC-PK1 cells with pRShMR, an MR expression plasmid, allowed at least 50-fold induction of luciferase with 10(-8) M aldosterone; the ED50 0.3 x 10(-9) M closely reflects the in vitro affinity of MR for aldosterone. Corticosterone only weakly induced luciferase (maximum of 6-fold induction).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:LLC-PK1 cells model 11 beta-hydroxysteroid dehydrogenase type 2 regulation of glucocorticoid access to renal mineralocorticoid receptors. 758 9

Although used for analytical purposes for more than 40 years it is only recently that biochemiluminescence (BCL) has found widespread acceptance. Methods employing BCL reactions now play an important role in biomedical research and laboratory medicine. The main attractions for the assay technology include exquisite sensitivity (attomole-zeptomole), high selectivity, speed and simplicity. In biomedical research, the most important applications of BCL are: (1) to estimate microbial numbers and to assess cellular states (e.g., after exposure to antibiotic or cytotoxic agents) and in reporter gene studies (firefly luciferase gene); (2) NAD(P)H involved in redox/dehydrogenase studies using Vibrio luciferase complex; (3) BCL labels and CL detection of enzyme labels in immunoassays are the most widespread routine application for this technology. BCL enzyme immunoassays represent the most active area of development, e.g., enhanced BCL method for peroxidase and BCL assays for alkaline phosphatase labels using adamantyl 1,2-dioxetane.
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PMID:Biochemiluminescence and biomedical applications. 769 95

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.
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PMID:NAD(P)H-flavin oxidoreductase from the bioluminescent bacterium, Vibrio fischeri ATCC 7744, is a flavoprotein. 803 96

NAD(P)H-flavin oxidoreductases (flavin reductases) from luminous bacteria catalyze the reduction of flavin by NAD(P)H and are believed to provide the reduced form of flavin mononucleotide (FMN) for luciferase in the bioluminescence reaction. By using an oligonucleotide probe based on the partial N-terminal amino acid sequence of the Vibrio harveyi NADPH-FMN oxidoreductase (flavin reductase P), a recombinant plasmid, pFRP1, was obtained which contained the frp gene encoding this enzyme. The DNA sequence of the frp gene was determined; the deduced amino acid sequence for flavin reductase P consists of 240 amino acid residues with a molecular weight of 26,312. The frp gene was overexpressed, apparently through induction, in Escherichia coli JM109 cells harboring pFRP1. The cloned flavin reductase P was purified to homogeneity by following a new and simple procedure involving FMN-agarose chromatography as a key step. The same chromatography material was also highly effective in concentrating diluted flavin reductase P. The purified enzyme is a monomer and is unusual in having a tightly bound FMN cofactor. Distinct from the free FMN, the bound FMN cofactor showed a diminished A375 peak and a slightly increased 8-nm red-shifted A453 peak and was completely or nearly nonfluorescent. The Kms for FMN and NADPH and the turnover number of this flavin reductase were determined. In comparison with other flavin reductases and homologous proteins, this flavin reductase P shows a number of distinct features with respect to primary sequence, redox center, and/or kinetic mechanism.
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PMID:Vibrio harveyi NADPH-flavin oxidoreductase: cloning, sequencing and overexpression of the gene and purification and characterization of the cloned enzyme. 820 32


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